Proteomic Portraits Reveal Evolutionarily Conserved and Divergent Responses to Spinal Cord Injury.

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of cerebrospinal fluid and serum samples from 111 patients with acute SCI and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at 6 months with an area under the receiver operating characteristic curve of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response and establish the cerebrospinal fluid abundance of glial fibrillary acidic protein as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

A cross-species validation of single-beat metrics of cardiac contractility.

The assessment of left ventricular (LV) contractility in animal models is useful in various experimental paradigms, yet obtaining such measures is inherently challenging and surgically invasive. In a cross-species study using small and large animals, we comprehensively tested the agreement and validity of multiple single-beat surrogate metrics of LV contractility against the field-standard metrics derived from inferior vena cava occlusion (IVCO). Fifty-six rats, 27 minipigs and 11 conscious dogs underwent LV and arterial catheterization and were assessed for a range of single-beat metrics of LV contractility. All single-beat metrics were tested for the various underlying assumptions required to be considered a valid metric of cardiac contractility, including load-independency, sensitivity to inotropic stimulation, and ability to diagnose contractile dysfunction in cardiac disease. Of all examined single-beat metrics, only LV maximal pressure normalized to end-diastolic volume (EDV), end-systolic pressure normalized to EDV, and the maximal rate of rise of the LV pressure normalized to EDV showed a moderate-to-excellent agreement with their IVCO-derived reference measure and met all the underlying assumptions required to be considered as a valid cardiac contractile metric in both rodents and large-animal models. Our findings demonstrate that single-beat metrics can be used as a valid, reliable method to quantify cardiac contractile function in basic/preclinical experiments utilizing small- and large-animal models KEY POINTS: Validating and comparing indices of cardiac contractility that avoid caval occlusion would offer considerable advantages for the field of cardiovascular physiology. We comprehensively test the underlying assumptions of multiple single-beat indices of cardiac contractility in rodents and translate these findings to pigs and conscious dogs. We show that when performing caval occlusion is unfeasible, single-beat metrics can be utilized to accurately quantify cardiac inotropic function in basic and preclinical research employing various small and large animal species. We report that maximal left-ventricular (LV)-pressure normalized to end-diastolic volume (EDV), LV end-systolic pressure normalized to EDV and the maximal rate of rise of the LV pressure waveform normalized to EDV are the best three single-beat metrics to measure cardiac inotropic function in both small- and large-animal models.

Duraplasty in Traumatic Thoracic Spinal Cord Injury: Impact on Spinal Cord Hemodynamics, Tissue Metabolism, Histology, and Behavioral Recovery Using a Porcine Model.

After acute traumatic spinal cord injury (SCI), the spinal cord can swell to fill the subarachnoid space and become compressed by the surrounding dura. In a porcine model of SCI, we performed a duraplasty to expand the subarachnoid space around the injured spinal cord and evaluated how this influenced acute intraparenchymal hemodynamic and metabolic responses, in addition to histological and behavioral recovery. Female Yucatan pigs underwent a T10 SCI, with or without duraplasty. Using microsensors implanted into the spinal cord parenchyma, changes in blood flow (ΔSCBF), oxygenation (ΔPO2), and spinal cord pressure (ΔSCP) during and after SCI were monitored, alongside metabolic responses. Behavioral recovery was tested weekly using the Porcine Injury Behavior Scale (PTIBS). Thereafter, spinal cords were harvested for tissue sparing analyses. In both duraplasty and non-animals, the ΔSCP increased ∼5 mm Hg in the first 6 h post-injury. After this, the SCP appeared to be slightly reduced in the duraplasty animals, although the group differences were not statistically significant after controlling for injury severity in terms of impact force. During the first seven days post-SCI, the ΔSCBF or ΔPO2 values were not different between the duraplasty and control animals. Over 12 weeks, there was no improvement in hindlimb locomotion as assessed by PTIBS scores and no reduction in tissue damage at the injury site in the duraplasty animals. In our porcine model of SCI, duraplasty did not provide any clear evidence of long-term behavioral or tissue sparing benefit after SCI.

Characterization of Lower Urinary Tract Dysfunction after Thoracic Spinal Cord Injury in Yucatan Minipigs.

There is an increasing need to develop approaches that will not only improve the clinical management of neurogenic lower urinary tract dysfunction (NLUTD) after spinal cord injury (SCI), but also advance therapeutic interventions aimed at recovering bladder function. Although pre-clinical research frequently employs rodent SCI models, large animals such as the pig may play an important translational role in facilitating the development of devices or treatments. Therefore, the objective of this study was to develop a urodynamics protocol to characterize NLUTD in a porcine model of SCI. An iterative process to develop the protocol to perform urodynamics in female Yucatan minipigs began with a group of spinally intact, anesthetized pigs. Subsequently, urodynamic studies were performed in a group of awake, lightly restrained pigs, before and after a contusion-compression SCI at the T2 or T9-T11 spinal cord level. Bladder tissue was obtained for histological analysis at the end of the study. All anesthetized pigs had bladders that were acontractile, which resulted in overflow incontinence once capacity was reached. Uninjured, conscious pigs demonstrated appropriate relaxation and contraction of the external urethral sphincter during the voiding phase. SCI pigs demonstrated neurogenic detrusor overactivity and a significantly elevated post-void residual volume. Relative to the control, SCI bladders were heavier and thicker. The developed urodynamics protocol allows for repetitive evaluation of lower urinary tract function in pigs at different time points post-SCI. This technique manifests the potential for using the pig as an intermediary, large animal model for translational studies in NLUTD.

Cardio-centric hemodynamic management improves spinal cord oxygenation and mitigates hemorrhage in acute spinal cord injury.

Chronic high-thoracic and cervical spinal cord injury (SCI) results in a complex phenotype of cardiovascular consequences, including impaired left ventricular (LV) contractility. Here, we aim to determine whether such dysfunction manifests immediately post-injury, and if so, whether correcting impaired contractility can improve spinal cord oxygenation (SCO2), blood flow (SCBF) and metabolism. Using a porcine model of T2 SCI, we assess LV end-systolic elastance (contractility) via invasive pressure-volume catheterization, monitor intraparenchymal SCO2 and SCBF with fiberoptic oxygen sensors and laser-Doppler flowmetry, respectively, and quantify spinal cord metabolites with microdialysis. We demonstrate that high-thoracic SCI acutely impairs cardiac contractility and substantially reduces SCO2 and SCBF within the first hours post-injury. Utilizing the same model, we next show that augmenting LV contractility with the ß-agonist dobutamine increases SCO2 and SCBF more effectively than vasopressor therapy, whilst also mitigating increased anaerobic metabolism and hemorrhage in the injured cord. Finally, in pigs with T2 SCI survived for 12 weeks post-injury, we confirm that acute hemodynamic management with dobutamine appears to preserve cardiac function and improve hemodynamic outcomes in the chronic setting. Our data support that cardio-centric hemodynamic management represents an advantageous alternative to the current clinical standard of vasopressor therapy for acute traumatic SCI.

Relationship between Early Vasopressor Administration and Spinal Cord Hemorrhage in a Porcine Model of Acute Traumatic Spinal Cord Injury.

Current practice guidelines for acute spinal cord injury (SCI) recommend augmenting mean arterial blood pressure (MAP) for the first 7 days post-injury. After SCI, the cord may be compressed by the bone/ligaments of the spinal column, limiting regional spinal cord blood flow. Following surgical decompression, blood flow may be restored, and can potentially promote a "reperfusion" injury. The effects of MAP augmentation on the injured cord during the compressed and decompressed conditions have not been previously characterized. Here, we used our porcine model of SCI to examine the impact of MAP augmentation on blood flow, oxygenation, hydrostatic pressure, metabolism, and intraparenchymal (IP) hemorrhage within the compressed and then subsequently decompressed spinal cord. Yucatan mini-pigs underwent a T10 contusion injury followed by 2 h of sustained compression. MAP augmentation of ∼20 mm Hg was achieved with norepinephrine (NE). Animals received MAP augmentation either during the period of cord compression (CP), after decompression (DCP), or during both periods (CP-DCP). Probes to monitor spinal cord blood flow (SCBF), oxygenation, pressure, and metabolic responses were inserted into the cord parenchyma adjacent to the injury site to measure these responses. The cord was harvested for histological evaluation. MAP augmentation increased SCBF and oxygenation in all groups. In the CP-DCP group, spinal cord pressure steadily increased and histological analysis showed significantly increased hemorrhage in the spinal cord at and near the injury site. MAP augmentation with vasopressors may improve blood flow and reduce ischemia in the injured cord but may also induce undesirable increases in IP pressure and hemorrhage.

Sensorimotor plasticity after spinal cord injury: a longitudinal and translational study.

Objective: The objective was to track and compare the progression of neuroplastic changes in a large animal model and humans with spinal cord injury. Methods: A total of 37 individuals with acute traumatic spinal cord injury were followed over time (1, 3, 6, and 12 months post-injury) with repeated neurophysiological assessments. Somatosensory and motor evoked potentials were recorded in the upper extremities above the level of injury. In a reverse-translational approach, similar neurophysiological techniques were examined in a porcine model of thoracic spinal cord injury. Twelve Yucatan mini-pigs underwent a contusive spinal cord injury at T10 and tracked with somatosensory and motor evoked potentials assessments in the fore- and hind limbs pre- (baseline, post-laminectomy) and post-injury (10 min, 3 h, 12 weeks). Results: In both humans and pigs, the sensory responses in the cranial coordinates of upper extremities/forelimbs progressively increased from immediately post-injury to later time points. Motor responses in the forelimbs increased immediately after experimental injury in pigs, remaining elevated at 12 weeks. In humans, motor evoked potentials were significantly higher at 1-month (and remained so at 1 year) compared to normative values. Conclusions: Despite notable differences between experimental models and the human condition, the brain's response to spinal cord injury is remarkably similar between humans and pigs. Our findings further underscore the utility of this large animal model in translational spinal cord injury research.

Optical Assessment of Spinal Cord Tissue Oxygenation Using a Miniaturized Near Infrared Spectroscopy Sensor.

Despite advances in the treatment of acute spinal cord injury (SCI), measures to mitigate permanent neurological deficits in affected patients are limited. Immediate post-trauma hemodynamic management of patients, to maintain blood supply and improve oxygenation to the injured spinal cord, is currently one aspect of critical care which clinicians can utilize to improve neurological outcomes. However, without a way to monitor the response of spinal cord hemodynamics and oxygenation in real time, optimizing hemodynamic management is challenging and limited in scope. This study aims to investigate the feasibility and validity of using a miniaturized multi-wavelength near-infrared spectroscopy (NIRS) sensor for direct transdural monitoring of spinal cord oxygenation in an animal model of acute SCI. Nine Yorkshire pigs underwent a weight-drop T10 contusion-compression injury and received episodes of ventilatory hypoxia and alterations in mean arterial pressure (MAP). Spinal cord hemodynamics and oxygenation were monitored throughout by a non-invasive transdural NIRS sensor, as well as an invasive intraparenchymal sensor as a comparison. NIRS parameters of tissue oxygenation were highly correlated with intraparenchymal measures of tissue oxygenation. In particular, during periods of hypoxia and MAP alterations, changes of NIRS-derived spinal cord oxygenated hemoglobin and tissue oxygenation percentage corresponded well with the changes in spinal cord oxygen partial pressures measured by the intraparenchymal sensor. Our data confirm that during hypoxic episodes and as changes occur in the MAP, non-invasive NIRS can detect and measure real-time changes in spinal cord oxygenation with a high degree of sensitivity and specificity.

Differences in Morphometric Measures of the Uninjured Porcine Spinal Cord and Dural Sac Predict Histological and Behavioral Outcomes after Traumatic Spinal Cord Injury.

One of the challenges associated with conducting experiments in animal models of traumatic spinal cord injury (SCI) is inducing a consistent injury with minimal variability in the degree of tissue damage and resultant behavioral and biochemical outcomes. We evaluated how the variability in morphometry of the spinal cord and surrounding cerebrospinal fluid (CSF) contributes to the variability in behavioral and histological outcomes in our porcine model of SCI. Using intraoperative ultrasound imaging, spinal cord morphometry was assessed in seven Yucatan minipigs undergoing a weight-drop T10 contusion-compression injury. Bivariate and multi-variate analysis and modeling were used to identify native morphometrical determinants of interanimal variability in histological and behavioral outcomes. The measured biomechanical impact parameters did not correlate with the histological measures or hindlimb locomotor behavior (Porcine Thoracic Injury Behavior Scale). In contrast, clear associations were revealed between CSF layer morphometry and the amount of white matter and tissue sparing. Specifically, the dorsoventral diameter of the dural sac and ventral CSF space were strong predictors of behavioral and histological outcome and together explained ≥95.0% of the variance in these parameters. In addition, a dorsoventral diameter of the spinal cord less than 5.331 mm was a strong contributing factor to poor behavioral recovery over 12 weeks. These results indicate that interanimal variability in cord morphometry provides a potential biological explanation for the observed heterogeneity in histological and behavioral outcomes. Such knowledge is helpful for appropriately balancing experimental groups, and/or varying impact parameters to match cord and CSF layer dimensions for future studies.

Review of the UBC Porcine Model of Traumatic Spinal Cord Injury.

Traumatic spinal cord injury (SCI) research has recently focused on the use of rat and mouse models for in vivo SCI experiments. Such small rodent SCI models are invaluable for the field, and much has been discovered about the biologic and physiologic aspects of SCI from these models. It has been difficult, however, to reproduce the efficacy of treatments found to produce neurologic benefits in rodent SCI models when these treatments are tested in human clinical trials. A large animal model may have advantages for translational research where anatomical, physiological, or genetic similarities to humans may be more relevant for pre-clinically evaluating novel therapies. Here, we review the work carried out at the University of British Columbia (UBC) on a large animal model of SCI that utilizes Yucatan miniature pigs. The UBC porcine model of SCI may be a useful intermediary in the pre-clinical testing of novel pharmacological treatments, cell-based therapies, and the "bedside back to bench" translation of human clinical observations, which require preclinical testing in an applicable animal model.

A Direct Comparison between Norepinephrine and Phenylephrine for Augmenting Spinal Cord Perfusion in a Porcine Model of Spinal Cord Injury.

Current clinical guidelines recommend elevating the mean arterial blood pressure (MAP) to increase spinal cord perfusion in patients with acute spinal cord injury (SCI). This is typically achieved with vasopressors such as norepinephrine (NE) and phenylephrine (PE). These drugs differ in their pharmacological properties and potentially have different effects on spinal cord blood flow (SCBF), oxygenation (PO2), and downstream metabolism after injury. Using a porcine model of thoracic SCI, we evaluated how these vasopressors influenced intraparenchymal SCBF, PO2, hydrostatic pressure, and metabolism within the spinal cord adjacent to the injury site. Yorkshire pigs underwent a contusion/compression SCI at T10 and were randomized to receive either NE or PE for MAP elevation of 20 mm Hg, or no MAP augmentation. Prior to injury, a combined SCBF/PO2 sensor, a pressure sensor, and a microdialysis probe were inserted into the spinal cord adjacent to T10 at two locations: a "proximal" site and a "distal" site, 2 mm and 22 mm from the SCI, respectively. At the proximal site, NE and PE resulted in little improvement in SCBF during cord compression. Following decompression, NE resulted in increased SCBF and PO2, whereas decreased levels were observed for PE. However, both NE and PE were associated with a gradual decrease in the lactate to pyruvate (L/P) ratio after decompression. PE was associated with greater hemorrhage through the injury site than that in control animals. Combined, our results suggest that NE promotes better restoration of blood flow and oxygenation than PE in the traumatically injured spinal cord, thus providing a physiological rationale for selecting NE over PE in the hemodynamic management of acute SCI.

Changes in Pressure, Hemodynamics, and Metabolism within the Spinal Cord during the First 7 Days after Injury Using a Porcine Model.

Traumatic spinal cord injury (SCI) triggers many perturbations within the injured cord, such as decreased perfusion, reduced tissue oxygenation, increased hydrostatic pressure, and disrupted bioenergetics. While much attention is directed to neuroprotective interventions that might alleviate these early pathophysiologic responses to traumatic injury, the temporo-spatial characteristics of these responses within the injured cord are not well documented. In this study, we utilized our Yucatan mini-pig model of traumatic SCI to characterize intraparenchymal hemodynamic and metabolic changes within the spinal cord for 1 week post-injury. Animals were subjected to a contusion/compression SCI at T10. Prior to injury, probes for microdialysis and the measurement of spinal cord blood flow (SCBF), oxygenation (in partial pressure of oxygen; PaPO2), and hydrostatic pressure were inserted into the spinal cord 0.2 and 2.2 cm from the injury site. Measurements occurred under anesthesia for 4 h post-injury, after which the animals were recovered and measurements continued for 7 days. Close to the lesion (0.2 cm), SCBF levels decreased immediately after SCI, followed by an increase in the subsequent days. Similarly, PaPO2 plummeted, where levels remained diminished for up to 7 days post-injury. Lactate/pyruvate (L/P) ratio increased within minutes. Further away from the injury site (2.2 cm), L/P ratio also gradually increased. Hydrostatic pressure remained consistently elevated for days and negatively correlated with changes in SCBF. An imbalance between SCBF and tissue metabolism also was observed, resulting in metabolic stress and insufficient oxygen levels. Taken together, traumatic SCI resulted in an expanding area of ischemia/hypoxia, with ongoing physiological perturbations sustained out to 7 days post-injury. This suggests that our clinical practice of hemodynamically supporting patients out to 7 days post-injury may fail to address persistent ischemia within the injured cord. A detailed understanding of these pathophysiological mechanisms after SCI is essential to promote best practices for acute SCI patients.

Serum MicroRNAs Reflect Injury Severity in a Large Animal Model of Thoracic Spinal Cord Injury.

Therapeutic development for spinal cord injury is hindered by the difficulty in conducting clinical trials, which to date have relied solely on functional outcome measures for patient enrollment, stratification, and evaluation. Biological biomarkers that accurately classify injury severity and predict neurologic outcome would represent a paradigm shift in the way spinal cord injury clinical trials could be conducted. MicroRNAs have emerged as attractive biomarker candidates due to their stability in biological fluids, their phylogenetic similarities, and their tissue specificity. Here we characterized a porcine model of spinal cord injury using a combined behavioural, histological, and molecular approach. We performed next-generation sequencing on microRNAs in serum samples collected before injury and then at 1, 3, and 5 days post injury. We identified 58, 21, 9, and 7 altered miRNA after severe, moderate, and mild spinal cord injury, and SHAM surgery, respectively. These data were combined with behavioural and histological analysis. Overall miRNA expression at 1 and 3 days post injury strongly correlates with outcome measures at 12 weeks post injury. The data presented here indicate that serum miRNAs are promising candidates as biomarkers for the evaluation of injury severity for spinal cord injury or other forms of traumatic, acute, neurologic injury.