Without blood flow, bone cannot maintain its integrity. Bone blood flow responds to various local and systemic factors, however, bone perfusion in humans remains relatively unstudied. The investigators will study key mechanisms that regulate bone perfusion in able-bodied and contrast responses to those with spinal cord injury (SCI). SCI is a model of chronic reduced loading with loss of sympathetic regulation. In tibial cortical bone, the investigators will: 1) determine the impact of compressive loading with and without muscle contractions; 2) determine the impact of vascular sympathetic activity and systemic perfusion pressure; 3) compare the response between able-bodied and those with SCI. Acute metabolic needs of bone due to loading increase flow substantially. In addition, the bone vasculature is innervated by a rich network of sympathetic nerves that serve a functional purpose in the control of blood flow. A critical limitation to the study of bone blood flow in humans has been the lack of non-invasive assessments. Previously, the investigators developed a near infrared spectroscopy (NIRS) device to non-invasively assess blood content in bone and assessed tibial perfusion in response to exercise. Here, the investigators will test the hypothesis that bone blood flow increases proportional to loading conditions in both able-bodied individuals and those with SCI. The investigators will also test the hypothesis that there are decreases in blood bone flow that are proportional to increases in leg vascular sympathetic outflow in the able-bodied, but that changes in bone blood flow are proportional to changes in blood pressure in those with SCI. The proposed research will be some of the first to determine the control of bone flow in humans.

All tissues of the human body require adequate perfusion to provide oxygen and nutrients to meet metabolic demands. It has long been known that the arterial system in bone is of overwhelming importance and that without blood flow, bone cannot maintain its integrity. Indeed, there is an extensive network of arteries, arterioles, and capillaries that supply human bone. Moreover, blood flow to bone is responsive to various local and systemic factors that can determine the overall health of bone. However, bone perfusion in humans remains relatively unstudied and so the underlying mechanisms that regulate bone blood flow are not well understood. The investigators propose to study key mechanisms that regulate bone perfusion in able-bodied individuals and to contrast them with spinal cord injured (SCI) individuals. SCI represents a human 'model' of chronic reduced loading with loss of sympathetic regulation below the level of injury that likely alters control of bone perfusion. Accordingly, our aims are to: 1) Determine the impact of compressive loading with and without associated muscle contractions on tibial perfusion; 2) Determine the impact of vascular sympathetic activity and systemic perfusion pressure on tibial perfusion; 3) Compare the changes in tibial perfusion in response to local and systemic factors between able-bodied and those with SCI.

The majority of work in bone blood flow has been in animals and/or has focused on the association between adequate or inadequate perfusion and bone health. For example, inadequate flow has been associated with bone loss, impaired growth, and delayed fracture healing. However, the acute metabolic needs of bone due to loading either with or without associated muscle contractions increase flow substantially. Indeed, within two minutes of isolated muscle contractions alone, tibial perfusion has been shown to increase significantly. Furthermore, when there is compressive loading with associated muscle contractions, flow to bone can double. Similarly, skeletal unloading for as short as ten minutes cuts femoral perfusion by half. Although it is unclear what specific local factors (e.g., metabolic by-products) with loading might be responsible for regulation of blood flow, these data strongly suggest that perfusion to bone is highly responsive to skeletal loading. Indeed, it appears that similar regulatory mechanisms may be at play in control of flow to bone and skeletal muscle during exercise. In addition, the bone vasculature is richly innervated by sympathetic nerves. Application of norepinephrine decreases blood flow to both intact bone and isolated bone. Likewise, sympathetic stimulation decreases flow to bone via alpha-adrenergic receptor activation. Moreover, smooth muscle of arterioles in bone respond as expected to vasodilators and vasoconstrictors. Hence, sympathetic innervation of the bone vasculature serves a functional purpose in control of flow. If this were not the case, independent of the link between bone metabolism and bone flow, the arterial network in bone would act as a simple pressure passive system.

A critical limitation to the study of bone flow in humans has been the lack of noninvasive assessments. Thus, it has been difficult to elucidate the mechanisms that control perfusion to bone. The dense nature of bone makes it difficult to investigate perfusion and the techniques used to quantify circulation in other tissues are either difficult or impossible to apply to bone in vivo. the investigators recently demonstrated the efficacy of a near infrared spectroscopy (NIRS) system to non-invasively detect changes in hemoglobin content in the tibia. Although our preliminary work showed the utility of NIRS, it was not designed to provide insight to blood flow regulation and disentangle the various possible contributors to bone perfusion. Here the investigators propose to study different mechanisms that control blood flow to bone in both able-bodied and spinal cord injured (SCI). The SCI population will offer valuable insights to the mechanisms of perfusion as several contributors (i.e. loading and vascular sympathetic control) are either reduced or disrupted.

Study completion update: Given the exploratory nature of this study, the scope has been adjusted to address the challenges and limitations that have occurred during the duration of this research. Nonetheless the work completed has provided unprecedented findings on key mechanisms of vascular regulation in bone in vivo in humans. We were not able to assess the impact of compressive loading with or without associated muscle contractions on tibial perfusion due to marked impact of motion artefacts on the near infrared spectroscopy technology used to assess tibial perfusion. In young healthy adults, we have assessed the impact of vascular sympathetic activity (in response to two stimuli: isometric handgrip exercise and cold pressor test) and increased perfusion pressure (in response to two stimuli: leg dependency and reactive hyperemia). In those with spinal cord injury, given the inherent limitations of working with this population (i.e., increased spasticity, autonomic dysreflexia), we have assessed the impact of lack of vascular sympathetic activity on tibial blood flow regulation. Furthermore, we have extended the initial work and in young healthy adults we have investigated an additional key regulation mechanism of the tibial vasculature, namely nitric oxide mediated vasodilation.

Even though we have obtained data during compressive loading in both able-bodied and adults with spinal cord injury, the data were not usable due to motion artefacts. In addition, the data obtained in those with SCI during leg dependency and reactive hyperemia were insufficient or unusable (due to increased spasticity and autonomic dysreflexia) to draw any meaningful conclusions.

Inclusion Criteria:

• healthy males and females
• individuals with spinal cord injuries, between 3 and 24 months post injury, with complete injuries according to the American Spinal Injury Association Impairment Scale A and B, with injuries at T6 and below

Exclusion Criteria:

• clinical signs or symptoms of heart disease
• hypertension
• coronary disease
• diabetes
• other neurological disease
• cancer
• recent weight change >15 pounds
• abnormal resting ECG
• pregnant and/or breastfeeding women
• underweight and obese individuals (body mass index between 18.5 and 29.9)
• use of amphetamines (Ritalin, Adderall, Concerta) in the past 48 hours
• tibial fracture or tibial stress fracture in the past year
• those with SCI will have no extreme spasticity to avoid spontaneous contractions
• use of baclofen for those with SCI

Adverse Events Time Frame - 1 week following study completion.

This represented an exploratory study of physiological mechanisms underlying bone blood flow regulation in humans. Given the exploratory nature of this work, several technical challenges resulted in unreliable or uninterpretable data which was not included in the final results reported. Technical difficulties included motion artifacts for total hemoglobin measurements, spasticity in adults with spinal cord injury, and inability to perform certain physiological stimuli.