Spinal cord injury impairs cardiac function due to impaired bulbospinal sympathetic control.

Spinal cord injury chronically alters cardiac structure and function and is associated with increased odds for cardiovascular disease. Here, we investigate the cardiac consequences of spinal cord injury on the acute-to-chronic continuum, and the contribution of altered bulbospinal sympathetic control to the decline in cardiac function following spinal cord injury. By combining experimental rat models of spinal cord injury with prospective clinical studies, we demonstrate that spinal cord injury causes a rapid and sustained reduction in left ventricular contractile function that precedes structural changes. In rodents, we experimentally demonstrate that this decline in left ventricular contractile function following spinal cord injury is underpinned by interrupted bulbospinal sympathetic control. In humans, we find that activation of the sympathetic circuitry below the level of spinal cord injury causes an immediate increase in systolic function. Our findings highlight the importance for early interventions to mitigate the cardiac functional decline following spinal cord injury.

Experimental high thoracic spinal cord injury impairs the cardiac and cerebrovascular response to orthostatic challenge in rats.

Spinal cord injury (SCI) impairs the cardiovascular responses to postural challenge, leading to the development of orthostatic hypotension (OH). Here, we apply lower body negative pressure (LBNP) to rodents with high-level SCI to demonstrate the usefulness of LBNP as a model for experimental OH studies, and to explore the effect of simulated OH on cardiovascular and cerebrovascular function following SCI. Male Wistar rats (n = 34) were subjected to a sham or T3-SCI surgery and survived into the chronic period postinjury (i.e., 8 wk). Cardiac function was tracked via ultrasound pre- to post-SCI to demonstrate the clinical utility of our model. At study termination, we conducted left-ventricular (LV) catheterization and insonated the middle cerebral artery to investigate the hemodynamic, cardiac, and cerebrovascular response to a mild dose of LBNP that is sufficient to mimic clinically defined OH in rats with T3-SCI but not sham animals. In response to mimicked OH, there was a greater decline in stroke volume, cardiac output, maximal LV pressure, and blood pressure in SCI compared with sham (P < 0.034), whereas heart rate was increased in sham but decreased in SCI (P < 0.029). SCI animals also had an exaggerated reduction in peak, minimum and mean middle cerebral artery flow, for a given change in blood pressure, in response to LBNP (P < 0.033), implying impaired dynamic cerebral autoregulation. Using a preclinical SCI model of OH, we demonstrate that complete high thoracic SCI impairs the cardiac response to OH and disrupts dynamic cerebral autoregulation.NEW & NOTEWORTHY This is the first use of LBNP to interrogate the cardiac and cerebrovascular responses to simulated OH in a preclinical study of SCI. Here, we demonstrate the utility of our simulated OH model and use it to demonstrate that SCI impairs the cardiac response to simulated OH and disrupts dynamic cerebrovascular autoregulation.

A porcine model for studying the cardiovascular consequences of high-thoracic spinal cord injury.

KEY POINTS: We have developed a novel porcine model of high-thoracic midline contusion spinal cord injury (SCI) at the T2 spinal level. We describe this model and the ensuing cardiovascular and neurohormonal responses, and demonstrate the model is efficacious for studying clinically relevant cardiovascular dysfunction post-SCI. We demonstrate that the high-thoracic SCI model, but not a low-thoracic SCI model, induces persistent hypotension along with a gradual reduction in plasma noradrenaline and increases in plasma aldosterone and angiotensin II. We additionally conducted a proof-of-concept long-term (12 weeks) survival study in animals with T2 contusion SCI demonstrating the potential utility of this model for not only acute experimentation but also long-term drug studies prior to translation to the clinic. ABSTRACT: Cardiovascular disease is a leading cause of morbidity and mortality in the spinal cord injury (SCI) population, especially in those with high-thoracic or cervical SCI. With this in mind, we aimed to develop a large animal (porcine) model of high-thoracic (T2 level) contusion SCI and compare the haemodynamic and neurohormonal responses of this injury against a low-thoracic (T10 level) model. Ten Yorkshire pigs were randomly subjected to 20 cm weight drop contusion SCI at either the T2 or the T10 spinal level. Systolic blood pressure (SBP), mean arterial pressure (MAP) and heart rate (HR) were continuously monitored until 4 h post-SCI. Plasma noradrenaline (NA), aldosterone and angiotensin II (ANGII) were measured pre-SCI and at 30, 60, 120 and 240 min post-SCI. Additionally, two Yucatan pigs were subjected to T2-SCI and survived up to 12 weeks post-injury to demonstrate the efficacy of this model for long-term survival studies. Immediately after T2-SCI, SBP, MAP and HR increased (P < 0.0001). Between decompression (5 min post-SCI) and 30 min post-decompression in T2-SCI, SBP and MAP were lower than pre-SCI (P < 0.038). At 3 and 4 h after T2-SCI, SBP remained lower than pre-SCI (P = 0.048). After T10-SCI, haemodynamic indices remained largely unaffected. Plasma NA was lower in T2- vs. T10-SCI post-SCI, whilst aldosterone and ANGII were higher. Both chronically injured pigs demonstrated a vast reduction in SBP at 12 weeks post-SCI. Our model of T2-SCI causes a rapid and sustained alteration in neurohormonal control and cardiovascular function, which does not occur in the T10 model.

Experimental Spinal Cord Injury Causes Left-Ventricular Atrophy and Is Associated with an Upregulation of Proteolytic Pathways.

Spinal cord injury (SCI) causes autonomic dysfunction, altered neurohumoral control, profound hemodynamic changes, and an increased risk of heart disease. In this prospective study, we investigated the cardiac consequences of chronic experimental SCI in rats by combining cutting edge in vivo techniques (magnetic resonance imaging [MRI] and left-ventricular [LV] pressure-volume catheterization) with histological and molecular assessments. Twelve weeks post-SCI, MRI-derived structural indices and in vivo LV catheterization-derived functional indices indicated the presence of LV atrophy (LV mass in Control vs. SCI = 525 ± 38.8 vs. 413 ± 28.6 mg, respectively; p = 0.0009), reduced ventricular volumes (left-ventricular end-diastolic volume in Control vs. SCI = 364 ± 44 vs. 221 ± 35 µL, respectively; p = 0.0004), and contractile dysfunction (end-systolic pressure-volume relationship in Control vs. SCI = 1.31 ± 0.31 vs. 0.76 ± 0.11 mm Hg/µL, respectively; p = 0.0045). Cardiac atrophy and contractile dysfunction in SCI were accompanied by significantly lower blood pressure, reduced circulatory norepinephrine, and increased angiotensin II. At the cellular level, we found the presence of reduced cardiomyocyte size and increased expression of angiotensin II type 1 receptors and transforming growth factor-beta receptors (TGF-ß receptor 1 and 2) post-SCI. Importantly, we found more than a two-fold increase in muscle ring finger-1 and Beclin-1 protein level following SCI, indicating the upregulation of the ubiquitin-proteasome system and autophagy-lysosomal machinery. Our data provide novel evidence that SCI-induced cardiomyocyte atrophy and systolic cardiac dysfunction are accompanied by an upregulation of proteolytic pathways, the activation of which is likely due to loss of trophic support from the sympathetic nervous system, neuromechanical unloading, and altered neurohumoral pathways.

Spinal Cord Injury Causes Systolic Dysfunction and Cardiomyocyte Atrophy.

Individuals with spinal cord injury (SCI) have been shown to exhibit systolic, and to a lesser extent, diastolic cardiac dysfunction. However, previous reports of cardiac dysfunction in this population are confounded by the changing loading conditions after SCI and as such, whether cardiac dysfunction per se is present is still unknown. Therefore, our aim was to establish if load-independent cardiac dysfunction is present after SCI, to understand the functional cardiac response to SCI, and to explore the changes within the cellular milieu of the myocardium. Here, we applied in vivo echocardiography and left-ventricular (LV) pressure-volume catheterization with dobutamine infusions to our Wistar rodent model of cardiac dysfunction 5 weeks following high (T2) thoracic contusion SCI, while also examining the morphological and transcriptional alterations of cardiomyocytes. We found that SCI significantly impairs systolic function independent of loading conditions (end-systolic elastance in control: 1.35 ± 0.15; SCI: 0.65 ± 0.19 mm Hg/µL). The reduction in contractile indices is accompanied by a reduction in width and length of cardiomyocytes as well as alterations in the LV extracellular matrix. Importantly, we demonstrate that the reduction in the rate (dP/dtmax) of LV pressure rise can be offset with beta-adrenergic stimulation, thereby experimentally implicating the loss of descending sympatho-excitatory control of the heart as a principle cause of LV dysfunction in SCI. Our data provide evidence that SCI induces systolic cardiac dysfunction independent of loading conditions and concomitant cardiomyocyte atrophy that may be underpinned by changes in the genes regulating the cardiac extracellular matrix.

Kynurenic acid downregulates IL-17/1L-23 axis in vitro.

Exploring the function of interleukin (IL) 17 and related cytokine interactions have been proven useful toward understanding the role of inflammation in autoimmune diseases. Production of the inflammatory cytokine IL-23 by dendritic cells (DC's) has been shown to promote IL-17 expression by Th17 cells. It is well established that Th17 cells play an important role in several autoimmune diseases including psoriasis and alopecia. Our recent investigations have suggested that Kynurenine-rich environment can shift a pro-inflammatory response to an anti-inflammatory response, as is the case in the presence of the enzyme Indoleamine 2,3 dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation and Kynurenine (Kyn) production. In this study, we sought to explore the potential role of kynurenic acid (KynA), in modulating the expression of IL-23 and IL-17 by DCs and CD4+ cells, respectively. The result of flow cytometry demonstrated that the frequency of IL-23-producing DCs is reduced with 100 µg/ml of KynA as compared with that of LPS-stimulated DCs. KynA (100 µg/ml) addition to activated T cells significantly decreased the level of IL-17 mRNA and frequency of IL-17+ T cells as compared to that of concanavalin (Con) A-activated T cells. To examine the mechanism of the suppressive role of KynA on IL-23/IL-17 in these cells, cells were treated with 3 µM G-protein-coupled receptor35 (GPCR35) inhibitor (CID), for 60 min. The result showed that the reduction of both adenylate cyclase (AC) and cyclic adenosine monophosphate (cAMP) by KynA is involved in suppression of LPS-induced IL-23p19 expression. Since GPCR35 is also detected on T cells; therefore, it is concluded that KynA plays an important role in modulating the expression of IL-23 and IL-17 in DCs and Th17 cells through inhibiting GPCR35 and downregulation of both AC and cAMP.

Development of a nanofibrous wound dressing with an antifibrogenic properties in vitro and in vivo model.

Dermal fibrosis, characterized by excessive extracellular matrix (ECM), is a pathological condition with limited effective therapeutic modalities. Lack of an antiscarring dressing further impedes the preventive measures for this condition. Here, we develop a new antiscarring dressing and investigate its potential as a slow-releasing vehicle for kynurenic acid (KynA), an antifibrotic agent. KynA was incorporated into polymethyl methacrylate (PMMA) nanofibers, containing increasing concentration of polyethylene glycol (PEG). Fibre morphology, water absorption capacity, surface hydrophilicity, in vitro drug release profile, and in vivo antifibrotic effects were investigated. Increasing concentrations of PEG (1-20%) significantly increased surface hydrophilicity, water absorption capacity, and drug release. Based on the obtained release profiles, PMMA + 10% PEG was the preferred formulation for sustained KynA release up to 120 hours. In vitro studies confirmed the preservation of KynA antifibrotic properties during electrospinning, indicated by fibroblasts proliferation suppression and ECM expression modulation. In vivo application of KynA-incorporated films significantly inhibited collagen (23.89 ± 4.79 vs. 6.99 ± 0.41, collagen-I/ß-actin mRNA expression, control vs. treated) and fibronectin expression (7.18 ± 1.09 vs. 2.31 ± 0.05, fibronectin/ß-actin mRNA expression, control vs. treated) and enhanced the production of an ECM-degrading enzyme (2.03 ± 0.88 vs. 11.88 ± 1.16 MMP-1/ß-actin mRNA expression, control vs. treated). The fabricated KynA-incorporated films can be exploited as antifibrotic wound dressings. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2334-2344, 2016.

Kynurenine Modulates MMP-1 and Type-I Collagen Expression Via Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts.

Dermal fibrosis is characterized by a high deposition of extracellular matrix (ECM) and tissue cellularity. Unfortunately all means of treating this condition are unsatisfactory. We have previously reported the anti-fibrotic effects of Kynurenine (Kyn), a tryptophan metabolite, in fibrotic rabbit ear model. Here, we report the mechanism by which Kyn modulates the expression of key ECM components in dermal fibroblasts. The results showed that Kyn activates aryl hydrocarbon receptor (AHR) nuclear translocation and up-regulates cytochrome-P450 (CYP1A-1) expression, the AHR target gene. A specific AHR antagonist, 6,2',4'-trimethoxyflavone, inhibited the Kyn-dependent modulation of CYP1A-1, MMP-1, and type-I collagen expression. Establishing the anti-fibrogenic effect of Kyn and its mechanism of action, we then developed nano-fibrous Kyn slow-releasing dressings and examined their anti-fibrotic efficacy in vitro and in a rat model. Our results showed the feasibility of incorporating Kyn into PVA/PLGA nanofibers, prolonging the Kyn release up to 4 days tested. Application of medicated-dressings significantly improved the dermal fibrosis indicated by MMP-1 induction, alpha-smooth muscle actin and type-I collagen suppression, and reduced tissue cellularity, T-cells and myofibroblasts. This study clarifies the mechanism by which Kyn modulates ECM expression and reports the development of a new slow-releasing anti-fibrogenic dressing. J. Cell. Physiol. 231: 2749-2760, 2016. © 2016 Wiley Periodicals, Inc.

An in-situ forming skin substitute improves healing outcome in a hypertrophic scar model.

Wound repair requires a sequential series of biological events that begins with the deposition of a temporary scaffold within which cells can repair the skin. Without a scaffold, repair is essentially impossible. Aberrant wound healing, such as hypertrophic scarring or nonhealing, has a tremendous burden on healthcare and quality of life. Timely wound closure dramatically reduces the risk of infection and scarring. Cellular skin substitutes are opportune to meet this need. Our goal was to create an in-situ forming scaffold that can be easily combined with cells to rapidly form a dermal substitute within the wound bed. In this study, we evaluated the application of a polyvinyl alcohol-collagen-glycosaminoglycan-based biohybrid scaffold system in full-thickness wounds on a rabbit fibrotic ear model. Punch wounds (6 mm) were either untreated or filled with an acellular scaffold, a scaffold containing xenofibroblasts, or a scaffold containing xenofibroblasts expressing indoleamine 2,3-dioxygenase (IDO). Results demonstrated that (1) both acellular and IDO-expressing fibroblast in-situ forming scaffolds significantly reduced scar elevation index (1.24±0.05 and 1.25±0.03; p<0.05) and improved overall healing quality compared with xenofibroblast scaffolds and untreated wounds; (2) IDO-expressing fibroblast scaffolds significantly reduced T-cell infiltration into the scaffold-engrafted area (p<0.05); and (3) both IDO-expressing and acellular in-situ forming scaffolds demonstrated increased vessel-like and nerve-like structures (p<0.05). The results demonstrated that the use of the in-situ forming scaffold, and even more so when delivering IDO-expressing cells, improved healing outcome in full-thickness hypertrophic rabbit ear wounds.

Effects of kynurenine on CD3+ and macrophages in wound healing.

As prolongation of the inflammation phase in a healing process frequently leads to wound impairment, here we queried whether kynurenine (Kyn) could modulate this phase of wound healing. To address this, a protein microarray, quantitative polymerase chain reaction (qPCR), flow cytometry for immune cells and immune cell proliferation in the presence and absence of Kyn were conducted and compared. The result of a protein microarray revealed that the expression of 12 pro-inflammatory cytokines and chemokines was modulated in Kyn-treated mouse splenocytes as compared with those of control. These findings were then evaluated by conducting a qPCR for the gene expression of these factors and showed a significant reduction in the gene expression of majority of these cytokines and chemokines (interleukin [IL]-2, IL-17, C-X-C motif chemokine ligand [CXCL] 10, CXCL1, C-C motif ligand [CCL] 12, CXCL9, CCL4, CXCL2, and CCL5) in response to Kyn treatment. To test the anti-inflammatory effect of Kyn in an animal model, dorsal surface wounds were generated in a mouse model and wounds received daily topical application of either nothing (control), dermal cream (second control), or Kyn cream using uninjured skin tissue as another control. The wounded tissues were harvested on days 3, 6, and 10 postwounding. As anticipated, the results of fluorescence-activated cell sorting analysis revealed that upon wounding, the number of total infiltrated CD3+ cells and macrophages (CD11b+) significantly increased on day 3, peaked on day 6, and reduced on day 10 post-wounding. Interestingly, as compared with those of uninjured and dermal cream alone-treated wounds, Kyn treatment significantly reduced the number of infiltrated CD3+ cells, but not CD11b+ cells, at different time intervals examined. These findings collectively suggest that Kyn, as a small molecule, can potentially be used to overcome the difficulties associated with persistency of inflammation in healing wounds.

Anti-scarring properties of different tryptophan derivatives.

Hypertrophic scars are associated with prolonged extracellular matrix (ECM) production, aberrant ECM degradation and high tissue cellularity. Routinely used antifibrotic strategies aim to reduce ECM deposition and enhance matrix remodeling. Our previous study investigating the antifibrotic effects of indoleamine2, 3 dioxygenase (IDO) led to the identification of kynurenine (Kyn) as an antiscarring agent. A topical antifibrogenic therapy using Kyn is very attractive; however, it is well established that Kyn passes the blood brain barrier (BBB) which causes complications including excitatory neuronal death. Here we investigated the antiscarring properties of kynurenic acid (KynA), a downstream end product of Kyn that is unlikely to pass the BBB, as an effective and safe replacement for Kyn. Our results indicated that while not having any adverse effect on dermal cell viability, KynA significantly increases the expression of matrix metalloproteinases (MMP1 and MMP3) and suppresses the production of type-I collagen and fibronectin by fibroblasts. Topical application of cream containing KynA in fibrotic rabbit ear significantly decreased scar elevation index (1.13±0.13 vs. 1.61±0.12) and tissue cellularity (221.38±21.7 vs. 314.56±8.66 cells/hpf) in KynA treated wounds compared to controls. KynA treated wounds exhibited lower levels of collagen deposition which is accompanied with a significant decrease in type-I collagen and fibronectin expression, as well as an increase in MMP1 expression compared to untreated wounds or wounds treated with cream only. The results of this study provided evidence for the first time that KynA is promising candidate antifibrogenic agent to improve healing outcome in patients at risk of hypertrophic scarring.

Passive hind-limb cycling improves cardiac function and reduces cardiovascular disease risk in experimental spinal cord injury.

Spinal cord injury (SCI) causes altered autonomic control and severe physical deconditioning that converge to drive maladaptive cardiac remodelling. We used a clinically relevant experimental model to investigate the cardio-metabolic responses to SCI and to establish whether passive hind-limb cycling elicits a cardio-protective effect. Initially, 21 male Wistar rats were evenly assigned to three groups: uninjured control (CON), T3 complete SCI (SCI) or T3 complete SCI plus passive hind-limb cycling (SCI-EX; 2 × 30 min day(-1), 5 days week(-1) for 4 weeks beginning 6 days post-SCI). On day 32, cardio-metabolic function was assessed using in vivo echocardiography, ex vivo working heart assessments, cardiac histology/molecular biology and blood lipid profiles. Twelve additional rats (n = 6 SCI and n = 6 SCI-EX) underwent in vivo echocardiography and basal haemodynamic assessments pre-SCI and at days 7, 14 and 32 post-SCI to track temporal cardiovascular changes. Compared with CON, SCI exhibited a rapid and sustained reduction in left ventricular dimensions and function that ultimately manifested as reduced contractility, increased myocardial collagen deposition and an up-regulation of transforming growth factor beta-1 (TGFß1) and mothers against decapentaplegic homolog 3 (Smad3) mRNA. For SCI-EX, the initial reduction in left ventricular dimensions and function at day 7 post-SCI was completely reversed by day 32 post-SCI, and there were no differences in myocardial contractility between SCI-EX and CON. Collagen deposition was similar between SCI-EX and CON. TGFß1 and Smad3 were down-regulated in SCI-EX. Blood lipid profiles were improved in SCI-EX versus SCI. We provide compelling novel evidence that passive hind-limb cycling prevents cardiac dysfunction and reduces cardiovascular disease risk in experimental SCI.

Immuno-regulatory function of indoleamine 2,3 dioxygenase through modulation of innate immune responses.

Successful long-term treatment of type-1 diabetes mainly relies on replacement of ß-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO-expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11 ± 1.47 vs. 70.5 ± 7.57 cells/HPF), T-cells (8.75 ± 1.03 vs. 75.75 ± 5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.