{"@context":{"@language":"en","Affiliation":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","AggregatedSourceRepository":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","Citation":"https:\/\/open.library.ubc.ca\/terms#identifierCitation","Contributor":"http:\/\/purl.org\/dc\/terms\/contributor","Creator":"http:\/\/purl.org\/dc\/terms\/creator","DateAvailable":"http:\/\/purl.org\/dc\/terms\/issued","DateIssued":"http:\/\/purl.org\/dc\/terms\/issued","Description":"http:\/\/purl.org\/dc\/terms\/description","DigitalResourceOriginalRecord":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","FullText":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","Genre":"http:\/\/www.europeana.eu\/schemas\/edm\/hasType","IsShownAt":"http:\/\/www.europeana.eu\/schemas\/edm\/isShownAt","Language":"http:\/\/purl.org\/dc\/terms\/language","PeerReviewStatus":"https:\/\/open.library.ubc.ca\/terms#peerReviewStatus","Provider":"http:\/\/www.europeana.eu\/schemas\/edm\/provider","Publisher":"http:\/\/purl.org\/dc\/terms\/publisher","PublisherDOI":"https:\/\/open.library.ubc.ca\/terms#publisherDOI","Rights":"http:\/\/purl.org\/dc\/terms\/rights","RightsURI":"https:\/\/open.library.ubc.ca\/terms#rightsURI","ScholarlyLevel":"https:\/\/open.library.ubc.ca\/terms#scholarLevel","Subject":"http:\/\/purl.org\/dc\/terms\/subject","Title":"http:\/\/purl.org\/dc\/terms\/title","Type":"http:\/\/purl.org\/dc\/terms\/type","URI":"https:\/\/open.library.ubc.ca\/terms#identifierURI","SortDate":"http:\/\/purl.org\/dc\/terms\/date"},"Affiliation":[{"@value":"Education, Faculty of","@language":"en"},{"@value":"Medicine, Faculty of","@language":"en"},{"@value":"Science, Irving K. Barber Faculty of (Okanagan)","@language":"en"},{"@value":"Biology, Department of (Okanagan)","@language":"en"},{"@value":"Cellular and Physiological Sciences, Department of","@language":"en"},{"@value":"Kinesiology, School of","@language":"en"}],"AggregatedSourceRepository":[{"@value":"DSpace","@language":"en"}],"Citation":[{"@value":"Biology 10 (10): 1006 (2021)","@language":"en"}],"Contributor":[{"@value":"University of British Columbia. Okanagan Campus. Centre for Chronic Disease Prevention and Management","@language":"en"},{"@value":"International Collaboration on Repair Discoveries","@language":"en"}],"Creator":[{"@value":"Wainman, Liisa","@language":"en"},{"@value":"Erskine, Erin","@language":"en"},{"@value":"Ahmadian, Mehdi","@language":"en"},{"@value":"Hanna, Thomas Matthew","@language":"en"},{"@value":"West, Christopher R.","@language":"en"}],"DateAvailable":[{"@value":"2021-11-04T21:03:15Z","@language":"en"}],"DateIssued":[{"@value":"2021-10-07","@language":"en"}],"Description":[{"@value":"As primary medical care for spinal cord injury (SCI) has improved over the last decades there are more individuals living with neurologically incomplete (vs. complete) cervical injuries. For these individuals, a number of promising therapies are being actively researched in pre-clinical settings that seek to strengthen the remaining spinal pathways with a view to improve motor function. To date, few, if any, of these interventions have been tested for their effectiveness to improve autonomic and cardiovascular (CV) function. As a first step to testing such therapies, we aimed to develop a model that has sufficient sparing of descending sympathetic pathways for these interventions to target yet induces robust CV impairment. Twenty-six Wistar rats were assigned to SCI (n = 13) or na\u00efve (n = 13) groups. Animals were injured at the T3 spinal segment with 300 kdyn of force. Fourteen days post-SCI, left ventricular (LV) and arterial catheterization was performed to assess in vivo cardiac and hemodynamic function. Spinal cord lesion characteristics along with sparing in catecholaminergic and serotonergic projections were determined via immunohistochemistry. SCI produced a decrease in mean arterial pressure of 17 \u00b1 3 mmHg (p < 0.001) and left ventricular contractility (end-systolic elastance) of 0.7 \u00b1 0.1 mmHg\/\u00b5L (p < 0.001). Our novel SCI model produced significant decreases in cardiac and hemodynamic function while preserving 33 \u00b1 9% of white matter at the injury epicenter, which we believe makes it a useful pre-clinical model of SCI to study rehabilitation approaches designed to induce neuroplasticity.","@language":"en"}],"DigitalResourceOriginalRecord":[{"@value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/80153?expand=metadata","@language":"en"}],"FullText":[{"@value":"biologyArticleDevelopment of a Spinal Cord Injury Model Permissive toStudy the Cardiovascular Effects of Rehabilitation ApproachesDesigned to Induce NeuroplasticityLiisa Wainman 1,2, Erin L. Erskine 1,2, Mehdi Ahmadian 1,2,3, Thomas Matthew Hanna 1,4and Christopher R. West 1,2,5,*\u0001\u0002\u0003\u0001\u0004\u0005\u0006\u0007\b\u0001\u0001\u0002\u0003\u0004\u0005\u0006\u0007Citation: Wainman, L.; Erskine, E.L.;Ahmadian, M.; Hanna, T.M.; West,C.R. Development of a Spinal CordInjury Model Permissive to Study theCardiovascular Effects ofRehabilitation Approaches Designedto Induce Neuroplasticity. Biology2021, 10, 1006. https:\/\/doi.org\/10.3390\/biology10101006Academic Editors: C\u00e9dric G. Geoffroyand Warren AlilainReceived: 1 August 2021Accepted: 29 September 2021Published: 7 October 2021Publisher\u2019s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: \u00a9 2021 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https:\/\/creativecommons.org\/licenses\/by\/4.0\/).1 Centre for Chronic Disease Prevention and Management, Faculty of Medicine, University of British Columbia,Kelowna, BC V1V 1V7, Canada; lwain27@student.ubc.ca (L.W.); erin.erskine@ubc.ca (E.L.E.);mahmadia@student.ubc.ca (M.A.); tmhanna7@student.ubc.ca (T.M.H.)2 International Collaboration of Repair Discoveries (ICORD), University of British Columbia,Vancouver, BC V5Z 1M9, Canada3 School of Kinesiology, Faculty of Education, University of British Columbia, Vancouver, BC V6T 1Z1, Canada4 Department of Biology, Faculty of Science, University of British Columbia Okanagan,Kelowna, BC V1V 1V7, Canada5 Department of Cell and Physiological Sciences, Faculty of Medicine, University of British Columbia,Vancouver, BC V6T 1Z3, Canada* Correspondence: chris.west@ubc.caSimple Summary: People living with high-level spinal cord injury experience worse cardiovascularhealth than the general population. In most spinal cord injuries, there are some remaining functioningpathways leading from the brain through the spinal cord to the organs and muscles, but not enough tosustain normal levels of function. Recently, therapies that aim to increase the strength of connectionsin these remaining pathways have shown great potential in restoring walking, hand, and breathingfunction in the spinal cord injured population. In order to test these therapies for their effects oncardiovascular function, we developed a new type of spinal cord injury rat model that spares enoughpathways for these therapies to act upon but still produces measurable reductions in heart and bloodvessel function that can be targeted with interventions\/treatments.Abstract: As primary medical care for spinal cord injury (SCI) has improved over the last decadesthere are more individuals living with neurologically incomplete (vs. complete) cervical injuries.For these individuals, a number of promising therapies are being actively researched in pre-clinicalsettings that seek to strengthen the remaining spinal pathways with a view to improve motorfunction. To date, few, if any, of these interventions have been tested for their effectiveness toimprove autonomic and cardiovascular (CV) function. As a first step to testing such therapies, weaimed to develop a model that has sufficient sparing of descending sympathetic pathways for theseinterventions to target yet induces robust CV impairment. Twenty-six Wistar rats were assigned to SCI(n = 13) or na\u00efve (n = 13) groups. Animals were injured at the T3 spinal segment with 300 kdyn of force.Fourteen days post-SCI, left ventricular (LV) and arterial catheterization was performed to assessin vivo cardiac and hemodynamic function. Spinal cord lesion characteristics along with sparing incatecholaminergic and serotonergic projections were determined via immunohistochemistry. SCIproduced a decrease in mean arterial pressure of 17 \u00b1 3 mmHg (p < 0.001) and left ventricularcontractility (end-systolic elastance) of 0.7 \u00b1 0.1 mmHg\/\u00b5L (p < 0.001). Our novel SCI modelproduced significant decreases in cardiac and hemodynamic function while preserving 33 \u00b1 9% ofwhite matter at the injury epicenter, which we believe makes it a useful pre-clinical model of SCI tostudy rehabilitation approaches designed to induce neuroplasticity.Keywords: cardiovascular; contusion; neuroplasticityBiology 2021, 10, 1006. https:\/\/doi.org\/10.3390\/biology10101006 https:\/\/www.mdpi.com\/journal\/biologyBiology 2021, 10, 1006 2 of 161. IntroductionSpinal cord injury (SCI) is a debilitating condition which, in addition to inducingsensorimotor dysfunction, also impairs autonomic function. Cardiovascular disease (CVD)has emerged as the primary cause of morbidity and mortality for individuals living withSCI [1]. SCI-induced dysregulation of the cardiovascular (CV) system occurs primarily as aresult of altered descending control of sympathetic preganglionic neurons (SPNs). In turn,such reduced medullary input to SPNs causes a host of CV complications including restinghypotension, orthostatic hypotension (OH; sudden decrease in BP upon changing posture),autonomic dysreflexia (AD; sudden episodic hypertension accompanied by reflex brady-cardia), and left-ventricular systolic function, which precipitate the early development ofCVD [2].In addition to changes in CV control, the sympathetic nervous system undergoesremarkable plasticity. These changes include decreased synaptic density accompanied byan increase in the number of inhibitory synapses rostral to the injury [3]. Caudal to thelesion, increased synaptogenesis [4] and changes in SPN morphology occurs, includingincreased arborization of SPNs [5] and axonal sprouting [6]. Historically, such sympatheticneuroplasticity has largely been considered detrimental due to the association of such plas-ticity with the expression of autonomic dysreflexia, immune suppression and neuropathicpain following SCI [7\u20139].In the wider field of SCI, a number of recent promising interventions have beenproposed that seeks to either leverage plasticity for functional benefit or alter such plasticityto offset functional decline. For example, the delivery of acute intermittent hypoxia (AIH)has been shown to enhance synaptic input onto spinal motor neurons and increase spinalexcitability, both of which increase synaptic strength [10,11] and subsequently improvemotor output in the acute [12,13] and chronic settings post-SCI [14,15]. Activity basedtherapy (ABT) is another intervention that has been demonstrated to facilitate the recoveryof specific tasks (i.e., swimming) [16,17], hind-limb [18,19], and forelimb function [20,21].These functional benefits are associated with increased spinal brain-derived neurotrophicfactor (BDNF) levels and synaptic plasticity [22]. In all the aforementioned studies, thebenefits of these therapies have been demonstrated in incomplete models of cervical and\/orlower-thoracic (i.e., T9\/10) SCI, wherein the injury is either not severe enough to induceCV dysfunction (i.e., the incomplete cervical models) or below the spinal level at whichinnervation to the key vascular beds and heart occurs (i.e., the low thoracic T9\/T10 models).For the CV system, a number of rat models have been developed to study the CVconsequences of SCI, as well as the efficacy of various therapeutics. Two of the modelsthat have received most traction are the T3\/T4 complete transection model or a verysevere midline contusion injury [23,24], though others also exist [25]. Both transection andsevere contusion injuries (i.e., 400 kdyn contusion model) have been effective in producingchanges to the CV function that mimic those observed clinically with high-lesion SCI, suchas the presence of pronounced hypotension, reduced systolic cardiac function, and thepresence of autonomic dysreflexia and orthostatic intolerance [23,26\u201329]. However, becausethese models either severed all pathways (in the case of transection injuries) or preservedsuch few medullary sympathetic pathways (i.e., <5% in the case of severe contusion) theyare likely to be inappropriate to test the application of interventions designed to strengthenspinal sympathetic pathways. Indeed, in the few studies that have investigated the effect ofABT on CV function using such models post-SCI it has been shown that ABT was ineffectivein restoring blood pressure control and systolic cardiac function, presumably because therewere not sufficient bulbo-spinal sympathetic pathways left for ABT to target [27]. Instead,any benefits of ABT in these settings appear to be limited to the peripheral circulationand\/or muscle.Here, we present an in vivo and histological validation of a new moderately severemid-line contusion injury model at the T3 level that we believe demonstrates an excellentbalance between sparing sufficient bulbo-spinal sympathetic pathways that can be targetedwith therapies, yet still induces a consistent and measurable decline in CV function thatBiology 2021, 10, 1006 3 of 16mimics that which occurs clinically. We propose that this model also more accurately reflectsthe changing demographic observed clinically, where the number of individuals withneurologically incomplete high-level injuries now outnumber those with neurologicallycomplete injuries.2. Materials and Methods2.1. Ethical ApprovalAll procedures were conducted in accordance with the Canadian Council for AnimalCare. Ethical approval was also obtained from the University of British Columbia (ACC-A18-0344).2.2. Experimental DesignA total of 26 male Wistar rats (Charles River Laboratories, 11 \u00b1 1 week old) wereassigned to either SCI (n = 13) or naive (n = 13) groups. Study endpoint was conductedat 2 weeks post-SCI. This timeframe was selected as reductions in BP fully manifest byday 6 [29] and cardiac dysfunction is present immediately following injury [30,31] andpersists into the chronic phase (i.e., 12 weeks post-SCI) [31,32]. Following in vivo measures,5 SCI animals were randomly selected for standard spinal cord immunohistochemistryquantification of the injury site, and the 3 animals had their spinal cords harvested andcut in the longitudinal axis to visualize descending catecholaminergic and serotonergicbulbo-spinal projections, both of which are known to play a key role in CV control in thechronic phase post-SCI [24,33].2.3. Spinal Cord Injury SurgeryRats were prepared for spinal cord contusion surgery as described in previous stud-ies [24,26,34,35]. The surgical preparation is depicted in Figure 1A. Briefly, on the day ofSCI animals were anesthetized (5% isofluorane chamber induction, maintenance on 1.5\u20132%isofluorane; Piramal Critical Care, Bethlehem, PA, USA) and administered enrofloxacin(10 mg\/kg; Bayer Animal Health, Shawnee, KS, USA), buprenorphine (0.5 mg\/kg; CevaAnimal Health, Cambridge, ON, Canada) and warmed lactated ringer\u2019s solution (5 mL sub-cutaneously; Baxter Corporation, Portland, OR, USA). A dorsal midline incision was madeand paraspinal musculature was bluntly dissected to expose C8\u2013T5 spinous processes. AT3 laminectomy was performed exposing the T3 dura. Rodents were then transportedand mounted on a plastic staging platform where the T2 and T4 spinous processes werestabilized with curved tip clamps. Using a high-definition camera secured to the mountingframe, the custom impactor tip (3 mm; Infinite Horizons (IH) Impactor; Precision Systemsand Instrumentation, Fairfax Station, VA, USA) was adjusted to track midline over theT3 dura. The impactor tip was dropped on the cord with 300 kdyn of predefined force(316 \u00b1 14 kdyn force, 1673 \u00b1 128 mm displacement, 124 \u00b1 5 mm\/s velocity). The muscleand the skin incisions were closed with 4-0 coated vicryl (Ethicon, Somerville, MA, USA).Velocity, force of impact, and distance travelled by the impactor were recorded. Animalswere recovered in an incubator for 30 min at 37 \u25e6C 50% humidity and received a subsequent5 mL lactated ringer\u2019s solution before they were returned to their home cages.2.4. Post-Surgical CareFor 4 days post injury, animals were administered subcutaneous lactated ringers (3\u00d7per day, 5 mL), buprenorphine (3\u00d7 per day, 0.02 mg\/kg) and enrofloxacin (1\u00d7 per day,10 mg\/kg). Bladders were manually voided 4\u00d7 per day until spontaneous voiding wasregained (4\u20136 days post-injury). Animals were pair-housed on oat bedding with rubbermatting to prevent the ingestion of woodchips due to opioid-induced pica and to aid inmobility. Animals were provided a supportive diet consisting of Hydrogel (ClearH2O),fruit, spinach, and cereal until mobility was regained and pica subsided.Biology 2021, 10, 1006 4 of 16Figure 1. (A) Rodent SCI surgery setup depicting laminectomy and contusion injury method.(B) In vivo terminal preparation consisting of an endotracheal tube and ventilator, left ventricularpressure-volume catheter, femoral artery pressure catheter, and femoral venous line.2.5. Outcome SurgeryAt 14 days post-SCI, echocardiography was performed to assess left ventricular (LV)structure, cardiac catheterization was performed to model LV pressure-volume relation-ships and assess LV contractility, arterial catheterization was performed to assess bloodpressure and a venous line was placed for intravenous fluid administration to maintainacid-base balance (Figure 1B).For the terminal in vivo assessments, animals were anesthetized with intraperitonealurethane (1.6 \u00b1 0.4 mg\/kg; Sigma-Aldrich, St. Louis, MO, USA). Animals were instru-mented with a rectal thermometer and all procedures were performed on a heating pad(RightTemp; Kent Scientific, Torrington, CT, USA) to maintain core body temperatureat 37 \u00b1 0.5 \u25e6C. Transthoracic echocardiography was used to obtain B-mode parasternallong axis images to measure LV volumes (Vevo 3100; VisualSonics, Toronto, ON, Canada).Next, the rat was placed supine, and a midline incision was performed from mandibleto manubrium. Sternohyoid muscle was bluntly dissected then trachea and right com-mon carotid artery (CCA) isolated. A tracheostomy was performed, an endotracheal tubewas secured, and the animal was ventilated on 100% O2 (VentElite; Harvard Apparatus,Holliston, MA, USA) using a standard tidal volume and breathing frequency calculationbased off the animal\u2018s mass [36]. The CCA was pierced, and a 1.9-French pressure-volume(PV) admittance catheter (Transonic Scisense, Ithaca, NY, US) advanced into the LV [36].Bilateral incisions along the inguinal ligament were performed and the femoral arteryand vein were isolated. A 1.6-French pressure catheter (Transonic Scisense, Ithaca, NY,USA) was placed into the left femoral artery and advanced into the abdominal aorta forcollection of hemodynamic data. The right femoral vein was cannulated with a fluiddelivery line (PE50 tubing) for constant infusion of lactated ringer\u2019s solution throughoutBiology 2021, 10, 1006 5 of 16the experiment (1.7 mL\/kg\/h; Pump 11 Elite, Harvard Apparatus, Holliston, MA, USA).Finally, a ventral laparotomy was performed and inferior vena cava isolated to performinferior vena cava occlusions (IVCOs) which enables venous return to be reduced and theslope of the end-systolic pressure-volume relationship to be obtained. The slope of thisrelationship is end-systolic elastance and is the reference standard for load-independentLV contractility [36].Following the completion of instrumentation, the animal was allowed to stabilize for15 min prior to the collection of a 5 min baseline for the assessment of hemodynamics andcardiac function. An IVCO was then performed to assess end-systolic elastance.2.6. Ethanasia and Tissue ProcessingFollowing the completion of all in vivo measures, 8 animals were selected at randomfor immunohistological preparation. Rats were perfused transcardially with 200\u2013300 mL of0.1 M phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MA, USA) and fixed with400\u2013500 mL 4% paraformaldehyde (PF; Sigma-Aldrich, St. Louis, MA, USA). Lesion sites(\u00b14 mm from epicenter; T1\u2013T5 segments) were dissected following perfusion and stored inPF for no more than 48 h followed by at least 24 h in 10% sucrose before being flash frozenin Shandon Cryomatrix (Thermo Scientific, Cat: 67-690-06, Waltham, MA, USA) and storedat \u221280 \u25e6C.2.7. Data AnalysisEchocardiography indices were obtained from an average of 3 end-systolic and end-diastolic images from each animal and used to correct PV estimates of volumes.All PV indices were analyzed using the PV loop analysis software in Labchart8 (ADInstruments). The following measures of LV systolic function were averaged across thefinal 60 s of baseline data: stroke volume (SV; calculated as end diastolic volume [EDV]-end systolic volume [ESV]), ejection fraction (EF; calculated as SV\/EDV\u00d7100%), end-systolic pressure (Pes), the maximal rate of rise of the LV pressure (dP\/dtmax), dP\/dtmaxnormalized to end-diastolic volume (dP\/dtmax\u2212EDV), stroke work (SW; area inside thePV loop), stroke work index (SWI; SWI = SW\/g), cardiac output (CO = SV\u00b7HR), cardiacindex (CI; CI = CO\/g), The following indexes of diastolic function were also measuredfrom the same loops: end-diastolic pressure (Ped), maximal rate of fall of the LV pressurewaveform (dP\/dtmin), and the time constant of LV pressure decay during isovolumetricrelaxation. Hemodynamic indices systolic blood pressure (SBP), diastolic blood pressure(DBP), pulse pressure (PP) (calculated as PP = SBP\u2013DBP), heart rate (HR), mean arterialpressure (MAP; calculated as MAP = 1\/3SBP + 2\/3 DBP) and systemic vascular resistance(SVR; SVR = MAP\/CO) were extracted from the same 60 s.Load-independent indices of LV contractility were calculated from one IVC occlusion.One 10-s section of the IVC occlusion was selected and loops that occurred during anexpiration were removed to prevent respiratory-induced changes in intrathoracic pressureright-shifting the PV loop. Preload-recruitable stroke work (PRSW) was evaluated as theslope of the linear regression of SW and EDV. End-systolic elastance (Ees) was taken as theslope of the end-systolic pressure-volume relationship. dP\/dtmax-EDV was calculated asthe slope of the linear regression of dP\/dtmax to EDV.Arterial elastance (Ea) was calculated as Ea = Pes\/SV. Ea\/Ees was calculated as thequotient of Ea divided by Ees.2.8. ImmunohistochemistrySpinal cords were cut using a cryostat (Leica, CM3050s, Wetzlar, Germany) in eitherthe transverse (n = 5) or longitudinal (n = 3) plane. Transverse sections were cut at 10 \u00b5mthickness with an inter-section distance of 1 mm. Longitudinal sections (n = 3) were cut at10 \u00b5m thickness and with an inter-section distance of 600 \u00b5m.Slides were thawed and dried at room temperature for 20 min then a hydrophobicbarrier was drawn. Slides were rehydrated with 3 10-min washes in PBS followed byBiology 2021, 10, 1006 6 of 16incubation in blocking solution (10% normal donkey serum) in PBS-Tx-Azd for 45 min.Slide were then incubated with primary antibodies over night. The next day the tissuewas washed three times (15 min each) with PBS, incubated with secondary antibodiesfor 2 h, and then washed with PBS three times (15 min each). Finally, the slides werecover-slipped using ProLong Gold antifade mounting medium (Invitrogen, LSP36930,Waltham, MA, USA).For transverse sections primary antibodies were used as follows; mouse GFAP (Glialfibrillary acidic protein; 1:1000, Sigma; G3893, Waltham, MA, USA), chicken polyclonalMBP (Myelin basic protein; 1:1000, Aves Labs; MBP), guineapig NeuN (Neuronal nuclei;1:500, Sigma; ABN90P, St. Louis, MA, USA). The following secondaries were used; donkeyanti-mouse Cy3 (1:800, Jackson Immunoresearch; 705-166-147, West Grove, PA, USA),donkey anti-chicken pAb Alexa647 (1:800, Jackson Immunoresearch; 7056-606-148, WestGrove, PA, USA), donkey anti-guineapig DyLight405 (1:800, Jackson Immunoresearch;711-475-152, West Grove, PA, USA).For longitudinal slides primary antibodies were used as follows; sheep TH (tyro-sine hydroxylase; 1:200, EMD Milipore; AB1542, Burlington, VT, USA), rabbit 5-HT (5-hydroxytryptamine; 1:2000, Immunostar; 20080. Hudson, NY, US). The following secon-daries were used; Donkey anti-sheep Cy3 (1:200, Jackson Immunoresearch; 713-166-147,West Grove, PA, USA) and donkey anti-rabbit DyLight488 (1:1000, Abcam; ab96899, Cam-bridge, UK).Immunofluorescence imaging was performed using an Axio Imager M2 microscope(Zeiss, Oberkochen, Germany) with an Axiocam 705 mono camera (Zeiss, Oberkochen,Germany) using ZEN 2 Blue software (Zeiss, Oberkochen, Germany). Images were digitallyprocessed using Zen 2 Blue software (Zeiss, Oberkochen, Germany).Analysis was performed in ImageJ (ImageJ, Rockville, MD, USA). Lesion area andwhite matter sparing were quantified every 400 \u00b5m from 2.0 mm rostral to 2.0 mm caudalto the injury epicenter. The injury epicenter section was based on the section with the leastintact GFAP signal. Lesion area was manually outlined based on the following definition:GFAP-negative or GFAP-positive area with disrupted or abnormal cytoarchitecture. Carewas taken to avoid inclusion of any artifacts. Myelin preservation (i.e., white mattersparing) was estimated by manually outlining MBP-positive area with normal or near-normal cytoarchitecture. Lesion volume was then calculated according to the followingformula: Volume = \u03a3 (area \u00b7 section thickness \u00b7 number of sections between samples) [24].For longitudinal sections, images were imported into ImageJ (ImageJ, Rockville, MD,USA) and converted to 8-bit. The backgrounds were then subtracted, and for each stain, theimages were set to a threshold only including pixels with intensity values from 20\u2013255. Theanalyzed regions were selected by tracing the epicenter and selecting 2 \u00d7 1 mm rectangles0.5 mm rostral and caudal to the border of the lesion. After measuring the positive pixeldensity of the enclosed areas, the density of the caudal area was divided by the densityof the rostral area to calculate the percent difference. The relative density of an anterior,posterior and central section of the cord for each animal was calculated and expressed asmeans and standard deviations calculated from 3 animals.2.9. StatisticsBetween-group differences in all in vivo physiological outcomes were analyzed usingan independent samples t-test in SPSS (IBM SPSS Statistics, Chicago, IL, USA). Data areexpressed as means \u00b1 standard deviation. Statistical significance was set at p < 0.05.Graphical representations of in vivo data were produced in MATLAB (MathWorks, Natick,MA, USA) and Prism (GraphPad Prism, San Diego, CA, USA). Histological images wereproduced in Zen Image Processing (Zeiss, Oberkochen, Germany).Biology 2021, 10, 1006 7 of 163. ResultsAt study termination SCI rats were significantly lighter than na\u00efve animals (p = 0.014;Figure 2) but there was no significant difference in body mass of SCI animals at day 14post-injury vs. pre-injury (p = 0.154).Figure 2. Body mass with time pre\/post-injury. Note there were no significant differences in bodymass across the 14-day study period. Black lines indicate mean mass \u00b1 SD, grey lines representindividual animals body mass.3.1. Resting Hemodynamics Are Impaired in T3 300 kdyn SCI RatsHemodynamic indices are presented in Table 1 and Figure 3. SBP, DBP, PP and MAPwere all significantly reduced among SCI compared to na\u00efve rats (all p < 0.001). HR wassignificantly higher among SCI rats compared to na\u00efve rats (p = 0.008). Systemic vascularresistance (SVR) was also reduced in SCI rats compared to na\u00efve (p = 0.038).Table 1. Anthropometric, hemodynamic and pressure-volume data for 2-week post T3 300 kdyn SCI and na\u00efve rats.Na\u00efve SCI p-ValueHemodynamic DataSBP (mmHg) 121 \u00b1 7 96 \u00b1 11 <0.001DBP (mmHg) 70 \u00b1 7 58 \u00b1 9 <0.001MAP (mmHg) 88 \u00b1 7 70 \u00b1 9 <0.001PP (mmHg) 50 \u00b1 4 38 \u00b1 6 <0.001HR (BPM) 413 \u00b1 38 462 \u00b1 48 0.008SVR(mmHg\u00b7min\u22121\u00b5L\u22121) * 0.89 \u00b1 0.20 0.74 \u00b1 0.12 0.038Pressure-Volume DataESV (\u00b5L) 66 \u00b1 18 59 \u00b1 11 0.256EDV(\u00b5L) 311 \u00b1 47 261 \u00b1 23 0.002Systolic FunctionSW (mmHg\u00b7mL) 33 \u00b1 7 21 \u00b1 4 <0.001SWI (mmHg\u00b7mL\u22121100 g\u22121) 10.90 \u00b1 2.53 7.52 \u00b1 1.64 <0.001CO (mL\/min) 102 \u00b1 20 93 \u00b1 12 0.201CI (mL\u00b7min\u22121100 g\u22121) 33.69 \u00b1 7.72 35.22 \u00b1 7.32 0.610SV (\u00b5L) 245 \u00b1 33 202 \u00b1 24 0.001Pes (mmHg) 98 \u00b1 11 75 \u00b1 10 <0.001EF (%) 79 \u00b1 4 77 \u00b1 5 0.272dP\/dtmax (mmHg\/s) 10316 \u00b1 809 6084 \u00b1 755 <0.001Biology 2021, 10, 1006 8 of 16Table 1. Cont.Na\u00efve SCI p-ValueEes (mmHg\/\u00b5L) ** 1.59 \u00b1 0.23 0.89 \u00b1 0.24 <0.001Ea (mmHg\/\u00b5L) 0.41 \u00b1 0.09 0.38 \u00b1 0.07 0.296Ea\/Ees ** 0.26 \u00b1 0.06 0.44 \u00b1 0.23 0.021PRSW (mmHg) * 131 \u00b1 30 94 \u00b1 17 0.001+dP\/dtmax\u2013EDV(mmHg\u00b7s\u22121 \u00b5L\u22121) * 34 \u00b1 7 27 \u00b1 4 <0.001Diastolic FunctiondP\/dtmin (mmHg\/s) \u22125890 \u00b1 449 \u22124021 \u00b1 630 <0.001Ped (mmHg) 3 \u00b1 2 4 \u00b1 4 0.231\u03c4 (ms) 7.32 \u00b1 0.77 8.03 \u00b1 3 0.416Data are presented as means \u00b1 SD. p-values represent significant difference following independent samples t test. SBP, systolic bloodpressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; PP, pulse pressure; HR, heart rate; SVR, systemic vascular resistance;ESV, end-systolic volume; EDV, end-diastolic volume; SW, stroke work; SWI, stroke work index; CO, cardiac output; CI, cardiac index; SV,stroke volume; Pes, end-systolic pressure; EF, ejection fraction; dP\/dtmax, maximum rate of rise of left ventricular pressure; Ees, end-systolicpressure-volume relationship; Ea, arterial elastance; PRSW, preload-recruitable stroke work; dP\/dtmin, maximum rate of decay of leftventricular pressure; Ped, end-diastolic pressure; \u03c4 time constant of left ventricular pressure decay. * denotes na\u00efve n = 12, SCI n = 12,** denotes na\u00efve n = 12, SCI n = 9 due to difficulties in performing IVC occlusions in some animals.Figure 3. Comparison of resting hemodynamic indices of na\u00efve (n = 13) and spinal cord injured (SCI)rats (n = 13) two weeks post-injury. Bars represent the means and standard deviations overlaid withindividual data. (A) Systolic blood pressure (SBP) was significantly lower in SCI compared to na\u00efveanimals (25 \u00b1 4 mmHg, p < 0.001). (B) Mean arterial pressure (MAP) was significantly lower amongSCI compared to na\u00efve animals (17 \u00b1 3 mmHg, p < 0.001). (C) Systemic vascular resistance (SVR)was significantly lower among SCI compared to na\u00efve animals (0.11 \u00b1 0.08 mmHg\u00b7min\u22121 \u00b5L\u22121,p = 0.034). (D): Heart rate (HR) was significantly higher among SCI compared to na\u00efve animals(49 \u00b1 17 beats per minute, BPM; p = 0.008).Biology 2021, 10, 1006 9 of 163.2. Left Ventricular Systolic Function Is Impaired in T3 300 kdyn SCI RatsLV measures of systolic and diastolic function are reported in Table 1 and selectindices are displayed in Figure 4. Among SCI rats, a decrease in EDV (p = 0.002) andSV (p = 0.001) was observed compared to na\u00efve rats, in the absence of changes to ESV(p = 0.256) and EF (p = 0.272). SW and SWI were significantly lower among SCI ratscompared to na\u00efve (both p < 0.001). Conversely, there was no difference in CO or CIbetween groups (p = 0.201; p = -0.610, respectively). Pes, Pmax and the maximum rate ofrise of LV pressure (dP\/dtmax) were lower among SCI compared to na\u00efve rats (all p < 0.001).Measures of load-independent function, Ees (p < 0.001), dP\/dtmax\u2212EDV (p < 0.001), andPRSW (p = 0.001) were significantly lower in SCI compared to na\u00efve rats. Ea was notsignificantly different between groups (p = 0.296), however Ea\/Ees was significantly higherin SCI rats compared to na\u00efve.Figure 4. At 2 weeks post-SCI, animals underwent left ventricular catheterization to assess cardiac function. Pressure-volume analysis revealed reduced pressure, volume, and contractile function among SCI compared to na\u00efve rats. (A) Anexample pressure-volume loop labelled with relevant indices acquired from pressure-volume analysis (ESV; end-systolicvolume, EDV; end-diastolic volume, Pes; end-systolic pressure, Ped; end-diastolic pressure, SV; stroke volume, SW;stroke work (area of the pressure volume loop), ESPVR; end systolic pressure volume relationship, Ees slope of ESPVR).(B) Example basal pressure volume loop from SCI and na\u00efve rats, overlaid with SEM bars, demonstrating diminished LVmaximum pressure (22 \u00b1 4 mmHg; p < 0.001) and EDV (50 \u00b1 15 ul; p = 0.002) in SCI compared to na\u00efve animals. C-D:Example inferior vena cava occlusions from na\u00efve (C) and SCI (D) groups demonstrating reduced Ees among SCI comparedto na\u00efve animals (0.7 \u00b1 0.1 mmHg\/ul; p < 0.001).Biology 2021, 10, 1006 10 of 16For diastolic function, dP\/dtmin was significantly lower among SCI rats compared tona\u00efve (p > 0.001) in the absence of differences in end diastolic pressure (p = 0.231) and timeconstant of LV pressure decay (tau, p = 0.416).3.3. Moderately-Severe T3 Midline Injury Interrupts Descending PathwaysFollowing moderately-severe T3 SCI the lesion area was 1.75 \u00b1 0.40 mm2 leaving21 \u00b1 6% tissue sparing. Lesion volume was 4.26 \u00b1 1.28 mm3. White matter sparing at theepicenter was 33 \u00b1 9% (Figure 5). The density of 5-HT+ fibres caudal to the epicenter wasreduced to 9 \u00b1 2% of rostral density (Figure 6). The density of TH+ fibres caudal to theepicenter were reduced to 18 \u00b1 9% of the rostral density (Figure 6).Figure 5. Lesion site characterization. (A) Representative immunohistological images of the rostral (top), epicenter (middle),and caudal (bottom) sections. Stains from left to right are Neuronal Nuclei (NeuN), Glial Fibrillary Protein (GFAP), MyelinBasic Protein (MBP), and the merged stain. Data were quantified every 400 \u00b5m from the lesion epicenter to a distanceof 2 mm rostrally and caudally (n = 5). (B) The GFAP signal was used to quantify lesion area which reached an area of1.75 \u00b1 0.40 mm2 at the epicenter. Data points and bars represent the mean and standard deviations, respectively. (C) Lesionvolume was calculated as Volume = \u03a3 (area \u00b7 section thickness \u00b7 number of sections between samples) [24] and was found tobe 4.26 \u00b1 1.28 mm3. Bars represent the means and standard deviations overlaid with individual data. (D) White mattersparing was quantified using the MBP signal and reached a minimum sparing at the epicenter of 33 \u00b1 9%. Data points andbars represent the mean and standard deviations, respectively.Biology 2021, 10, 1006 11 of 16Figure 6. Representative immunohistological images of longitudinal spinal cord sections, anterior (top), central (middle),and posterior (bottom) (A\u2013C). Stains from left to right are 5-HT+, TH+ and merged. Each quantified section was 500 \u00b5mremoved from the one previous (D) Schematic depicting the anatomical location of where densities of 5-HT+ and TH+ weremeasured 0.5 mm rostral and caudal to the epicenter with the area of study being 2 mm wide by 1 mm tall. The associateddensity plots comparing the caudal to the rostral stain density across anterior, central and posterior sections for 5-HT+(E) TH+ (F). Caudal sparing was quantified as 9 \u00b1 2% and 18 \u00b1 9% relative to rostral for 5-HT+ and TH+, respectively(n = 3). Data are presented as means \u00b1 standard error.4. DiscussionWe have developed a novel moderately severe high-thoracic midline contusion SCImodel that produces robust and clinically relevant impairment in cardiac and hemody-namic function whilst preserving 33 \u00b1 9% of white matter at the injury epicenter. Thoughour model also robustly reduces the density of 5-HT+ and TH+ fibres at and below theinjury epicenter, we were able to clearly visualize both TH+ and 5-HT+ fibres projectingthrough and below the injury site. As such we believe this model provides a nice balancebetween producing a clinically relevant decline in CV function yet sparing sufficient bulbo-spinal sympathetic and serotonergic pathways that can be targeted with therapies designedto induce\/alter spinal neuroplasticity with a view to improving CV function.4.1. Resting Hemodynamics Are Impaired in T3 300 kdyn SCI RatsReduced SBP and MAP have been demonstrated in a variety of high-thoracic con-tusion, clip compression and transection SCI models. Though it is difficult to compareBP across studies due to heterogeneity in measurement technique and rodent strain, themagnitude of decline in SBP and MAP is typically in the 15\u201325 mmHg range with completetransection or severe contusion at the T2\u2013T4 spinal level [24,26,27,32,34,37]. We found asimilar 25 mmHg decline in the present study despite our model being less severe andexhibiting more sparing at the injury epicenter (see below) than those typically used toinduce CV dysfunction. It has been recently shown that the major \u02ddhot-spot\u02dd for blood pres-sure control are the splanchnic projecting SPNs that exit the cord at the T11\u2013T13 level [28].Biology 2021, 10, 1006 12 of 16SPNs in this region of the cord are under the control of both descending bulbo-spinalcatecholaminergic and serotonergic [33] pathways originating in the RVLM and Raphe,respectively. Histological analyses of TH+ and 5-HT+ fibres in longitudinal sections of thespinal cord revealed our injury significantly reduces the density of both sets of fibers atand below (vs. above) the injury site. In turn, this loss of catecholaminergic and seroton-ergic excitatory input to SPNs reduces vascular tone, leading to splanchnic pooling andhypotension [38]. Persistent hypotension is of clinical importance as it contributes to thedisproportionate burden of ischemic stroke heart disease observed in SCI [2]. Notably, a20 mmHg decline in SBP and MAP is typical of that observed in individuals with chronichigh-level SCI [39,40], thus increasing the potential for translation of findings using thismodel. Importantly, whilst TH+ and 5-HT+ fibre density was reduced post-SCI we wereable to clearly visualize both TH+ and 5-HT+ fibres traversing the injury site. We believethe presence of such fibres, whilst insufficient to offset hypotension, can act as a target forneurotherapeutic interventions that aim to strengthen synaptic input.4.2. Left Ventricular Systolic Function Is Impaired in T3 300 kdyn SCI RatsAnother major finding of the present was that almost all pressure- and volume-related indices of resting LV function were significantly decreased in SCI vs. na\u00efverats, with the exception of ESV and EF. We have previously reported similar findingsin more severe models of SCI (i.e., T3 400 kdyn contusion with 5 s dwell, or T3 completetransection) [24,26,27,34,37], but not in less-severe models of SCI (T3 200 kdyn contusion,5 s dwell) [34]. Reductions in volumetric function post-SCI also occurs clinically withhigh-level SCI. Although there was no difference in ESV, SV was lower in SCI rats likelydue to reduced preload (i.e., EDV). Interestingly, this decrease in SV was compensated byincreases in HR resulting in no statistical difference in CO between groups. A SCI-inducedincrease in HR has also been demonstrated in other studies with injuries at the T3\u2013T5 spinallevel, and has been hypothesized to result from increased sympathetic activity above thelevel of injury [41] as this injury model spares some sympathetic input to T1 level SPNs. Itis equally possible, however, that SCI induces changes in cardiovagal balance such thatHR can increase via vagal withdrawal sufficiently to normalize CO. Whilst the reductionin resting pressure and volume indices of LV systolic function are likely due to reducedcatecholaminergic and serotonergic input to the SPNs in the T2\u2013T5 level of the spinalcord [42], these indices are also critically dependent on changes in pre-load and afterload,both of which are impacted by SCI, as evidenced by reduced EDV and systemic bloodpressure in this present study. Unlike resting pressure-volume indices, dP\/dtmax\u2212EDV,PRSW and Ees obtained from IVC occlusions are largely insensitive to changes in loador rate [43] and as such are considered the reference metrics for LV systolic function [36].We found all 3 metrics were significantly reduced in SCI vs. na\u00efve rats, presumably dueto the reduced density of 5-HT+ and TH+ fibres at and below the injury epicenter. Themagnitude of reduction in these indices of contractility is similar to that observed in ourmore severe injury models [26,27,32,35] despite our current model sparing more whitematter and there being a clear visualization of both TH+ and 5-HT+ fibres traversing theinjury site. The reduction in Ees precipitated an increase in the Ea\/Ees ratio, implying thisSCI model impairs cardiac efficiency.4.3. T3 Moderately-Severe Contusion Injury Interrupts Descending PathwaysModerately-severe T3 spinal cord contusion injury resulted in substantial white matterdamage at the epicenter and a lesion that extended at least 2 mm rostral and caudallyencompassing the T2 spinal level thus interrupting supraspinal control to SPNs criticalfor CV regulation at the spinal levels described above. These histological findings aresupported by the reduced density of 5-HT+ and TH+ fibres below the lesion suggestingthat serotonergic and catecholaminergic input to SPNs is reduced by this model of SCI.Serotonergic [44,45] and catecholaminergic [46,47] bulbo-spinal fibres densely innervateareas of the cord associated with input to SPNs. Loss of supraspinal 5-HT+ fibres play aBiology 2021, 10, 1006 13 of 16key role in inducing CV dysregulation including contributing to the development of ADand hypotension observed post-SCI [33,48]. Other models of SCI which have exploredthe relationship between 5-HT+ preservation and CV function post-SCI include completecrush [25], and partial transection [49] models of SCI at the T4 level. Neither crush injurynor partial transection reported reduced MAP despite showing reduced\/absent densityof serotonergic fibres caudal to the injury. While these SCI models were performed ata more caudal spinal level, CV dysfunction is seen in severe injuries as low as T6. Thisobservation suggests, perhaps, that the mechanism of injury is not severe enough to seversufficient pathways to impair CV function, given that it is necessary to decrease whitematter sparing substantially to induce a decline in CV function post-SCI [24]. Other modelsof SCI which demonstrated motor recovery following ABT utilized a T10 contusive injurywhich resulted in 8\u201322% white matter sparing at the epicenter [17] or T10 hemisectionmodels with approximately 35% white matter sparing [18]. Such levels of \u2019required\u2019sparing suggests that our model maintains sufficient pathways for successful applicationof therapies to induce neuroplasticity.4.4. Comparison to Other Rodent Models of CV InstabilityA number of rodent models of CV dysfunction have now been proposed in theliterature and we have summarized the findings and gaps in knowledge from these modelsin Table 2. To aid in this comparison, we selected a number of key findings that we believeare critical for an animal model to exhibit when studying the efficacy of interventionsaimed at inducing neuroplasticity. Our selected indices included reduced blood pressure,cardiac pressures and cardiac volumes, as well as sufficient tissue and white matter sparingat the epicentre and the presence of catecholaminergic and serotonergic fibres traversingthe injury site. In the studies conducted to date that we are aware of, whilst almost allof the high-thoracic models induce reductions in blood pressure and\/or heart functionit is likely that only the current T3 contusion model has sufficient tissue sparing andcatecholaminergic\/serotonergic projections for such therapies to work. As such we believeour model achieves a feat not previously achieved in prior models; that is, modest tissuesparing at the injury epicenter yet a severe reduction in cardiac and hemodynamic function.Table 2. Comparisons with previous models of SCI that have been used to induce cardiovascular dysfunction.Injury ModelT3 300 kdynContusionT2 400 kdynContusionT2 200 kdynContusionT2-3TransectionT4Complete CrushT10 400 kdynContusion\u2193 Blood pressure 4 4 4 4 6 6\u2193 Cardiac pressures 4 4 ? 4 ? ?\u2193 Cardiac output 6 4 6 4 ? ?>15% tissue sparing 4 6 6 6 ? ?>20% white matterPreservation 4 6 6 6 ? 6Preservedsub-lesionalserotonergic\/catecholaminergicpathways4 6 4 ? 6 N\/AReferences [24,26,27,34] [24,34] [23,30,32] [25] [50,51]An additional benefit of this model is that animal health was greatly improved overour typical experience with more severe contusion and transection injury models. Notably,animals regained spontaneous voiding within 5 days post-injury compared to the typical10 days seen among transected rats [52], returned to pre-injury health scores and pre-surgical body mass by 14 days-post injury and mortality was remarkably low with 93% ofall animals surviving the initial injury surgery and no mortality across the 14-day period.We believe this model, therefore, improves upon animal welfare and decreases the burdenof care on researchers.Biology 2021, 10, 1006 14 of 164.5. LimitationsThis model has yet to be tested in female rats to account for sex differences in auto-nomic function. Although we know that cardiac dysfunction manifests within the first4 h post-SCI [31], and there are similar impairments at 3 and 7 days [53], 5 weeks [34],and 12 weeks post-SCI [32] the time course of CV function beyond 12 weeks in contusivemodels of SCI has not yet been characterized.5. ConclusionsHere, we have presented a high-thoracic contusion model of SCI which demonstratedmarked CV decline and modest tissue sparing at the epicenter, a feat not achieved byprevious SCI models. Given the recent impetus of the field to move towards interventionsthat aim to enhance neuroplasticity (i.e., ABT and\/or AIH) we believe this model will beuseful to test the efficacy of these interventions to improve CV and autonomic function.Author Contributions: Conceptualization, L.W. and C.R.W.; Data curation, L.W.; formal analysis,L.W., E.L.E. and T.M.H., Funding acquisition, C.R.W., Investigation, L.W. and M.A., methodology,L.W., M.A., E.L.E., T.M.H. and C.R.W.; Project administration, E.L.E.; Resources, E.L.E., Supervision,C.R.W.; visualization, L.W., E.L.E. and T.M.H. writing\u2014original draft preparation, L.W.; writing\u2014review and editing, L.W., E.L.E., T.M.H. and C.R.W. All authors have read and agreed to the publishedversion of the manuscript.Funding: This research was funded by Praxis as a part of the Blusson Integrated Cares Partner-ship, number GR004076. Research in the lab of Christopher West is funded by an infrastructuregrant from the Canadian Foundation for Innovation and BC Knowledge Development Fund: grantnumber 34803.Institutional Review Board Statement: The study was conducted according to the guidelines ofthe Declaration of Helsinki and approved by the clinical Research Ethics Board of the University ofBritish Columbia (ACC-A18-0344; 24 January 2019).Informed Consent Statement: Not applicable.Data Availability Statement: Data available upon request.Acknowledgments: The authors thank Ryan L. Hoiland and Liam C. Stewart for their excellenttechnical assistance.Conflicts of Interest: The authors declare no conflict of interest.References1. Garshick, E.; Kelley, A.; A Cohen, S.; Garrison, A.; Tun, C.G.; Gagnon, D.; Brown, R. 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