Cardioplegia at subnormothermia facilitates rapid functional resuscitation of hearts preserved in SOMAH for transplants

Objectives: Hearts preserved ex vivo at 4°C undergo time-dependent irreversible injury due to extreme hypothermia. Studies using novel organ preservative solution SOMAH, suggest that hearts are optimally ‘preserved’ at subnormothermic temperature of 21°C. Present study evaluates relative efficacy of SOMAH ‘cardioplegia’ at 4 and 21°C in preservation of optimum heart function after in vitro storage at subnormothermia. Methods: Porcine hearts arrested with SOMAH cardioplegia at 4 or 21°C were stored in SOMAH for 5-hour at 21°C (n = 5). At the end of storage, the weight of hearts was recorded and biopsies taken for cardiac tissue high energy phosphate level measurements. The hearts were then attached to a reperfusion apparatus and biochemical parameters including cardiac enzyme release and myocardial oxygen consumption and lactate production were determined in perfusate samples at regular intervals during ex vivo perfusion experiment. Functional evaluation of the hearts intraoperatively and ex vivo was performed by 2D echocardiography using trans-esophageal echocardiography probe. Results: Post-storage heart weights were unaltered in both groups, while available high-energy phosphates (HEP) were greater in the 21°C group. Upon ex vivo reperfusion, coronary flow was significantly greater (p < 0.05) in 21°C group. 2D echo revealed a greater cardiac output, fractional area change and ejection fraction in 21°C group that was not significantly different than the 4°C group. However, unlike 4°C hearts, 21°C hearts did not require inotropic intervention. Upon reperfusion, rate of cardiac enzyme release temporally resolved in 21°C group, but not in the 4°C group. 21°C working hearts maintained their energy state during the experimental duration but not the 4°C group; albeit, both groups demonstrated robust metabolism and function during this period. Conclusions: Rapid metabolic switch, increased synthesis of HEP, decreased injury and optimal function provides evidence that hearts arrested at 21°C remain viably and functionally superior to those arrested at 4°C when stored in SOMAH at ambient temperature pre-transplant. Ultramini-abstract Cardioplegic arrest and preservation of hearts in SOMAH at ambient temperature efficiently conserves metabolism and function in in vitro porcine model of heart transplant. Electronic supplementary material The online version of this article (doi:10.1186/s13019-014-0155-z) contains supplementary material, which is available to authorized users

Evaluation of endoscopic vein extraction on structural and functional viability of saphenous vein endothelium

<p>Abstract</p> <p>Objectives</p> <p>Endothelial injury during harvest influences graft patency post CABG. We have previously shown that endoscopic harvest causes structural and functional damage to the saphenous vein (SV) endothelium. However, causes of such injury may depend on the extraction technique. In order to assess this supposition, we evaluated the effect of VirtuoSaph endoscopic SV harvesting technique (VsEVH) on structural and functional viability of SV endothelium using multiphoton imaging, biochemical and immunofluorescence assays.</p> <p>Methods</p> <p>Nineteen patients scheduled for CABG were prospectively identified. Each underwent VsEVH for one portion and "No-touch" open SV harvesting (OSVH) for another portion of the SV. A two cm segment from each portion was immersed in GALA conduit preservation solution and transported overnight to our lab for processing. The segments were labeled with fluorescent markers to quantify cell viability, calcium mobilization and generation of nitric oxide. Morphology, expression, localization and stability of endothelial caveolin, eNOS, von Willebrand factor and cadherin were evaluated using immunofluorescence, Western blot and multiphoton microscopy (MPM).</p> <p>Results</p> <p>Morphological, biochemical and immunofluorescence parameters of viability, structure and function were well preserved in VsEVH group as in OSVH group. However, tonic eNOS activity, agonist-dependent calcium mobilization and nitric oxide production were partially attenuated in the VsEVH group.</p> <p>Conclusions</p> <p>This study indicates that VirtuoSaph endoscopic SV harvesting technique preserves the structural and functional viability of SV endothelium, but may differentially attenuate the vasomotor function of the saphenous vein graft.</p> <p>Ultramini-Abstract</p> <p>Endoscopic extraction preserved the structure and function, but attenuated the calcium mobilization and nitric oxide generation in human SV endothelium.</p

Imaging of Nitric Oxide in Nitrergic Neuromuscular Neurotransmission in the Gut

Background: Numerous functional studies have shown that nitrergic neurotransmission plays a central role in peristalsis and sphincter relaxation throughout the gut and impaired nitrergic neurotransmission has been implicated in clinical disorders of all parts of the gut. However, the role of nitric oxide (NO) as a neurotransmitter continues to be controversial because: 1) the cellular site of production during neurotransmission is not well established; 2) NO may interacts with other inhibitory neurotransmitter candidates, making it difficult to understand its precise role. Methodology/Principal Findings: Imaging NO can help resolve many of the controversies regarding the role of NO in nitrergic neurotransmission. Imaging of NO and its cellular site of production is now possible. NO forms quantifiable fluorescent compound with diaminofluorescein (DAF) and allows imaging of NO with good specificity and sensitivity in living cells. In this report we describe visualization and regulation of NO and calcium ($Ca^{2+}$) in the myenteric nerve varicosities during neurotransmission using multiphoton microscopy. Our results in mice gastric muscle strips provide visual proof that NO is produced de novo in the nitrergic nerve varicosities upon nonadrenergic noncholinergic (NANC) nerve stimulation. These studies show that NO is a neurotransmitter rather than a mediator. Changes in NO production in response to various pharmacological treatments correlated well with changes in slow inhibitory junction potential of smooth muscles. Conclusions/Significance: Dual imaging and electrophysiologic studies provide visual proof that during nitrergic neurotransmission NO is produced in the nerve terminals. Such studies may help define whether NO production or its signaling pathway is responsible for impaired nitrergic neurotransmission in pathological states

Visualization of myenteric nerve varicosities by their Ca²⁺ signals.

<p>These studies were done after EFS of the muscle strips preloaded with the indicator calcium orange. The fluorescent image was superimposed on the image in the transmission mode (a) is a low power view fluorescent (orange-red) varicosities. Note the varicosities along the axon running in the long axis of the circular muscles. (b) is magnified view of one of the axons with varicosities along its length . Downward pointing arrows show the varicosities and upward pointing arrows indicate the inter-varicosity axon. (c) shows intensity profiles of the fluorescent varicosities. Note that there was no Ca⁺ signal in smooth muscle cells.</p

Colocalization NO signals with nNOS immunoreactive varicosities.

<p>In this experiment the muscle strip that was preloaded with DAF-2 under NANC conditions, applied EFS and then fixed was immunostained with anti-nNOS antibody. The NO signals are long lasting as the reaction of NO with DAF-2 to form DAF-2T is irreversible. Top panel shows imaging for NO and reveals spots of green fluorescence, representing NO in the varicosities. Note that smooth muscle cells showed no NO signals. The middle panel shows the same section imaged for nNOS immunofluorescence. The bottom panel shows the merged image revealing that the NO and nNOS signals are superimposed indicating that NO was generated in the nNOS immunoreactive nerves. Note that no NO signals were seen in neighboring smooth muscle cells. (320× magnification).</p

Colocalization of NO and Ca²⁺ in the varicosities.

<p>The muscle strip simultaneously preloaded with calcium orange and DAF-2A were electrically stimulated and the varicosities were examined for Ca²⁺ and NO signals. Top panel shows green NO positive varicosities. Middle panel shows orange-red calcium positive varicosities. Bottom panel shows colocalized NO and Ca²⁺ signals. Neither NO nor Ca²⁺signals were seen in the neighboring smooth muscle cells. (320× magnification).</p

Influence of various treatments on relative quantification of NO and Ca²⁺ signals.

<p>Values represent mean±SEM of normalized fluorescence intensity in arbitrary units after EFS from three independent experiments.</p