Jessica A. Inskip

Recurrent autonomic dysreflexia exacerbates vascular dysfunction after spinal cord injury

BACKGROUND CONTEXT Individuals with high spinal cord injury (SCI) are prone to significant fluctuation in blood pressure with episodes of very high and low blood pressure during autonomic dysreflexia (AD) and orthostatic hypotension, respectively. We do not know how such blood pressure lability affects the vasculature. PURPOSE We used a well-characterized animal model of AD to determine whether increasing the frequency of AD during recovery from SCI would exacerbate injury-induced dysfunction in resistance vessels. STUDY DESIGN/SETTING Experimental animal study. International Collaboration On Repair Discoveries (ICORD), University of British Columbia, Canada. METHODS Complete transection of the T3 spinal cord was performed in male Wistar rats. For 14 days after injury, AD was induced via colorectal distension (CRD; 30 minutes per day) in the experimental group (SCI-CRD). One month after SCI, baseline cardiovascular parameters and severity of CRD-induced AD were assessed in SCI-CRD animals and SCI-only controls. Mesenteric arteries were harvested for in vitro myography to characterize vasoactive responses to phenylephrine (PE) and acetylcholine (ACh). RESULTS Mesenteric arteries from SCI-CRD animals exhibited larger maximal responses to PE than arteries from SCI-only controls. Hyperresponsiveness to PE was not a product of endothelial dysfunction because mesenteric arteries from both groups had similar vasodilator responses to ACh. Both SCI-only controls and SCI-CRD animals exhibited CRD-evoked AD 1 month after SCI; however, CRD-induced hypertension was less pronounced in animals that were previously exposed to CRD. CONCLUSIONS Injury-induced changes within the vasculature may contribute to the development of AD after SCI. Here, we provide evidence that AD itself has significant and long-lasting effects on vascular function. This finding has implications for the medical management of AD and provides an impetus for maintaining stable blood pressure.

Plasticity of TRPV1-Expressing Sensory Neurons Mediating Autonomic Dysreflexia Following Spinal Cord Injury.

Spinal cord injury (SCI) triggers profound changes in visceral and somatic targets of sensory neurons below the level of injury. Despite this, little is known about the influence of injury to the spinal cord on sensory ganglia. One of the defining characteristics of sensory neurons is the size of their cell body: for example, nociceptors are smaller in size than mechanoreceptors or proprioceptors. In these experiments, we first used a comprehensive immunohistochemical approach to characterize the size distribution of sensory neurons after high- and low-thoracic SCI. Male Wistar rats (300 g) received a spinal cord transection (T3 or T10) or sham-injury. At 30 days post-injury, dorsal root ganglia (DRGs) and spinal cords were harvested and analyzed immunohistochemically. In a wide survey of primary afferents, only those expressing the capsaicin receptor (TRPV1) exhibited somal hypertrophy after T3 SCI. Hypertrophy only occurred caudal to SCI and was pronounced in ganglia far distal to SCI (i.e., in L4-S1 DRGs). Injury-induced hypertrophy was accompanied by a small expansion of central territory in the lumbar spinal dorsal horn and by evidence of TRPV1 upregulation. Importantly, hypertrophy of TRPV1-positive neurons was modest after T10 SCI. Given the specific effects of T3 SCI on TRPV1-positive afferents, we hypothesized that these afferents contribute to autonomic dysreflexia (AD). Rats with T3 SCI received vehicle or capsaicin via intrathecal injection at 2 or 28 days post-SCI; at 30 days, AD was assessed by recording intra-arterial blood pressure during colo-rectal distension (CRD). In both groups of capsaicin-treated animals, the severity of AD was dramatically reduced. While AD is multi-factorial in origin, TRPV1-positive afferents are clearly involved in AD elicited by CRD. These findings implicate TRPV1-positive afferents in the initiation of AD and suggest that TRPV1 may be a therapeutic target for amelioration or prevention of AD after high SCI.

Care of Rats with Complete High-Thoracic Spinal Cord Injury

The complications of spinal cord injury (SCI) increase in number and severity with the level of injury. A recent survey of SCI researchers reveals that animal models of high SCI are essential. Despite this consensus, most laboratories continue to work with mid- or low-thoracic SCI. The available data on cervical SCI in animals characterize incomplete injuries; for example, nearly all studies published in 2009 examine discrete, tract-specific lesions that are not clinically-relevant. A primary barrier to developing animal models of severe, higher SCI is the challenge of animal care, a critical determinant of experimental outcome. Currently, many of these practices vary substantially between laboratories, and are passed down anecdotally within institutions. The care of animals with SCI is complex, and becomes much more challenging as the lesion level ascends. In our experience, the care of animals with high-thoracic (T3) SCI is much more demanding than the care of animals with low-thoracic SCI, even though both injuries result in paraplegia. We have developed an animal care regimen for rats with complete high-thoracic SCI. Our practices have been refined over the past 7 years, in collaboration with animal care centre staff and veterinarians. During this time, we have cared for more than 300 rats with T3 complete transection SCI, with experimental end-points of up to 3 months. Here we provide details of our animal care procedures, including acclimatization, housing, diet, antibiotic prophylaxis, surgical procedures, post-operative monitoring, and prevention of complications. In our laboratory, this comprehensive approach consistently produces good outcomes following T3 complete transection SCI: using body weight as an objective indicator of animal health, we have found that our rats typically return to pre-operative weights within 10 days of T3 complete SCI. It is our hope that the information provided here will improve care of experimental animals, and facilitate adoption of models that directly address the complications associated with higher level injuries.

Cardiometabolic Risk Factors in Experimental Spinal Cord Injury

Cardiometabolic risk factors are sorely underreported after spinal cord injury (SCI), despite the high prevalence of metabolic disorders and cardiovascular mortality in this population. Body-composition analysis and serum-lipid profiling are two assessments that are beginning to be more widely used to document metabolic changes after clinical SCI. Individuals with SCI have been reported to carry increased visceral fat and to exhibit altered serum-lipid levels. However, little is known about the development of these cardiometabolic risk factors in animal models. Using a combination of magnetic resonance imaging (MRI) and adipose tissue dissection, we show that visceral and subcutaneous adipose tissue were both increased at 1 month, but not at 1 week, after complete T3 SCI in rats. Additionally, at 1 month post injury, T3 SCI rats exhibited nonfasting serum hypertriglyceridemia, a result obtained using both standard clinical methods and a home cholesterol monitoring device (CardioChek). Interestingly, at 1 month post injury, rats with complete T10 SCI did not show an increase in either visceral adiposity or serum triglyceride levels. The fact that complete high-thoracic SCI disrupts lipid metabolism and perturbs fat storage in the subacute period, while low-thoracic SCI does not, suggests that differences in descending sympathetic control of adipose tissue might play a role in these changes. These results provide the first evidence of cardiometabolic risk factors in experimental animals with SCI, and are a starting point for investigations of the etiology of obesity and metabolic dysfunctions that often accompany SCI.

A New Organellar Complex in Rat Sympathetic Neurons

Membranous compartments of neurons such as axons, dendrites and modified primary cilia are defining features of neuronal phenotype. This is unlike organelles deep to the plasma membrane, which are for the most part generic and not related directly to morphological, neurochemical or functional specializations. However, here we use multi-label immunohistochemistry combined with confocal and electron microscopy to identify a very large (∼6 microns in diameter), entirely intracellular neuronal organelle which occurs singly in a ubiquitous but neurochemically distinct and morphologically simple subset of sympathetic ganglion neurons. Although usually toroidal, it also occurs as twists or rods depending on its intracellular position: tori are most often perinuclear whereas rods are often found in axons. These ‘loukoumasomes’ (doughnut-like bodies) bind a monoclonal antibody raised against beta-III-tubulin (SDL.3D10), although their inability to bind other beta-III-tubulin monoclonal antibodies indicate that the responsible antigen is not known. Position-morphology relationships within neurons and their expression of non-muscle heavy chain myosin suggest a dynamic structure. They associate with nematosomes, enigmatic nucleolus-like organelles present in many neural and non-neural tissues, which we now show to be composed of filamentous actin. Loukoumasomes also separately interact with mother centrioles forming the basal body of primary cilia. They express gamma tubulin, a microtubule nucleator which localizes to non-neuronal centrosomes, and cenexin, a mother centriole-associated protein required for ciliogenesis. These data reveal a hitherto undescribed organelle, and depict it as an intracellular transport machine, shuttling material between the primary cilium, the nematosome, and the axon.

Spectral analyses of cardiovascular control in rodents with spinal cord injury.

The severity of injury to cardiovascular autonomic pathways following clinical spinal cord injury (SCI) can be evaluated with spectral analyses. Whether this technique provides a translatable assessment of cardiovascular autonomic function in rodent SCI is unknown. Beat-to-beat blood pressure and pulse interval were measured in male rats 1 month after complete T3 or T10 SCI, and in uninjured control animals. Univariate autoregressive spectral analyses were performed and the power of the low frequency (LF), high frequency (HF), and very low frequency (VLF) peaks identified. Frequency domain variables were correlated with the severity of orthostatic hypotension (OH) and the severity of hypertension during autonomic dysreflexia (AD). Total heart rate variability (HRV) and blood pressure variability (BPV) were reduced in animals with T3, but not T10, SCI. VLF and LF HRV were reduced and HF HRV was increased in animals with T3 SCI compared to controls; there were no changes in animals with T10 SCI. BPV in the VLF and LF range was reduced in animals with T3 SCI, but not T10 SCI. In all animals with SCI, severity of OH was positively correlated with LF BPV, and negatively correlated with HF BPV. Severity of AD was positively correlated with HF BPV and HF HRV, and negatively correlated with VLF HRV. Spectral analyses can detect alterations in cardiovascular autonomic function in animals with SCI at rest. These parameters underscore the distinct cardiovascular ramifications of high- versus low-thoracic SCI, and correlate with the severity of AD and OH, clinically-relevant measures of abnormal blood pressure control.

Dynamic wheelchair seating positions impact cardiovascular function after spinal cord injury.

Background Innovative wheelchairs allow individuals to change position easily for comfort and social situations. While these wheelchairs are beneficial in multiple ways, the effects of position changes on blood pressure might exacerbate hypotension and cerebral hypoperfusion, particularly in those with spinal cord injury (SCI) who can have injury to autonomic nerves that regulate cardiovascular control. Conversely, cardiovascular benefits may be obtained with lowered seating. Here we investigate the effect of moderate changes in wheelchair position on orthostatic cardiovascular and cerebrovascular reflex control. Methods Nineteen individuals with SCI and ten neurologically-intact controls were tested in supine and seated positions (neutral, lowered, and elevated) in the Elevation™ wheelchair. Participants with SCI were stratified into two groups by the severity of injury to cardiovascular autonomic pathways. Beat-to-beat blood pressure, heart rate and middle cerebral artery blood flow velocity (MCAv) were recorded non-invasively. Results Supine blood pressure and MCAv were reduced in individuals with lesions to autonomic pathways, and declined further with standard seating compared to those with preserved autonomic control. Movement to the elevated position triggered pronounced blood pressure and MCAv falls in those with autonomic lesions, with minimum values significantly reduced compared to the seated and lowered positions. The cumulative duration spent below supine blood pressure was greatest in this group. Lowered seating bolstered blood pressure in those with lesions to autonomic pathways. Conclusions Integrity of the autonomic nervous system is an important variable that affects cardiovascular responses to orthostatic stress and should be considered when individuals with SCI or autonomic dysfunction are selecting wheelchairs. Sponsorship This work was supported in part by the Heart and Stroke Foundation of British Columbia and the Yukon (V.E.C).