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.

A systematic review of cellular transplantation therapies for spinal cord injury.

Cell transplantation therapies have become a major focus in pre-clinical research as a promising strategy for the treatment of spinal cord injury (SCI). In this article, we systematically review the available pre-clinical literature on the most commonly used cell types in order to assess the body of evidence that may support their translation to human SCI patients. These cell types include Schwann cells, olfactory ensheathing glial cells, embryonic and adult neural stem/progenitor cells, fate-restricted neural/glial precursor cells, and bone-marrow stromal cells. Studies were included for review only if they described the transplantation of the cell substrate into an in-vivo model of traumatic SCI, induced either bluntly or sharply. Using these inclusion criteria, 162 studies were identified and reviewed in detail, emphasizing their behavioral effects (although not limiting the scope of the discussion to behavioral effects alone). Significant differences between cells of the same "type" exist based on the species and age of donor, as well as culture conditions and mode of delivery. Many of these studies used cell transplantations in combination with other strategies. The systematic review makes it very apparent that cells derived from rodent sources have been the most extensively studied, while only 19 studies reported the transplantation of human cells, nine of which utilized bone-marrow stromal cells. Similarly, the vast majority of studies have been conducted in rodent models of injury, and few studies have investigated cell transplantation in larger mammals or primates. With respect to the timing of intervention, nearly all of the studies reviewed were conducted with transplantations occurring subacutely and acutely, while chronic treatments were rare and often failed to yield functional benefits.

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.

Arterialization of a vein graft promotes cell cycle progression through Akt and p38 mitogen-activated protein kinase pathways: impact of the preparation procedure.

BACKGROUND: Vein arterialization following bypass surgery often leads to graft occlusion, but the underlying cellular mechanisms have been poorly studied. OBJECTIVES: Cell cycle progression and the activation of proliferation signalling were compared in arterialized grafts prepared either according to the conventional procedure or using pharmacological relaxation with the native vein. METHODS: Using the porcine carotid-jugular bilateral interposition graft model on one side, a segment of porcine jugular vein was prepared for grafting using the conventional procedure, with pressure distention at 300 mmHg; the segment grafted on the other side was treated with a combination of pharmacological vasodilators. Both veins were grafted into the carotid artery for two weeks. RESULTS: On the immunolabelling of proliferation cell nuclear antigen, a greater number of proliferating cells was found in the conventionally prepared grafts compared with pharmacologically prepared grafts. Cyclin D1 expression and phosphorylation of retinoblastoma increased after implantation, coinciding with nuclear accumulation of beta-catenin, activation of the Akt and mitogen-activated protein kinase cascades, and upregulated phosphatase and tensin homologue phosphorylation. Replacement of distention with pharmacological relaxation reduced the increase in cyclin D1 expression, phosphorylation of retinoblastoma, Akt-Thr(308), glycogen synthase kinase 3 beta and p38, but not extracellular signal-regulated kinases. This technique preserved the active phosphatase and tensin homologue, as well as the expression of cyclin-dependent kinase inhibitor p21(Cip1), while elevating the expression of p27(Kip1). CONCLUSIONS: It was concluded that two-week arterial implantation stimulates proliferation signalling and promotes the cell cycle in vein grafts. Replacement of the conventional preparation procedures with pharmacological vasorelaxation restricts the activation of proliferation and cell cycle progression, and can be beneficial for improving vein graft patency.

Enhanced cell cycle entry and mitogen-activated protein kinase-signaling and downregulation of matrix metalloproteinase-1 and -3 in human diabetic arterial vasculature.

Diabetic patients have a strong predilection for atherosclerosis and postangioplasty restenosis. Accelerated cell proliferation and excessive extracellular matrix deposition are believed to contribute to the development of atherosclerotic plaques and neointima. We investigated the effect of diabetes on cell cycle, proliferation signaling, and the activation of matrix metalloproteinases (MMPs). Segments of internal mammary arteries from 26 type 2 diabetic and 26 non-diabetic patients undergoing coronary artery bypass grafting surgery were compared. Increased levels of cyclin D1 mRNA (by 135+/-14%) and protein expression (by 93.8+/-7.0%), retinoblastoma protein phosphorylation (by 45.9+/-4.8%), and beta-catenin nuclear localization (by 176+/-16%) indicated the enhanced cell cycle entry in the diabetic arteries. Diabetes increased phosphorylation of extracellular signal-regulated kinase-1/2 and p-38-mitogen-activated protein kinase (MAPK) by 76.0+/-6.8 and 62.3+/-4.3%. Increased collagen deposition was evidenced in the diabetic arteries. mRNA levels of MMP-1 and MMP-3 were decreased in the diabetic tissue to 55 and 82%, respectively, compared to the non-diabetic group; protein levels were also decreased accompanied with decreased enzymatic activities by 21 and 50%, respectively. In conclusion, enhanced cell cycle entry, increased MAPK signaling, and downregulated MMP-1 and MMP-3 were characteristic of diabetic arterial vasculature, and could contribute to the progressive atherosclerosis and postangioplasty restenosis in diabetic patients.

Pharmacologic relaxation of vein grafts is beneficial compared with pressure distention caused by upregulation of endothelial nitric oxide synthase and nitric oxide production.

OBJECTIVE: Pressure distention of veins during preparation for bypass surgery is believed to impair vascular integrity and reduce graft patency. We previously suggested a combination of pharmacologic vasodilatators as an alternative to distention. Vascular homeostasis is largely regulated by nitric oxide. We investigated the role of distention in comparison with pharmacologic vasorelaxation in the regulation of nitric oxide synthases, nitric oxide bioavailability, and vascular reactivity in vein grafts. METHODS: In a porcine model the internal jugular vein from either side received pressure distention or the combination of vasodilators (alpha-adrenergic antagonist, phenoxybenzamine, 10 micromol/L; Rho-kinase inhibitor, HA-1077 [fasudil], 50 mumol/L; calcium blocker, nicardipine, 1 micromol/L) and then was grafted into the carotid artery. Regulation of nitric oxide synthase, as well as nitrate and nitrite levels, were examined in vein grafts after 2 weeks of implantation. RESULTS: Distention of jugular veins resulted in reduction of vasoconstriction in response to depolarization and agonist stimulation. Arterial grafting doubled inducible nitric oxide synthase expression in both grafts but caused a pronounced upregulation of endothelial nitric oxide synthase protein (by 57.3% +/- 5%) only in drug-treated grafts, whereas in distended grafts the endothelial nitric oxide synthase level was decreased by 27.5% +/- 2.7%. The downregulated endothelial nitric oxide synthase level in the distended grafts was accompanied by a 45.2% +/- 3.1% reduction of phospho-endothelial nitric oxide synthase Ser1177 levels and by a significant reduction in nitric oxide synthase activity (12.1% +/- 1.2%) and nitrate production (48.9% +/- 5.6%) in comparison with that seen in drug-treated grafts. CONCLUSIONS: Pharmacologic preparation of the vein grafts results in upregulation of endothelial nitric oxide synthase and increased nitric oxide production in the vein grafts after arterial implantation. This might provide greater clinical benefit than conventional pressure-distention methods.

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.

Pressure distention compared with pharmacologic relaxation in vein grafting upregulates matrix metalloproteinase-2 and -9.

OBJECTIVE: Autogenous vein bypasses are a common and effective method to treat occlusive disease. During surgical preparation, veins are routinely pressure distended to overcome vasospasm and twists. Distention, however, is believed to promote vascular remodeling and contribute to decreased graft patency. Pharmacologic vasorelaxation with a combination of effective vasodilators has been suggested as an alternative to pressure distention. The extracellular matrix (ECM)-degrading matrix metalloproteinases (MMPs) have been implicated in vascular remodeling and neointima formation. The purpose of the present study was to compare the effects of pressure distention with pharmacologic vasorelaxation on graft remodeling and regulation of MMP-2 and MMP-9 in porcine vein grafts. METHODS: Carotid artery bypass utilizing internal jugular veins was performed in eight female white pigs. Jugular veins were randomized to receive pressure distention (300 mm Hg for 2 minutes) or a combination of vasodilators (the alpha-adrenergic antagonist phenoxybenzamine, 10 micromol/L; the Rho-kinase inhibitor HA-1077 [fasudil], 50 micromol/L; and the calcium-channel blocker nicardipine, 1 micromol/L) for 30 minutes and then were grafted into the carotid arteries. Two weeks after surgery, vein graft samples were analyzed for vessel intimal and medial area, lumen diameter, and ECM composition. Molecular analysis using reverse transcription-polymerase chain reaction, Western immunoblotting, gelatin zymography, and reverse zymography were performed to study the expression and activation of MMP-2 and MMP-9, and tissue inhibitors of MMP (TIMP)-1 and TIMP-2. RESULTS: Pressure distention irreversibly overstretched the porcine jugular vein and increased MMP-2 and MMP-9 proteolytic activity by 40% and 77%, respectively. Two weeks of vein grafting in the carotid arterial bed induced vessel wall thickening, ECM modification, and neointima formation, which were more pronounced in the distended grafts (P < .05) and accompanied by an increase in MMP expression and activity. Distended grafts demonstrated higher percentages of active MMP-9 (17.8% +/- 1.0%) and higher activities of latent (35.5% +/- 3.3%) and active MMP-2 (69.6% +/- 8.8%) than the pharmacologically treated grafts. Protein expression of TIMP-1 and TIMP-2 was downregulated after arterial grafting, but the pharmacologically treated grafts expressed significantly more TIMP-1 protein (by 36.8% +/- 4.1%) than the distended ones. The activities of TIMPs were markedly decreased after grafting, contributing to the upregulated MMP activity. CONCLUSIONS: Pressure distention of vein grafts before implantation, compared with pharmacologic vasodilatation, stimulates neointima formation and augments MMP activities. Pharmacologic vasorelaxation may be clinically superior to distention in attenuating graft remodeling and possibly improving graft patency. CLINICAL RELEVANCE: Autogenous vein bypasses are a common and effective method to treat occlusive disease. This study demonstrated that pressure distention, a common preparatory procedure in bypass surgery, upregulates extracellular matrix-degrading matrix metalloproteinases, which predisposes vein grafts to extensive remodeling and contributes to neointima formation and graft occlusion. The topical application of a combination of vasodilators to the vein graft before implantation may be clinically superior to pressure distention in attenuating graft remodeling and may possibly improve graft patency and reduce secondary surgical interventions.

Compromised arterial function in human type 2 diabetic patients.

Diabetes is associated with a perturbation of signaling pathways in vascular tissue, which causes vasomotor dysfunction such as hypertension and accelerated atherosclerosis. In the present study, the mechanisms of vasomotor dysfunction, Akt (Thr308 and Ser473) phosphorylation and expression of endothelial NO (nitric oxide) synthase, and inducible NO synthase were investigated in human diabetic internal mammary arteries. The phospho-Akt (Thr308) level in arteries from diabetic patients was reduced to about one-half of the level in nondiabetic patients, suggesting impaired insulin signaling in human diabetic vascular tissue. Augmented vasoconstriction was observed in diabetic arteries, due in part to deficiency of basal and stimulated NO production. This correlated with decreased endothelial NO synthase expression and activity in diabetic vessels. The sensitivity of diabetic vessels to the NO donor, sodium nitroprusside, was reduced as well, suggesting that NO breakdown and/or decreased sensitivity of smooth muscle to NO are also responsible for abnormal vasoconstriction. In addition, the abnormal vasoconstriction in diabetic vessels was not completely abolished in the presence of Nomega-nitro-L-arginine methyl ester, revealing that NO-independent mechanisms also contribute to vasomotor dysfunction in diabetes. In conclusion, diabetes downregulates the Akt-signaling pathway and compromises human arterial function through a decrease in NO availability as well as through NO-independent mechanisms.

Effect of moderate pressure distention on the human saphenous vein vasomotor function.

BACKGROUND: Manual pressure distension, which is commonly applied to the human saphenous vein graft for coronary artery bypass, is believed to have detrimental consequences for the graft patency. The vasomotor function of the vein after distention during surgical preparation for grafting and after distention in laboratory conditions at pressure of 50 to 600 mm Hg was studied. The effect of a combination of vasodilative agents to prevent vasospasm was also tested. METHODS: The contractile and dilatory responses of distended and undistended human saphenous veins and those after drug treatment were examined in organ baths under isometric conditions. RESULTS: Distention at the pressure range 100 to 300 mm Hg resulted in an increased contractile response of the saphenous vein to both alpha-adrenergic activation with 50 micromol/L phenylephrine (153.73% +/- 15.69%) and depolarization with 80 mmol/L K(+) (141.03% +/- 15.13%) in comparison with the undistended vein and did not impair the relaxation. In contrast manual distention during surgical preparation abolished the contractile response and impaired the relaxation. The application of a combination of vasodilative drugs (alpha-adrenergic antagonist phenoxybenzamine, 10 micromol/L, Rho-kinase inhibitor HA-1077, 50 micromol/L, and calcium blocker nicardipine, 1 micromol/L) eliminated the contractile response of the vein to phenylephrine and 80 mmol/L K(+). This effect was sustained more than 20 hours after the washout of the drugs. CONCLUSIONS: The distention of the human saphenous vein at moderate pressure combined with the application of the effective combination of vasodilative drugs before grafting into the arterial circulation could be a beneficial alternative to the current practice of uncontrolled pressure distension.

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.

1. 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. 2. The contractions elicited by 10 micro M CPA required an intact endothelium, were dependent upon external Ca(2+) and were prevented by 10 micro M indomethacin, the inhibitor of prostaglandin synthesis, or 1 micro M SQ29548, the specific prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker. 3. A blocker of receptor/store operated Ca(2+) channels and voltage gated calcium channels (VGCC), SK&F 96365 (10 micro M), completely abolished the contractions, while a specific blocker of VGCC nifedipine (1 micro M) inhibited them by one third. 4. Dichlorobenzamyl hydrochloride, a blocker of Na(+)/Ca(2+) exchange effectively prevented return of tension to baseline value. 5. At higher concentrations (30-100 micro M) 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. 6. PGH2/TXA2 receptors in mouse aorta are highly sensitive to the thromboxane analogue U46619 (EC(50) : 1.93 nM). This compound stimulates contractions even in the absence of external Ca(2+), which are abolished by the Rho-kinase inhibitor HA-1077. 7. The results suggest that 10 micro M CPA induced capacitive Ca(2+) entry in endothelial cells stimulating the release of PGH2/TXA2, which subsequently caused smooth muscle contraction dependent on Ca(2+) influx and myofilament sensitization by Rho-kinase. Higher concentrations of CPA (30-100 micro M) directly induced contraction of mouse aortic smooth muscle.