**5. Methods**

## *5.1. Ethical Approval*

The study was approved by the Institutional Review Board of Hualien Tzu Chi Hospital (IRB 110-033-B). Patients' informed consent was waived due to the retrospective nature of this study.

## *5.2. Patient Enrollment*

We retrospectively reviewed the medical records of SCI patients who reported AD and received either detrusor or urethral sphincter injection with Botox from 1998 to 2022. All patients had either storage symptoms, such as urgency and urgency incontinence; emptying symptoms, such as difficult urination or large postvoid residual volume; or both storage and emptying symptoms. Thus, all were ready for detrusor or urethral Botox injection. All patients also had symptoms of AD, such as headache, hypertension, increased reflexes, profuse sweating, bradycardia, and other systemic symptoms either associated with bladder fullness or occurring during urination. These AD symptoms were considered moderate to severe, causing discomfort to the patients and requiring medication to alleviate them. Patient data included age, sex, level of SCI, bladder management, videourodynamic study (VUDS) profiles, and subjective improvement in AD.

#### *5.3. Botulinum Toxin A Injection*

The techniques for Botox injection were described previously [12,26,27]. The treatment was performed in an operating room under light intravenous general anesthesia. For detrusor injection, 200 U of onabotulinumtoxinA (Botox, Allergan, Irvine, CA, USA) was diluted with 20 mL of normal saline and injected into 20 well-distributed sites in the bladder wall, sparing the trigone. All cystoscopic injections were performed using a rigid injection instrument (22-Fr, Richard Wolf, Knittlingen, Germany) and a 23-gauge injection needle. For urethral sphincter injection, a total of 100 U Botox was given. A single vial of 100 U Botox was dissolved in 5 mL of normal saline, resulting in a concentration equivalent to 20 U/mL. Each 1 mL of Botox solution was injected transurethrally under cystoscopy into the urethral sphincter at the 2, 4, 8, 10, and 12 o'clock positions in men, and transcutaneously into the urethral sphincter along the urethral lumen at the sides of the urethral meatus in women. The selection of Botox injection to the detrusor or the urethral sphincter was based on the individual patient's main lower urinary tract symptoms. Detrusor Botox injection was performed for NDO with urinary incontinence and AD. Urethral sphincter Botox injection was performed for detrusor sphincter dyssynergia (DSD) to facilitate spontaneous voiding, ease self-catheterization, and improve AD.

## *5.4. VUDS Parameters*

VUDS was performed under fluoroscopy and pressure flow study [28]. The VUDS parameters were defined as follows. Bladder sensation was evaluated by first sensation of filling (FSF), full sensation (FS), and urge sensation (US). Cystometric bladder capacity (CBC) was calculated by adding post-voiding residual (PVR) and voided volume (Vol). Bladder compliance was calculated by dividing the volume at full sensation (FS) by the detrusor pressure (Pdet). Voiding efficiency (VE) was defined as voided volume divided by bladder capacity. Maximal flow rate (Qmax) and detrusor pressure at the maximum flow rate (Pdet@Qmax) were recorded. The bladder outlet obstruction index (BOOI) was

calculated by (Pdet@Qmax -2 × Qmax). Detrusor overactivity (DO) was defined as any involuntary detrusor contraction during the filling phase of the pressure flow study [29].

#### *5.5. Outcome Measurement and Statistics Analysis*

The primary endpoint was the subjective improvement in AD, as defined by the global response assessment (GRA). The scale we used was as follows: −3 for markedly worse, −2 for moderately worse, −1 for mildly worse, 0 for no change, 1 for mildlimprovement, 2 for moderate improvement, and 3 for marked improvement [30]. The GRA assessments were taken within 1 month after Botox, and VUDS was carried out within 3 months after Botox. GRA and VUDS were evaluated separately. The secondary endpoint was the change in VUDS parameters before and after Botox injection. Either the Student's *t*-test or Mann–Whitney U test was performed to compare numerical data, and the Chi-square test was performed to compare categorical data. Statistical analyses were performed using free software (R version 4.0.0). All statistical tests were two-tailed, with *p* < 0.05 indicating significance.

**Author Contributions:** Conceptualization, H.-C.K.; methodology, P.-M.C.; software, P.-M.C.; formal analysis, P.-M.C.; investigation, P.-M.C.; resources, H.-C.K.; data curation, H.-C.K.; writing—original draft preparation, P.-M.C.; writing—review and editing, H.-C.K.; visualization, P.-M.C.; supervision, H.-C.K.; project administration, H.-C.K.; funding acquisition, H.-C.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Buddhist Tzu Chi Medical Foundation (TCMF-MP 110-03-01), National Taiwan University Hospital (112-S0151), and National Science and Technology Council (109-2314-B-002-173-MY3).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Hualien Tzu Chi Hospital Institutional Review Board (IRB 110-033-B, 7 March 2021).

**Informed Consent Statement:** Patient consent was waived due to the retrospective nature of this study.

**Data Availability Statement:** All data were presented with the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
