Mar 26, 2024

Public workspaceBladder responses to thoracolumbar epidural stimulation in female urethane-anesthetized rats with graded contusion spinal cord injuries

DOI
dx.doi.org/10.17504/protocols.io.5jyl82nm6l2w/v1
  • 1University of Louisville School of Medicine
  • Charles H. Hubscher: Corresponding Author
Natasha Lorraine Wilkins
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl82nm6l2w/v1
Protocol Citation: Natasha Lorraine Wilkins, Daniel Medina Aguinaga, Robert F. Hoey, Jason Fell, Susan J. Harkema, Charles H. Hubscher 2024. Bladder responses to thoracolumbar epidural stimulation in female urethane-anesthetized rats with graded contusion spinal cord injuries. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82nm6l2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 26, 2024
Last Modified: March 26, 2024
Protocol Integer ID: 97404
Keywords: spinal cord injury, epidural stimulation, bladder, lower urinary tract
Funders Acknowledgement:
SPARC
Grant ID: OT2OD024898
Abstract
The current experiment utilizes epidural stimulation at T13, L3, or L6 to modulate urinary function in rats with graded spinal cord contusion injuries. Female Wistar rats with mild, moderate or severe T8 spinal cord injuries underwent epidural stimulation mapping procedures at 6 weeks post-injury. The mapping procedure included 10 and 30 Hz frequencies and intensities of 10 µA to 60 µA intensities to determine the most effective stimulus parameters to impact lower urinary tract function with simulation at T13, L3 or L6. End points included: external urethral sphincter electromyography, bladder pressure changes, voiding volume, and muscle movement thresholds. By recording these responses, and the subsequent changes in lower urinary tract function, these experiments will provide insight into optimal stimulation parameters to influence bladder activity in individuals with anatomically incomplete spinal cord injury.
Materials

ABCD
Itemcompanycatalog numberwebsite link
KetamineDechra Vet ProductsVINV-CIII-0016https://northamerica.covetrus.com/Product?sku=080896
XylazineCovetrus1XYL006https://northamerica.covetrus.com/Product?sku=034049
wound clips (9 mm autoclip)MikRon PrecisionACS-CShttps://www.braintreesci.com/surgical-tools-supplies/wound-closure/appliers-removers-clips/9mm-autoclip/
MeloxicamCovetrus6451603845https://northamerica.covetrus.com/Product?sku=049756
Gentafuse, gentamicinCovetrusVINB-0069-1300https://northamerica.covetrus.com/Product?sku=006913
penicilin GBimeda1PRO304https://northamerica.covetrus.com/Product?sku=069322
IsofluraneCovetrus11695067772https://northamerica.covetrus.com/Product?sku=029405
UrethaneThermo Scientific ChemicalsAC325542500https://www.fischersci.com/shop/products/urethane-97-thermo-scientific/AC325542500#?keyword=urethane
water-heated padGaymarTP-700https://www.braintreesci.com/temperature-measurement-control/heat-therapy-pumps-pads/gaymar-heating-cooling-t-pump-back-in-stock/
PE-60 tubingBD Intramedical PE TubingBD 427416https://www.fischersci.com/shop/products/intramedic-pe-tubing-16/1417012C?keyword=true
Infusion PumpBraintree Scientific, Braintree, MA, USABS-300https://www.braintreesci.com/syringe-surgical-pumps/programmable-syringe-pumps/just-infusion-syringe-pump/
BLPR2 TransducerWorld Precision Instruments [WPI,LLC];Sarasota, FL, USA503067https://www.wpiinc.com/503067-blpr2-tranducer-without-cable.html
Differential amplifierA-M systems, Sequim, WA, USAModel 3000 AC/DC differential amplifierhttps://a-msystems.com/p-254-model-3000-acdc-differential-amplifier.aspx
balanceResearch Products International Corp (Ohaus)SPX123https://www.fischersci.com/shop/products/ohaus-scout-spx-spx123/502114168#?keyword+ohaus%20scout%20spx123
USB device interface cableStarTechICUSB232PROChttps://www.staples.com/StarTech-USB-C-to-serial-adapter-with-com-port-retention-usb-c-toDB9-Cable-or-RS232-Cable-ICUSB232PROC/product_IM12DG846
software for balanceResearch Products International Corp (Ohaus)Serial Port Data Collection, SPDC, Ohaus, V2.01https://us.ohaus.com/en-us/support/software-and-drivers
FORT100 weight transducerWPI, LLCFORT100https://www.wpiinc.com/var-2858-large-fort-force-transducer-amplifer.html
Transbridge 4 M amplifierWPI, LLCSYS-TBM4Mhttps://www.wpiinc.com/systbm4m-4-channel-transducer-amplifier.html
Software to record dataCambridge Electronic DeviceSpike 2, V8.21https://ced.co.uk/products/spkovin
CED Micro3 1401 unitCambridge Electronic DeviceMicro3, 1401 unithttps://ced.co.uk/products/mic4in
4-channel differential AC amplifierAM-Systems,model 1700Model 1700 Differential AC Amplifierhttps://www.a-msystems.com/p-202-model-1700-differential-ac-amplifer.aspx
Pressure probes for anorectal manometry Millar SPR-524AD instrumentsSPR-524https://www.adinstruments.com/products/pressure-catheters
Infinite Horizon Impactor (IH-0400) for contusion SCIPrecision Systems and InstrumentationIH-0400https://psiimpactors.com/product/ih400/

Before start
Prior to terminal testing procedures, animals are implanted with a jugular catheter, bladder catheter, bilateral fine wire electrodes in the External Urethral Sphincter, and a MicroLeads 15-electrode epidural stimulation array.
Spinal cord contusion
Spinal cord contusion
Under aseptic conditions, the body temperature was maintained within the range of 36–37°C via a warm water recirculator.
All animals were anesthetized with a mix of ketamine/xylazine (80/10 mg/kg, intraperitoneally).
A dorsal longitudinal incision was made to expose the T7 vertebra, and a laminectomy was made to expose the T8 spinal cord.
The animal was stabilized below the IH-impactor and a mild (150 kdyn), moderate (215 kdyn), or severe (215 kdyn, 1 sec dwell) was delivered to the exposed spinal cord.
The muscular layer and the skin were closed with wound clips (Mikrotek, 9 mm autoclip), and the animals were allowed to recover from anesthesia.
Post-surgical care
Post-surgical care
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.
After surgery, the rat's urinary bladder was manually emptied 3/day until voiding reflexively.
Terminal mapping-anesthesia
Terminal mapping-anesthesia
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.
A sagittal mid-line incision was made in the neck to expose the jugular vein and the trachea.
A PE-60 (Intramedic, Clay Adams) jugular catheter was inserted into the vessel and secured with silk suture to urethane infusion.
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.
The skin was closed with a silk suture securing jugular catheter with the same suture.
Terminal mapping-electromyography and cystometrogram sensors surgical placement
Terminal mapping-electromyography and cystometrogram sensors surgical placement
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.
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.
The bladder catheter and electrodes were tunneled and exteriorized through the skin on the back of the neck.
The muscular abdominal wall and the skin were closed with a silk suture.
Terminal mapping-mapping procedure
Terminal mapping-mapping procedure
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.
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.
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.
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.
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.
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.
The animal, perfusion pump, and table are grounded to the electrophysiology cabinet containing the stimulator and associated electrical components.
Data acquisition unit is a CED 1401 micro 3 system. The software used is Spike 2 version 8.
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).
Electrical stimulation equipment is connected to a breadboard which controls the electrode implant. A grass stimulator (S88) with a current isolation unit provides the electrical stimulation.
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.
The animal is allowed to have several fill-void cycles until there is a consistent time in between voids.
Three baseline periods of activity are collected. A timer is used to ensure 2 minute baseline period measurements.
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).
Stimulation parameters are changed after each presentation.
  • Frequency parameter: 10, 30 Hz.
  • Intensity parameter: 10, 20, 30, 40, 50, 60 µA.
  • Once changes in lower urinary function were detected with stimulation (change in inter-contractile interval, void-volume, or EUS activity) the cohorts for stimulation were changed in order to decrease the active surface area of the epidural stimulator.
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.
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.
In an animal underwent mapping on two spinal levels, the wound clips were removed and the stimulator was moved to the next stimulation target (T13, L3 or L6). The muscle and skin were then closed with wound clips and the mapping procedure was repeated for the new stimulation location.
Protocol references
Medina-Aguiñaga, D., Hoey, R.F., Wilkins, N.L. et al. Mid-lumbar (L3) epidural stimulation effects on bladder and external urethral sphincter in non-injured and chronically transected urethane-anesthetized rats. Sci Rep 13, 12258 (2023). https://doi.org/10.1038/s41598-023-39388-9