TITLE: Mid-lumbar (L3) epidural stimulation effects on bladder and external urethral sphincter in non-injured and chronically transected urethane-anesthetized rats
AUTHORS: Daniel Medina Aguinaga, Daniel Medina Aguinaga, Charles H. Hubscher, Natasha L. Wilkins, Robert Hoey, Jason Fell, Beatrice Ugiliweneza, Susan J. Harkema

[DESCRIPTION]
Current experiments are utilizing epidural stimulation, and the measurement of numerous systems, after either sham transection or full T9 transection. Transected Wistar rats (male and female) are tested after 6 weeks of recovery. Initial mapping experiments have targeted the L3 segment of the spinal cord for epidural stimulation. The testing paradigm subjects this segment of the spinal cord to combinations of frequency (5, 10, 30, 45, 60 Hz) and intensity (50, 75, 100, 150, 300μA) to determine the most effective stimulus parameters for external urethral sphincter modulation. Endpoints include: rectal and distal colon pressure activity, external urethral sphincter electromyography (EUS EMG), external anal sphincter electromyography (EAS EMG), bladder pressure changes, voiding and drop patterns, volume of voided fluid, and muscle movement thresholds. By recording these responses, and the changes due to stimulation, these experiments will provide insight into the best stimulus parameters to influence bladder activity after spinal cord injury. 

Before starting

Prior to terminal testing procedures, animals are implanted with: a jugular catheter, a tracheal cannula, a bladder catheter, bilateral fine wire electrodes in the External Urethral Sphincter, and a modified Medtronic 5-6-5 array electrode. 


[STEPS]
SECTION: Post-surgical care
6. After surgery, the rat's urinary bladder was manually emptied 3/day until voiding reflexively.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
25. The rat is placed on their ventrum throughout testing. The hindlimbs are taped down to the platform
as the electrical stimulation can cause motor movements that may move the animal out of position. The tail is held upright and out of line of sight by a movable arm anchored to the table by a magnet.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
26. Bilateral fine wire electrodes are implanted into the external anal sphincter, using 27g needles.
External anal sphincter (EAS) electrodes are implanted at an oblique angle so as to travel from midline to lateral aspect of the sphincter. 
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
27. SPR-524 pressure sensors (AD instruments) are inserted into the rectum (2 cm from anal verge) and the distal colon (10 cm from the anal verge) and secured to the base of the tail using tape. These probes have their own control unit which is then fed into our data acquisition unit.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
28. A perfusion pump is connected to the catheter hub and set to deliver saline at a rate of 0.25 ml
per minute. The pump syringe has a pressure sensor attached so that pressures in the bladder can be detected during filling. A 60 ml syringe is used to ensure enough saline for the entire testing procedure.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
29. All wire electrodes (ground wire, bilateral external urethral sphincter (EUS), bilateral EAS) are connected to wires fitted with
copper duck bill clip connectors. The end of the electrode wire is bared and clipped with the connectors. Only strip as much insulation as necessary for a good hold by the clip as any extra wire with cause noise in the signal. Electrode wires are amplified (AM Systems, 4 channel, differential amplifier) and then sent to the data acquisition unit.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
30. A balance (OhausScout) is placed underneath the surgical platform to collect voided material
and to relay that information to our acquisition computer via RS-232 connector and a Serial Port Data Collect (SPDC) software.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
31. The animal, perfusion pump, and table are grounded to the electrophysiology cabinet containing
the stimulator and associated electrical components.

SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
32. Data acquisition unit is a CED 1401 micro 3 system. The software used is Spike 2 version 8.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
33. Spike 2 is opened and a configuration file is loaded that contains the setup for all of the channels
being recorded (EUS, EAS, 2 cm probe, 10 cm probe, leaks, stim marker, keyboard
input).
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
34. Electrical stimulation equipment is connected to the Medtronic interface which controls
the electrode implant. A grass stimulator (S88) with a current isolation unit provides the electrical stimulation.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
35. Once the animal has all necessary components setup, the acquisition software starts recording.
Shortly afterward, the perfusion pump is turned on and bladder pressure begins to rise.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
36. The animal is allowed to have several fill-void cycles until there is a consistent time in between
voids.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
37. Five baseline periods of activity are collected. A timer is used to ensure 2 minute baseline
period measurements.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
38. After the baseline periods the stimulation is turned on for either: 2 minutes (if fill-void cycle
is shorter than 2 minutes or there is a dripping pattern) or until one void occurs (with a longer than 2 minute interval).
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
39. Stimulation parameters are changed after each presentation. Frequency parameter: 5, 10, 30,
45, 60 Hz. Intensity parameter: 50, 75, 100, 150, 300, 500 µA. The frequency-intensity pairings are presented in an increasing fashion. If frequency is varied first, the stimulations would proceed: 5 Hz 50 µA, 10 Hz 50 µA, etc. through all frequency-intensity pairings. If intensity is varied first, the stimulations would proceed: 5 Hz 50 µA, 5 Hz 75 µA, etc. Each stimulation is followed by an off period of 2 minutes to allow for any residual energy to dissipate and the system to return to baseline.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
40. During this period, data is collected on bowel function (rectal and distal colon), urethral sphincter activity (EUS EMG), external anal sphincter activity (EAS EMG), bladder pressure, when urine is expelled via marker button, exact electrical stimulation markers, volume of urine voided, and any notes made via keyboard input.
SECTION: Spinal cord transections
4. The muscular layer and the skin were closed with wound clips (Mikrotek, 9 mm autoclip), and the animals were allowed to recover from anesthesia.
SECTION: Spinal cord transections
1. Under aseptic conditions, the body temperature was maintained within the range of 36–37°C via a warm water recirculator. 
SECTION: Spinal cord transections
3. A dorsal longitudinal incision was made to expose the T7 vertebra, and a laminectomy was made to expose the T8 spinal cord. The dura mater was lateralized and the spinal cord was transected using surgical scissors. Gelfoam was placed in the lesion cavity as hemostatic.
SECTION: Spinal cord transections
2. All animals were anesthetized with a mix of ketamine/xylazine (80/10 mg/kg, intraperitoneally).
SECTION: Post-surgical care
5. Pain medication (Meloxicam,1/day for 3 days intramuscular) and antibiotics (Gentafuse, gentamicin, 1/day for 3 days; Penject, penicillin G, 1/day for 3 days, subcutaneous) were applied to all subjects.
SECTION: Terminal mapping - Anesthesia and airway care
7. Animals were initially anesthetized with Isoflurane (3l/min 5% for induction, and 2% for maintenance) and placed in supine on a surgical table conditioned with a water-heated pad (Gaymar) to maintain body temperature.
SECTION: Terminal mapping - Anesthesia and airway care
8. A sagittal mid-line incision was made in the neck to expose the jugular vein and the trachea.
SECTION: Terminal mapping - Anesthesia and airway care
9. A PE-60 (Intramedic, Clay Adams) jugular catheter was inserted into the vessel and secured with silk suture to urethane infusion.

SECTION: Terminal mapping - Anesthesia and airway care
10. A polyethylene tubing was placed into the trachea to ensure airway opening.
SECTION: Terminal mapping - Anesthesia and airway care
11. Anesthesia was switched from isoflurane to 50% urethane solution (1.2 g/kg), reducing the isoflurane percentage and slowly infusing the urethane, maintaining continuous surgical depth of anesthesia.
SECTION: Terminal mapping - Anesthesia and airway care
12. The skin was closed with a silk suture securing the tracheal and jugular catheters with the same suture.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
14. The external urethral sphincter (EUS) was exposed and implanted bilaterally with two thin wires (A-M Systems, 0.002″ diameter, stainless steel). A third electrode (A-M Systems, 0.003″ diameter, stainless steel) was inserted into the abdominal wall as a reference.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
15. The bladder catheter and electrodes were tunneled and exteriorized through the skin on the back of the neck.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
24. 
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
13. The urinary bladder was exposed by a midline abdominal incision and a PE-60 tubing with a heat-flared end) inserted into the vesical lumen through a dome incision and secured with a collar of silk suture.
SECTION: Terminal mapping - Epidural electrode implant
17. The spinal cord was exposed by a midline abdominal incision and quadruple laminectomy. A modified multi-electrode epidural stimulation array (Specify 5–6–5, Medtronic, Minneapolis, MN) modified for use as one electrode row was placed on the epidural surface, one over L3-L2 segment.
SECTION: Terminal mapping - Epidural electrode implant
18. The muscular layer and the skin were closed with a silk suture and the wire of the electrode was secured with the same suture.
SECTION: Terminal mapping - Mapping procedure
20. Bilateral fine wire electrodes are implanted into the external anal sphincter (EAS), using 27g needles. EAS electrodes are implanted at an oblique angle so as to travel from midline to lateral aspect of the sphincter.
SECTION: Terminal mapping - Mapping procedure
19. The rat is placed on their ventrum throughout testing. The hindlimbs are taped down to the platform as the electrical stimulation can cause motor movements that may move the animal out of position. The tail is held upright and out of line of sight by a movable arm anchored to the table by a magnet.
SECTION: Terminal mapping - Mapping procedure
21. SPR-524 pressure sensors (AD instruments) are inserted into the rectum (2 cm from anal verge) and the distal colon (10 cm from the anal verge) and secured to the base of the tail using tape. These probes have their own control unit which is then fed into our data acquisition unit.
SECTION: Terminal mapping - Electromyography and cystometrogram sensors surgical placement
16. The muscular abdominal wall and the skin were closed with a silk suture.
SECTION: Terminal mapping - Mapping procedure
22. A perfusion pump is connected to the urinary bladder catheter hub and set to deliver saline at a rate of 0.25 ml per minute. The pump syringe has a pressure sensor attached so that pressures in the bladder can be detected during filling. A 60 ml syringe is used to ensure enough saline for the entire testing procedure.
SECTION: Terminal mapping - Mapping procedure
23. All wire electrodes (ground wire, bilateral EUS, and EAS) are connected to wires fitted with copper duck bill clip connectors. The end of the electrode wire is borne and clipped with the connectors. Only strip as much insulation as necessary for a good hold by the clip, as any extra wire with cause noise in the signal. Electrode wires are amplified (A-M Systems, 4 channel, differential amplifier) and then sent to the data acquisition unit.
23. 
SECTION: Terminal mapping - Mapping procedure
23. 
SECTION: Terminal mapping - Mapping procedure
24. A balance (OhausScout) is placed underneath the surgical platform to collect voided material and to relay that information to our acquisition computer via RS-232 connector and a Serial Port Data Collect (SPDC) software.
SECTION: Terminal mapping - Mapping procedure
25. The animal, perfusion pump, and table are grounded to the electrophysiology cabinet containing the stimulator and associated electrical components.
SECTION: Terminal mapping - Mapping procedure
26. Data acquisition unit is a CED 1401 micro 3 system. The software used is Spike 2 version 8.
SECTION: Terminal mapping - Mapping procedure
27. Spike 2 is opened and a configuration file is loaded that contains the setup for all of the channels being recorded (EUS, EAS, 2 cm probe, 10 cm probe, leaks, stim marker, keyboard input).
SECTION: Terminal mapping - Mapping procedure
28. Electrical stimulation equipment is connected to the Medtronic interface which controls the electrode implant. A grass stimulator (S88) with a current isolation unit provides the electrical stimulation.
SECTION: Terminal mapping - Mapping procedure
29. Once the animal has all necessary components set up, the acquisition software starts recording. Shortly afterward, the perfusion pump is turned on and bladder pressure begins to rise.
SECTION: Terminal mapping - Mapping procedure
30. The animal is allowed to have several fill-void cycles until there is a consistent time in between voids.
SECTION: Terminal mapping - Mapping procedure
31. Five baseline periods of activity are collected. A timer is used to ensure 2 minute baseline period measurements.
SECTION: Terminal mapping - Mapping procedure
32. After the baseline periods, the stimulation is turned on for either: 2 minutes (if fill-void cycle is shorter than 2 minutes or there is a dripping pattern) or until one void occurs (with a longer than 2-minute interval).
SECTION: Terminal mapping - Mapping procedure
33. Stimulation parameters are changed after each presentation.
Frequency parameter: 5, 10, 30, 45, 60 Hz.
Intensity parameter: 50, 75, 100, 150, 300, 500 µA.

The frequency-intensity pairings are presented in an increasing fashion. 
If frequency is varied first, the stimulations would proceed: 5 Hz 50 µA, 10 Hz 50 µA, etc. through all frequency-intensity pairings.
If intensity is varied first, the stimulations would proceed: 5 Hz 50 µA, 5 Hz 75 µA, etc.

Each stimulation is followed by an off period of 2 minutes to allow for any residual energy to dissipate and the system to return to baseline.

SECTION: Terminal mapping - Mapping procedure
34. During this period, data is collected on bowel function (rectal and distal colon), urethral sphincter activity (EUS EMG), external anal sphincter activity (EAS EMG), bladder pressure, when urine is
expelled via marker button, exact electrical stimulation markers, volume of urine voided, and any notes made via keyboard input.