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Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.
Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.
Spinal cord injury induced by ischemia/reperfusion (SCI) is one of the most devastating complications of aortic repair associated with reduced outcome1,2,3,4. Current prevention and treatment options for SCI include the optimization of macrohemodynamic parameters as well as the normalization of cerebrospinal fluid pressure (CSP) to improve spinal cord perfusion pressure2,5,6,7,8,9. Despite the implementation of these maneuvers, incidence of SCI still ranges between 2% and 31% depending on the complexity of aortic repair10,11,12.
Recently, microcirculation has gained increased attention13,14. Microcirculation is the area of cellular oxygen uptake and metabolic exchange and therefore, plays a critical role in organ function and cellular integrity13. Impaired microcirculatory blood flow is a major determinant of tissue ischemia associated with increased mortality15,16,17,18,19. Impairment of spinal cord microcirculation is associated with reduced neurological function and outcome20,21,22,23. Therefore, optimization of microperfusion for the treatment of SCI is a most promising approach. Persistence of microcirculatory disturbances, despite macrocirculatory optimization, has been described26,27,28,29. This loss of hemodynamic coherence occurs frequently in various conditions including ischemia/reperfusion, emphasizing the need for direct microcirculatory evaluation and microcirculation-targeted therapies26,27,30.
So far, only few studies have used laser-Doppler probes for real-time assessment of spinal cord microcirculatory behavior20,31. Existing studies have often used microsphere injection techniques, which are limited by intermittent use and post-mortem analysis32,33. The number of different measurements using microsphere injection technique is limited by the availability of microspheres with different wavelengths. Moreover, in contrast to Laser-Doppler techniques, real-time assessment of microperfusion is not possible, as post-mortem tissue processing and analysis is needed for this method. Here, we present an experimental protocol for the real-time assessment of spinal cord microcirculation in a porcine large animal model of ischemia/reperfusion.
This study was part of a large animal project combining a randomized study comparing the influence of crystalloids vs. colloids on microcirculation in ischemia/reperfusion as well as an explorative randomized study on the effects of fluids vs. vasopressors on spinal cord microperfusion. Flow probe 2-point calibration as well as pressure-tip catheter calibration has been previously described34. In addition to the reported protocol, fluorescent microspheres were used for the measurement of spinal cord microperfusion, as previously described, using 12 samples of spinal cord tissue for each animal, with samples 1-6 representing the upper spinal cord and 7-12 representing the lower spinal cord35,36. Microsphere injection was performed for each measurement step after the completion of Laser-Doppler recordings and macrohemodynamic evaluation. Histopathological evaluation was performed using the Kleinman-Score as previously described37.
The study was approved by the Governmental Commission on the Care and Use of Animals of the City of Hamburg (Reference-No. 60/17). The animals received care in compliance with the 'Guide for the Care and Use of Laboratory Animals' (NIH publication No. 86-23, revised 2011) as well as FELASA recommendations and experiments were carried out according to the ARRIVE guidelines24,25. This study was an acute trial, and all animals were euthanized at the end of protocol.
NOTE: The study was performed in six three-month-old male and female pigs (German Landrace) weighing approximately 40 kg. Animals were brought to the animal care facilities at least 7 days prior to the experiments and were housed in accordance to animal welfare recommendations. Animals were provided food and water ad libitum, and their health status was regularly assessed by the responsible veterinarian. A fasting time of 12 h was maintained prior to the experiments. The entire experimental procedure and handling of the animals was supervised by the responsible veterinarian.
1. Anesthesia induction and maintenance of anesthesia
2. Probe placement
3. Catheter placement
4. Surgical preparation
5. Assessment and data acquisition
6. Experimental protocol
7. Euthanasia
8. Organ harvesting
9. Statistical analysis
All six animals survived until the completion of the protocol. Animal weight was 48.2 ± 2.9 kg; five animals were male, and one animal was female. Spinal cord needle probe insertion as well as spinal cord Flux measurement was feasible in all animals.
Examples of real-time spinal cord microcirculatory recordings in combination with cerebral microcirculatory and macrohemodynamic recordings during aortic cross-clamping for isc...
SCI induced by spinal cord ischemia is a major complication of aortic repair with tremendous impact on patient outcome1,2,3,4,10,11,12. Microcirculation-targeted therapies to prevent and treat SCI are most promising. The protocol provides a reproducible method for real-time spinal cord micro...
Constantin J. C. Trepte has received an honorary award for lectures by Maquet. All other authors declare no conflicts of interest.This study was supported by the European Society of Anaesthesiology Young Investigator Start-Up Grant 2018.
The authors would like to thank Lena Brix, V.M.D, Institute of Animal Research, Hannover Medical School, as well as Mrs. Jutta Dammann, Facility of Research Animal Care, University Medical Center Hamburg-Eppendorf, Germany, for providing pre- and perioperative animal care and their technical assistance on animal handling. The authors would further like to thank Dr. Daniel Manzoni, Department of Vascular Surgery, Hôpital Kirchberg, Luxembourg, for his technical assistance.
Name | Company | Catalog Number | Comments |
CardioMed Flowmeter | Medistim AS, Oslo, Norway | CM4000 | Flowmeter for Flow-Probe Femoral Artery |
CardioMed Flow-Probe, 5mm | Medistim AS, Oslo, Norway | PS100051 | Flow-Probe Femoral Artery |
COnfidence probe, | Transonic Systems Inc., Ithaca, NY, USA | MA16PAU | Flow-Probe Aorta |
16 mm liners | |||
DIVA Sevoflurane Vapor | Dräger Medical, Lübeck, Germany | Vapor | |
Hotline Level 1 Fluid Warmer | Smiths Medical Germany GmbH, Grasbrunn, Germany | HL-90-DE-230 | Fluid Warmer |
Infinity Delta | Dräger Medical, Lübeck, Germany | Basic Monitoring Hardware | |
Infinity Hemo | Dräger Medical, Lübeck, Germany | Basic Pressure Monitoring and Pulmonary Thermodilution Hardware | |
LabChart Pro | ADInstruments Ltd., Oxford, UK | v8.1.16 | Synchronic Laser-Doppler, Blood Pressure, ECG and Blood-Flow Aquisition Software |
LiquoGuard 7 | Möller Medical GmbH, Fulda, Germany | Cerebrospinal Fluid Drainage System | |
Millar Micro-Tip Pressure Catheter (5F, Single, Curved, 120cm, PU/WD) | ADInstruments Ltd., Oxford, UK | SPR-350 | Pressure-Tip Catheter Aorta |
moor VMS LDF | moor Instruments, Devon, UK | Designated Laser-Doppler Hardware | |
moor VMS Research Software | moor Instruments, Devon, UK | Designated Laser-Doppler Software | |
Perivascular Flow Module | Transonic Systems Inc., Ithaca, NY, USA | TS 420 | Flow-Module for Flow-Probe Aorta |
PiCCO 2, Science Version | Getinge AB, Göteborg, Sweden | v. 6.0 | Blood Pressure and Transcardiopulmonary Monitoring Hard- and Software |
PiCCO 5 Fr. 20cm | Getinge AB, Göteborg, Sweden | Thermistor-tipped Arterial Line | |
PowerLab | ADInstruments Ltd., Oxford, UK | PL 3516 | Synchronic Laser-Doppler, Blood Pressure, ECG and Blood-Flow Aquisition Hardware |
QuadBridgeAmp | ADInstruments Ltd., Oxford, UK | FE 224 | Four Channel Bridge Amplifier for Laser-Doppler and Invasive Blood Pressure Aquisition |
Silverline | Spiegelberg, Hamburg, Germany | ELD33.010.02 | Cerebrospinal Fluid Drainage |
SPSS statistical software package | IBM SPSS Statistics Inc., Armonk, New York, USA | v. 27 | Statistical Software |
Twinwarm Warming System | Moeck & Moeck GmbH, Hamburg, Germany | 12TW921DE | Warming System |
Universal II Warming Blanket | Moeck & Moeck GmbH, Hamburg, Germany | 906 | Warming Blanket |
VP 3 Probe, 8mm length (individually manufactured) | moor Instruments, Devon, UK | Laser-Doppler Probe | |
Zeus | Dräger Medical, Lübeck, Germany | Anesthesia Machine |
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