Porcine Kidney Tissue Research Articles

Normothermic Machine Perfusion Reconstitutes Porcine Kidney Tissue Metabolism But Induces an Inflammatory Response, Which Is Reduced by Complement C5 Inhibition.

Normothermic machine perfusion (NMP) is a clinical strategy to reduce renal ischemia-reperfusion injury (IRI). Optimal NMP should restore metabolism and minimize IRI induced inflammatory responses. Microdialysis was used to evaluate renal metabolism. This study aimed to assess the effect of complement inhibition on NMP induced inflammatory responses. Twenty-two pig kidneys underwent 18h of static cold storage (SCS) followed by 4h of NMP using a closed-circuit system. Kidneys were randomized to receive a C5-inhibitor or placebo during SCS and NMP. Perfusion resulted in rapidly stabilized renal flow, low renal resistance, and urine production. During SCS, tissue microdialysate levels of glucose and pyruvate decreased significantly, whereas glycerol increased (p < 0.001). In the first hour of NMP, glucose and pyruvate increased while glycerol decreased (p < 0.001). After 4h, all metabolites had returned to baseline. Inflammatory markers C3a, soluble C5b-9, TNF, IL-6, IL-1β, IL-8, and IL-10 increased significantly during NMP in perfusate and kidney tissue. C5-inhibition significantly decreased perfusate and urine soluble C5b-9 (p < 0.001; p = 0.002, respectively), and tissue IL-1β (p = 0.049), but did not alter other inflammatory markers. Microdialysis can accurately monitor the effect of NMP on renal metabolism. Closed-circuit NMP induces inflammation, which appeared partly complement-mediated. Targeting additional immune inhibitors should be the next step.

Optimized protocol for the multiomics processing of cryopreserved human kidney tissue.

Biobanking of tissue from clinically obtained kidney biopsies for later analysis with multiomic approaches, such as single-cell technologies, proteomics, metabolomics, and the different types of imaging, is an inevitable step to overcome the need of disease model systems and toward translational medicine. Hence, collection protocols that ensure integration into daily clinical routines by the usage of preservation media that do not require liquid nitrogen but instantly preserve kidney tissue for both clinical and scientific analyses are necessary. Thus, we modified a robust single-nucleus dissociation protocol for kidney tissue stored snap-frozen or in the preservation media RNAlater and CellCover. Using at first porcine kidney tissue as a surrogate for human kidney tissue, we conducted single-nucleus RNA sequencing with the widely recognized Chromium 10X Genomics platform. The resulting datasets from each storage condition were analyzed to identify any potential variations in transcriptomic profiles. Furthermore, we assessed the suitability of the preservation media for additional analysis techniques such as proteomics, metabolomics, and the preservation of tissue architecture for histopathological examination including immunofluorescence staining. In this study, we show that in daily clinical routines, the preservation medium RNAlater facilitates the collection of highly preserved human kidney biopsies and enables further analysis with cutting-edge techniques like single-nucleus RNA sequencing, proteomics, and histopathological evaluation. Only metabolome analysis is currently restricted to snap-frozen tissue. This work will contribute to build tissue biobanks with well-defined cohorts of the respective kidney disease that can be deeply molecularly characterized, opening up new horizons for the identification of unique cells, pathways and biomarkers for the prevention, early identification, and targeted therapy of kidney diseases.NEW & NOTEWORTHY In this study, we addressed challenges in integrating clinically obtained kidney biopsies into everyday clinical routines. Using porcine kidneys, we evaluated preservation media (RNAlater and CellCover) versus snap freezing for multi-omics processing. Our analyses highlighted RNAlater's suitability for single-nucleus RNA sequencing, proteome analysis and histopathological evaluation. Only metabolomics are currently restricted to snap-frozen biopsies. Our research established a cryopreservation protocol that facilitates tissue biobanking for advancing precision medicine in nephrology.

Durability Test of Microtome Blades with the High-Precision Tissue-Sectioning Machine

In this study, the durability of microtome blades, used for sectioning paraffin blocks, was evaluated with the goal of improving the quality of sections in pathology tests. First, for the durability test of microtome blades, a sectioning test device that realizes stable sectioning operations was developed. This device comprised precise stages supported by cross-roller guides, achieving sufficient rigidness. This device allowed automated repetitive sectioning and simultaneously measured the principal and thrust cutting forces. Samples embedding porcine kidney and rib tissues were used for the durability test. Two types of blades with different blade edge angles were used. Additionally, the rake face and cross-section of blades, as well as H&amp;E-stained sections, were observed. In the durability test with porcine kidney tissue, good quality sections were obtained even after 100 times of sectioning with both microtome blades, showing sufficient durability. However, in sectioning porcine rib tissue, the microtome blade with a large blade edge angle produced good-quality sections in the initial phase of the durability test; however, defects such as overlapping of folds were observed after 100 times of sectioning. Meanwhile, the microtome blade with a small blade edge angle experienced blade damage from the early phase of the durability test, resulting in the production of unsuitable preparations for pathology tests. These results indicated that the microtome blade with a small blade edge angle lacked durability against hard tissues such as porcine ribs.

Expanding the toolset of fluorescent covalent staining of biological samples by labeling carboxylate and phosphate groups.

Recently, fluorescent covalent staining methods have been developed for visualization of anatomical structures in cells and tissues. Coupled with expansion microscopy, these stains revealed various ultrastructural details. However, the covalently stainable chemical groups have been limited to amines, carbohydrates, and thiols. Here, we developed procedures for covalently labeling tissues for carboxylate and phosphate groups, utilizing carbodiimide crosslinker chemistry. In porcine kidney tissues, the carboxylate and phosphate stain provides 1.8-4.8-fold higher signal intensity than those from the three existing stains. In cancer cells, such stain allows 2-8-fold more accurate identification of nucleoli than the amine stain. In expansion microscopy samples, such stain reveals a variety of sub-cellular structures in tissues when combined with the amine stain. Such stain also allows imaging of lipid-based structures in cultured cells. With these advantages, this new covalent staining method further expands the toolset for fluorescent visualization of histology.

Measurement of Thermal Conductivity and Thermal Diffusivity of Porcine and Bovine Kidney Tissues at Supraphysiological Temperatures up to 93 °C.

This experimental study aimed to characterize the thermal properties of ex vivo porcine and bovine kidney tissues in steady-state heat transfer conditions in a wider thermal interval (23.2-92.8 °C) compared to previous investigations limited to 45 °C. Thermal properties, namely thermal conductivity (k) and thermal diffusivity (α), were measured in a temperature-controlled environment using a dual-needle probe connected to a commercial thermal property analyzer, using the transient hot-wire technique. The estimation of measurement uncertainty was performed along with the assessment of regression models describing the trend of measured quantities as a function of temperature to be used in simulations involving heat transfer in kidney tissue. A direct comparison of the thermal properties of the same tissue from two different species, i.e., porcine and bovine kidney tissues, with the same experimental transient hot-wire technique, was conducted to provide indications on the possible inter-species variabilities of k and α at different selected temperatures. Exponential fitting curves were selected to interpolate the measured values for both porcine and bovine kidney tissues, for both k and α. The results show that the k and α values of the tissues remained rather constant from room temperature up to the onset of water evaporation, and a more marked increase was observed afterward. Indeed, at the highest investigated temperatures, i.e., 90.0-92.8 °C, the average k values were subject to 1.2- and 1.3-fold increases, compared to their nominal values at room temperature, in porcine and bovine kidney tissue, respectively. Moreover, at 90.0-92.8 °C, 1.4- and 1.2-fold increases in the average values of α, compared to baseline values, were observed for porcine and bovine kidney tissue, respectively. No statistically significant differences were found between the thermal properties of porcine and bovine kidney tissues at the same selected tissue temperatures despite their anatomical and structural differences. The provided quantitative values and best-fit regression models can be used to enhance the accuracy of the prediction capability of numerical models of thermal therapies. Furthermore, this study may provide insights into the refinement of protocols for the realization of tissue-mimicking phantoms and the choice of tissue models for bioheat transfer studies in experimental laboratories.

O102 Rapid evaporative ionisation mass spectrometry (REIMS) coupled with the hydrosurgical debridement system and the carbon dioxide surgical laser: a proof of concept study in porcine tissues

Abstract Introduction Rapid evaporative ionisation mass spectrometry (REIMS) is a tissue profiling technology that allows for rapid information. The successful integration of this technology with surgical devices can be beneficial for diagnostic, prognostic, and individualised treatment purposes. Objectives To access the feasibility of coupling REIMS with the hydrosurgical debridement device and the carbon dioxide surgical laser. Methods The 3D printing attachments and circuit connections were designed for device couplings. The aerosols generated from the hydrosurgical debridement device in terms of mist (applying to porcine liver vs kidney tissue samples), and the carbon dioxide laser in terms of smoke (applying to porcine liver vs kidney cortex vs kidney medulla) were collected for REIMS analysis. The abstract models for tissue classification were built from the obtained mass spectrometry data using the linear discriminant analysis (LDA) dimensional reduction technique. The abstract models were cross-validated and the real-time classifications of different tissue types were tested in both coupling platforms. Results The real-time spectral data signals were reliably achieved from both coupling platforms. For REIMS-hydrosurgical debridement coupling, the abstract model was successfully built from 804 spectra obtained from multiple porcine liver and kidney samples. The cross-validation resulted in 98.01% correct classification rate between the two types of tissue. For REIMS-carbon dioxide laser platform, the abstract model built from 1,454 spectra of porcine tissue samples (liver vs kidney cortex vs kidney medulla) demonstrated 94.36% correct classification rate of the three tissue types. Conclusion REIMS can be coupled with the hydrosurgical debridement device and the carbon dioxide surgical laser with accurate real-time classification of porcine tissues. These platforms have the potential for future translational applications in the clinical arena.

Карбоксикриоабляция почки. Экспериментальное исследование

The aim of this experimental study was to evaluate the effect of low temperatures of carbon dioxide on a "living" blood-supplying organ (pig kidney), to determine the possibility of performing cryoablation of kidney tissue with carbon dioxide (carboxycryoablation), as well as to establish experimentally modes of carboxycryoablation of the kidney.To carry out this experimental study, a female of the mini-pig line was used. We performed laparoscopic access to the kidney for carboxycryoablation. During the freezing of the kidney, three modes were compared: 60, 90, and 120 s on one kidney of one animal. Immediately after the completion of cryoablation, nephrectomy was performed, and the removed kidney was sent for histological examination. According to the histological study, the results of the use of carbon dioxide during cryoablation of porcine kidney tissue were evaluated.Cryoablation based on carbon dioxide (carboxycryoablation) leads to irreversible death (necrosis) and destruction of the affected tissue. A direct relationship between the exposure mode and the size of the cryonecrosis zone was noted. Thus, the most extensive zone of necrosis with a diameter of 10 mm was achieved when performing cryoablation in the exposure mode of 120 s.This experiment showed that carbon dioxide-based cryoablation remains a feasible procedure that leads to irreversible death (necrosis) and destruction of the affected tissue. However, further studies on the safety and efficacy of cryoablation of kidney tissue with carbon dioxide are required.

Liquid Nitrogen-Based Cryoablation in In Vivo Porcine Tissue: A Pilot Study.

Introduction:Liquid nitrogen-based cryoablation induces freezing evenly throughout the probe tip surface, resulting in larger ablation volumes and faster treatment times. The purpose of this preliminary investigation is to determine the efficacy of the liquid nitrogen-based Visica2 Cryoablation System (Sanarus Technologies, Pleasanton, CA) in in vivo porcine kidney, liver, and fibro-fatty tissue. Methods:Ablations were performed under ultrasound guidance in 4 Yorkshire pigs. The target lesion cross-section width (W) and depth (D) were 1 cm for liver (n=8), kidney (n=4), and head-neck (n=5) and 2 cm for kidney (n=4). Expected axial length (L) of the resulting lesion is approximately 4 cm. After three-day survival, the ablated tissue was harvested and histologically analysed. The mean width and depth were compared with the target diameter using a one-sample t-test. Results:All animals survived the procedure. For the 1 cm target, mean dimensions (L x W x D) were 3.8±1.5 x 1.7±0.3 x 1.7±0.7 for liver, 3.0±0.5 x 2.0±0.4 x 1.7±0.6 for kidney, and 3.3±0.8 x 1.8±0.4 x 1.8±0.4 for head-neck. Mean width and depth were significantly greater than desired dimension. For the 2 cm target, mean dimensions were 3.2±0.5 x 3.1±0.8 x 1.9±0.7. Mean width and depth were not significantly different to desired target. Conclusion:Our preliminary results show that the Visica2 liquid nitrogen-based cryoablation system can efficiently and reproducibly create ablation volumes in liver, kidney, and fibro-fatty tissue within 4 minutes and 12 minutes for 1cm and 2cm targeted diameters, respectively. Further investigation is necessary to determine the optimal freeze-thaw-freeze protocol for larger ablation volumes.

Abstract LB-285: Rapid needle biopsy assessment at point of care to advance personalized cancer therapy

Abstract Rationale: Although core needle biopsies (CNBs) are the preferred minimally invasive procedure for breast cancer diagnostics, the standard of care H&amp;E histopathologic analysis of core biopsies is both tissue destructive and labor intensive. Our novel millifluidic instrument, CoreView, is designed to evaluate fresh CNBs to make a real time cancer diagnosis or adequacy determination, conserve tissue for downstream diagnostics and assist in timely treatment planning. Technology and Methods: CoreView is designed to handle, stain, and transport CNBs with the use of millifluidics. The first stage of the CoreView removes biopsy procured tissue from side-cut and end-cut needle-type biopsy guns (14-16 gauge). The reliability of the novel low pressure fluidic process in removing intact CNBs was tested using freshly-excised (ex-vivo) porcine breast (N = 28), kidney (N = 21), liver (N = 42), lung (N = 25), and lymph node (N = 30) tissue. The shear rate, provided by the driving pressure of saline solution, was increased in increments until the CNB was removed and the resulting intactness was qualitatively analyzed by preserved overall shape and size. For optical imaging, a second stage streams fluorescence dye over the CNB for H&amp;E equivalent staining. High resolution imaging with 285 nm excitation is currently performed using microscopy with UV-surface excitation [MUSE, Levenson Group, UC Davis] for anatomical imaging. Previous studies have shown the optical sectioning thickness of MUSE is about 3x thicker than microtome-sectioned specimens. To improve the quality of images produced by MUSE within CoreView, we are limiting UV tissue penetration by steric hindrance of dye molecules using conjugated nanoparticles, such as 525-705 nm CdSe Qdots [ThermoFisher], as well as optical blocking dyes as counterstains. Results: The CNB removal stage was 3D-printed in a patent pending disposable design which is optically clear for monitoring the process. CNBs were fully released and structurally intact for analysis in 93% breast, 100% kidney, 100% liver, 84% lung, and 90% lymph node CNB samples. The average volume per time (µL/ms), an analog to shear rate, for each tissue type was 2.19 (σ;; = 1.04), 1.97 (σ;; =0.71), 1.74 (σ;; = 0.87), 2.05 (σ;; = 0.87), and 2.01 (σ;; = 0.87) for breast, kidney, liver, lung, and lymph node, respectively. The CNB tissue was removed in under 15 seconds, some as quickly as 2 seconds. Furthermore, the device's round channel, which holds a CNB with diameter 10-20% less than the channel, results in inherent structural preservation. Conclusions: The novel millifluidic biopsy removal device is an essential first stage to CoreView's automated biopsy handling and future MUSE imaging and adequacy analysis. The customizable CoreView system design allows optimization for specific CNB needle, tissue, and imaging procedure. We predict an adequacy check at nearly 100% reliability in CNB release and integrity within a few minutes at the point of care. Citation Format: David J. Cooper, Mark E. Fauver, Suzanne M. Dintzis, Eric J. Seibel. Rapid needle biopsy assessment at point of care to advance personalized cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-285.

Line scanning, fiber bundle fluorescence HiLo endomicroscopy with confocal slit detection.

.Fiber bundle fluorescence endomicroscopy is an effective method for in vivo imaging of biological tissue samples. Line-scanning confocal laser endomicroscopy realizes confocal imaging at a much higher frame rate compared to the point scanning system, but with reduced optical sectioning. To address this problem, we describe a fiber bundle endomicroscopy system that utilizes the HiLo technique to enhance the optical sectioning while still maintaining high image acquisition rates. Confocal HiLo endomicroscopy is achieved by synchronizing the scanning hybrid-illumination laser line with the rolling shutter of a CMOS camera. An evident improvement of axial sectioning is achieved as compared to the line-scanning confocal endomicroscopy without the HiLo technique. Comparisons are also made with epifluorescence endomicroscopy with and without HiLo. The optical sectioning enhancement is demonstrated on lens tissue as well as porcine kidney tissue.

Mechanical and functional validation of a perfused, robot-assisted partial nephrectomy simulation platform using a combination of 3D printing and hydrogel casting.

There is a scarcity of high-fidelity, life-like, standardized and anatomically correct polymer-based kidney models for robot-assisted partial nephrectomy (RAPN) simulation training. The purpose of this technical report is to present mechanical and functional testing data as evidence for utilizing a perfused hydrogel kidney model created utilizing 3D printed injection casts for RAPN simulation and training. Anatomically correct, tumor-laden kidney models were created from 3D-printed casts designed from a patient's CT scan and injected with poly-vinyl alcohol (PVA). A variety of testing methods quantified Young's modulus in addition to comparing the functional effects of bleeding and suturing among fresh porcine kidneys and various formulations of PVA kidneys. 7% PVA at three freeze-thaw cycles (7%-3FT) was found to be the formula that best replicates the mechanical properties of fresh porcine kidney tissue, where mean(± SD) values of Young's modulus of porcine tissue vs 7%-3FT samples were calculated to be 85.97(± 35) kPa vs 80.97(± 9.05) kPa, 15.7(± 1.6) kPa vs 74.56(± 10) kPa and 87.46(± 2.97) kPa vs 83.4(± 0.7) kPa for unconfined compression, indentation and elastography testing, respectively. No significant difference was seen in mean suture tension during renorrhaphy necessary to achieve observable hemostasis and capsular violation during a simulated perfusion at 120mmHg. This is the first study to utilize extensive material testing analyses to determine the mechanical and functional properties of a perfused, inanimate simulation platform for RAPN, fabricated using a combination of image segmentation, 3D printing and PVA casting.

Development of a two-stage model system to investigate the mineralization mechanisms involved in idiopathic stone formation: stage 2 in vivo studies of stone growth on biomimetic Randall's plaque.

Idiopathic stone formers often form calcium oxalate (CaOx) stones that are attached to calcium phosphate (CaP) deposits in the renal tissue, known as Randall's plaques (RP). Plaques are suggested to originate in the renal tubular basement membrane and spread into the interstitial regions where collagen fibrils and vesicles become mineralized; if the epithelium is breached, the RP becomes overgrown with CaOx upon exposure to urine. We have developed a two-stage model system of CaP-CaOx composite stones, consisting of Stage (1) CaP mineralized plaque, followed by Stage (2) CaOx overgrowth into a stone. In our first paper in this series (Stage 1), osteopontin (and polyaspartate) were found to induce a non-classical mineralization of porcine kidney tissues, producing features that resemble RP. For the Stage 2 studies presented here, biomimetic RPs from Stage 1 were implanted into the bladders of rats. Hyperoxaluria was induced with ethylene glycol for comparison to controls (water). After 4weeks, rats were sacrificed and the implants were analyzed using electron microscopy and X-ray microanalyses. Differences in crystal phase and morphologies based upon the macromolecules present in the biomimetic plaques suggest that the plaques have the capacity to modulate the crystallization reactions. As expected, mineral overgrowths on the implants switched from CaP (water) to CaOx (hyperoxaluric). The CaOx crystals were aggregated and mixed with organic material from the biomimetic RP, along with some amorphous and spherulitic CaOx near the "stone" surfaces, which seemed to have become compact and organized towards the periphery. This system was successful at inducing "stones" more similar to human idiopathic kidney stones than other published models.

Mechanical fractionation of tissues using microsecond-long HIFU pulses on a clinical MR-HIFU system

Purpose: High intensity focussed ultrasound (HIFU) can non-invasively treat tumours with minimal or no damage to intervening tissues. While continuous-wave HIFU thermally ablates target tissue, the effect of hundreds of microsecond-long pulsed sonications is examined in this work. The objective of this study was to characterise sonication parameter-dependent thermomechanical bioeffects to provide the foundation for future preclinical studies and facilitate clinical translation.Methods and materials: Acoustic power, number of cycles/pulse, sonication time and pulse repetition frequency (PRF) were varied on a clinical magnetic resonance imaging (MRI)-guided HIFU (MR-HIFU) system. Ex vivo porcine liver, kidney and cardiac muscle tissue samples were sonicated (3 × 3 grid pattern, 1 mm spacing). Temperature, thermal dose and T2 relaxation times were quantified using MRI. Lesions were histologically analysed using H&E and vimentin stains for lesion structure and viability.Results: Thermomechanical HIFU bioeffects produced distinct types of fractionated tissue lesions: solid/thermal, paste-like and vacuolated. Sonications at 20 or 60 Hz PRF generated substantial tissue damage beyond the focal region, with reduced viability on vimentin staining, whereas H&E staining indicated intact tissue. Same sonication parameters produced dissimilar lesions in different tissue types, while significant differences in temperature, thermal dose and T2 were observed between the parameter sets.Conclusion: Clinical MR-HIFU system was utilised to generate distinct types of lesions and to produce targeted thermomechanical bioeffects in ex vivo tissues. The results guide HIFU research on thermomechanical tissue bioeffects, inform future studies and advice sonication parameter selection for direct tumour ablation or immunomodulation using a clinical MR-HIFU system.

Comparative characterization of decellularized renal scaffolds for tissue engineering

Native extracellular matrix (ECM) provides scaffolds for tissue engineering with natural architecture and biochemical composition. Maintaining the native ECM in decellularized tissues provides cues for cells, which promote their tissue specific arrangement and function. Several approaches have been used to decellularize ECM from the kidney in order to reestablish renal tissue but their comparability is hampered because methods for decellularization and assessment of ECM vary widely. Therefore, we applied a standardized immersion protocol to decellularize porcine kidney tissue with three detergents Triton X-100, SDS and sodium deoxycholate (SDC) at variable temperatures. For comparative analysis decellularization efficacies, structural preservation, composition and cell attachment and viability were analyzed. Structural ECM-conservation is strongly dependent on decellularization temperature, while preservation of glycosaminoglycans (GAG), collagens and cytokines was affected by the detergents used. GAG and collagens were best maintained by 1% SDS at 4 °C, whereas cytokines were best maintained in 1% SDC at 4 °C. Viability and attachment of human induced pluripotent stem cell derived renal precursor cells were best in SDC-ECM and thus not associated with the degree of GAG and collagen maintenance but the cytokine preservation. Based on structural and functional characteristics, we developed a scoring system that allows intra- and inter-study comparison of decellularization strategies. Application of the scoring system to our experimental data showed that decellularization with 1% SDS at 4 °C provided the highest structural and composition scores, while 1% SDC at 4 °C had lower structural and composition but a significantly better cell performance score. Inclusion of multiple published studies in the scoring matrix for comparison identified the highest structural and composition scores when decellularization was performed with SDS at low concentration, for a short period of time and at low temperature. Furthermore, the scoring system indicated that cell attachment and viability cannot be concluded from any other parameter and should therefore always be included in evaluation of decellularization strategies.

Indentation of heterogeneous soft tissue: Local constitutive parameter mapping using an inverse method and an automated rig.

In the domain of soft tissue biomechanics, the development of numerical simulations has raised the experimental challenge of identifying local internal mechanical constitutive data of heterogeneous organs (e.g. brain tissue). In this context, this paper presents an ex-vivo alternative characterization method to full-field imaging techniques. It is based on automated, multiple indentations of an organ section using a custom-built rig, effectively allowing to map the viscoelastic and hyperelastic constitutive parameters of the tissue at the millimetre scale, under dynamic conditions. In this paper, this technique is described and used to map the constitutive data of three sections from porcine liver, kidney and brain tissues. The results of this mapping present strong evidence of correlation between the organ constituents (e.g. white/grey matter distribution) and the identified constitutive parameters. It was also found that brain and kidney tissues are highly heterogeneous in terms of identified properties, suggesting that such a technique is essential for fully characterizing their mechanical behaviour. This method opens the way to 3D mapping of constitutive parameters to feed finite element models of the organs with region-specific properties.

Histotripsy Produced by Hundred-Microsecond-Long Focused Ultrasonic Pulses: A Preliminary Study

A new strategy is proposed in this study to rapidly generate mechanical homogenized lesions using hundred-microsecond-long pulses. The pulsing scheme was divided into two stages: generating sufficient bubble seed nuclei via acceleration by boiling bubbles and efficiently forming a mechanically homogenized and regularly shaped lesion with a homogenate inside via inertial cavitation. The duty cycle was set at 4.9%/3.9% in stage 1 and 1%/0.88% in stage 2 by changing the pulse duration (PD) and off-time independently. The pulse sequence was 500-μs/400-μs PD with a 100-Hz pulse repetition frequency (PRF) in stage 1, followed by 500-μs/400-μs PD with a 100-Hz PRF and 200-μs PD with a 200-Hz PRF in stage 2. Experiments were conducted on polyacrylamide phantoms with bovine serum albumin and on ex vivo porcine kidney tissues using a single-element 1.06-MHz transducer at an 8-MPa peak negative pressure with shock waves. The lesion evolution and dynamic elastic modulus variation in the phantoms and the histology in the tissue samples were investigated. The results indicate that the two-stage treatment using hundred-microsecond-long pulses can efficiently produce mechanically homogenized lesions with smooth borders, long tear shapes and the total homogenate inside. The time to generate a single mechanically homogenized lesion is shortened from >50 s to 17.1 s.

PU.1 antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes.

Antisense long non-coding RNAs (AS lncRNAs) play important roles in refined regulation of animal gene expression. However, their functions and molecular mechanisms for domestic animal adipogenesis are largely unknown. Here, we found a novel AS lncRNA transcribed from the porcine PU.1 gene (also known as SPI1) by strand-specific RT-PCR. Results showed that PU.1 AS lncRNA was expressed and generally lower than the level of PU.1 mRNA in porcine subcutaneous adipose, heart, liver, spleen, lympha, skeletal muscle and kidney tissues. We further found that the levels of PU.1 mRNA and PU.1 protein were significantly lower in subcutaneous and intermuscular adipose than in mesenteric and greater omentum adipose, whereas the levels of PU.1 AS lncRNA showed no difference in porcine adipose tissues from four different parts of the body. During porcine adipogenesis, levels of PU.1 mRNA increased at day 2 and then gradually decreased. Meanwhile, PU.1 AS lncRNA exhibited an expression trend similar to PU.1 mRNA but sharply decreased after day 2. Interestingly, PU.1 protein level rose during differentiation. In addition, at day 6 after differentiation, knockdown of endogenous PU.1 promoted adipogenesis, whereas knockdown of endogenous PU.1 AS lncRNA had the opposite effect. Moreover, peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid synthase (FASN) were significantly upregulated in the PU.1 shRNA treatment group (P < 0.05), whereas they were downregulated in the PU.1 AS shRNA treatment group (P < 0.05). Adipose triglyceride lipase [ATGL; also known as patatin-like phospholipase domain containing 2 (PNPLA2)] and hormone-sensitive lipase [HSL; also known as lipase, hormone-sensitive (LIPE)] contrasted with PPARG and FASN. Finally, the PU.1 mRNA/PU.1 AS lncRNA duplex was detected by an endogenous ribonuclease protection assay combined with RT-PCR. Based on the above results, we suggest that PU.1 AS lncRNA (vs. its mRNA translation) promotes adipogenesis through the formation of a sense-antisense RNA duplex with PU.1 mRNA.

Development and validation of determinative and confirmatory LC-MS/MS methodologies for total florfenicol and tulathromycin residues in bovine, equine and porcine kidney, liver and muscle tissues.

Separate methods for the quantitation and confirmation of regulatory relevant residue concentrations of total florfenicol and tulathromycin residues in multiple tissue matrices were developed and validated. Total florfenicol residues, determined and expressed as florfenicol amine (FFA) equivalents, were quantified and confirmed over a concentration range of 100-4000ng/g, with an LOD of 33ng/g, while total tulathromyicn residues, determined as CP-60,300 and expressed as tulathromycin equivalents, were quantified and confirmed over a concentration range of 500-10,000ng/g, with an LOD of 300ng/g. A 2 or 1h acid digestion for the FFA and tulathromycin methods, respectively, followed by extraction, cleanup, and concentration using mixed-mode strong cation-exchange SPE cartridges was used. Quantitation and confirmation were accomplished using commercially available tri-deuterated FFA (FFA-D3) as internal standard and multiple reaction monitoring (MRM) of three transitions per target analyte. Mean recoveries and matrix effects were 60% and 25%; and 100% and 110%, respectively for the target analytes florfenicol amine and CP-60,300. Intra-lab method reproducibilities ranged from 7 to 11% RSD, which are within the AOAC recommended HORRATr guidelines for method reproducibilities estimated from single laboratory validation studies. Blind spikes showed that method bias was generally less than 15% for both methods within the calibration range. Both methods have been shown to meet requirements for use in national chemical residue monitoring programs.

Irreversible electroporation of the porcine kidney: Temperature development and distribution

ObjectiveAlthough tissue ablation by irreversible electroporation (IRE) has been characterized as nonthermal, the application of frequent repetitive high-intensity electric pulses has the potential of substantially heating the targeted tissue and causing thermal damage. This study evaluates the risk of possible thermal damage by measuring temperature development and distribution during IRE of porcine kidney tissue. MethodsThe animal procedures were conducted following an approved Institutional Animal Ethics Committee protocol. IRE ablation was performed in 8 porcine kidneys. Of them, 4 kidneys were treated with a 3-needle configuration and the remaining 4 with a 4-needle configuration. All IRE ablations consisted of 70 pulses with a length 90µs. The pulse frequency was set at 90 pulses/min, and the pulse intensity at 1,500V/cm with a spacing of 15mm between the needles. The temperature was measured internally using 4 fiber-optic temperature probes and at the surface using a thermal camera. ResultsFor the 3-needle configuration, a peak temperature of 57°C (mean = 49±10°C, n = 3) was measured in the core of the ablation zone and 40°C (mean = 36±3°C, n = 3) at 1cm outside of the ablation zone, from a baseline temperature of 33±1°C. For the 4-needle configuration, a peak temperature of 79°C (mean = 62±16°C, n = 3) was measured in the core of the ablation zone and 42°C (mean = 39±3°C, n = 3) at 1cm outside of the ablation zone, from a baseline of 35±1°C. The thermal camera recorded the peak surface temperatures in the center of the ablation zone, reaching 31°C and 35°C for the 3- and 4-needle configuration IRE (baseline 22°C). ConclusionsThe application of repetitive high-intensity electric pulses during IRE ablation in porcine kidney causes a lethal rise in temperature within the ablation zone. Temperature monitoring should be considered when performing IRE ablation near vital structures.

Evaluation of thermal injury to liver, pancreas and kidney during irreversible electroporation in an in vivo experimental model.

Irreversible electroporation (IRE) is a new technique for tumour cell ablation that is reported to involve non-thermal-based energy using high voltage at short microsecond pulse lengths. In vivo assessment of the thermal energy generated during IRE has not been performed. Thermal injury can be predicted using a critical temperature model. The aim of this study was to assess the potential for thermal injury during IRE in an in vivo porcine model. In vivo continuous temperature assessments of 86 different IRE procedures were performed on porcine liver, pancreas, kidney and retroperitoneal tissue. Tissue temperature was measured continuously throughout IRE by means of two thermocouples placed at set distances (0·5 cm or less, and 1 cm) from the IRE probes within the treatment field. Thermal injury was defined as a tissue temperature of 54°C lasting at least 10 s. Tissue type, pulse length, probe exposure length, number of probes and retreatment were evaluated for associations with thermal injury. In addition, IRE ablation was performed with metal clips or metal stents within the ablation field to determine their effect on thermal injury. An increase in tissue temperature above the animals' baseline temperature (median 36·0°C) was generated during IRE in all tissues studied, with the greatest increase found at the thermocouple placed within 0·5 cm in all instances. On univariable and multivariable analysis, ablation in kidney tissue (maximum temperature 62·8°C), ablation with a pulse length setting of 100 µs (maximum 54·7°C), probe exposure of at least 3·0 cm (maximum 52·0°C) and ablation with metal within the ablation field (maximum 65·3°C) were all associated with a significant risk of thermal injury. IRE can generate thermal energy, and even thermal injury, based on tissue type, probe exposure lengths, pulse lengths and proximity to metal. Awareness of probe placement regarding proximity to critical structures as well as probe exposure length and pulse length are necessary to ensure safety and prevent thermal injury. A probe exposure of 2·5 cm or less for liver IRE, and 1·5 cm or less for pancreas, with maximum pulse length of 90 µs will result in safe and non-thermal energy delivery with spacing of 1·5-2·3 cm between probe pairs.