Earl Steward

Prevention of chronic rejection by pravastatin in a rat kidney transplant model.

Background. Pravastatin when administered with cyclosporine (CsA) has been shown to ameliorate transplant vasculopathy in the clinical setting. Previously we showed that pravastatin prevents chronic rejection in rat cardiac and liver transplant models. Here we determine whether pravastatin prevents chronic rejection in a rat renal allograft model. Methods. Orthotopic renal transplantations were performed using Fisher 344 rats as donors and Lewis rats as recipients. Recipients were treated with low-dose CsA for 10 days to prevent acute rejection. Recipients were divided into three groups: CsA, CsA + pravastatin, and syngeneic. Renal function was assessed by serum creatinine level at day 130. Allografts were evaluated by histology and immunohistochemistry. Serum levels of alloantibodies were measured by flow cytometry. Intragraft cytokine mRNA expression was determined by semiquantitative reverse transcriptase-polymerase chain reaction. Intragraft levels of the antiapoptotic Bag-1 gene were measured by Western blot. Results. Unlike allografts from the pravastatin group, control allografts demonstrated glomerulosclerosis, vascular obliteration, tubular atrophy, and interstitial fibrosis. Serum creatinine levels and graft infiltration of T cells and macrophages in the pravastatin-treated animals were significantly lower. Intragraft cytokines showed a T helper 2 polarization and decreased transforming growth factor-&bgr; in the pravastatin group. Intragraft expression of Bag-1 was increased in the pravastatin group. Conclusion. This study demonstrates the ability of pravastatin to inhibit chronic rejection in rat renal allografts. Pravastatin’s pleiotropic effects of reducing intragraft inflammatory cytokines, inhibiting immune cell infiltration, and causing up-regulation of the antiapoptotic gene Bag-1 suggest that its ability to prevent transplant chronic rejection may be multifactorial.

Ultrafast optical-ultrasonic system and miniaturized catheter for imaging and characterizing atherosclerotic plaques in vivo

Atherosclerotic coronary artery disease (CAD) is the number one cause of death worldwide. The majority of CAD-induced deaths are due to the rupture of vulnerable plaques. Accurate assessment of plaques is crucial to optimize treatment and prevent death in patients with CAD. Current diagnostic techniques are often limited by either spatial resolution or penetration depth. Several studies have proved that the combined use of optical and ultrasonic imaging techniques increase diagnostic accuracy of vulnerable plaques. Here, we introduce an ultrafast optical-ultrasonic dual-modality imaging system and flexible miniaturized catheter, which enables the translation of this technology into clinical practice. This system can perform simultaneous optical coherence tomography (OCT)-intravascular ultrasound (IVUS) imaging at 72 frames per second safely in vivo, i.e., visualizing a 72 mm-long artery in 4 seconds. Results obtained in atherosclerotic rabbits in vivo and human coronary artery segments show that this ultrafast technique can rapidly provide volumetric mapping of plaques and clearly identify vulnerable plaques. By providing ultrafast imaging of arteries with high resolution and deep penetration depth simultaneously, this hybrid IVUS-OCT technology opens new and safe opportunities to evaluate in real-time the risk posed by plaques, detect vulnerable plaques, and optimize treatment decisions.

Cardiac function after eight hour storage by using polyethylene glycol hemoglobin versus crystalloid perfusion.

Efforts to extend myocardial preservation for transplantation by crystalloid perfusion have been limited by edema and compromised function. We hypothesized that hypothermic perfusion preservation with a polyethylene glycol (PEG) conjugated hemoglobin solution may extend preservation times. The purpose of this study was to compare cardiac function after continuous perfusion by using a hypocalcemic, normokalemic crystalloid perfusate with and without the addition of PEG-hemoglobin (Hb). The hearts of 20 anesthetized and ventilated New Zealand White rabbits were harvested after cold cardioplegic arrest. Group I (n = 10) hearts were continuously perfused with a hypocalcemic, normokalemic 3% bovine PEG-Hb solution at 20°C and 30 mm Hg for 8 hours. Group II (n = 10) hearts were continuously perfused with an identical crystalloid solution without PEG-Hb for 8 hours under the same conditions as group I hearts. Cardiac function was measured with a left ventricular force transducer after transfer to a standard crystalloid Langendorff circuit at 37°C and an aortic root pressure of 59 mm Hg. After 8 hours of perfusion preservation, heart rate was similar for groups I and II (p = not significant [NS]). Coronary blood flow after and during preservation was similar between PEG-Hb and crystalloid preserved hearts (p = NS). Left ventricular developed pressure, peak dP/d t, and peak −dP/d t were superior in hearts preserved with PEG-Hb. Percent water of total ventricular weight was 82.0% for group I and 81.6% for group II (p = NS). Continuous perfusion preservation of rabbit hearts for 8 hours with a hypocalcemic normokalemic PEG-Hb based solution at 30 mm Hg and 20°C yields left ventricular function that is superior to perfusion with a similar crystalloid solution without PEG-Hb, despite similar myocardial edema and coronary flow. Extended cardiac perfusion preservation with this PEG-Hb based solution deserves further study, including comparison with traditional cardioplegic preservation solutions.

Laser Doppler Imaging for Early Detection of Hemorrhage

BACKGROUND Laser Doppler Imaging (LDI) is a noninvasive means to measure blood flow through the superficial skin capillary plexus using flux units. Our objective was to determine the ability of LDI of the skin to detect and quantify rapid, severe hemorrhage. METHODS Five Yucatan mini-pigs (25-35 kg) underwent controlled hemorrhage of 25 mL/kg blood for 20 minutes. Median flux of a 10 cm × 10 cm area of the lower abdomen was measured at 2-minute intervals from initiation of hemorrhage to resuscitation with concurrent measurement of heart rate (HR), systolic blood pressure (SBP), and mean arterial pressure (MAP). RESULTS Average time to a change of 5 U in flux following start of hemorrhage was 2.4 minutes. This was significantly faster than time to change in HR (19.2 minutes, p < 0.05) and showed a trend toward more rapid identification of hemorrhage relative to changes in SBP (3.2 minutes, p = 0.157) and MAP (3.6 minutes, p = 0.083). Flux changes occurred at smaller % total blood volume lost than HR (3.94% vs. 28.8%, p < 0.05) and trended toward smaller volume identification than SBP (4.88%, p = 0.180) and MAP (5.36%, p = 0.102). Average correlation (ρ) of blood volume lost to flux was -0.974; HR, 0.346; SBP, -0.978; and MAP, -0.975. A change of 5 flux units was significantly more sensitive for hemorrhage than a change of 5 beats per minute in HR or 5 mm Hg in SBP or MAP (0.596 vs. 0.169, 0.438, and 0.287 respectively, all p < 0.05). CONCLUSION LDI is a sensitive, specific, and early means to detect and quantify severe hemorrhage.

Inhibition of intraluminal pancreatic enzymes with nafamostat mesilate improves clinical outcomes after hemorrhagic shock in swine

BACKGROUND Recent studies suggest that intraluminal pancreatic enzymes play a major role in the initiation of the inflammatory cascade by the gut after hemorrhagic shock. Previous animal models have shown that the inhibition of enteral pancreatic enzymes with a serine protease inhibitor, nafamostat mesilate (NM), decreases leukocyte activation and transfusion requirements after hemorrhagic shock. The objective of this study was to determine whether enteroclysis with NM would improve the clinical outcomes in swine after hemorrhagic shock and intestinal hypoperfusion. METHODS Thirty-three male Yucatan minipigs weighing 25 kg to 30 kg underwent a controlled hemorrhage of 25 mL/kg with mesenteric clamp for further gut ischemia. Animals were allocated to three groups: (1) shock only (n = 15), (2) shock + enteroclysis with 100 mL/kg GoLYTELY (GL) as a carrier (n = 11), and (3) shock + enteroclysis with GL + 0.37 mmol/L NM (GL+NM, n = 7). Animals were resuscitated, recovered from anesthesia, observed for 3 days, and graded on a daily 4-point clinical scoring system. A score of 0 indicated a moribund state or early death, and a score of 4 indicated normal behavior. RESULTS Pigs treated with GL + NM had significantly higher mean postoperative recovery scores (3.8 +/- 0.4, essentially normal behavior with no early deaths) compared with animals within the shock only and shock + GL groups (2.1 +/- 1 with one early death and 2.2 +/- 1.2 with two early deaths, respectively, analysis of variance p < 0.003). CONCLUSION The inhibition of intraluminal pancreatic enzymes using enteroclysis with the serine protease inhibitor, NM, after hemorrhagic shock significantly improves the clinical outcome.

Optimal flushing agents for integrated optical and acoustic imaging systems

Abstract. An increasing number of integrated optical and acoustic intravascular imaging systems have been developed and hold great promise for accurately diagnosing vulnerable plaques and guiding atherosclerosis treatment. However, in any intravascular environment, the vascular lumen is filled with blood, a high-scattering source for optical and high-frequency ultrasound signals. Blood must be flushed away to provide clearer images. To our knowledge, no research has been performed to find the ideal flushing agent for combined optical and acoustic imaging techniques. We selected three solutions as potential flushing agents for their image-enhancing effects: mannitol, dextran, and iohexol. Testing of these flushing agents was performed in a closed-loop circulation model and in vivo on rabbits. We found that a high concentration of dextran was the most useful for simultaneous intravascular ultrasound and optical coherence tomography imaging.

Extending vaterite microviscometry to ex vivo blood vessels by serial calibration

The endothelial glycocalyx layer is a ~2 µm thick glycosaminoglycan rich pericellular matrix expressed on the luminal surface of vascular endothelial cells, which has implications in vessel mechanics and mechanotransduction. Despite its role in vascular physiology, no direct measurement has of yet been made of vessel glycocalyx material properties. Vaterite microviscometry is a laser tweezers based microrheological method, which has been previously utilized to measure the viscosity of linear and complex fluids under flow. This form of microrheology has until now relied on complete recollection of the forward scattered light. Here we present a novel method to extend vaterite microviscometry to relatively thick samples. We validate our method and its assumptions and measure the apparent viscosity as a function of distance from the vascular endothelium. We observe a differential response in conditions designed to preserve the EGL in comparison to those designed to collapse it.

Recovery of cardiac function after standard hypothermic storage versus preservation with Peg-hemoglobin.

Preservation of the heart for transplantation after infusion of cardioplegia and extirpation of a cardiac allograft results in an ischemic insult to the myocardium. This ischemic insult may lead to a loss of function in the transplanted heart. Hypothermic perfusion preservation with an oxygen hemoglobin carrying solution may avert ischemic injury and lead to improved recovery of cardiac function. The purpose of this study was to compare cardiac function after 8 hours of continuous hypothermic perfusion with a unique polyethylene-glycol-hemoglobin (PEG-Hb) solution to hearts preserved by 4 hours of hypothermic ischemic storage. Freshly extirpated hearts served as functional controls. The hearts of 26 anesthetized and intubated New Zealand white rabbits were harvested after cold cardioplegic arrest. Group I (n = 12) hearts were perfused with a PEG-Hb solution at 20°C and 30 mm Hg for 8 hours. PO2 was maintained ≥ 500 mm Hg. Group II (n = 7) hearts were preserved by cold ischemic storage for 4 hours at 4°C. Group III (n = 7) were tested immediately after harvest. Left ventricular (LV) function was measured in the nonworking state at 15 minutes, 1 hour, and 2 hours after transfer to a standard crystalloid Langendorff circuit. Measurement of LV developed pressure, peak + dP/dt and −dP/dt revealed a superior trend between Group I and Group II hearts in comparison with freshly extirpated hearts. Heart rate was similar among all groups throughout testing (p = ns). Coronary blood flow was not significantly different between groups. Continuous perfusion preservation of rabbit hearts for 8 hours with PEG-Hb solution at 30 mm Hg and 20°C yielded LV function that was similar to 4 hours of ischemic hypothermic storage. Furthermore, return of cardiac function after 8 hours of perfusion preservation using this PEG-Hb solution may be superior to that obtained in freshly extirpated hearts. These data suggest that some recovery of myocardial function may occur during perfusion preservation with this PEG-Hb solution after the ischemic insult of cardioplegic arrest. Continuous perfusion preservation using this PEG-Hb solution deserves further investigation in large animal transplant models.

Ideal flushing agents for integrated optical acoustic imaging systems

An increased number of integrated optical acoustic intravascular imaging systems have been researched and hold great hope for accurate diagnosing of vulnerable plaques and for guiding atherosclerosis treatment. However, in any intravascular environment, vascular lumen is filled with blood, which is a high-scattering source for optical and high frequency ultrasound signals. Blood must be flushed away to make images clear. To our knowledge, no research has been performed to find the ideal flushing agent that works for both optical and acoustic imaging techniques. We selected three solutions, mannitol, dextran and iohexol, as flushing agents because of their image-enhancing effects and low toxicities. Quantitative testing of these flushing agents was performed in a closed loop circulation model and in vivo on rabbits.