TITLE: Single-fill cystometry at varying bladder infusion rates in urethane anesthetized rats
AUTHORS: Daniel J Jaskowak, Zachary C Danziger

[DESCRIPTION]
Cystometry is a common method to observe the function of the lower urinary tract (LUT) in in vivo animal models where an infusion pump fills the bladder at a set rate with physiological saline until it evokes a bladder contraction. This protocol was created to determine if the rate of bladder infusion impacts the bladder volume or pressure that is coincident with the onset of cystometry induced bladder contractions. The effect of infusion rate on LUT reflex activation was investigated in 25 female Sprague-Dawley rats. Rats were anesthetized through subcutaneous injection of 1.2 g/kg urethane. A catheter in series with a pressure transducer and compter-controlled infusion pump was inserted into the dome of the bladder. Silver electrode wires were inserted into the external urethral sphincter percutaneously for electromyography. Each animal's bladder capacity was estimated prior to data collection with a series of 3-5 initial fill-to-void cycles. A subset of bladder infusion rates tested were computed as a percentage of this initial bladder capacity estimate (i.e., relative rates) to ensure the full infusion rate range was sampled across all bladder sizes.  The remaining rates used were drawn from a predetermined set without reference to bladder size and selected to span a very broad range. The predetermined rates fell into three categories, 1) those similar to the average rate of fluid added from the ureters, 2) typical infusion rates used in prior rat cystometry studies, or 3) extremely high infusion rates. Infusion rates were sampled differently in different groups of animals to maximize the amount of overall infusion rates tested. A set of 10 infusion rates composed of relative and predefined rates were randomly presented during experiments such that each infusion rate was used at least 3 times. 

[STEPS]
SECTION: Animal Preparation
1. Urethane Anesthesia Protocol
SECTION: Animal Preparation
2. Suprapubic bladder catheterization
SECTION: Animal Preparation
1.1. Anesthetize rat via inhalation of isoflurane
SECTION: Animal Preparation
1.2. Weigh Animal
SECTION: Animal Preparation
1.3. Use animal weight to determine urethane dosage (1.2 g of Urethane per kg of body weight [BW]). We use a 0.2 g/mL solution. Example calculation: 0.250 kg BW * 1.2 g/kg BW ÷ 0.2 g/mL urethane solution = 1.5 mL urethane dose.
SECTION: Animal Preparation
1.4. Subcutaneous injection of urethane at the lower back towards to the midline of the body.
SECTION: Animal Preparation
1.5. Monitor toe-pinch reflex every half hour to determine depth of anesthesia. 
SECTION: Animal Preparation
1.6. Supplementary subcutaneous doses of urethane (0.1 g/kg BW) were administered if needed in 30-minute intervals following initial injection.
SECTION: Animal Preparation
2.1. Make a midline incision at the lower abdomen of the rat to open the pelvic cavity.
SECTION: Animal Preparation
2.2. Make a small incision in the dome of the bladder.
SECTION: Animal Preparation
2.3. Insert catheter (polyethylene tubing, PE-90) with a flared tip into the bladder dome. 
SECTION: Animal Preparation
2.4. Secure catheter with purse string suture to the bladder dome grabbing a minimal amount of bladder tissue. 
SECTION: Animal Preparation
2.5. Fill bladder with infusion pump to ensure that there is a water tight seal around the catheter.
SECTION: Animal Preparation
2.6. Suture the rat's abdomen together.
SECTION: Animal Preparation
3. Place electrode wires percutaneously into the external urethral sphincter
SECTION: Bladder Capacity Estimation
5. Perform 3 to 5 single cystometrograms.
SECTION: Bladder Capacity Estimation
5.2. Empty the bladder.
SECTION: Animal Preparation
4. Lay rat prone with urethral meatus positioned over voided volume collection cup.
SECTION: Determination of Infusion rates
6. Relative Infusion Rates (Qi_rel)
SECTION: Determination of Infusion rates
6.1. Calculate the average bladder capacity (BC_avg) from the bladder capacity estimation procedure.
SECTION: Determination of Infusion rates
6.2. Add 0.19 mL to average bladder capacity (BC_avg'=BC_avg+0.19)to account for bladder capacity drift in preliminary experiments
SECTION: Determination of Infusion rates
6.3. The relative infusion rates (Qi_rel) used in the study were calculated by the following equation: Qi_rel=BC_avg'/300*R, where 300 is a fill duration in seconds and R is a 1x5 matrix written as [0.25 0.75 1.25 1.75 2.25]. 
SECTION: Determination of Infusion rates
6.4. There were group differences in the infusion rates used during an experiment which allowed us to expand sampling of infusion rates. We show here how the matrix R changed with each group.
subject experimental group-G1: R=[0.25 0.75 1.25 1.75 2.25]
subject experimental group-G2: R=[0.75 1.25 1.75 2.25]
subject experimental group-G3: R=[1.25 1.75 2.25]
subject experimental group-G4: R=[1.25 1.75 2.25]
SECTION: Determination of Infusion rates
7. Predefined Infusion Rates  (Qi_pre)
SECTION: Determination of Infusion rates
8. Pseudorandom order of experiment infusion rates (Qi_exp)
SECTION: Determination of Infusion rates
7.1. We wanted to sample the infusion rates that are suggested for cystometry in rats.1,2 The range includes infusion rates of 2.4-10.8 ml/hr. We sampled infusion rates of 2.4, 3.93, 5, 6.86, and 10.8 ml/hr. 
SECTION: Determination of Infusion rates
7.2. Larger infusion rates were selected to ensure that there was no "upper bound" where the bladder capacity would start to decrease. We sampled infusion rates of 16.2, 21.6, 32.7, and 65.5 ml/hr.
SECTION: Determination of Infusion rates
7.3. The rates in steps 7.1 and 7.2 are larger than the average addition of fluid from the ureters per hour. Therefore, rates of 0.92 and 1.3 ml/hr were sampled.
SECTION: Determination of Infusion rates
7.4. Let Qi_pre be a matrix of predefined infusion rates. Qi_pre changed with each group to expand the sampling of infusion rates. 
subject experimental group-G1: Qi_pre=[2.4 3.93 5 6.86 10.8]
subject experimental group-G2: Qi_pre=[3.93 5 6.86 10.8 16.2 21.6]
subject experimental group-G3: Qi_pre=[0.92 1.3 10.8 16.2 21.3 32.7 65.5]
subject experimental group-G4: Qi_pre=[2.4 3.93 5 6.86 10.8 32.7 65.5]
SECTION: Determination of Infusion rates
8.1. Let Qi_exp be a 1x10 matrix of the infusion rates used in the experiment. Each group had 10 infusion rates composed of Qi_rel and Qi_pre. 
subject experimental group-G1: Qi_exp=[2.4 3.93 5 6.86 10.8 0.25*Qi_rel 0.75*Qi_rel 1.25*Qi_rel 1.75*Qi_rel 2.25*Qi_rel]
subject experimental group-G2: Qi_exp=[3.93 5 6.86 10.8 16.2 21.6 0.75*Qi_rel 1.25*Qi_rel 1.75*Qi_rel 2.25*Qi_rel]
subject experimental group-G3: Qi_exp=[0.92 1.3 10.8 16.2 21.3 32.7 65.5 1.25*Qi_rel 1.75*Qi_rel 2.25*Qi_rel]
subject experimental group-G4: Qi_exp=[2.4 3.93 5 6.86 10.8 32.7 65.5 1.25*Qi_rel 1.75*Qi_rel 2.25*Qi_rel]
SECTION: Determination of Infusion rates
8.2. The matrix Qi_exp was replicated 7 times. Each replicate of Qi_exp was pseudorandomly ordered.  The pseudorandomly ordered replicates of Qi_exp were appended together into a 1x70 matrix. Infusion rates were presented according to the index in the 1x70 matrix.  
SECTION: Data Collection
9. Experiment Sessions
SECTION: Data Collection
9.1. A session was a set of five cystometrograms. For each cystometrogram, the bladder was emptied prior to the infusion pump turning on. The infusion pump was turned on and the bladder filled until a reflex voiding contraction occurred. The pump was turned off and the residual volume was collected.  Between cystometrograms of the same session the rest was ~1 minute. 
SECTION: Bladder Capacity Estimation
5.5. Rest for 20 minutes between cystometrograms.
SECTION: Data Collection
9.2. Between sessions there was a 15 minute rest period.
SECTION: Data Collection
9.3. Sessions were continued until there were at least 3 cystometrograms for every infusion rate tested for an individual animal (at least 30 cystometrograms).
SECTION: Bladder Capacity Estimation
5.4. Turn the pump off, measure the residual volume and voided volume.
SECTION: Bladder Capacity Estimation
5.3. Set infusion pump rate to 5 ml/hr and fill until a voiding contraction occurs.
SECTION: Bladder Capacity Estimation
5.1. Connect infusion pump to MATLAB. Pump commands (on, off, and setting the infusion rate) were executed from a computer.