Intact Porcine Research Articles

Investigation into the biomechanics of lumbar spine micro-dynamic pedicle screw

BackgroundNumerous reports have shown that rigid spinal fixation contributes to a series of unwanted complications in lumbar fusion procedure. This innovative micro-dynamic pedicle screw study was designed to investigate the biomechanical performance of lumbar implants using numerical simulation technique and biomechanical experiment.MethodsInstrumented finite element models of three configurations (dynamic fixation, rigid fixation and hybrid fixation) using a functional L3-L4 lumbar unit were developed, to compare the range of motion of the lumbar spine and stress values on the endplate and implants. An in vitro experiment was simultaneously conducted using 18 intact porcine lumbar spines and segmental motion analyses were performed as well.ResultsSimulation results indicated that the dynamic fixation and the hybrid fixation models respectively increased the range of motion of the lumbar spine by 95 and 60% in flexion and by 83 and 55% in extension, compared with the rigid fixation model. The use of micro-dynamic pedicle screw led to higher stress on endplates and lower stress on pedicle screws. The outcome of the in vitro experiment demonstrated that the micro-dynamic pedicle screw could provide better range of motion at the instrumented segments than a rigid fixation.ConclusionThe micro-dynamic pedicle screw has the advantage of providing better range of motion than conventional pedicle screw in flexion-extension, without compromising stabilization, and has the potential of bringing the load transfer behavior of fusional segment closer to normal and also lowers the stress values of pedicle screws.

Effects of supercritical fluid CO2 and 25kGy gamma irradiation on the initial mechanical properties and histological appearance of tendon allograft.

Tendon allografts, when autograft options are limited or when obtaining an autograft is not aligned with the patients' best interest, play an important role in tendon and ligament reconstruction. To minimize the risk of infectious disease transmission tissue banks perform screening tests and the allografts cleaned are sterilized. The current study examines and compares the initial mechanical properties and histological appearance of supercritical CO2 (SCCO2)-treated and gamma-irradiated porcine extensor tendons. Thirty intact porcine forelimb extensor tendons randomized equally into three groups: control group, gamma-irradiation group, and SCCO2-treated group. Once treated, histological assessment and histomorphologic measurements were made on the histological sections obtained from each tendon while stiffness and ultimate failure loads were evaluated from tensile testing. Histological evaluation of gamma-irradiated tendons showed significant disruption to the hierarchical morphology of the fascicle bundles, which was not evident in SCCO2-treated specimens. Histomorphologic measurements showed a significant increase for measured dead space (void) between tendon fibrils of the gamma-irradiated group comparing to both control and SCCO2 treated groups (p < 0.01). There was a significant reduction in the ultimate failure load for tendons treated by gamma-irradiation compared to the control group (p < 0.05). No statistically significant difference was detected between control and SCCO2-treated tendons in the ultimate failure load. Stiffness values were not significantly different between three-study groups. This study suggests that while gamma-irradiation has a deleterious effect on mechanical properties of tendon tissue, SCCO2 does not alter the biomechanical properties and the histological structure of porcine extensor tendons.

CD8+ single positive and CD4+CD8+ double positive cells constitute a significant proportion of the total lymphocytes in the anterior chamber of the normal porcine eye

Abstract This study investigated whether CD4− and CD8-expressing cells are present in the anterior chamber of the normal porcine eye. Intact porcine globes were obtained from local slaughterhouses. They did not show any abnormalities or signs of disease. The cornea was incised and then aqueous humor (AH) from both globes of the same animal was collected. The lymphocyte population in peripheral blood was gated on the basis of forward and side scatter properties. The positioning of this gate served as a reference for setting the lymphocyte gate in AH samples. The number of lymphocytes in the anterior chamber was extremely low; only about 5000 events in the lymphocyte gate were collected for the entire volume of AH samples. Single positive (SP) CD8+ cells constituted almost 40% of the total AH lymphocyte population. In contrast, the mean percentage of SP CD4+ cells in the lymphocyte pool was only 1.77. It is well known that the peripheral blood lymphocyte (PBL) population in pigs contains a large share of CD4+(high)CD8+(low) double positive (DP) cells. Likewise, a large contribution of these cells was found in the anterior chamber, namely their mean percentage was 17.45. However, in contrast to their PBL counterpart, the majority of AH DP cells exhibited low expression of CD4. The results suggest that the anterior chamber in swine is an element in the normal migratory pathway of SP CD4+, SP CD8+ and DP cells. However, the trafficking of these cells into this compartment seems to be relatively poor. Moreover, the study demonstrated a limited presence of SP CD4+ cells in the anterior chamber compared to SP CD8+ and DP cells. Such selective homing of SP CD8+ and CD4+ cells to the anterior chamber may be involved in the induction and maintenance of ocular immune privilege.

Effect of increasing flow when grooving during phacoemulsification

To determine optimum flow settings on the Centurion Vision System during the grooving step in cataract surgery. John A. Moran Eye Center Laboratory, University of Utah, Salt Lake City, Utah, USA. Experimental study. Intact porcine lenses hardened by formalin and placed in a chamber designed to simulate the anterior chamber of the eye were used to test flow rate settings at 20mL/min, 40mL/min, and 60mL/min. Vacuum was set at 400mm Hg, longitudinal power at 80%, torsional power at 80%, and intraocular pressure at 50mm Hg. A balanced phaco tip with a 20-degree tip and a 30-degree bevel was used. Efficiency (time to groove the lens in half) was determined. Increasing flow from 20 to 40mL/min during grooving increased efficiency by 17% (P=.05), with no significant improvement shown at 60mL/min. A flow rate of 40mL/min was determined to be most efficient during the grooving step of cataract surgery. Further increases in flow rate showed no statistically significant improvement in efficiency, and with only 17% improvement flow rates less than 40mL/min might be almost as efficient and might be safer.

TRPV1-dependent ERK1/2 activation in porcine lens epithelium

Recently we determined that the Transient Receptor Potential Vanilloid 4 ion channel (TRPV4) has a crucial signaling role in a pathway that regulates various aspects of lens epithelium function. Here, we report on a different TRPV channel, TRPV1, in porcine lens. The presence of TRPV1 in the lens was evident from RT-PCR studies and Western blot analysis of MAPK signaling pathway activation caused by the TRPV1 agonist capsaicin. TRPV1 mRNA was detected in the epithelium of porcine as well as human lens. Transient ERK1/2 and p38 MAPK phosphorylation was detected within 1 min in the epithelium isolated from intact porcine lenses exposed to capsaicin (100 nM), a selective TRPV1 agonist, and the response was significantly inhibited by A889245 (1.0 μM), a TRPV1 antagonist. A similar ERK 1/2 and p38 response in the epithelium, also inhibitable by A889245, was evident in lenses treated with hyperosmotic solution (350 vs 300 mOsm). Lenses pre-treated with either the cytosolic Ca2+ chelator BAPTA-AM or the PKC inhibitor sotrastaurin (1.0 μM) had a diminished ERK1/2 activation response to capsaicin and hyperosmotic solution. Taken together the findings support the notion that TRPV1 functions as a plasma membrane ion channel that, when activated, permits the entry of extracellular calcium into the lens epithelium, leading to activation of PKC, ERK1/2 and p38 MAPK. It is significant that the findings confirm earlier proposals that hyperosmotic stress is linked to TRPV1 channel activation in the mouse lens. Further studies are ongoing to determine what functional changes are triggered by the TRPV1-linked signaling pathways and how they might relate to lens volume homeostasis.

Short-duration ocular iontophoresis of ionizable aciclovir prodrugs: A new approach to treat herpes simplex infections in the anterior and posterior segments of the eye

The objective was to investigate (trans)corneal and transscleral iontophoresis of biolabile amino acid ester prodrugs of aciclovir (ACV-X, X = Arg, Gly and Trp) as a means to increase ocular bioavailability of ACV. Prodrugs displayed tissue-dependent susceptibility to hydrolysis. Iontophoresis of ACV-Arg, ACV-Gly and ACV-Trp (5 mM, 0.5 mA/cm2) for 5 min followed by 55 min passive diffusion resulted in appreciable corneal deposition (21.5 ± 5.1, 14.1 ± 2.0 and 5.3 ± 0.6 nmol/cm2, respectively) and transcorneal permeation (13.9 ± 1.6, 10.9 ± 1.8 and 5.7 ± 0.5 nmol/cm2, respectively) of ACV species. In contrast, passive delivery of ACV across porcine cornea after 1 h was < LOQ (i.e. <0.125 nmol/cm2). Transscleral permeation of ACV-Arg, ACV-Gly and ACV-Trp (9 mM, 1.25 mA/cm2) after iontophoresis for 5 min was 20.4 ± 3.8, 12.3 ± 0.3 and 8.4 ± 0.4 nmol/cm2, respectively – far superior to passive delivery which was again < LOQ. Using intact porcine eye globes, 5 min transscleral iontophoresis of ACV-Gly at 3.75 mA/cm2 resulted in considerable delivery of ACV species to the choroid/retina and vitreous humour (5.7 ± 2.3 and 11.7 ± 3.7 nmol/cm2, respectively). Furthermore, the average concentration of ACV species in the whole eyeball (4.5 ± 1.6 nmol/cm3) was significantly higher than the IC50 of ACV against HSV-1 (<0.22 nmol/cm3), demonstrating the potential application for the treatment of ocular HSV infections.

A Standardized Dissection Protocol to Generate Aortic Valvular Scaffolds from Porcine Hearts.

To describe a particular harvesting procedure for isolating intact porcine aortic heart valve roots as potential sources for biologic scaffolds. Fresh porcine hearts were brought to the Tissue Engineering and Regenerative Medicine Laboratory at the University of Medicine and Pharmacy in Targu Mures. The aortic roots were extracted from the porcine hearts by anatomical dissection. For this purpose, we used a basic surgical instrument kit. This initial phase was the first step in obtaining acellular extracellular matrix as a biologic scaffold material. Aortic roots were isolated with preservation of the ascending aorta as well as the intact aortic sinus and coronaries together with the adjacent myocardial tissue and anterior leaflet of the mitral valve. This approach allowed for safe mounting of roots into mounting rings for perfusion decellularization. The described procedure is a feasible protocol for obtaining intact biological valvular scaffolds from porcine hearts. Reduced requirements regarding tools and personnel underline the easiness of aortic root harvesting using this particular procedure.

Imaging microscopic distribution of antifungal agents in dandruff treatments with stimulated Raman scattering microscopy.

Treatment of dandruff condition usually involves use of antidandruff shampoos containing antifungal agents. Different antifungal agents show variable clinical efficacy based on their cutaneous distribution and bioavailability. Using stimulated Raman scattering (SRS), we mapped the distribution of unlabeled low-molecular weight antifungal compounds zinc pyrithione (ZnPT) and climbazole (CBZ) on the surface of intact porcine skin with cellular precision. SRS has sufficient chemical selectivity and sensitivity to detect the agents on the skin surface based on their unique chemical motifs that do not occur naturally in biological tissues. Moreover, SRS is able to correlate the distribution of the agents with the morphological features of the skin using the CH 2 stretch mode, which is abundant in skin lipids. This is a significant strength of the technique since it allows the microscopic accumulation of the agents to be correlated with physiological features and their chemical environment without the use of counter stains. Our findings show that due to its lower solubility, ZnPT coats the surface of the skin with a sparse layer of crystals in the size range of 1 to 4 ?? ? m . This is consistent with the current understanding of the mode of action of ZnPT. In contrast, CBZ being more soluble and hydrophobic resulted in diffuse homogeneous distribution. It predominantly resided in microscopic lipid-rich crevasses and penetrated up to 60 ?? ? m into the infundibular spaces surrounding the hair shaft. The ability of the SRS to selectively map the distribution of agents on the skin’s surface has the potential to provide insight into the mechanisms underpinning the topical application of antifungal or skin-active agents that could lead to the rational engineering of enhanced formulations.

Development of a novel ex vivo porcine laparoscopic Heller myotomy and Nissen fundoplication training model (Toronto lap-Nissen simulator).

Surgical trainees are required to develop competency in a variety of laparoscopic operations. Developing laparoscopic technical skills can be difficult as there has been a decrease in the number of procedures performed. This study aims to develop an inexpensive and anatomically relevant model for training in laparoscopic foregut procedures. An ex vivo, anatomic model of the human upper abdomen was developed using intact porcine esophagus, stomach, diaphragm and spleen. The Toronto lap-Nissen simulator was contained in a laparoscopic box-trainer and included an arch system to simulate the normal radial shape and tension of the diaphragm. We integrated the use of this training model as a part of our laparoscopic skills laboratory-training curriculum. Afterwards, we surveyed trainees to evaluate the observed benefit of the learning session. Twenty-five trainees and five faculty members completed a survey regarding the use of this model. Among the trainees, only 4 (16%) had experience with laparoscopic Heller myotomy and Nissen fundoplication. They reported that practicing with the model was a valuable use of their limited time, repeating the exercise would be of additional benefit, and that the exercise improved their ability to perform or assist in an actual case in the operating room. Significant improvements were found in the following subjective measures comparing pre- vs. post-training: (I) knowledge level (5.6 vs. 8.0, P<0.001); (II) comfort level in assisting (6.3 vs. 7.6, P<0.001); and (III) comfort level in performing as the primary surgeon (4.9 vs. 7.1, P<0.001). The trainees and faculty members agreed that this model was of adequate fidelity and was a representative simulation of actual human anatomy. We developed an easily reproducible training model for laparoscopic procedures. This simulator reproduces human anatomy and increases the trainees' comfort level in performing and assisting with myotomy and fundoplication.

Glucocorticoid receptor number and affinity differ between peripheral blood mononuclear cells and granulocytes in domestic pigs

The aim of the present study was to characterize the number and affinity of glucocorticoid receptors (GR) in peripheral blood mononuclear cells (PBMC) and granulocytes of domestic pigs because glucocorticoid signaling is considered important for animal health and welfare. To investigate GR binding characteristics in intact porcine immune cells, blood samples of 6 castrated male pigs were collected via indwelling vein catheters. Porcine PBMC and granulocytes were isolated using two-layer density gradients, followed by radioligand binding assays to determine the number of GR sites per cell and the dissociation constant Kd as a measure for GR binding affinity. The present study revealed a greater number of GR sites per cell (P = 0.039) in PBMC (mean ± SEM: 1,953 ± 207 sites/cell) compared to granulocytes (1,561 ± 159 sites/cell) in domestic pigs. Furthermore, porcine PBMC had a higher GR binding affinity than porcine granulocytes (P = 0.003) as the dissociation constant Kd of PBMC (1.8 ± 0.2 nM) was lower than that of granulocytes (3.5 ± 0.4 nM). Our results point to differences in underlying mechanisms of glucocorticoid signaling in different porcine leukocyte populations.

Novel 3D ultrasound system for midline single-operator epidurals: a feasibility study on a porcine model

BackgroundWe developed a real-time 3D ultrasound thick slice rendering technique and innovative Epiguide needle-guide as an adjunct to single-operator midline epidural needle insertions. Study goals were to determine feasibility of the technique in a porcine model and compare the visibility of standard and echogenic needles. MethodsThirty-four lumbar needle insertions were performed on six intact porcine spines ex vivo. Ultrasound scanning identified the insertion site and, using an Epiguide, the needle was guided into the epidural space through the ligamentum flavum in the midline plane, watched in real-time on the 3D ultrasound. Entry into the epidural space was judged by a loss-of-resistance technique. Needle visibility was rated by the anesthesiologist performing the technique using a 4-point scale; (0=cannot see, 1=poor, 2=satisfactory, 3=excellent), and later by an independent assessor viewing screenshots. The procedure was repeated at all lumbar levels using either the standard or echogenic needle. ResultsSuccessful loss-of-resistance to fluid was achieved in 76% of needle insertions; needle visibility with echogenic needles (94.2% rated satisfactory/excellent) was significantly better than with standard needles (29.4% satisfactory/excellent, P<0.0001). Successful loss-of-resistance was 93% when mean needle visibility was rated as ‘excellent’. Inter-observer agreement between assessors was ‘near-perfect’ (weighted kappa=0.83). ConclusionIt is feasible to perform 3D ultrasound-guided real-time single-operator midline epidural insertions, in a porcine model. Echogenic needles were found to consistently improve needle visibility; and improved needle visibility tended to increase successful entry into epidural space.

Development of Topical Delivery Systems for Flightless Neutralizing Antibody

Flightless I (Flii) is an actin remodeling protein important for cytoskeletal regulation and cellular processes including migration, proliferation, and adhesion. Previous studies have clearly identified Flii as a novel therapeutical target for improved wound repair and have demonstrated Flii regulation using Flii neutralizing antibodies (FnAb) in different models of wound healing in vivo. Here we describe the development of an optimized topical delivery system that can neutralize Flii activity in the epidermis. Topical delivery of FnAb is an attractive approach as it provides a convenient application, sustained release, localized effect, and reduced dosage. Three successful formulations were developed, and their physical and chemical stability examined. The in vitro release revealed prolonged and sustained release of FnAb in all the tested formulations. Additionally, penetration studies using intact porcine skin showed that FnAb penetrated the epidermis and upper papillary dermis. The penetrated FnAb significantly reduced Flii expression compared to dosed matched IgG controls. This study has successfully developed a topical delivery system for FnAb that could serve as a potential platform for future localized wound treatments.

A videofluoroscopy-based tracking algorithm for quantifying the time course of human intervertebral displacements

The motions of individual intervertebral joints can affect spine motion, injury risk, deterioration, pain, treatment strategies, and clinical outcomes. Since standard kinematic methods do not provide precise time-course details about individual vertebrae and intervertebral motions, information that could be useful for scientific advancement and clinical assessment, we developed an iterative template matching algorithm to obtain this data from videofluoroscopy images. To assess the bias of our approach, vertebrae in an intact porcine spine were tracked and compared to the motions of high-contrast markers. To estimate precision under clinical conditions, motions of three human cervical spines were tracked independently ten times and vertebral and intervertebral motions associated with individual trials were compared to corresponding averages. Both tests produced errors in intervertebral angular and shear displacements no greater than 0.4° and 0.055 mm, respectively. When applied to two patient cases, aberrant intervertebral motions in the cervical spine were typically found to correlate with patient-specific anatomical features such as disc height loss and osteophytes. The case studies suggest that intervertebral kinematic time-course data could have value in clinical assessments, lead to broader understanding of how specific anatomical features influence joint motions, and in due course inform clinical treatments.

Correlation of IOP with Corneal Acoustic Impedance in Porcine Eye Model.

Purpose The aim of this study is to correlate the intraocular pressure (IOP) change with the acoustic impedance of the cornea, in order to propose a noncontact and noninvasive method for IOP monitoring. Methods and Materials A highly focused transducer (frequency 47-MHz; bandwidth 62%) was made to measure the echo from the anterior and posterior surfaces of intact porcine eyes, respectively. A multilayered transmission and reflection model was used to calculate the acoustic impedance. The linear relationship between acoustic impedance and intraocular pressure was analyzed by statistical method. ResultDuring pressure elevation from 10 mm Hg to 50 mm Hg, the mean acoustic impedance of the posterior cornea increased from 1.5393 to 1.5698 MRayl, which showed a strong linear correlation (R = 0.9849; P = 0.0022). Meanwhile, the mean value of the anterior cornea increased from 1.5399 to 1.5519 MRayl, and a less significant correlation was observed (R = 0.7378; P = 0.0025). Conclusion This study revealed a linear correlation between intraocular pressure and acoustic impedance of the cornea, thus demonstrating a potentially important method to noninvasively measure the intraocular pressure in vivo.

Adrenomedullin Stimulates Porcine Sinus Node Automaticity

Adrenomedullin (AM) is a vasodilatory and chronotropic peptide that has cardioprotective properties in settings of ischemia-reperfusion. Specifically, the 52-amino acid peptide hormone has been shown to reduce infarct size and potentially be protective against malignant arrhythmias. AM reduces L-type calcium currents in rabbit ventricular myocytes, and can possibly be utilized as an antiarrhythmic drug. However, at present it is unclear whether AM has cardiac electrophysiological effects in vivo. In the present study, we investigated whether AM affects sinus and atrioventricular nodal functions, cardiac pacing thresholds, refractory properties and intracardiac conduction intervals in an intact porcine model (Norwegian landrace and Yorkshire hybrids; n=12). Hemodynamic and electrophysiological parameters were recorded at baseline and following 60 min of AM-infusion (100 ng/kg/min). The hemodynamic effects of AM were characterized by a reduced mean arterial pressure (76 ± 9 vs. 57 ± 6; p<0.05; mmHg), tachycardia (91 ± 13 vs. 112 ± 15; p<0.05; beats/min) and an augmented cardiac index (131 ± 20 vs. 176 ± 28; p<0.05; ml/min/kg). The sinus cycle length was reduced (674 ± 89 vs. 546 ± 73; p<0.05; ms), sinoatrial conduction time was unaltered (79 ± 24 vs. 72 ± 19; ns; ms), and the sinus node recovery time (SNRT) was reduced at paced cycle lengths of 425 ms, 375 ms and 325 ms (average SNRT 1034 ± 449 vs.704 ± 141; p<0.05; ms). Diastolic pacing thresholds, effective refractory periods, Wenckebach cycle length and intracardiac conduction intervals were unaffected by AM. In conclusion, AM stimulates sinus node function with a reduced sinus cycle length and increased automaticity but has no further detectable intracardiac electrophysiological effects. Whether the increased automaticity is a direct effect on the sinus node or an indirect effect mediated through the autonomic nervous system remains to be determined.

In Situ Mechanical Characterization of Multilayer Soft Tissue Using Ultrasound Imaging.

In this paper, we report the development of a technique to characterize layer-specific nonlinear material properties of soft tissue in situ with the potential for in vivo testing. A soft tissue elastography robotic arm system comprising of a robotically manipulated 30MHz high-resolution ultrasound probe, a custom designed compression head, and load cells has been developed to perform compression ultrasound imaging on the target tissue and measure reaction forces. A multilayer finite element model is iteratively optimized to identify the material coefficients of each layer. Validation has been performed using tissue mimicking agar-based phantoms with a low relative error of ∼7% for two-layer phantoms and ∼10% error for three layer phantoms when compared to known ground-truth values obtained using a commercial material testing system. The technique has then been used to successfully determine the in situ layer-specific mechanical properties of intact porcine stomach. The mean C10 and C20 for a second-order reduced polynomial material model were determined for the muscularis (6.41 ± 0.60, 4.29 ± 1.87 kPa), submucosal (5.21 ± 0.57, 3.68 ± 3.01 kPa), and mucosal layers (0.06 ± 0.02, 0.09 ± 0.24 kPa). Such a system can be utilized to perform in vivo mechanical characterization, which is left as future work.

Effect of radial meniscal tear on in situ forces of meniscus and tibiofemoral relationship.

To clarify the effect of the radial tear of the lateral meniscus on the in situ meniscus force and the tibiofemoral relationship under axial loads and valgus torques. Ten intact porcine knees were settled to a 6-degree of freedom robotic system, while the force and 3-dimensional path of the knees were recorded via Universal Force Sensor (UFS) during 3 cycles of 250-N axial load and 5-Nm valgus torque at 15°, 30°, 45°, and 60° of knee flexion. The same examination was performed on the following 3 meniscal states sequentially; 33, 66, and 100% width of radial tears at the middle segment of the lateral meniscus, while recording the force and path of the knees via UFS. Finally, all paths were reproduced after total lateral meniscectomy and the in situ force of the lateral meniscus were calculated with the principle of superposition. The radial tear of 100% width significantly decreased the in situ force of the lateral meniscus and caused tibial medial shift and valgus rotation at 30°-60° of knee flexion in both testing protocols. Under a 250-N axial load at 60° of knee flexion, the in situ force decreased to 36±29N with 100% width of radial tear, which was 122±38N in the intact state. Additionally, the tibia shifted medially by 2.1±0.9mm and valgusrotated by 2.5±1.9° with the complete radial tear. However, the radial tear of 33 or 66% width had little effect on either the in situ force or the tibial position. A radial tear of 100% width involving the rim significantly decreased the in situ force of the lateral meniscus and caused medial shift and valgus rotation of the tibia, whereas a radial tear of up to 66% width produced only little change. The clinical relevance is that loss of meniscal functions due to complete radial tear can lead to abnormal stress concentration in a focal area of cartilage and can increase the risk of osteoarthritis in the future.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.

Eleclazine, an inhibitor of the cardiac late sodium current, is superior to flecainide in suppressing catecholamine-induced ventricular tachycardia and T-wave alternans in an intact porcine model

The capacity of catecholamines to induce ventricular tachycardia (VT) is well documented. The effectiveness of the novel cardiac late sodium inhibitor eleclazine in suppressing catecholamine-induced VT in a large animal model was compared with that of flecainide. In 13 closed-chest anesthetized Yorkshire pigs, spontaneous VT and surges in T-wave alternans (TWA) level measured using the Modified Moving Average method were induced by epinephrine (2.0 µg/kg, i.v., bolus over 1 minute). Effects of eleclazine (0.3 mg/kg, i.v., infused over 15 minutes; n = 6) or flecainide (1 mg/kg, i.v., bolus over 2 minutes followed by 1 mg/kg/hr, i.v., for 1 hour; n = 7) on VT incidence and TWA level were measured from right intraventricular electrogram recordings. Epinephrine reproducibly elicited hemodynamically significant spontaneous VT in all 13 pigs and increased TWA level by 33-fold compared to baseline (P < .001). Eleclazine reduced the incidence of epinephrine-induced ventricular premature beats and couplets by 51% (from 31.3 ± 1.91 to 15.2 ± 5.08 episodes; P = .038) and the incidence of 3- to 7-beat VT by 56% (from 10.8 ± 3.45 to 4.7 ± 3.12 episodes; P = .004). Concurrently, the drug reduced the peak epinephrine-induced TWA level by 64% (from 217 ± 22.2 to 78 ± 15.3 µV; P < .001). Flecainide also reduced the incidence of epinephrine-induced ventricular premature beats and couplets by 53% (from 40.4 ± 6.37 to 19.0 ± 2.73 episodes; P = .024) but did not affect the incidence of VT (from 15.0 ± 3.08 to 11.6 ± 2.93 episodes; P = .29) or the peak TWA level (from 207 ± 30.6 to 172 ± 26.2 µV; P = .34). Selective inhibition of cardiac late sodium current with eleclazine is more effective than flecainide in reducing catecholamine-induced VT and TWA in an intact porcine model.

Delayed Gadolinium-enhanced MR Imaging of Cartilage: A Comparative Analysis of Different Gadolinium-based Contrast Agents in an ex Vivo Porcine Model

Purpose To compare the delayed gadolinium-enhanced magnetic resonance (MR) imaging of cartilage (dGEMRIC) indexes acquired with different gadolinium-based contrast agents (GBCAs), with emphasis on the difference in electrical charge, and to evaluate the feasibility of the use of GBCAs other than gadopentetate dimeglumine with a double negative charge (Gd-DTPA2-) as alternatives at dGEMRIC. Materials and Methods Intact porcine patellae (n = 44) were divided into four groups according to GBCA used: Gd-DTPA2-, double negative gadobenate dimeglumine (Gd-BOPTA2-), single negative gadoterate meglumine (Gd-DOTA-), and nonionic gadobutrol (Gd-DT-DO3A). Patellae in each group were further assigned to control (n = 3) or trypsin-treated (n = 8) groups and were immersed in GBCA solutions prepared at a concentration of 2.5 mmol/L. T1 maps were acquired at 10-minute intervals at 0-120 minutes. The difference between postcontrast R1 and precontrast R1 (ΔR) and the time ΔR curves were plotted. Patellae were stained with safranin-O to evaluate the proteoglycan content of the cartilage. A linear mixed-effects model was used to analyze the time ΔR curves, and Student t tests and Mann-Whitney U tests were used to compare dGEMRIC indexes between groups. Results The difference in the estimated slopes of the time ΔR curves between control and trypsin-treated groups were greatest with Gd-BOPTA2-, followed by Gd-DTPA2-, Gd-DOTA-, and Gd-DT-DO3A, with differences in the estimated slopes of 0.037, 0.022, 0.018, and 0.011, respectively. The slope difference between control and trypsin-treated groups was significantly greater with Gd-BOPTA2- (P < .001) and significantly smaller with Gd-DT-DO3A (P = .004) in comparison with that with Gd-DTPA2-. Only the GBCAs with double negative charges showed significant differences in both the T1 measured after equilibration of cartilage with GBCA solution and the ΔR at 90 and 120 minutes between the control and trypsin-treated groups. Conclusion Double negative GBCAs produced better contrast between normal and degenerated cartilage than did those with a single negative charge and nonionic GBCAs at the same concentration for dGEMRIC. Because Gd-BOPTA2-, a high-relaxivity GBCA, showed higher contrast than did Gd-DTPA2-, Gd-BOPTA2-may be useful as an alternative GBCA for dGEMRIC. © RSNA, 2016 Online supplemental material is available for this article.