Sep 27, 2023

Public workspaceMid-lumbar (L3) epidural stimulation effects on bladder and external urethral sphincter in non-injured and chronically transected urethane-anesthetized rats V.2

DOI
dx.doi.org/10.17504/protocols.io.bp2l6xdk5lqe/v2
  • 1Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine;
  • 2Department of Neurological Surgery, University of Louisville School of Medicine, Louisville, KY, USA;
  • 3Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, KY, USA;
  • 4Department of Health Management and Systems Science, School of Public Health and Information Science, University of Louisville, Louisville, KY, USA
Daniel Medina Aguinaga
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6xdk5lqe/v2
Protocol Citation: Daniel Medina Aguinaga, Robert Hoey, Natasha L. Wilkins, Beatrice Ugiliweneza, Jason Fell, Susan J. Harkema, Charles H. Hubscher 2023. Mid-lumbar (L3) epidural stimulation effects on bladder and external urethral sphincter in non-injured and chronically transected urethane-anesthetized rats. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xdk5lqe/v2Version created by Daniel Medina Aguinaga
Manuscript citation:
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
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: September 27, 2023
Last Modified: September 27, 2023
Protocol Integer ID: 88493
Funders Acknowledgement:
SPARC
Grant ID: OT2OD024898
Abstract
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.

Materials

ABCD
ITEMCompanycatalog numberwebsite link
ketamineDechra Vet Products VINV-CIII-0016https://northamerica.covetrus.com/Product?sku=080524
xylazineCovetrus 1XYL006https://northamerica.covetrus.com/Product?sku=080896
4–0 suture Ethicon1629Hhttps://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/
MeloxicamCovetrus 6451603845https://northamerica.covetrus.com/Product?sku=049756
Gentafuse, gentamicinCovetrus VINB-0069-1300https://northamerica.covetrus.com/Product?sku=006913
penicilin GBimeda1PRO304https://northamerica.covetrus.com/Product?sku=069322
IsofluraneCovetrus 11695067772https://northamerica.covetrus.com/Product?sku=029405
urethaneThermo Scientific ChemicalsAC325542500 https://www.fishersci.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 Intramedic‱ PE TubingBD 427416https://www.fishersci.com/shop/products/intramedic-pe-tubing-16/1417012C?keyword=true
electrodes (0.002″ diameter, stainless steel)A-M Systems790500https://www.a-msystems.com/p-809-pfa-coated-stainless-steel-wire.aspx
multi-electrode epidural stimulation array (Specify 5–6–5)(Medtronic, Minneapolis, MN) multi-electrode epidural stimulation array (Specify 5–6–5)https://www.medtronic.com/us-en/healthcare-professionals/services/innovation-lab.html
infussion 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-transducer-without-cable.html
differential amplifierA-M Systems, Sequim, WA, USAModel 3000 AC/DC Differential Amplifierhttps://www.a-msystems.com/p-254-model-3000-acdc-differential-amplifier.aspx
balanceResearch Products International Corp (Ohaus)SPX123https://www.fishersci.com/shop/products/ohaus-scout-spx-spx123/502114168#?keyword=ohaus%20scout%20spx123
USB device interface cable (RS232)StarTechICUSB232PROChttps://www.staples.com/StarTech-USB-C-to-Serial-Adapter-with-COM-Port-Retention-USB-C-to-DB9-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.html
Transbridge 4 M amplifierWPI LLCSYS-TBM4Mhttps://www.wpiinc.com/sys-tbm4m-4-channel-transducer-amplifier.html
Software to record dataCambridge Electronic DeviceSpike 2, V8.21https://ced.co.uk/products/spkovin
CED Mircro3 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-amplifier.aspx
Pressure probes for anorectal manometry Millar SPR-524AD InstrumentsSPR-524https://www.adinstruments.com/products/pressure-catheters

Spinal cord transections
Spinal cord transections
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 dura mater was lateralized and the spinal cord was transected using surgical scissors. Gelfoam was placed in the lesion cavity as hemostatic.
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 and airway care
Terminal mapping - Anesthesia and airway care
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.
A polyethylene tubing was placed into the trachea to ensure airway opening.
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 the tracheal and jugular catheters 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 - Epidural electrode implant
Terminal mapping - Epidural electrode implant
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.
The muscular layer and the skin were closed with a silk suture and the wire of the electrode was secured with the same 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 the Medtronic interface 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.
Five 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: 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.
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.