Elena B. Okon

A Systematic Review of Non-Invasive Pharmacologic Neuroprotective Treatments for Acute Spinal Cord Injury

An increasing number of therapies for spinal cord injury (SCI) are emerging from the laboratory and seeking translation into human clinical trials. Many of these are administered as soon as possible after injury with the hope of attenuating secondary damage and maximizing the extent of spared neurologic tissue. In this article, we systematically review the available pre-clinical research on such neuroprotective therapies that are administered in a non-invasive manner for acute SCI. Specifically, we review treatments that have a relatively high potential for translation due to the fact that they are already used in human clinical applications, or are available in a form that could be administered to humans. These include: erythropoietin, NSAIDs, anti-CD11d antibodies, minocycline, progesterone, estrogen, magnesium, riluzole, polyethylene glycol, atorvastatin, inosine, and pioglitazone. The literature was systematically reviewed to examine studies in which an in-vivo animal model was utilized to assess the efficacy of the therapy in a traumatic SCI paradigm. Using these criteria, 122 studies were identified and reviewed in detail. Wide variations exist in the animal species, injury models, and experimental designs reported in the pre-clinical literature on the therapies reviewed. The review highlights the extent of investigation that has occurred in these specific therapies, and points out gaps in our knowledge that would be potentially valuable prior to human translation.

Augmented contractile response of vascular smooth muscle in a diabetic mouse model.

The vasomotor properties of isolated aortae and mesenteric arteries of insulin-resistant ob/ob and 57CBL/6J mice were compared in organ bath studies. Vessels from ob/ob mice were more sensitive to phenylephrine. Pretreatment with L-NAME caused similar leftward shifts of the phenylephrine concentration response curves in diabetic and non-diabetic vessels. The ob/ob aortae contracted in response to phenylephrine with roughly twice the force while they were not stiffer than control aortae. L-NAME caused a greater percentage increase in maximal force in the control than in the ob/ob tissue. Denudation potentiated force in the control aortae, but not in the ob/ob aortae. Endothelium-dependent relaxation in the ob/ob aortae and mesenteric arteries was impaired as manifested by a decreased sensitivity and maximal relaxation to acetylcholine, while the aortic basal eNOS mRNA levels did not differ between the two strains. In addition, ob/ob aortae were less sensitive to the nitric oxide donor sodium nitroprusside. Inhibition of endogenous prostaglandin synthesis with indomethacin (10 µM) partly normalized the contractile response of the ob/ob aortae and enhanced their endothelium-dependent relaxation. Neither blockade of endothelin-1 receptors (bosentan, 10 µM) nor PKC inhibition (calphostin, 1 µM) affected the contractile response to phenylephrine in the mouse aortae of either strain. In conclusion, vascular dysfunction in the aorta and mesenteric artery of ob/ob mice are due to increased smooth muscle contractility and impaired dilation but not to changes in elasticity of the vascular wall. Endothelium-produced prostaglandins contribute to the increased vasoconstriction.

Reduced Expression of Vascular Endothelial Growth Factor Paralleled With the Increased Angiostatin Expression Resulting From the Upregulated Activities of Matrix Metalloproteinase-2 and -9 in Human Type 2 Diabetic Arterial Vasculature

Impaired angiogenesis could contribute to the increased incidence of coronary and peripheral artery disease in diabetic patients. Angiogenesis is initiated by vascular endothelial growth factor (VEGF), a potent angiogenic cytokine, and suppressed by angiostatin, which is generated by matrix metalloproteinase (MMP)-2 and -9 through proteolytic cleavage of plasminogen. We hypothesized that MMP-2 and -9 were upregulated in the diabetic vasculature, resulting in increased angiostatin production and reduced blood vessel formation. In diabetic internal mammary artery samples (n=32) collected from patients undergoing coronary artery bypass grafting surgery, capillary density was only 30% of that in the nondiabetic vessels (n=32), whereas VEGF expression was reduced by 48%. Diabetes upregulated the expression and the gelatinolytic activity of MMP-2 and -9. Active MMP-2 and -9 were released from diabetic arteries, but not from nondiabetic vessels, during phenylephrine-induced vasoconstriction. Diabetes enhanced transcription and protein expression of tissue inhibitor of MMP (TIMP)-1 but had an opposite effect on TIMP-2. In diabetic vessels angiostatin was increased by 62% and was positively correlated with the activities of MMP-2 and -9 (r2=0.806 and 0.742, respectively). This report indicated a strong correlation between the upregulation of MMP-2 and MMP-9 and the increased angiostatin expression in the human diabetic arterial vasculature. The enhanced angiostatin production with a reduced VEGF formation may explain the pathogenesis of impaired angiogenesis in diabetes mellitus.

A Systematic Review of Directly Applied Biologic Therapies for Acute Spinal Cord Injury

An increasing number of therapies for spinal cord injury (SCI) are emerging from the laboratory and seeking translation into human clinical trials. Many of these are administered as soon as possible after injury with the hope of attenuating secondary damage and maximizing the extent of spared neurologic tissue. In this article, we systematically reviewed the available preclinical research on such neuroprotective therapies that are administered in a non-invasive manner for acute SCI. Specifically, we reviewed treatments that have a relatively high potential for translation due to the fact that they are already used in human clinical applications or are available in a form that could be administered to humans. These included: erythropoietin, NSAIDs, anti-CD11d antibodies, minocycline, progesterone, estrogen, magnesium, riluzole, polyethylene glycol, atorvastatin, inosine, and pioglitazone. The literature was systematically reviewed to examine studies in which an in vivo animal model was utilized to assess the efficacy of the therapy in a traumatic spinal cord injury paradigm. Using these criteria, 122 studies were identified and reviewed in detail. Wide variations exist in the animal species, injury models, and experimental designs reported in the preclinical literature on the therapies reviewed. The review highlights the extent of investigation that has occurred in these specific therapies, and points out gaps in our knowledge that would be potentially valuable prior to human translation.

A Novel Porcine Model of Traumatic Thoracic Spinal Cord Injury

Spinal cord injury (SCI) researchers have predominately utilized rodents and mice for in vivo SCI modeling and experimentation. From these small animal models have come many insights into the biology of SCI, and a growing number of novel treatments that promote behavioral recovery. It has, however, been difficult to demonstrate the efficacy of such treatments in human clinical trials. A large animal SCI model that is an intermediary between rodent and human SCI may be a valuable translational research resource for pre-clinically evaluating novel therapies, prior to embarking upon lengthy and expensive clinical trials. Here, we describe the development of such a large animal model. A thoracic spinal cord injury at T10/11 was induced in Yucatan miniature pigs (20-25 kg) using a weight drop device. Varying degrees of injury severity were induced by altering the height of the weight drop (5, 10, 20, 30, 40, and 50 cm). Behavioral recovery over 12 weeks was measured using a newly developed Porcine Thoracic Injury Behavior Scale (PTIBS). This scale distinguished locomotor recovery among animals of different injury severities, with strong intra-observer and inter-observer reliability. Histological analysis of the spinal cords 12 weeks post-injury revealed that animals with the more biomechanically severe injuries had less spared white matter and gray matter and less neurofilament immunoreactivity. Additionally, the PTIBS scores correlated strongly with the extent of tissue sparing through the epicenter of injury. This large animal model of SCI may represent a useful intermediary in the testing of novel pharmacological treatments and cell transplantation strategies.

In the presence of L-NAME SERCA blockade induces endothelium-dependent contraction of mouse aorta through activation of smooth muscle prostaglandin H2/thromboxane A2 receptors

The mechanism of transient contractions induced by the sarcoplasmic–endoplasmic reticulum calcium ATPase (SERCA) blocker cyclopiazonic acid (CPA) in the presence of L‐NAME was investigated in mouse aorta. The contractions elicited by 10 μM CPA required an intact endothelium, were dependent upon external Ca2+ and were prevented by 10 μM indomethacin, the inhibitor of prostaglandin synthesis, or 1 μM SQ29548, the specific prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker. A blocker of receptor/store operated Ca2+ channels and voltage gated calcium channels (VGCC), SK&F 96365 (10 μM), completely abolished the contractions, while a specific blocker of VGCC nifedipine (1 μM) inhibited them by one third. Dichlorobenzamyl hydrochloride, a blocker of Na+/Ca2+ exchange effectively prevented return of tension to baseline value. At higher concentrations (30–100 μM) CPA induced indomethacin‐resistant tonic contractions of mouse aorta. The CPA dose response curve for tonic contractions is shifted to the right compared to the transient contractions suggesting that smooth muscle is less sensitive to CPA than endothelium. PGH2/TXA2 receptors in mouse aorta are highly sensitive to the thromboxane analogue U46619 (EC50 : 1.93 nM). This compound stimulates contractions even in the absence of external Ca2+, which are abolished by the Rho‐kinase inhibitor HA‐1077. The results suggest that 10 μM CPA induced capacitive Ca2+ entry in endothelial cells stimulating the release of PGH2/TXA2, which subsequently caused smooth muscle contraction dependent on Ca2+ influx and myofilament sensitization by Rho‐kinase. Higher concentrations of CPA (30–100 μM) directly induced contraction of mouse aortic smooth muscle.

Human Vascular Smooth Muscle Cells From Diabetic Patients Are Resistant to Induced Apoptosis Due to High Bcl-2 Expression

An emerging body of evidence suggests that vascular remodeling in diabetic patients involves a perturbation of the balance between cell proliferation and cell death. Our aim was to study whether arteries and vascular smooth muscle cells (VSMCs) isolated from diabetic patients exhibit resistance to apoptosis induced by several stimuli. Internal mammary arteries (IMAs) were obtained from patients who had undergone coronary artery bypass graft surgery. Arteries from diabetic patients showed increasing levels of Bcl-2 expression in the media layer, measured by immunofluorescence and by Western blotting. Human IMA VSMCs from diabetic patients showed resistance to apoptosis, measured as DNA fragmentation and caspase-3 activation, induced by C-reactive protein (CRP) and other stimuli, such as hydrogen peroxide and 7β-hydroxycholesterol. The diabetic cells also exhibited overexpression of Bcl-2. Knockdown of Bcl-2 expression with Bcl-2 siRNA in cells from diabetic patients reversed the resistance to induced apoptosis. Consistent with the above, we found that pretreatment of nondiabetic VSMCs with high glucose abolished the degradation of Bcl-2 induced by CRP. Moreover, cell proliferation was increased in diabetic compared with nondiabetic cells. This differential effect was potentiated by glucose. We conclude that the data provide strong evidence that arterial remodeling in diabetic patients results from a combination of decreased apoptosis and increased proliferation.

Diabetes modulates capacitative calcium entry and expression of transient receptor potential canonical channels in human saphenous vein.

Diabetes is associated with a perturbation of signaling pathways in vascular tissue, which causes vasomotor dysfunction such as hypertension. We have previously demonstrated that vessels from diabetic patients were more contractile than those from non-diabetic. However, in human vessels, the receptor-stimulated contraction is mainly due to enzymatic, rather than calcium signaling pathway. In this study, we hypothesized that the differential contractile response between diabetic and non-diabetic human vessels could be due to the receptor signaling to sarcoplasmic reticulum and the regulation of capacitative calcium entry. In saphenous vein samples (n=20) collected from diabetic patients undergoing bypass surgery, the contraction initiated by the addition of the sarco-endoplasmatic reticulum calcium ATPase blocker, cyclopiazonic acid, was significantly higher than that in the vessels from non-diabetic patients (n=26) (84.0+/-14.9% vs 44.2+/-9.2%), and this contraction was inhibited by SKF-96365, an inhibitor of store-operated calcium channels. Pre-incubation with indomethacin reduced the cyclopiazonic acid-induced contraction in the non-diabetic veins, but had no effect on the diabetic ones. The gene expression of transient receptor potential canonical channels (TRPC)4 was upregulated by 22% in the diabetic vessels compared with the non-diabetic ones. However, the protein expression of TRPC1 and TRPC6 was downregulated in the diabetic group by 50%. We concluded that diabetes would modulate the capacitative calcium entry likely through the store-operated calcium channel specifically via the regulation of TRPC.

Hyperglycemia and hyperlipidemia are associated with endothelial dysfunction during the development of type 2 diabetes

Diabetes mellitus impairs endothelial function, which can be considered as the hallmark in the development of cardiovascular diseases. Hyperglycemia, hyperinsulinemia, and hyperlipidemia are believed to contribute to endothelial dysfunction. In the present study, we investigated the possible links among these plasma metabolic markers and endothelial function in a mouse model during the development of type 2 diabetes. C57BL/6J-Lepob/ob mice at 8, 12, and 16 weeks were used to study endothelial function during the establishment of type 2 diabetes. Endothelial function was accessed in vitro in the thoracic aorta by measuring acetylcholine (ACh)-stimulated vasodilatation. Blood plasma was obtained for the measurements of glucose, insulin, triglycerides, and cholesterol levels. Correlation and multiple regression analysis revealed strong negative associations between the ACh responsiveness and the plasma levels of glucose, insulin, and lipid profiles at the age of 8 weeks. Associations were observed at neither older age nor in C57BL/6J mice. In conclusion, the increase in plasma levels of glucose, insulin, and lipids is associated with the impairment of the endothelial function during the early stage of the development of type 2 diabetes. The loss of correlation at an older age suggests multifactorial regulation of endothelial function and cardiovascular complications at later stages of the disease.

Intraparenchymal Microdialysis after Acute Spinal Cord Injury Reveals Differential Metabolic Responses to Contusive versus Compressive Mechanisms of Injury

In animal models, spinal cord injury (SCI) is typically imparted by contusion alone (e.g., weight drop) or by compression alone (e.g., clip compression). In humans, however, the cord is typically injured by a combination of violent contusion followed by varying degrees of ongoing mechanical compression. Understanding how the combination of contusion and compression influences the early pathophysiology of SCI is important for the pre-clinical development of neuroprotective therapies that are applicable to the human condition. Disturbances in the metabolism of energy-related substrates such as lactate, pyruvate, and glucose are important aspects of secondary damage. In this study, we used a porcine model of traumatic SCI to determine the extent to which these metabolites were influenced by contusion followed by sustained compression, using the microdialysis technique. Following contusion injury, lactate and pyruvate levels near the epicenter both increased, while glucose remained quite stable. When the contusion injury was followed by sustained compression, we observed a transient rise in lactate, while pyruvate and glucose levels dropped rapidly, which may reflect decreased regional spinal cord blood flow. Furthermore, contusion with sustained compression produced a prolonged and dramatic increase in the lactate-pyruvate (L/P) ratio as a marker of tissue hypoxia, whereas after contusion injury alone, a transient and less significant elevation of the L/P ratio was observed. In this study, we demonstrate that disturbances in energy metabolism within the injured spinal cord vary greatly depending upon the biomechanical nature of the injury. Such differences are likely to be relevant to the applicability of novel therapies targeting specific aspects of the early secondary injury cascade after acute human SCI.