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Segel, L. D., J. L. Ensunsa, and W. A. Boyle. "Prolonged support of working rabbit hearts using Fluosol-43 or erythrocyte media." American Journal of Physiology-Heart and Circulatory Physiology 252, no. 2 (February 1, 1987): H349—H359. http://dx.doi.org/10.1152/ajpheart.1987.252.2.h349.
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We compared the perfluorochemical emulsion Fluosol-43 and an erythrocyte-based solution as support media for ex vivo working rabbit hearts functioning with a physiological workload. Both groups of hearts (n = 5/group) exhibited stable function (left ventricular peak systolic pressure, peak rates of left ventricular pressure rise and relaxation, aortic flow, peak aortic flow rate, stroke work, and peak power) for the first 6 h of perfusion. Coronary flow, coronary venous O2 content, and O2 supply-to-demand ratio declined similarly in both groups during the first 6 h. Both groups of hearts preferentially utilized pyruvate to glucose. The Fluosol-43-perfused hearts had higher heart rate, left ventricular peak systolic pressure, peak rate of left ventricular pressure rise, aortic flow, coronary flow, and myocardial O2 consumption compared with the erythrocyte-perfused hearts. The Fluosol-43 hearts produced more lactate and released more creatine phosphokinase than did the erythrocyte-perfused hearts, but the rates were low and constant throughout perfusion, indicating that the hearts were not progressively ischemic. After the first 6 h, function of the Fluosol-43 hearts declined, resulting in their earlier failure compared with the erythrocyte-perfused hearts. The data indicate that Fluosol-43 had sufficient O2- carrying capacity to support stable function of a rabbit heart at a physiological workload for 6 h, and differences in function and ex vivo longevity of the two groups of hearts suggested that a component or contaminant of Fluosol-43 altered sarcolemmal function and/or that a component needed for membrane integrity was lacking in the Fluosol-43 perfusate.
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Hnat, Michael, and Roger E. Bawdon. "Transfer of Meropenem in the ex Vivo Human Placenta perfusion Model." Infectious Diseases in Obstetrics and Gynecology 13, no. 4 (2005): 223–27. http://dx.doi.org/10.1155/2005/961356.
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Objectives.To determine maternal-fetal transplacental passage of meropenem in the ex vivo human perfusion model.Study design.Term placentae (n= 6) were collected immediately after delivery. A single cotyledon was localized, perfused and stabilized with physiologic Eagles minimal essential medium containing 3% bovine albumin and heparin as described by Chalier (Chalier JC. Criteria for evaluating perfusion experiments and presentation results. Contrib Gynecol Obstet 1985; 13:32–39). Meropenem was added to the maternal medium in concentrations similar to maternal serum peak and trough levels, then perfused through the maternal circulation of the cotyledon. To assess transfer and accumulation, fluid aliquots from both the maternal and fetal compartments were collected over an hour at defined intervals in an open and closed system. AntipyrineC14was added to the medium in order to calculate the transport fraction and clearance indexes. Meropenem and antipyrineC14concentrations were determined by High-pressure Liquid Chromatography and liquid scintillation, respectively.Results.Mean antipyrine transport fraction was 2.33 + 0.25. Maternal and fetal mean meropenem peak concentrations were 54.3 + 3.3μg/ml and 2.2 + 0.18μg/ml, respectively. Whereas, maternal and fetal mean trough concentrations were 12.7 + 1.3μg/ml and 0.41 + 0.10μg/ml, respectively. Mean peak clearance index was 0.077 + 0.007 and the mean trough was 0.052 + 0.015. Mean accumulation for the peak and trough concentrations of meropenem were 0.9 and 2.95μg/ml, respectively.Conclusions.Transplacental passage of meropenem was incomplete in the ex vivo human placental perfusion model. Accumulation was also noted in the fetal compartment
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Espanol, Maryceline T., Lawrence Litt, Lee-Hong Chang, Thomas L. James, Philip R. Weinstein, and Pak H. Chan. "Adult Rat Brain-Slice Preparation for Nuclear Magnetic Resonance Spectroscopy Studies of Hypoxia." Anesthesiology 84, no. 1 (January 1, 1996): 201–10. http://dx.doi.org/10.1097/00000542-199601000-00022.
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Background When perfused neonatal brain slices are studied ex vivo with nuclear magnetic resonance (NMR) spectroscopy, it is possible to use 31P detection to monitor levels of intracellular adenosine triphosphate (ATP), cytosolic pH, and other high-energy phosphates and 1H detection to monitor lactate and glutamate. Adult brain slices of high metabolic integrity are more difficult to obtain for such studies, because the adult cranium is thicker, and postdecapitation revival time is shorter. A common clinical anesthesia phenomenon--loss of temperature regulation during anesthesia, with surface cooling and deep hypothermia, was used to obtain high-quality adult rat cerebrocortical slices for NMR studies. Methods Spontaneously breathing adult rats (350 g), anesthetized with isoflurane in a chamber, were packed in ice and cooled until rectal temperatures decreased to approximately 30 degrees C. An intraaortic injection of heparinized saline at 4 degrees C further cooled the brain to approximately 18 degrees C. Slices were obtained and then recovered at 37 degrees C in oxygenated medium. Interleaved 31P/1H NMR spectra were acquired continually before, during, and after 20 min of no-flow hypoxia (PO2 approximately 0 mmHg). Histologic (Nissl stain) measurements were made from random slices removed at different times in the protocol. Three types of pretreatment were compared in no-flow hypoxia studies. The treatments were: (1) hyperoxia; (2) hypercapnia (50% CO2); and (3) hypoxia, which was accomplished by washing the slices with perfusate equilibrated with 100% N2 and maintaining a 100% N2 gas flow in the air space above the perfusate. Results During hyperoxia, 31P NMR metabolite ratios were identical to those seen in vivo in adult brains, except that, in vitro, the Pi peak was slightly larger than in vivo. A lactate peak was seen in in vitro 1H spectra of slices after metabolic recovery from decapitation, although lactate is barely detectable in vivo in healthy brains. The in vitro lactate peak was attributed to a small population of metabolically impaired cells in an injury layer at the cut edge. NMR spectral resolution from the solenoidal coil exceeded that obtained in vivo in surface coil experiments. Phosphocreatine and ATP became undetectable during oxygen deprivation, which also caused a three- to sixfold increase in the ratio of lactate to N-acetyl-aspartate. Within experimental error, all metabolite concentrations except pHi recovered to control values within 2 h after oxygen restoration. Nissl-stained sections suggested that pretreatment with hypercapnia protected neurons from cell swelling during the brief period of no-flow oxygen deprivation. Conclusions Perfused, respiring adult brain slices having intact metabolic function can be obtained for NMR spectroscopy studies. Such studies have higher spectral resolution than can be obtained in vivo. During such NMR experiments, one can deliver drugs or molecular probes to brain cells and obtain brain tissue specimens for histologic and immunochemical measures of injury. Important ex vivo NMR spectroscopy studies that are difficult or impossible to perform in vivo are feasible in this model.
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Hefler, Joshua, Sanaz Hatami, Aducio Thiesen, Carly Olafson, Kiarra Durand, Jason Acker, Constantine J. Karvellas, David L. Bigam, Darren H. Freed, and Andrew Mark James Shapiro. "Model of Acute Liver Failure in an Isolated Perfused Porcine Liver—Challenges and Lessons Learned." Biomedicines 10, no. 10 (October 6, 2022): 2496. http://dx.doi.org/10.3390/biomedicines10102496.
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Acute liver failure (ALF) is a rare but devastating disease associated with substantial morbidity and a mortality rate of almost 45%. Medical treatments, apart from supportive care, are limited and liver transplantation may be the only rescue option. Large animal models, which most closely represent human disease, can be logistically and technically cumbersome, expensive and pose ethical challenges. The development of isolated organ perfusion technologies, originally intended for preservation before transplantation, offers a new platform for experimental models of liver disease, such as ALF. In this study, female domestic swine underwent hepatectomy, followed by perfusion of the isolated liver on a normothermic machine perfusion device. Five control livers were perfused for 24 h at 37 °C, while receiving supplemental oxygen and nutrition. Six livers received toxic doses of acetaminophen given over 12 h, titrated to methemoglobin levels. Perfusate was sampled every 4 h for measurement of biochemical markers of injury (e.g., aspartate aminotransferase [AST], alanine aminotransferase [ALT]). Liver biopsies were taken at the beginning, middle, and end of perfusion for histological assessment. Acetaminophen-treated livers received a median dose of 8.93 g (8.21–9.75 g) of acetaminophen, achieving a peak acetaminophen level of 3780 µmol/L (3189–3913 µmol/L). Peak values of ALT (76 vs. 105 U/L; p = 0.429) and AST (3576 vs. 4712 U/L; p = 0.429) were not significantly different between groups. However, by the end of perfusion, histology scores were significantly worse in the acetaminophen treated group (p = 0.016). All acetaminophen treated livers developed significant methemoglobinemia, with a peak methemoglobin level of 19.3%, compared to 2.0% for control livers (p = 0.004). The development of a model of ALF in the ex vivo setting was confounded by the development of toxic methemoglobinemia. Further attempts using alternative agents or dosing strategies may be warranted to explore this setting as a model of liver disease.
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Averkiou, Michalakis, Christina Keravnou, Maria Izamis, and Edward Leen. "Evaluation of Perfusion Quantification Methods with Ultrasound Contrast Agents in a Machine-Perfused Pig Liver." Ultraschall in der Medizin - European Journal of Ultrasound 39, no. 01 (May 3, 2016): 69–79. http://dx.doi.org/10.1055/s-0042-104645.
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Abstract Purpose To evaluate dynamic contrast-enhanced ultrasound (DCEUS) as a tool for measuring blood flow in the macro- and microcirculation of an ex-vivo machine-perfused pig liver and to confirm the ability of DCEUS to accurately detect induced flow rate changes so that it could then be used clinically for monitoring flow changes in liver tumors. Materials and Methods Bolus injections of contrast agents in the hepatic artery (HA) and portal vein (PV) were administered to 3 machine-perfused pig livers. Flow changes were induced by the pump of the machine perfusion system. The induced flow rates were of clinical relevance (150 – 400 ml/min for HA and 400 – 1400 ml/min for PV). Quantification parameters from time-intensity curves [rise time (RT), mean transit time (MTT), area under the curve (AUC) and peak intensity (PI)] were extracted in order to evaluate whether the induced flow changes were reflected in these parameters. Results A linear relationship between the image intensity and the microbubble concentration was confirmed first, while time parameters (RT and MMT) were found to be independent of concentration. The induced flow changes which propagated from the larger vessels to the parenchyma were reflected in the quantification parameters. Specifically, RT, MTT and AUC correlated with flow rate changes. Conclusion Machine-perfused pig liver is an excellent test bed for DCEUS quantification approaches for the study of the hepatic vascular networks. DCEUS quantification parameters (RT, MTT, and AUC) can measure relative flow changes of about 20 % and above in the liver vasculature. DCEUS quantification is a promising tool for real-time monitoring of the vascular network of tumors.
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Froghi, Saied, Andrew Hall, Arif Hanafi Bin Jalal, Matheus Oliveira de Andrade, Layla Mohammad Hadi, Hassan Rashidi, Pierre Gélat, Nader Saffari, Brian Davidson, and Alberto Quaglia. "Ultrasound Histotripsy on a Viable Perfused Whole Porcine Liver: Histological Aspects, Including 3D Reconstruction of the Histotripsy Site." Bioengineering 10, no. 3 (February 21, 2023): 278. http://dx.doi.org/10.3390/bioengineering10030278.
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Non-invasive therapeutic-focused ultrasound (US) can be used for the mechanical dissociation of tissue and is described as histotripsy. We have performed US histotripsy in viable perfused ex vivo porcine livers as a step in the development of a novel approach to hepatocyte cell transplantation. The histotripsy nidus was created with a 2 MHz single-element focused US transducer, producing 50 pulses of 10 ms duration, with peak positive and negative pressure values of P+ = 77.7 MPa and P− = –13.7 MPaat focus, respectively, and a duty cycle of 1%. Here, we present the histological analysis, including 3D reconstruction of histotripsy sites. Five whole porcine livers were retrieved fresh from the abattoir using human transplant retrieval and cold static preservation techniques and were then perfused using an organ preservation circuit. Whilst under perfusion, histotripsy was performed to randomly selected sites on the live. Fifteen lesional sites were formalin-fixed and paraffin-embedded. Sections were stained with Haematoxylin and Eosin and picro-Sirius red, and they were also stained for reticulin. Additionally, two lesion sites were used for 3D reconstruction. The core of the typical lesion consisted of eosinophilic material associated with reticulin loss, collagen damage including loss of birefringence to fibrous septa, and perilesional portal tracts, including large portal vein branches, but intact peri-lesional hepatic plates. The 3D reconstruction of two histotripsy sites was successful and confirmed the feasibility of this approach to investigate the effects of histotripsy on tissue in detail.
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Batchu, Sri Nagarjun, Veera Ganesh Yerra, Youan Liu, Suzanne L. Advani, Thomas Klein, and Andrew Advani. "The Dipeptidyl Peptidase-4 Inhibitor Linagliptin Directly Enhances the Contractile Recovery of Mouse Hearts at a Concentration Equivalent to that Achieved with Standard Dosing in Humans." International Journal of Molecular Sciences 21, no. 16 (August 11, 2020): 5756. http://dx.doi.org/10.3390/ijms21165756.
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Despite a similar mechanism of action underlying their glucose-lowering effects in type 2 diabetes, dipeptidyl peptidase-4 (DPP-4) inhibitors have diverse molecular structures, raising the prospect of agent-specific, glucose-independent actions. To explore the issue of possible DPP-4 inhibitor cardiac heterogeneity, we perfused different DPP-4 inhibitors to beating mouse hearts ex vivo, at concentrations equivalent to peak plasma levels achieved in humans with standard dosing. We studied male and female mice, young non-diabetic mice, and aged diabetic high fat diet-fed mice and observed that linagliptin enhanced recovery after ischemia-reperfusion, whereas sitagliptin, alogliptin, and saxagliptin did not. DPP-4 transcripts were not detected in adult mouse cardiomyocytes by RNA sequencing and the addition of linagliptin caused ≤0.2% of cardiomyocyte genes to be differentially expressed. In contrast, incubation of C166 endothelial cells with linagliptin induced cell signaling characterized by phosphorylation of Akt and endothelial nitric oxide synthase, whereas the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine increased serine 16 phosphorylation of the calcium regulatory protein, phospholamban in cardiomyocytes. Furthermore, linagliptin increased cardiomyocyte cGMP when cells were co-cultured with C166 endothelial cells, but not when cardiomyocytes were cultured alone. Thus, at a concentration comparable to that achieved in patients, linagliptin has direct effects on mouse hearts. The effects of linagliptin on cardiomyocytes are likely to be either off-target or indirect, mediated through NO generation by the adjacent cardiac endothelium.
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Harkema, S. J., G. R. Adams, and R. A. Meyer. "Acidosis has no effect on the ATP cost of contraction in cat fast- and slow-twitch skeletal muscles." American Journal of Physiology-Cell Physiology 272, no. 2 (February 1, 1997): C485—C490. http://dx.doi.org/10.1152/ajpcell.1997.272.2.c485.
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Studies of skinned fibers suggest that the rate of ATP turnover in skeletal muscle is depressed by acidosis. To examine whether this occurs in intact muscles, the ATP cost of isometric contractions was measured in ex vivo, arterially perfused cat biceps (predominantly fast-twitch) and soleus (slow-twitch) muscles under normocapnic (5% CO2) and hypercapnic (70% CO2) conditions. Hypercapnia decreased extracellular pH from 7.4 to 6.7 and intracellular pH from 7.1 to 6.5 (soleus) or 6.6 (biceps) but had no significant effect on the phosphocreatine (PCr)-to-ATP ratio in muscles at rest. The ATP cost of contraction was estimated from PCr changes, measured by gating the acquisition of 31P-nuclear magnetic resonance spectra to times before and after brief tetani (1 s at 100 Hz and 2 s at 25 Hz for biceps and soleus, respectively) or 10-s trains of twitches (2 and 1 Hz, respectively). Peak isometric force and the ATP cost of tetanic contraction (PCr/force x time integral) were not significantly different under hypercapnic compared with normocapnic conditions in either muscle (mean: 7.97 and 2.44 micromol x kg(-1) x s(-1) for biceps and soleus, respectively). Twitch force and the ATP cost per twitch decreased by nearly 50% during hypercapnic perfusion in both muscle types. The results indicate that hypercapnic acidosis has no significant effect on the ATPase rate per active myosin head in intact mammalian skeletal muscle.
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Roy, Arijit, Fatemeh Derakhshan, and Richard J. A. Wilson. "Stress peptide PACAP engages multiple signaling pathways within the carotid body to initiate excitatory responses in respiratory and sympathetic chemosensory afferents." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 304, no. 12 (June 15, 2013): R1070—R1084. http://dx.doi.org/10.1152/ajpregu.00465.2012.
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Consistent with a critical role in respiratory and autonomic stress responses, the carotid bodies are strongly excited by pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide implicated in stress responses throughout the sympathetic nervous system. PACAP excites isolated carotid body glomus cells via activation of PAC1 receptors, with one study suggesting PAC1-induced excitation is due entirely to protein kinase A (PKA)-mediated inhibition of TASK channels. However, in other systems, PAC1 is known to be coupled to multiple intracellular signaling pathways, including PKA, phospholipase C (PLC), phospholipase D (PLD), and protein kinase C (PKC), that trigger multiple downstream effectors including increased Ca2+ mobilization, inhibition of various K+ channels, and activation of nonselective cation channels. This study tests if non-PKA/TASK channel signaling helps mediate the stimulatory effects of PACAP on the carotid body. Using an ex vivo arterially perfused rat carotid body preparation, we show that PACAP-38 stimulates carotid sinus nerve activity in a biphasic manner (peak response, falling to plateau). PKA blocker H-89 only reduced the plateau response (∼41%), whereas the TASK-1-like K+ channel blocker/transient receptor potential vanilloid 1 channel agonist anandamide only inhibited the peak response (∼48%), suggesting involvement of additional pathways. The PLD blocker CAY10594 significantly inhibited both peak and plateau responses. The PLC blocker U73122 decimated both peak and plateau responses. Brefeldin A, a blocker of Epac (cAMP-activated guanine exchange factor, reported to link Gs-coupled receptors with PLC/PLD), also reduced both phases of the response, as did blocking signaling downstream of PLC/PLD with the PKC inhibitors chelerythrine chloride and GF109203X. Suggesting the involvement of non-TASK ion channels in the effects of PACAP, the A-type K+ channel blocker 4-aminopyridine, and the putative transient receptor potential channel (TRPC)/T-type calcium channel blocker SKF96365 each significantly inhibited the peak and steady-state responses. These data suggest the stimulatory effect of PACAP-38 on carotid body sensory activity is mediated through multiple signaling pathways: the PLC-PKC pathways predominates, with TRPC and/or T-type channel activation and Kv channel inactivation; only partial involvement is attributable to PKA and PLD activation.
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Kashihara, N., Y. Watanabe, H. Makino, and Y. S. Kanwar. "Interleukin-1-induced alterations in glomerular proteoglycans: biochemical and tissue autoradiographic aspects." Journal of the American Society of Nephrology 3, no. 2 (August 1992): 203–13. http://dx.doi.org/10.1681/asn.v32203.
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The effect of interleukin-1 alpha (IL-1) on the synthesis of glomerular basement membrane heparan sulfate-proteoglycan (HS-PG) was investigated. An ex vivo recirculating organ perfusion system was used. Kidneys were perfused with a medium containing (35S) sulfate and IL-1 (0.625 to 6.25 ng/mL). After radiolabeling was performed, a small cortical piece was saved for tissue autoradiography; the remaining kidney and perfusion medium were used for biochemical studies. Renal cortices were dissected out, and glomeruli were isolated; the PG were extracted and characterized. With exposure to IL-1 (5 ng/mL), an approximately twofold increase of the radioactivity in the glomerular fraction was noted. The increase was unaffected by indomethacin treatment. By Sepharose CL-6B chromatography, a single peak of radioactivity with Kav = 0.25 (macromolecular form of PG) was observed in the control group. The IL-treated group had two peaks of radioactivity with Kav = 0.25 and 0.45: the first peak contained PG, and the second peak consisted of free glycosaminoglycan (GAG) chains. Elution profiles of hydrolyzed tissue GAG chains were similar. No change in the ratio of chondroitin to heparan sulfate was observed. By DEAE-Sephacel chromatography, the glomerular PG/GAG of the IL-treated group eluted at a relatively lower salt concentration, suggesting a change in the charge density characteristics. No differences in the elution profiles of media PG/GAG were observed. (35S)methionine labeling of proteins showed no significant increase of the total radioincorporation in the IL-treated group. Immunoprecipitation studies revealed an approximately 88% increase in the de novo synthesis of PG. Tissue autoradiography revealed an approximately twofold increase of (35S) sulfate radioactivity over the glomerular mesangium, epithelium, and basement membranes. These results indicate that IL-1 enhances the synthesis of the macromolecular form of PG and the generation of free chains. Conceivably, such alterations may lead to defective macromolecular interactions among various components of the glomerular basement membrane and compromise the integrity of the ultrafiltration unit of the glomerulus.
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Libonati, Joseph R., Abdelkarim Sabri, Canhua Xiao, Scott M. MacDonnell, and Brian F. Renna. "Exercise training improves systolic function in hypertensive myocardium." Journal of Applied Physiology 111, no. 6 (December 2011): 1637–43. http://dx.doi.org/10.1152/japplphysiol.00292.2011.
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The general purpose of this study was to test the effect of exercise training on the left ventricular (LV) pressure-volume relationship (LV/PV) and apoptotic signaling markers in normotensive and hypertensive hearts. Four-month-old female normotensive Wistar-Kyoto rats (WKY; n = 37) and spontaneously hypertensive rats (SHR; n = 38) were assigned to a sedentary (WKY-SED, n = 21; SHR-SED, n = 19) or treadmill-trained (WKY-TRD, n = 16; SHR-TRD, n = 19) group (∼60% V̇o2 peak, 60 min/day, 5 days/wk, 12 wk). Ex vivo LV/PV were established in isovolumic Langendorff-perfused hearts, and LV levels of Akt, phosphorylated Akt (AktPi), Bad, phosphorylated Bad (BadPi) c-IAP, x-IAP, calcineurin, and caspases 3, 8, and 9 were measured. Heart-to-body weight ratio was increased in SHR vs. WKY ( P < 0.05), concomitant with increased calcineurin mRNA ( P < 0.05). There was a rightward shift in the LV/PV ( P < 0.05) and a reduction in systolic elastance (Es) in SHR vs. WKY. Exercise training corrected Es in SHR ( P < 0.05) but had no effect on the LV/PV in WKY. Caspase 3 was increased in SHR-SED relative to WKY-SED, while BadPi, c-IAP, and x-IAP were significantly lower in SHR relative to WKY ( P < 0.05). Exercise training increased BadPi in both WKY and SHR but did not alter caspase 9 activity in either group. While caspase 3 activity was increased with training in WKY ( P < 0.05), it was unchanged with training in SHR. We conclude that moderate levels of regular aerobic exercise attenuate systolic dysfunction early in the compensatory phase of hypertrophy, and that a differential phenotypical response to moderate-intensity exercise exists between WKY and SHR.
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Collins, Brendan J., Matthew G. Blum, Richard E. Parker, Andrew C. Chang, Kelly S. A. Blair, George L. Zorn, Brian W. Christman, and Richard N. Pierson. "Thromboxane mediates pulmonary hypertension and lung inflammation during hyperacute lung rejection." Journal of Applied Physiology 90, no. 6 (June 1, 2001): 2257–68. http://dx.doi.org/10.1152/jappl.2001.90.6.2257.
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The role of thromboxane (Tx) in hyperacute rejection of pig lung by human blood was studied in an ex vivo model, wherein lungs from juvenile piglets were perfused with fresh heparinized human blood. In this model, hyperacute lung rejection was characterized by an abrupt rise in pulmonary vascular resistance (PVR; >1 cmH2O · ml−1· min) and prolific Tx elaboration (>15 ng/ml) within 5 min and loss of function within 10 min. Although papaverine significantly blunted the rise in PVR (<0.2 cmH2O · ml−1· min), Tx production was not inhibited (>20 ng/ml), and florid tracheal edema was usually evident within 20 min. In contrast, both inhibition of Tx synthesis (Tx < 3 ng/ml) with OKY-046 and blockade of the Tx receptor with SQ-30741 (Tx > 20 ng/ml) were not only associated with significantly lower peak PVRs (<0.2 cmH2O · ml−1· min) but also with attenuated increase in lung wet-to-dry ratio and airway edema. In concert, elaboration of histamine and tumor necrosis factor was blunted, and median survival increased >10-fold to 2 h (SQ-30741) and >4 h (OKY-046). Depletion of the pig lung macrophages with dichloromethyl bisphosphonate in liposomes, but not Pall filtration of the human blood or liposomes alone, significantly inhibited Tx elaboration (<0.2 vs. >8 ng/ml for Pall filtration or liposomes) and blunted PVR elevation (<0.3 cmH2O · ml−1· min) during initial perfusion. C3a and histamine elaboration were inhibited, and median survival was significantly prolonged (>4 h). These findings implicate Tx in the inflammation associated with hyperacute lung rejection and demonstrate that pulmonary intravascular macrophages are critical to its elaboration.
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Hou, Yan, Xi Lei, Benjamin Xiaoyi Li, Xiangrong Dai, Zhongqiang Yang, Fang Qian, Guohui Zhang, et al. "The First In Vitro and In Vivo Assessment Of Anfibatide, a Novel Glycoprotein Ib Antagonist, In Mice and In a Phase I Human Clinical Trial." Blood 122, no. 21 (November 15, 2013): 577. http://dx.doi.org/10.1182/blood.v122.21.577.577.
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Abstract Background Platelet adhesion and subsequent aggregation at the site of vascular injury are critical for hemostasis and thrombosis. It has been well accepted that interaction between the GPIb complex and von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear. Platelet surface integrin αIIbβ3, through interaction with fibrinogen or other ligands, then mediates platelet aggregation to form a stable hemostatic plug or thrombus. Recently, the indispensable role of the GPIb-VWF interaction in platelet aggregation at extremely high shear (e.g. > 10,000s-1; areas of stenosis following arteriosclerosis and/or thrombus growth) has been highlighted. Therefore, both the GPIb complex and αIIbβ3 are considered major targets for antithrombotic therapies. Interestingly, although several inhibitors of αIIbβ3 have been developed for antithrombotic therapies, no drug has been developed to target the GPIb complex even though there are limitations for anti-αIIbβ3 therapies. The GPIb complex is, therefore, an attractive target for anti-thrombotic therapy. Here, we evaluated the efficacy and safety in vitro and in vivo of Anfibatide, a novel GPIb antagonist, in mice and in a phase I clinical trial. Methods Anfibatide was purified from venom of the Agkistrodon acutus snake and its purity was analyzed by mass spectrometry. The effect of Anfibatide on murine platelet function was assessed by in vitro platelet aggregometry, ex vivo perfusion chamber, and two complementary in vivo intravital microscopy models. The effects of Anfibatide on human platelet aggregation and thrombus formation were studied in vitro, and thrombealastography (TEG) was also performed. Most importantly, we evaluated the safety and efficacy of Anfibatide on platelet function and coagulation in a total of 94 healthy human volunteers in a phase I clinical trial. Results MALDI-TOF mass spectrometry of Anfibatide showed only one peak and the mass to charge ratio is 29799.7. Anfibatide specifically inhibited ristocetin-induced human platelet aggregation. Interestingly, Anfibatide was not able to inhibit botrocetin-induced murine platelet aggregation in plate-rich plasma (PRP), suggesting that its binding site may differ from other snake venom-derived GPIb antagonists. We found Anfibatide did not affect ADP-, TRAP- or collagen-induced aggregation in PRP, suggesting its specificity to GPIb. In ex vivo perfusion, Anfibatide strongly inhibited murine and human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface at both high and low shear flow conditions although it is far more sensitive at high shear. Importantly, Anfibatide effectively dissolved the preformed thrombi when we continuously perfused Anfibatide-treated whole blood through perfusion chambers, demonstrating its potential as an anti-thrombotic therapy. In the mesenteric arteriole thrombosis model, Anfibatide strongly inhibited platelet adhesion, thrombus formation, and prevented vessel occlusion in response to FeCl3 injury (P<0.05). At sites of laser-injured cremaster arterioles, Anfibatide also dramatically inhibited platelet accumulation and thrombus growth. Anfibatide did not cause significant murine platelet activation in vitro and had no significant change in coagulation parameters in TEG when we treated human whole blood with Anfibatide, suggesting it had minimal side effects. In the phase I clinical trial, results showed that Anfibatide can occupy approximately 95% of GPIb and inhibit up to 90% of ristocetin specific platelet aggregation. The inhibitory effect was undetectable four hours after Anfibatide was withdrawn. There were no serious adverse events, or deaths that occurred during the study. Anfibatide did not significantly prolong bleeding time, activated partial thromboplastin time (APTT), prothrombin time (PT), or thrombin time (TT). There was also no spontaneous bleeding or bleeding from blood collection sites. Anfibatide did not significantly affect platelet count and no anti-Anfibatide antibodies were detected in the subjects, suggesting that Anfibatide is well-tolerated in healthy individuals. Conclusion These comprehensive studies in mice and human subjects and in the first clinical trial clearly demonstrated that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy. Disclosures: Hou: Lee’s pharmaceutical holdings limited: Research Funding. Lei:Lee’s pharmaceutical holdings limited: Research Funding. Zhao:Lee’s pharmaceutical holdings limited: Research Funding. Shen:Lee’s pharmaceutical holdings limited: Research Funding. Zhou:Lee’s pharmaceutical holdings limited: Research Funding. Wang:Lee’s pharmaceutical holdings limited: Research Funding. Marshall:Lee’s pharmaceutical holdings limited: Research Funding. Ni:Lee’s pharmaceutical holdings limited: Research Funding.
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Tanguary, Hario, Gaëtan Jasmin, Gilbert Blaise, and Louis Dumont. "Resistance of the failing dystrophic hamster heart to the cardioprotective effects of diltiazem and clentiazem: evidence of coronary vascular dysfunctions." Canadian Journal of Physiology and Pharmacology 73, no. 8 (August 1, 1995): 1108–17. http://dx.doi.org/10.1139/y95-158.
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Although hypothermia and cardioplegic cardiac arrest provide effective protection during cardiac surgery, ischemia of long duration, poor preoperative myocardial function, and ventricular hypertrophy may lead to heterogeneous delivery of cardioplegic solutions, incomplete protection, and impaired postischemic recovery. Calcium antagonists are potent cardioprotective agents, but their efficacy in the presence of cold cardioplegia is still controversial, especially in heart failure, since it is often believed that failing hearts are more sensitive to their negative inotropic and chronotropic actions. However, recent data have demonstrated that the benzothiazepine-like calcium antagonists diltiazem and clentiazem, in selected dose ranges, elicit significant cardioprotection independently of intrinsic cardiodepression, thus lending support to their use in cardioprotective maneuvers involving the failing heart. We therefore evaluated the cardioprotective interaction of diltiazem, clentiazem, and cold cardioplegia in both normal and failing ischemic hearts. Hearts were excised from 200- to 225-day-old cardiomyopathic hamsters (CMHs) of the UM-X7.1 line and age-matched normal healthy controls. Ex vivo perfusion was performed at a constant pressure (140 cmH2O; 1 cmH2O = 98.1 Pa) according to the method of Langendorff. Heart rate, left ventricular developed pressure (LVDP), and coronary flow were monitored throughout the study. Global ischemia was produced for 90 min by shutting down the perfusate flow, followed by reperfusion for 30 min. Normal and failing CMH hearts were either untreated (control) or perfused at the onset of global ischemia with one of the following combinations: cold cardioplegia alone (St. Thomas' Hospital cardioplegic solution, 4 °C, infused for 2 min), cold cardioplegia + 10 nM diltiazem, or cold cardioplegia + 10 nM clentiazem. The cardiac and coronary dilator properties of 10 nM diltiazem and 10 nM clentiazem alone were investigated in separate groups of isolated preparations. Failing CMH hearts had lower basal LVDP (42 ± 2 vs. 77 ± 2 mmHg (1 mmHg = 133.3 Pa) for normal hearts, p < 0.05), while coronary flow was only slightly reduced (5.6 ± 0.2 vs. 6.2 ± 0.2 mL/min for normal hearts). Following 90 min global ischemia, coronary flow was increased in both groups, but the peak hyperemic response declined only in failing CMH hearts (+50 ± 17 vs. +82 ± 17% in normal hearts). In normal hearts, LVDP virtually recovered within 5 min of reperfusion but steadily decreased thereafter (−37 ± 4% at 30 min). In contrast, in failing CMH hearts, LVDP significantly decreased early during reperfusion but improved over time (−19 ± 7% at 30 min). In normal hearts, the addition of diltiazem or clentiazem to cold cardioplegic solutions resulted in improved postischemic contractile function for the duration of reperfusion (85 ± 4% vs. only 71 ± 6% for cardioplegia, p < 0.05). The post-ischemic increase in coronary flow was similar in all groups. In failing CMH hearts, the addition of diltiazem or clentiazem afforded no significant contractile benefit at reperfusion. In nonischemic normal hearts, infusion of diltiazem or clentiazem (10 nM) alone increased coronary flow (+6 ± 1% for diltiazem and +24 ± 3% for clentiazem) without significant negative inotropic or chronotropic effects. In nonischemic failing CMH hearts, infusion of diltiazem or clentiazem did not elicit cardiodepression. In contrast their coronary dilator actions reverted to vasoconstriction (diltiazem) or were significantly attenuated (clentiazem). From these experiments we can conclude that, compared with the normal heart, the failing CMH heart adapted differently to global ischemia. In addition to potential alterations in membrane integrity and changes in calcium handling, attenuation of the coronary dilator response to diltiazem and clentiazem rather than an increased sensitivity to their intrinsic cardiodepressant actions appears as a potential contributor to the lack of cardioprotection by these calcium antagonists in the failing CMH hearts.Key words: heart failure, cardioplegia, diltiazem, clentiazem, calcium antagonists, coronary flow, contractility.
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Nandurkar, Harshal, Warwick Nesbitt, Rose Brazilek, Francisco Tovar-Lopez, Angus Wong, Huyen Tran, Amanda Davis, and Arnan Mitchell. "A Shear Micro-Gradient Microfluidic to Monitor Platelet Aggregation Dynamics in the Context of Von Willebrand Disease." Blood 128, no. 22 (December 2, 2016): 3753. http://dx.doi.org/10.1182/blood.v128.22.3753.3753.
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Abstract Background: Virchow's triad identifies the three principle parameters driving haemostasis and thrombosis as: i. Changes in vessel wall properties and exposure of subendothelial matrix proteins; ii. Presentation of blood borne chemical activators (hypercoaguability); and iii.Blood-flow dependent mechanical factors (haemodynamics). Studies identifying a key role for micro-scale shear gradients in driving the earliest stages of platelet thrombus formation have informed the development of a novel set of microfluidic devices that have potential utility as rapid and efficient screening tools of shear dependent platelet function. Aim: The aim of this project was to undertake a small scale clinical and laboratory based characterisation study of a microfluidic platform designed by us and to assess its ability to identify differences in platelet aggregation dynamics in citrated whole blood taken from control subjects and subjects with clinically diagnosed or undiagnosed von Willebrand disease (Types 1, 2 and 3). Method: Patients with VWD were recruited from the haemophilia outpatient clinic, Alfred hospital. Whole blood samples (250mL) or samples treated ex vivo to block the canonical platelet amplification loop pathways were perfused at a defined flow rate (45ml/mL) through a set of well defined micro-shear gradient geometries pre-coated with purified VWF to initiate platelet capture. Microfluidic geometries were characterised by an entry shear rate of 1,800.s-1, that was accelerated to a peak shear rate of either 45,000.s-1 or 150,000.s-1, returning to an exit shear rate of 1,800.s-1. The rate of initial shear acceleration was varied using a series of geometries with contraction entry angles varying from 15 - 85o. Results: The microfluidic platform was able to identify patients with Types 1 (VWF antigen < 30%), 2A and 3 VWD. ROC analysis of control versus VWD samples determined that the device sensitivity approached 94.4%, with a specificity of 100% for VWD. A statistically significant difference (p< 0.05) was observed when comparing control blood samples to type 1VWD (p< 0.001) and type 2A VWD samples (p=0.004), with both subtypes showing minimal to no platelet aggregation in the device. Patients presenting with bleeding symptoms but found not to have VWD (normal VWF:Ag levels) showed no significant difference (p=0.907) to controls. Furthermore, exogenous titration of Type 3 (n=2) VWD blood samples with purified VWF (10 - 100mg/mL) recapitulated platelet aggregation in a concentration dependent manner. Head to head comparison with standard laboratory based tests including, VWF:Ag, VWF:CB and VWF:RCo demonstrated a strong linear correlation with device output. In addition, time-course (0, 2 and 4 Hrs) trials demonstrate that the device is a sensitive measure of DDAVP treatment of VWD. Conclusion: The Studies presented demonstrate that haemodynamically sensitive platelet aggregation within our prototype platform is critically dependent on blood VWF antigen levels and demonstrates good proof-of-concept that the microfludic can selectively identify VWD dependent defects in whole blood platelet aggregation. Taken together these data suggest that a microfluidic platform with discrete haemodynamic control can operate as a sensitive screen for VWD. Future studies will focus on defining the particle and key haemodynamic parameters that shift both device selectivity and sensitivity. Disclosures No relevant conflicts of interest to declare.
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Ono, Yukako, Yumiko Matsubara, Tatsuya Tanaka, Shinichiro Okamoto, Yasuo Ikeda, and Mitsuru Murata. "Pre-Adipocytes Differentiate Into Megakaryocytes (MK) by a Paracrine Thrombopoietin (TPO) Loop." Blood 120, no. 21 (November 16, 2012): 4742. http://dx.doi.org/10.1182/blood.v120.21.4742.4742.
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Abstract Abstract 4742 The regulation of megakaryopoiesis and thrombopoiesis are incompletely understood. Better understanding of the underlying mechanisms would be of biological import, but may also lead to novel approaches for generating of platelets for clinical application. One cell line that undergoes terminal differentiation into megakaryocytes is pre-adipocytes. These cells represent a potential candidate cell for ex vivo generation of megakaryocytes. Here we demonstrate that pre-adipocytes differentiate into MKs and platelets by using an endogenous paracrine loop involving TPO, the primary cytokine involved in megakaryopoiesis, and its receptor c-mpl. We previously reported that pre-adipocytes differentiate into MKs and form platelets when the culture medium is switched from maintenance medium to MK lineage induction (MKLI) medium. Neither media include TPO. Based on these observations, the present study tested the hypothesis that pre-adipocytes might differentiate into MKs and platelets by endogenous TPO and c-mpl expression of sufficient magnitude to drive megakaryopoiesis. We used primary mouse pre-adipocytes (CD45-, CD117-, Sca1+, CD29+, CD73+, CD90+, CD105+, CD106+) from subcutaneous adipose tissues and also the mouse pre-adipocyte line 3T3-L1 cells. The TPO levels, as assessed by ELISA, in the supernatant from 106 pre-adipocytes cultured in 2 ml MKLI medium without exogenous TPO (TPO-) were unmeasurable level on Day 0, 29±14 pg/ml on Day 7 and 8±2 pg/ml on Day 12. Similar results were obtained in the supernatant from 3T3-L1 during MK differentiation. We did not observe measureable TPO in supernatants from pre-adipocytes and 3T3-L1 cells cultured in maintenance medium on Days 0, 7 and 12. We then compared MK differentiation from pre-adipocytes in MKLI media in the absence (TPO-) or addition (final concentration, 50 ng/ml; TPO+) of TPO. The frequency of CD41-postive pre-adipocytes in culture after 6 days was 58±11% for TPO+ and 70±7% for TPO- (p>0.05), consistent with endogenous TPO being sufficient under these circumstances to stimulate MK differentiation of pre-adipocytes. DNA ploidy and c-mpl expression assessed by immunohistochemistry were also similar with or without added exogenous TPO. We next examined the number of CD41-expressing large-sized (>15 μm) cells beginning with 1.2 × 104 preadipocytes in the absence or presence of the c-mpl inhibitor rat anti-mouse c-mpl inhibitor AMM2 (final concentration, 10 μg/ml). In the absence of AMM2, 10-fold more CD41-positive, large cells were seen (6.8±1.7×103) than in its presence (0.6±0.2×103, p<0.05). These observations support that pre-adipocytes differentiate into MKs using endogenous TPO stimulation via c-mpl. We next examined the ability to release platelets from these treated pre-adipocytes in each experimental condition (pre-adipocytes and 3T3-L1 cells, each ± exogenous TPO). Infusion of 2 × 105 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled MKs into irradiated thrombocytopenic mice (7 days post-2.0Gy exposure) led to a time-dependent appearance of CFSE+/CD41+ platelet-sized particles with a peak at 4 hours after the infusion reaching ∼2.5% of the total circulating platelet population (∼30 platelets/infused MK) under all tested experimental conditions. For platelet function, blood samples from each of these mice were perfused over a collagen-coated chip for 10 minutes, and the incorporation of CFSE+ particles into thrombi on the chip was determined (Total Thrombus-formation Analyzing System). A similar degree of platelet incorporation was observed under all experimental conditions and each was with an efficiency similar to that seen when CFSE+ control platelets were infused. These findings demonstrate that pre-adipocytes differentiate into MKs and subsequent platelets by an endogenous TPO release and a paracrine loop involving c-mpl. We propose that such pre-adipocytes could be used as a model of a continuously replicating cell line that upon switching media simultaneous expresses TPO and its receptor c-mpl to establish a paracrine loop leading to terminal differentiation into functional MKs. The basis of why this media switch induces this change needs to be clarified to further develop this pre-adipocyte strategy as a donor-independent source for platelet transfusion as well as for studying mechanism underlying megakaryopoiesis and thrombopoiesis. Disclosures: Matsubara: Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.
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Zimmer, Maximilian, Karl Herbert Hillebrandt, Nathalie Nora Roschke, Steffen Lippert, Oliver Klein, Grit Nebrich, Joseph Maria George Vernon Gassner, et al. "Distinctive protein expression in elderly livers in a Sprague–Dawley rat model of normothermic ex vivo liver machine perfusion." European Journal of Medical Research 29, no. 1 (July 11, 2024). http://dx.doi.org/10.1186/s40001-024-01961-x.
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Abstract Background Liver grafts are frequently declined due to high donor age or age mismatch with the recipient. To improve the outcome of marginal grafts, we aimed to characterize the performance of elderly vs. young liver grafts in a standardized rat model of normothermic ex vivo liver machine perfusion (NMP). Methods Livers from Sprague–Dawley rats aged 3 or 12 months were procured and perfused for 6 h using a rat NMP system or collected as a reference group (n = 6/group). Tissue, bile, and perfusate samples were used for biochemical, and proteomic analyses. Results All livers cleared lactate during perfusion and continued to produce bile after 6 h of perfusion (614 mg/h). Peak urea levels in 12-month-old animals were higher than in younger animals. Arterial and portal venous pressure, bile production and pH did not differ between groups. Proteomic analysis identified a total of 1477 proteins with oxidoreductase and catalytic activity dominating the gene ontology analysis. Proteins such as aldehyde dehydrogenase 1A1 and 2-Hydroxyacid oxidase 2 were significantly more present in livers of older age. Conclusions Young and elderly liver grafts exhibited similar viability during NMP, though proteomic analyses indicated that older grafts are less resilient to oxidative stress. Our study is limited by the elderly animal age, which corresponds to mature but not elderly human age typically seen in marginal human livers. Nevertheless, reducing oxidative stress could be a promising therapeutic target in the future.
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Zhou, Yirong, Junyoung Kim, Guillermo Tearney, and Aaron Aguirre. "Abstract 4139162: Optical coherence tomography measures mechanical contractile properties of the beating murine heart." Circulation 150, Suppl_1 (November 12, 2024). http://dx.doi.org/10.1161/circ.150.suppl_1.4139162.
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Introduction: Optical imaging techniques can characterize properties of tissue at high temporal and spatial resolution, offering unique insights into electrically excitable tissues such as nerve and muscle. We hypothesized that the gated high-speed optical coherence tomography (OCT) could profile the contractile properties of mouse myocardial tissue and facilitate investigations of microscale mechanical function in the beating heart. Methods: C57BL/6 male mice (n=6) underwent a sternotomy while anesthetized and ventilated and were imaged at baseline with OCT. Another cohort of C57BL/6 male mice (n=6) were sacrificed and the hearts were ex vivo perfused with crystalloid using a Langendorff system. These hearts were imaged at baseline and after inhibition of contractile function with blebbistatin. OCT was performed using a custom tissue stabilizer and a Thorlabs TEL 321 OCT system, which was synchronized to physiologic signals using custom hardware and software and cardiac pacing protocols (Fig. 1A and 1B). OCT cross-sectional B-scan images (Fig. 1C) were obtained throughout multiple cardiac cycles in both in vivo and ex vivo preparations. Data were processed in MATLAB and ImageJ using the workflow in Fig. 1D. Results: Highly reproducible cross-sectional mean intensity changes were observed over the contractile cycle in vivo and ex vivo (Fig. 1E, left and middle). With mechanical contraction inhibited by blebbistatin, this cyclical intensity extinguished, linking the optical scattering change measured by OCT to myocardial contraction (Fig. 1E, right). B-scan curves averaged over multiple mice and temporally aligned with the ECG waveform (Fig. 1F) demonstrate a signal intensity waveform that reproducibly varies with the contractile cycle for both in vivo and ex vivo preparations. In vivo and ex vivo baseline waveforms both had peak intensity during systole and minimum in end-diastole (Fig. 1F). Reproducibility in ex vivo beating heart preparations without blood perfusion provides evidence that myocardial mechanical properties, not red blood cell flow, generate the intensity changes. Conclusion: Gated high-speed OCT imaging can quantify the mechanical contraction wave in the myocardium. Combined with gated OCT angiography, OCT techniques will provide high resolution imaging of both blood flow and mechanical contractility of the beating heart in small animal models of heart disease.
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El-Kurdi, Mohammed S., Jeffrey S. Vipperman, and David A. Vorp. "Control of Circumferential Wall Stress and Luminal Shear Stress Within Intact Vascular Segments Perfused Ex Vivo." Journal of Biomechanical Engineering 130, no. 5 (July 10, 2008). http://dx.doi.org/10.1115/1.2948419.
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Proportional, integral, and derivative (PID) controllers have proven to be robust in controlling many applications, and remain the most widely used control system architecture. The purpose of this work was to use this architecture for designing and tuning two PID controllers. The first was used to control the physiologic arterial circumferential wall stress (CWS) and the second to control the physiologic arterial shear stress (SS) imposed on intact vascular segments that were implanted into an ex vivo vascular perfusion system (EVPS). In order to most accurately control the stresses imposed onto vascular segments perfused ex vivo, analytical models were derived to calculate the CWS and SS. The mid-vein-wall CWS was calculated using the classical Lamé solution for thick-walled cylinders in combination with the intraluminal pressure and outer diameter measurements. Similarly, the SS was calculated using the Hagen–Poiseuille equation in combination with the flow rate and outer diameter measurements. Performance of each controller was assessed by calculating the root mean square of the error (RMSE) between the desired and measured process variables. The performance experiments were repeated ten times (N=10) and an average RMSE was reported for each controller. RMSE standard deviations were calculated to demonstrate the reproducibility of the results. Sterile methods were utilized for making blood gas and temperature measurements in order to maintain physiologic levels within the EVPS. Physiologic blood gases (pH, pO2, and pCO2) and temperature within the EVPS were very stable and controlled manually. Blood gas and temperature levels were recorded hourly for several (N=9)24h perfusion experiments. RMSE values for CWS control (0.427±0.027KPa) indicated that the system was able to generate a physiologic CWS wave form within 0.5% error of the peak desired CWS over each cardiac cycle. RMSE values for SS control (0.005±0.0007dynes∕cm2) indicated that the system was able to generate a physiologic SS wave form within 0.3% error of the peak desired SS over each cardiac cycle. Physiologic pH, pO2, pCO2, and temperature levels were precisely maintained within the EVPS. The built-in capabilities and overall performance of the EVPS described in this study provide us with a novel tool for measuring molecular responses of intact vascular segments exposed to precisely simulated arterial biomechanical conditions.
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Buttar, Sana N., Hasse Møller-Sørensen, Michael Perch, Hannelouise Kissow, Thomas N. B. Lilleør, Rene H. Petersen, and Christian H. Møller. "Porcine lungs perfused with three different flows using the 8-h open-atrium cellular ex vivo lung perfusion technique." Frontiers in Bioengineering and Biotechnology 12 (June 25, 2024). http://dx.doi.org/10.3389/fbioe.2024.1357182.
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The number of lung transplantations is limited due to the shortage of donor lungs fulfilling the standard criteria. The ex vivo lung perfusion (EVLP) technique provides the ability of re-evaluating and potentially improving and treating marginal donor lungs. Accordingly, the technique has emerged as an essential tool to increase the much-needed donor lung pool. One of the major EVLP protocols, the Lund protocol, characterized by high pulmonary artery flow (100% of cardiac output [CO]), an open atrium, and a cellular perfusate, has demonstrated encouraging short-EVLP duration results. However, the potential of the longer EVLP duration of the protocol is yet to be investigated, a duration which is considered necessary to rescue more marginal donor lungs in future. This study aimed to achieve stable 8-h EVLP using an open-atrium cellular model with three different pulmonary artery flows in addition to determining the most optimal flow in terms of best lung performance, including lung electrolytes and least lung edema formation, perfusate and tissue inflammation, and histopathological changes, using the porcine model. EVLP was performed using a flow of either 40% (n = 6), 80% (n = 6), or 100% (n = 6) of CO. No flow rate demonstrated stable 8-h EVLP. Stable 2-h EVLP was observed in all three groups. Insignificant deterioration was observed in dynamic compliance, peak airway pressure, and oxygenation between the groups. Pulmonary vascular resistance increased significantly in the 40% group (p < .05). Electrolytes demonstrated an insignificant worsening trend with longer EVLP. Interleukin-8 (IL-8) in perfusate and tissue, wet-to-dry weight ratio, and histopathological changes after EVLP were insignificantly time dependent between the groups. This study demonstrated that stable 8-h EVLP was not feasible in an open-atrium cellular model regardless of the flow of 40%, 80%, or 100% of CO. No flow was superior in terms of lung performance, lung electrolytes changes, least lung edema formation, minimal IL-8 expression in perfusate and tissue, and histopathological changes.
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Fogg, Ryan W., Mina P. Ghatas, Brendan McCormack, Michael Shields, Ashley N. Matthew, Gabrielle Grob, Nat Araia, Linda Burkett, John E. Speich, and Adam P. Klausner. "Increased Afferent Nerve Firing Is Correlated With the Detection of Bladder Wall Micromotion in a Perfused Ex‐Vivo Porcine Model." Neurourology and Urodynamics, January 13, 2025. https://doi.org/10.1002/nau.25661.
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ABSTRACTIntroduction and ObjectiveObservable autonomous rhythmic changes in intravesical pressure, termed bladder wall micromotion, is a phenomenon that has been linked to urinary urgency, the key symptom in overactive bladder (OAB). However, the mechanism through which micromotion drives urinary urgency is poorly understood. In addition, micromotion is inherently difficult to study in human urodynamics due to challenges distinguishing it from normal cyclic physiologic processes such as pulse rate, breathing, rectal contractions, and ureteral jetting. Therefore, the goal of this study was to create a reproducible model of micromotion using an ex‐vivo perfused porcine bladder, as well as to describe the relationship between micromotion and afferent nerve signaling.MethodsPorcine bladders were reanimated using ex‐vivo perfusion with a physiologic buffer. The pelvic nerve adjacent to the bladder was dissected, grasped with micro‐hook electrodes and electroneurogram (ENG) signals were recorded at 20 kHz. Bladders were catheterized and intravesical pressure measurements were taken using a Laborie XT Urodynamics system. Bladders were filled to a fixed volume of 300 mL and control measurements were recorded. The bladders were then washed with 0.001 M carbachol (CCh) solution and refilled to 300 mL to induce micromotion, which was detected as rhythmic changes in intravesical pressure. ENG amplitude was calculated in μV, and nerve firing rate was calculated as number of spikes above baseline threshold per minute.ResultsMicromotion was induced by carbachol in 12/25 (48.4%) of trials as rhythmic changes in intravesical pressure after the instillation of carbachol but not in any control period. A fast Fourier transform (FFT) algorithm showed average peak dominant frequency component amplitude was significantly higher during the carbachol period when compared to the control period (0.47 vs. 0.01 cm‐H2O, p < 0.0001). Peak waveform frequency (1.13 vs. 1.54 cycles/min, p > 0.05) did not differ between control and carbachol periods. With regard to afferent nerve signaling, normalized average amplitude (0.66 ± 0.24 vs. 0.05 ± 0.08 μV) and firing rate (0.68 ± 0.28 vs. 0.18 ± 0.22 spike/min) for all bladders was significantly greater in the carbachol period when compared to the control period (p < 0.001).ConclusionsMicromotion can be induced using instillation of carbachol in a perfused ex‐vivo porcine bladder. Increased afferent nerve firing is observed during periods of micromotion. Thus, micromotion may drive afferent nerve signaling and may potentially contribute to urinary urgency, detrusor overactivity, and OAB. The development of an experimental ex‐vivo porcine model for micromotion provides a reproducible method to study bladder micromotion and its potential role in the pathophysiology of urinary urgency and voiding dysfunction.
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King, D. Ryan, Rachel L. Padget, Justin Perry, Gregory Hoeker, James W. Smyth, David A. Brown, and Steven Poelzing. "Elevated perfusate [Na+] increases contractile dysfunction during ischemia and reperfusion." Scientific Reports 10, no. 1 (October 14, 2020). http://dx.doi.org/10.1038/s41598-020-74069-x.
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Abstract Recent studies revealed that relatively small changes in perfusate sodium ([Na+]o) composition significantly affect cardiac electrical conduction and stability in contraction arrested ex vivo Langendorff heart preparations before and during simulated ischemia. Additionally, [Na+]o modulates cardiomyocyte contractility via a sodium-calcium exchanger (NCX) mediated pathway. It remains unknown, however, whether modest changes to [Na+]o that promote electrophysiologic stability similarly improve mechanical function during baseline and ischemia–reperfusion conditions. The purpose of this study was to quantify cardiac mechanical function during ischemia–reperfusion with perfusates containing 145 or 155 mM Na+ in Langendorff perfused isolated rat heart preparations. Relative to 145 mM Na+, perfusion with 155 mM [Na+]o decreased the amplitude of left-ventricular developed pressure (LVDP) at baseline and accelerated the onset of ischemic contracture. Inhibiting NCX with SEA0400 abolished LVDP depression caused by increasing [Na+]o at baseline and reduced the time to peak ischemic contracture. Ischemia–reperfusion decreased LVDP in all hearts with return of intrinsic activity, and reperfusion with 155 mM [Na+]o further depressed mechanical function. In summary, elevating [Na+]o by as little as 10 mM can significantly modulate mechanical function under baseline conditions, as well as during ischemia and reperfusion. Importantly, clinical use of Normal Saline, which contains 155 mM [Na+]o, with cardiac ischemia may require further investigation.
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Rassam, Rassam, Emily Mihalko, Shahid M. Nimjee, and Susan M. Shea. "Abstract TMP116: A Novel Microfluidic Model Of Ischemic Stroke Recapitulating Arterial Occlusion And Prolonged Retraction." Stroke 54, Suppl_1 (February 2023). http://dx.doi.org/10.1161/str.54.suppl_1.tmp116.
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To our knowledge, an ex vivo model of thrombotic occlusion that (1) mimics arterial setting, (2) allows true occlusion, and (3) drug delivery without disturbing the thrombus does not exist. A platform with these capabilities would allow for high-throughput mechanistic study of thrombosis and thrombolysis. Here we present a microfluidic system that achieves these goals, providing a biofidelic ex vivo platform to study ischemic stroke. A mold with an upstream Y and a region of stenosis was fabricated and used to manufacture microfluidic chambers. Channels were coated with collagen at the stenosis. Heparinized blood was collected from healthy human donors (N=3) and platelets were labelled with fluorescent anti-CD41. Samples were perfused at 10000/s and outflow mass collected on a scale. Occlusion was indicated by mass plateau, and thrombi were left to retract in situ for 0 or 6 hrs. Kinetic immunofluorescent images were captured and used to derive surface area and the image profile by averaging MFI in each pixel column. Sample from the same donor dosed with 0.7 or 7nM alteplase (ALT) was perfused using the Y inlet using a constant pressure head (1000/s; mimicking middle cerebral artery) for 120 min. After 6 hours of post-occlusion retraction, thrombi substantially remodeled. The MFI curves (Fig 1) demonstrate even distribution of MFI post-occlusion that shifts to a central peak after 6 hr. Dosed sample from the same donor was able to be delivered upstream of the thrombus without disruption. Immediate reperfusion resulted in a reduction in thrombus surface area of (mean (SD)) 2% (10%) with 0.7nM ALT, 23% (7%) with 7nM, and 15% (1%) with vehicle. Reperfusion after 6 hrs resulted in a reduction of 14% (14%) with 0.7nM, 45% (12%) with 7nM, and 17% (8%) with vehicle. We have established an ex vivo high throughput system capable of recapitulating arterial occlusion, thrombus retraction in situ , drug delivery, and thrombolysis. This system can be used for mechanistic inquiry and drug discovery.
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Law, Michelle L., and Joseph M. Metzger. "Cardiac myocyte intrinsic contractility and calcium handling deficits underlie heart organ dysfunction in murine cancer cachexia." Scientific Reports 11, no. 1 (December 2021). http://dx.doi.org/10.1038/s41598-021-02688-z.
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AbstractCachexia is a muscle wasting syndrome occurring in many advanced cancer patients. Cachexia significantly increases cancer morbidity and mortality. Cardiac atrophy and contractility deficits have been observed in patients and in animal models with cancer cachexia, which may contribute to cachexia pathophysiology. However, underlying contributors to decreased in vivo cardiac contractility are not well understood. In this study, we sought to distinguish heart-intrinsic changes from systemic factors contributing to cachexia-associated cardiac dysfunction. We hypothesized that isolated heart and cardiac myocyte functional deficits underlie in vivo contractile dysfunction. To test this hypothesis, isolated heart and cardiac myocyte function was measured in the colon-26 adenocarcinoma murine model of cachexia. Ex vivo perfused hearts from cachectic animals exhibited marked contraction and relaxation deficits during basal and pacing conditions. Isolated myocytes displayed significantly decreased peak contraction and relaxation rates, which was accompanied by decreased peak calcium and decay rates. This study uncovers significant organ and cellular-level functional deficits in cachectic hearts outside of the catabolic in vivo environment, which is explained in part by impaired calcium cycling. These data provide insight into physiological mechanisms of cardiomyopathy in cachexia, which is critical for the ultimate development of effective treatments for patients.
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Hamed, M. O., A. D. Barlow, N. Dolezalova, S. Khosla, A. Sagar, F. M. Gribble, S. Davies, et al. "Ex vivo normothermic perfusion of isolated segmental porcine bowel: a novel functional model of the small intestine." BJS Open 5, no. 2 (March 2021). http://dx.doi.org/10.1093/bjsopen/zrab009.
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Abstract Background There is an unmet need for suitable ex vivo large animal models in experimental gastroenterology and intestinal transplantation. This study details a reliable and effective technique for ex vivo normothermic perfusion (EVNP) of segmental porcine small intestine. Methods Segments of small intestine, 1.5–3.0 m in length, were retrieved from terminally anaesthetized pigs. After a period of cold ischaemia, EVNP was performed for 2 h at 37°C with a mean pressure of 80 mmHg using oxygenated autologous blood diluted with Ringer’s solution. The duration of EVNP was extended to 4 h for a second set of experiments in which two segments of proximal to mid-ileum (1.5–3.0 m) were retrieved from each animal and reperfused with whole blood (control) or leucocyte-depleted blood to examine the impact of leucocyte depletion on reperfusion injury. Results After a mean cold ischaemia time of 5 h and 20 min, EVNP was performed in an initial group of four pigs. In the second set of experiments, five pigs were used in each group. In all experiments bowel segments were well perfused and exhibited peristalsis during EVNP. Venous glucose levels significantly increased following luminal glucose stimulation (mean(s.e.m.) basal level 1.8(0.6) mmol/l versus peak 15.5(5.8) mmol/l; P < 0.001) and glucagon-like peptide 1 (GLP-1) levels increased in all experiments, demonstrating intact absorptive and secretory intestinal functions. There were no significant differences between control and leucocyte-depleted animals regarding blood flow, venous glucose, GLP-1 levels or histopathology at the end of 4 h of EVNP. Conclusions This novel model is suitable for the investigation of gastrointestinal physiology, pathology and ischaemia reperfusion injury, along with evaluation of potential therapeutic interventions.
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Shah, Sushrut, Marilen Federico, Yuexing Yuan, Alexis Shatzman, Shey-Shing Sheu, and Deepak Deshpande. "Abstract 4141674: Inhibiting Detrimental Cardiac Functions Using Biased Allosteric Modulators Of β 2 -Adrenergic Receptor Modulating The Canonical Signaling." Circulation 150, Suppl_1 (November 12, 2024). http://dx.doi.org/10.1161/circ.150.suppl_1.4141674.
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BACKGROUND: Cardiac diseases such as heart failure (HF) are characterized by irregular cardiac contraction and relaxation due to repeated damage to the myocardium. β-adrenergic receptors (βAR) on cardiomyocytes (CMs) are predominant regulators of cardiac contractility and hence β-blockers are utilized in the treatment of HF. Biasing βAR-mediated signaling in CMs presents an opportunity for developing alternative therapies. We have recently discovered a new allosteric modulators (AMs) that preferentially diminish βAR-Gαs signaling, not affecting β-arrestin mediated signaling. Considering the functional significance of βAR-mediated Gαs- and β-arrestin signaling in CMs, our objective is to investigate the impact of our recently identified β 2 AR AMs on CM contractility and heart function. We hypothesize that Negative AMs (NAMs) of β 2 AR will inhibit the βAR-Gαs signaling induced by β-agonists thereby decreasing cardiac contractility in vitro and in vivo. METHODS: In isolated adult ventricular murine CMs (AVCM), we assessed the effect of NAM on isoproterenol-induced contractility and calcium transients using the IonOptix System. A subset of cells were pre-treated with β 1 AR-antagonist. To examine the effect in the whole heart, we treated perfused hearts in Langendorff Apparatus with ISO (100nM) and vehicle/NAM. Finally, to assess effects of AMs on heart functions in vivo , we performed electrocardiogram (ECG) and echocardiogram in mice treated with ISO. RESULTS: In AVCMs, ISO augmented CM contractility and calcium. Concomitant NAM treatment significantly attenuated ISO-induced cell contractility, elevation of calcium peak and decreased time-to-relaxation. Interestingly, pretreatment of CMs with β 1 AR-antagonist reversed the inhibitory effect of NAM on ISO-mediated contractility. Our ex vivo perfused heart experiments demonstrated an inhibition of ISO-induced cardiac contractility by the β 2 AR NAM. ECG studies demonstrated an attenuation of ISO-induced increase in parameters including heart rate and PVCs. Echocardiogram recording confirmed the decrease in heart rate and revealed alleviation of ISO-induced reduction in ejection fraction and fractional shortening and inhibition of ISO-induced isovolumic contraction time and myocardial performance index. CONCLUSIONS: These data collectively demonstrate that our recently discovered biased NAM could preferentially ameliorate cardiotoxic Gαs signaling and function in CMs.
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Dengu, Fungai. "Ischaemia reperfusion induces the release of donor derived Passenger Leukocytes (PLs) during normothermic machine perfusion (NMP) of the liver- a new opportunity for ex situ graft leukodepletion?" Journal of the Nuffield Department of Surgical Sciences 2, no. 4 (October 12, 2021). http://dx.doi.org/10.37707/jnds.v2i4.202.
Full textAbstract:
Fungai Dengu1, Tamsyn Clark1,3, Hussain Abbas1, Etohan Ann Ogbemudia1, Faysal El Gilani1,David Nasralla1, Peter Friend1, James Fildes2
1. Oxford Organ Perfusion Lab, Nuffield Department of Surgical Sciences and Oxford Biomedical ResearchCentre, University of Oxford, Oxford, UK2. The Ex-Vivo Lab, Division of Cell Matrix Biology and Regenerative Medicine, School of BiologicalSciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester AcademicHealth Science Centre, Manchester, UK3. Institute of Biomedical Engineering, University of Oxford, Oxford, UK
Background Passenger Leukocytes (PLs) are implicated in both the direct and semi-direct pathways of allorecognition which is the process that underpins acute allograft rejection1. The majority of liver-derived PLs are short lived and predominantly impact early recipient immune responses2. Removal of PLs has been shown in kidney, lung and vascularised composite allografts to reduce early allograft damage and abrogate ejection3. We aimed to assess the use normothermic machine perfusion (NMP) to investigate PL kinetics and explore PL depletion strategies in donor livers.
Methods Porcine livers (N=4) procured in a donation after circulatory death (DCD) model were preserved with sequential static cold storage then NMP. During NMP, livers were subjected to repeated 20 min warm ischaemic hits (IH) followed by 30mins of NMP using a leukocyte depleted autologous RBC based perfusate. Leukocytes were quantified using the Sysmex® cell counter system and samples stored for flow cytometric analysis.
Results In total, 3.4x106 PLs are effluxed into the circuit immediately after initiation of NMP, this falls rapidly to 1.35x106 by 30 mins. Following the first IH, a further efflux of occurs with a peak of 3.74x106 occurring. The second IH also induced an efflux of cells (1.61x106) with lymphocytes representing the predominant leukocyte sub-type in each efflux.
Discussion During NMP, there is an inducible and reproducible efflux of graft derived PLs into the circuit that is composed of predominantly lymphocytes with unexpectedly low numbers of monocytes. Removal of these PLs from the perfusate during NMP may therefore be feasible using an in-line leukocyte-filter.
References
1. Alsughayyir, J., Motallebzadeh, R. & Pettigrew, G. J. Are donor lymphocytes a barrier to transplantation tolerance? Curr. Opin. Organ Transplant. 23, 90–96 (2018).2. Mastoridis, S. et al. Impact of donor extracellular vesicle release on recipient cell “cross-dressing” following clinical liver and kidney transplantation. Am. J. Transplant. ajt.16123 (2020). doi:10.1111/ajt.161233. Stone, J. P. et al. Mechanical removal of dendritic cell–generating non-classical monocytes via ex vivo lung perfusion. J. Hear. Lung Transplant. 33, 864–869 (2014).
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Angelini, Marina, Arash Pezhouman, Nicoletta Savalli, Marvin G. Chang, Federica Steccanella, Kyle Scranton, Guillaume Calmettes, et al. "Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current." Journal of General Physiology 153, no. 12 (October 26, 2021). http://dx.doi.org/10.1085/jgp.202012584.
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Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation–contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.
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Masse, S., R. D. Anderson, H. Gonna, M. A. Azam, P. F. H. Lai, D. C. Deno, and K. Nanthakumar. "Multi-modal physiological parameter for mapping ablation lesions." Europace 25, Supplement_1 (May 24, 2023). http://dx.doi.org/10.1093/europace/euad122.752.
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Abstract Funding Acknowledgements Type of funding sources: None. Background In addition to variety of force time indices, electrophysiologist use peak to peak voltage (Vpp) and or S wave on unipolar electrogram to assess RF lesions they deliver. This strategy ignores cardiac conduction speed, direction, and wave curvature, all of which are affected by delivered lesion. Electrode array catheters such as the Advisor HD Grid allow individual cardiac wavefronts from sites to be characterized simultaneously in two dimensions from which vectorcardiograms can be derived. This underappreciated attribute of electrograms when integrated into activation mapping may provide new insights into the electrophysiologic state of tissue including wavefront amplitude, direction and velocity. Objective We tested the hypothesis that intracardiac vectorcardiogram loops contain aspects of properties that may better detect lesions compared to Vpp reduction and that a novel multi-modal approach will provide additional insights on wavefront interaction with substrate. Methods In 5 ex-vivo Langendorff perfused swine hearts, 56-pole (7 x 8 configuration) electrode arrays were sutured to the anterior epicardial left ventricle. Unipolar electrograms were acquired with UHN mapping system during programmed stimulation from all cardinal directions before and after creating RF ablation lesions. Vector loop maps were derived from orthogonal bipole pairs to study wavefront organization and voltage. Isochrone maps were generated from unipolar local activation times. Vector loop eccentricity was defined for each 4-electrode group as a function of loop’s major and minor axis lengths. Low values denote less eccentricity (more circular). Results An illustrative example of vector, loop and isochrone maps before and after ablation is shown in figures 1 and 2. Pre-ablation conditions show activation from a point stimulation on the left side with slender vector loops indicative of a relatively homogeneous linear conduction. In contrast, post-ablation vector loops are much less eccentric near the ablated area and smaller reflecting attenuated amplitude. Interestingly, near border zone locations large round loops are observed, suggesting curved or nonlinear wave propagation. Figure 2 shows that vector loops recorded from scar areas were diminished in size following ablation with a mean Vpp of 3.76 vs 2.18mV (p=0.0001) and exhibited less mean eccentricity, 0.925 vs 0.848 (p<0.0001), with more round loops (eccentricity < 0.7) found after ablation (12% of sites vs 4% pre-ablation, p < 0.0001). Conclusions Significant changes in loop characteristics were observed even when ablation induced voltage attenuation was minimal. This suggests that vectorial loop eccentricity may provide information in addition to omnipolar Vpp. Wavefront propagation disruption near ablation border zones is thus potentially valuable to detect lesion gaps. This information along with wave amplitude and direction can now be integrated into a novel multi-modal map.
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Crompton, Michael, Judy J. Watson, Elizabeth Colby, Wilmelenne Clapper, Celia P. Jenkinson, Bruce Hendry, Radko Komers, et al. "#457 Sparsentan has direct effects on the glomerular capillary wall to attenuate increased permeability after exposure to nephrotic syndrome plasma." Nephrology Dialysis Transplantation 39, Supplement_1 (May 2024). http://dx.doi.org/10.1093/ndt/gfae069.015.
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Abstract Background and Aims The glomerular endothelial glycocalyx (eGlx), a luminal layer of proteoglycans, glycoproteins and glycolipids, forms the first part of the glomerular filtration barrier (GFB). Nephrotic syndrome (NS) describes a group of pathologies of the renal glomerulus that result in proteinuria and is associated with glomerular endothelial dysfunction. Current treatments are broad and non-specific. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist that has received accelerated approval in the United States for the reduction of proteinuria in adults with IgA nephropathy at high risk of disease progression. Our validated glomerular permeability assay directly measures the albumin permeability (Ps’alb)of capillary loops within individually trapped glomeruli [1]. This ex vivo assay is independent of haemodynamic factors and tubular albumin handling – factors known to affect urine protein concentrations. This work examines whether sparsentan could reduce glomerular albumin permeability in NS, by preserving the eGlx to maintain the GFB. Method Human NS plasma samples were collected under ethical consent from 3 patients that had undergone plasma exchange from periods when they were in relapse (RL), or subsequent remission (RM). Adult male Sprague Dawley rats (175–200 g) were perfused with 4% Ringer BSA solution. Glomeruli were isolated on ice by graded sieving from the cortex of each kidney. Glomeruli were incubated, in the presence of AF488-conjugated BSA (AF488-BSA, 30 μg/ml) and R18 (36.5 μg/ml), with 10% plasma from NS patients for 1 hour, and simultaneously treated with sparsentan (0.1 μM, 1 μM and 10 μM) or vehicle. The glomerular Ps’alb assay was used to measure changes in albumin permeability. We applied a fluorescence profile peak-to-peak confocal imaging technique to treated glomeruli to assess glomerular eGlx thickness [2]. Results Human NS patients in RL had a significantly greater proteinuria compared to RM (RL, 10, 000 ± 1, 354 mg/g; RM, 95.3 ± 59.7 mg/g, P = 0.017). Incubation of rat glomeruli with RL plasma induced a significant increase in Ps’alb (RM+vehicle, 6.0 × 10−7 ± 0.12 × 10−7 cm/s; RL+vehicle, 10.9 × 10−7 ± 0.84 × 10−7 cm/s, P < .001) with a significant reduction in glomerular eGlx thickness (RM+vehicle, 210 ± 21.2 nm; RL+vehicle, 109 ± 7.9 nm, P = .027), compared to rat glomeruli incubated with paired RM plasma. Sparsentan-treated RL incubated glomeruli were protected from both the increase in Ps’alb (RL+spars 10 µM, 6.3 × 10−7 ± 0.19 × 10−7 cm/s, P < .001) and the loss in glomerular eGlx (RL+spars 10 µM, 222 ± 8.5 nm, P = .013), to a level comparable to RM incubated glomeruli. The effect of sparsentan on Ps’alb was dose dependent (RL+spars 1 µM, 8.0 × 10−7 ± 0.46 × 10−7 cm/s; RL+spars 0.1 µM, 9.6 × 10−7 ± 0.22 × 10−7 cm/s) with Ps’alb changes correlating inversely with glomerular eGlx thickness (r² = 0.75, P < .0001). In RM glomeruli, sparsentan alone had no effect on Ps’alb compared with vehicle (RM+spars 10 µM, 6.2 × 10−7 ± 0.25 × 10−7 cm/s, P = .590), suggesting no effect on otherwise healthy capillaries. Conclusion We have shown that dual inhibition of endothelin and angiotensin receptors, with sparsentan, preserves the glomerular eGlx resulting in normalised glomerular permeability in NS. These findings suggest that the direct action of sparsentan on the GFB could help maintain barrier integrity in NS, in particular by glycocalyx protection.