Effects of Delivering Guanidinoacetic Acid or Its Prodrug to the Neural Tissue: Possible Relevance for Creatine Transporter Deficiency

The creatine precursor guanidinoacetate (GAA) was used as a dietary supplement in humans with no adverse events. Nevertheless, it has been suggested that GAA is epileptogenic or toxic to the nervous system. However, increased GAA content in rodents affected by guanidinoacetate methyltransferase (GAMT) deficiency might be responsible for their spared muscle function. Given these conflicting data, and lacking experimental evidence, we investigated whether GAA affected synaptic transmission in brain hippocampal slices. Incubation with 11.5 μM GAA (the highest concentration in the cerebrospinal fluid of GAMT-deficient patients) did not change the postsynaptic compound action potential. Even 1 or 2 mM had no effect, while 4 mM caused a reversible decrease in the potential. Guanidinoacetate increased creatine and phosphocreatine, but not after blocking the creatine transporter (also used by GAA). In an attempt to allow the brain delivery of GAA when there was a creatine transporter deficiency, we synthesized diacetyl guanidinoacetic acid ethyl ester (diacetyl-GAAE), a lipophilic derivative. In brain slices, 0.1 mM did not cause electrophysiological changes and improved tissue viability after blockage of the creatine transporter. However, diacetyl-GAAE did not increase creatine nor phosphocreatine in brain slices after blockage of the creatine transporter. We conclude that: (1) upon acute administration, GAA is neither epileptogenic nor neurotoxic; (2) Diacetyl-GAAE improves tissue viability after blockage of the creatine transporter but not through an increase in creatine or phosphocreatine. Diacetyl-GAAE might give rise to a GAA–phosphoGAA system that vicariates the missing creatine–phosphocreatine system. Our in vitro data show that GAA supplementation may be safe in the short term, and that a lipophilic GAA prodrug may be useful in creatine transporter deficiency.

In vitro impact preliminary assessment of airborne particulate from metalworking and woodworking industries

Inhalation is the main route of exposure to airborne pollutants. To evaluate the safety and assess the risks of occupational hazards different testing approaches are used. 3D airway epithelial tissues allow to mimic exposure conditions in vitro, generates human-relevant toxicology data, allows to elucidate the mode of action of pollutants. Gillian3500 pumps were used to collect the airborne particulate from woodworking and metalworking environments. EpiAirway tissues were used to model half working day (4 h), full working day (8 h), and 3 working day exposures to occupational pollutants. Tissue viability was assessed using an MTT assay. For preliminary assessment, RT-qPCR analyses were performed to analyze the expression of gelsolin, caspase-3, and IL-6. Tissue morphology was assessed by hematoxylin/eosin staining. An effect on the proliferation of lung epithelial cell line A549 was assessed. Acute exposure to workspace pollutants slightly affected tissue viability and did not change the morphology. No inhibiting effect was observed on the proliferation of A549 cells. Preliminary analysis showed that both types of particles suppressed the expression of gelsolin, with the effect of metalworking samples being more pronounced. A slight reduction in caspase-3 expression was observed. Particles from metalworking suppressed IL-6 expression.

Cyclic Variation of Spectral Parameters for the Differentiation of Atrial Myocardium Before and Immediately Following Radiofrequency Ablation

Radiofrequency ablation (RFA) is a common treatment of atrial fibrillation. However, current treatment is associated with a greater than 20% recurrence rate, in part due to inadequate monitoring of tissue viability during ablation. Spectral parameters, in particular cyclic variation of integrated backscatter (CVIB), have shown promise as early indicators of myocardial recovery from ischemia. Our aim was to demonstrate the use of spectral parameters to differentiate atrial myocardium before and after radiofrequency ablation. An AcuNav 10 F catheter was used to collect radiofrequency signals from the posterior wall of the left atrium of patients before and immediately after RFA for AF. The normalized power spectrum was obtained and three spectral parameters (integrated backscatter [IB], slope, and intercept) were extracted across two continuous heart cycles. Parameters were gated for ventricular end-diastole and compared before and after ablation. Additionally, the cyclic variation of each of these three parameters was generated as an average of the variation across the two recorded heart cycles. Data from 14 patients before and after ablation demonstrated a significant difference in the magnitude of the cyclic variation of integrated backscatter (9.0 vs. 6.0 dB, p < .001) and cyclic variation of the intercept (14.0 vs. 11.5 dB, p = .04). No significant difference was noted in the magnitude of the cyclic variation of the slope. Among spectral parameters gated for end-diastole, significant differences were noted in the slope (−4.39 vs. −3.73 dB/MHz, p = .002) and intercept (16.8 vs. 11.9 dB, p = .002). No significant difference was noted in the integrated backscatter. Spectral parameters are able to differentiate atrial myocardium before and immediately following ablation and may be useful in monitoring atrial ablations.

Investigating the Benefits of Using 3D Camera Technology to Improve Wound Measurements in a Tissue Viability Service: Outcomes of a Pilot Implementation Study

Sub-optimal experience and outcomes for people with stalled wounds is common. Clinicians have limited methods for reliably and accurately measure wounds. Depth measurement is an important indicator of healing, and digital methods of imaging the wound may offer increased accuracy and enable clinical decision-making. This study aimed to implement a Panasonic FZ-M1 toughpad with WoundCareLite software version 1.5.0.0, to enable three-dimensional measurements in Tissue Viability (TV) service. Length, width, and depth measurement were compared with usual manual measurement using a paper ruler alongside a 2D photographic image. Statistical analysis included the comparison of wound dimension measures and a presentation of visual healing trajectories over 4 weeks using run-charts. 30 patients were recruited over five weeks (13 female and 17 male), representing 4% of the usual caseload. Manual measurement and 3D software automatic method demonstrated that the width and depth 3D auto measures were more accurate than manual measures but depth measures were often wrong thus making volumetric measures inaccurate. Consistent wound size measurement was feasible, and healing trajectories provide a useful means of continuous assessment. Technology guided measurement has potential benefits over manual measurement as a means of more accurately monitoring healing. In this case, depth measurement could not be accurately assessed in practice and further software innovation is indicated to enable outcome measurement in tissue viability services.

Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50–300μM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9–13.4μl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU) –ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300μM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

In Vitro Impact Preliminary Assessment of Airborne Particulate From Metalworking and Woodworking Industries

Inhalation is the main route of exposure to airborne pollutants. To evaluate the safety and assess the risks of occupational hazards different testing approaches are used. 3D airway epithelial tissues allow to mimic exposure conditions in vitro, generates human-relevant toxicology data, allows to elucidate mode of action of pollutants. Gilian 3500 pumps equipped with Standard Midget Impingers were used to collect the airborne particulate from woodworking and metalworking environments. EpiAirway™ tissues were used to model half working day (4 h), full working day (8 h), and 3 working day exposures to occupational pollutants. Tissue viability was assessed using MTT assay. RT-qPCR analyses performed to analyze the expression of gelsolin, caspase-3, and IL-6. Tissue morphology was assessed by hematoxylin/eosin staining. Acute exposure to workspace pollutants slightly affected tissue viability and did not change the morphology. Both types of particles suppressed expression of gelsolin, with metalworking samples showing the most pronounced effect. A slight reduction in caspase-3 expression was observed. Particles from metalworking suppressed IL-6 expression. 3D Epithelial tissues can be used to model exposures to airborne pollutants. Exposure to particles from woodworking and metalworking had a minor effect on tissue viability but affected the expression of inflammation and apoptosis-related genes.