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Materials and Methods: expanded version

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Subjects

This study was conducted with the approval of the local IRB. Subjects signed written informed consent prior to enrollment. Subjects were adults of age = 20 y, either sex, and either handedness. Exclusion criteria included obesity, mobility or mentation problems that precluded being scanned; history of claustrophobia; history or suspicion of implanted metal or electronic object; overt neurological disease; spinal cord injury or malformation resulting in obvious deficit; current urinary tract infection; history of pelvic irradiation; or bladder cancer. No subject was taking antimuscarinic medication for bladder problems. Half of the subjects demonstrated detrusor overactivity (involuntary bladder contraction) during prior urodynamics[22] and gave a corresponding history (precipitant voiding, urge incontinence, increased voiding frequency and/or nocturia). The other half of the subjects demonstrated no detrusor overactivity, in spite of provocation, and denied any such symptoms. Potential subjects who did not fit either of these categories were not included. An advantage of this procedure was that subjects had become accustomed to catheterization before scanning and so were not greatly troubled by the urethral catheters, which may introduce artifacts into brain activity.[15] None of the subjects had overt cognitive impairment, but a test sensitive to slight changes in mentation was performed (Hopkins test[23] of recall of a word list after a delay to perform a trailmaking task[24]).

Measurements during scanning

To monitor bladder behavior during fMRI, intravesical pressure was recorded. It proved adequate to recognize or rule out detrusor contractions in subjects lying quietly in the scanner, without measurement of abdominal pressure. Subjects voided before entering the scanner and were catheterized with 2 soft (8 French gauge) catheters, one to record the intravesical pressure and one to fill and empty the bladder. The bladder was drained of any residual urine. Subjects then lay supine in the scanner (GE Signa with 3T magnet), on a pad to absorb possible leakage. The catheters were connected via two 10-m long saline-filled Nalgene tubes (ID 3 mm, OD 6 mm) to urodynamic equipment (Laborie Avanti) situated in the control room, outside the strong magnetic field of the scanner. Preliminary bench tests had shown that these tubes transmitted typical bladder pressure signals without significant damping. Intravesical pressure was zeroed to atmosphere and the transducer placed at the level of the symphysis pubis, albeit in a different room. The filling tube was connected to a reversible peristaltic pump (Cole-Parmer Masterflex) via a pressure transducer that provided a signal to indicate the direction of flow and its timing. In most studies, part of the filling tube was immersed in warm water in an endeavour to warm the infused saline to near body temperature. Subjects were given a pushbutton to signal strong desire to void. The pushbutton signal, the flow direction/timing signal, the intravesical pressure, and the filling volume were recorded by the urodynamic equipment.
During scanning the subject's head was enclosed in a standard head coil and held in place by cushions. Earplugs were worn to protect against scanner noise, but voice communication between subject and control room was still possible. A trial MR scan was performed and the subject's position adjusted if necessary. Three longitudinal relaxation time (T1)-weighted scouts (in the axial, coronal, and sagittal planes) were made for anatomic reference.
The functional scanning protocol was then carried out as shown in Fig. 1 of J Urol paper. The bladder was filled, with saline solution at 60 ml/min, to a volume of approximately 100 ml. In accordance with good urodynamic practice
[25] the subject was asked to cough once to check pressure measurement quality. fMRI data on BOLD contrast were then acquired using transverse relaxation time (T2*)-weighted single-shot spiral scans.26-28 The field of view was equal to 20 cm, resolution = 3.12 mm, repetition time (TR) = 1.5 s, echo time (TE) = 26 ms, and flip angle (FA) = 60°, allowing for full volume acquisition every 1.5 s. Thirty oblique 3.2 mm thick slices were acquired in each volume, typically with the eleventh slice from the bottom oriented along the line connecting the anterior and posterior commissure, but adjusted individually to include the presumed location of the pontine L-region.
During scanning a small amount of saline solution was repeatedly infused into and withdrawn from the bladder, in 2 blocks of 4 repetitions each (Fig. 1 in J Urol paper). Each repetition started with a pause (10.5 s = 7 scans), followed by infusion, pause and withdrawal. Each block was preceded by 6 dummy data cycles to allow for signal stabilization and was completed by a 10.5 s pause during which scanning continued, to enable the delay between pumping and hemodynamic response to be allowed for when the results were analysed. During each infusion period approximately 22 ml of saline was infused, and during each withdrawal period 15 ml was withdrawn, so that accommodation to the repeated infusions was avoided. Synchronization of scans and infusion/withdrawal was accomplished by a laptop-computer program that provided timing instructions to the experimenter controlling the pump.
After completion of these 2 measurement blocks, the bladder was further filled at 60 ml/min until the subject signalled strong desire to void (Fig. 1 in J Urol paper). Third and fourth measurement blocks were then recorded. If the subject agreed, two more measurement blocks were recorded. After catheter removal the subject voided in private into a container to establish the final volume in the bladder. Residual urine was measured by ultrasound if there was reason to suspect that the bladder had not been emptied.


Analysis

Structural images were reconstructed by standard techniques and functional images (scans) were reconstructed with in-house algorithms, yielding an effective voxel size of 1.5 x 1.5 x 1.5 mm. Statistical Parametric Mapping (SPM2)[29] was used to align the functional images in each measurement block so as to compensate for head movement; to coregister the functional images with the subject's anatomical structure; and to normalize the functional and structural images to the standard brain (ICBM152) provided by SPM2. The images were then smoothed with an 8 mm gaussian kernel, to reduce both the impact of remaining between-subject anatomic variability and the within-subject variability of the fMRI signal. The mean normalized structural image was calculated for the 12 subjects, for use as a template.
To determine brain responses to infusion overall, all measurement blocks from each subject were analyzed so as to maximize statistical power. In order to examine the dependence of responses on bladder volume, just the first two and the last two measurement blocks from each subject were included, so as to create a balanced design. The first two blocks had been measured with only a small volume in the bladder, before the subject had signalled a strong desire to void. The last two blocks had been measured after strong desire to void had been signalled and with close to the maximum tolerable amount in the bladder.
Statistical analysis was based initially on a random-effects model.
[30] For each subject, for a given choice of measurement blocks, and at each voxel, the value of Student's t for the signal contrast representing (infusion - withdrawal) was calculated. Standard SPM functions were used to implement a high pass filter (period 84 s) to suppress low-frequency noise; to implement a dummy covariate to absorb any remaining linear temporal variation; to allow for the delayed and smoothed hemodynamic response to neuronal activity; and to allow for temporal correlation between successive scans. Thus for each subject the many scans in a group of measurement blocks were reduced to a single 3-dimensional map showing the t-value at each voxel. With these first-level t-values as input data, second-level t-tests of the differences or similarities among groups of subjects, or correlations with other variables, were performed. Maps showing the second level t-values were superimposed on the mean structural image. For display purposes maps were thresholded at P<0.0001 to P<0.01, suppressing regions with fewer than 16 contiguous activated voxels. Within the regions listed above, based on a priori expectation of activation, significance was assessed for clusters of contiguous activated voxels (P<0.05, corrected for multiple comparisons).[31] For the smallest regions listed a priori (e.g., L-region), significance was assessed based on P<0.01 (uncorrected).
Because random-effects models are less well suited to small numbers of subjects, comparisons involving subgroups with good and poor bladder control were analysed by fixed-effects models also, in which all included measurement blocks were treated as equivalent, irrespective of subject.
Locations of activations were expressed in the SPM stereotaxic coordinate system based on the standard brain ICBM152.

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References

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