-
American Journal of Transplantation :... May 2022Pancreas transplantation improves and extends the life of patients with insulin-dependent diabetes. Pancreata from extended criteria donors have been increasingly used...
Pancreas transplantation improves and extends the life of patients with insulin-dependent diabetes. Pancreata from extended criteria donors have been increasingly used due to the scarcity of available grafts. Normothermic ex situ pancreas perfusion (NESPP) can keep grafts metabolically active, potentially allowing for assessment and organ repair, and could improve outcomes of marginal grafts. A novel NESPP technique was developed and tested. Porcine pancreata were removed after a short period of warm ischemia and subjected to 6 h of NESPP. Perfusion parameters, potential graft assessment markers and graft injury were measured. Next, pancreata subjected to 3 h of NESPP were transplanted and animals were followed for up to 3 days. Graft function and injury post-transplantation were evaluated. Using this novel system of perfusion, pancreata were perfused for an extended period of time with minimal edema. Histology at the end of perfusion showed intact islet cells with only mild signs of tissue injury. NESPP transplanted grafts showed immediate function after transplantation, with glucose levels in normal range. NESPP maintains a physiologic environment and excellent graft function without causing significant graft injury. Porcine pancreas transplantation is feasible and allows for in vivo graft assessment of pancreas function and injury after NESPP.
Topics: Animals; Humans; Organ Preservation; Pancreas; Pancreas Transplantation; Perfusion; Swine; Warm Ischemia
PubMed: 35258859
DOI: 10.1111/ajt.17019 -
Imaging Photoplethysmography for Noninvasive Anastomotic Perfusion Assessment in Intestinal Surgery.The Journal of Surgical Research Mar 2023Anastomotic leakage after gastrointestinal surgery has a high impact on patient's quality of life and its origin is associated with inadequate perfusion. Imaging...
INTRODUCTION
Anastomotic leakage after gastrointestinal surgery has a high impact on patient's quality of life and its origin is associated with inadequate perfusion. Imaging photoplethysmography (iPPG) is a noninvasive imaging technique that measures blood-volume changes in the microvascular tissue bed and detects changes in tissue perfusion.
MATERIALS AND METHODS
Intraoperative iPPG imaging was performed in 29 patients undergoing an open segment resection of the small intestine or colon. During each surgery, imaging was performed on fully perfused (true positives) and ischemic intestines (true negatives) and the anastomosis (unknowns). Imaging consisted of a 30-s video from which perfusion maps were extracted, providing detailed information about blood flow within the intestine microvasculature. To detect the predictive capabilities of iPPG, true positive and true negative perfusion conditions were used to develop two different perfusion classification methods.
RESULTS
iPPG-derived perfusion parameters were highly correlated with perfusion-perfused or ischemic-in intestinal tissues. A perfusion confidence map distinguished perfused and ischemic intestinal tissues with 96% sensitivity and 86% specificity. Anastomosis images were scored as adequately perfused in 86% of cases and 14% inconclusive. The cubic-Support Vector Machine achieved 90.9% accuracy and an area under the curve of 96%. No anastomosis-related postoperative complications were encountered in this study.
CONCLUSIONS
This study shows that noninvasive intraoperative iPPG is suitable for the objective assessment of small intestine and colon anastomotic perfusion. In addition, two perfusion classification methods were developed, providing the first step in an intestinal perfusion prediction model.
Topics: Humans; Photoplethysmography; Quality of Life; Anastomosis, Surgical; Digestive System Surgical Procedures; Anastomotic Leak; Perfusion; Indocyanine Green
PubMed: 36462380
DOI: 10.1016/j.jss.2022.10.086 -
Experimental Eye Research Jul 2022The key risk factor for glaucoma is elevation of intraocular pressure (IOP) and alleviating it is the only effective therapeutic approach to inhibit further vision loss....
The key risk factor for glaucoma is elevation of intraocular pressure (IOP) and alleviating it is the only effective therapeutic approach to inhibit further vision loss. IOP is regulated by the flow of aqueous humour across resistive tissues, and a reduction in outflow facility, is responsible for the IOP elevation in glaucoma. Measurement of outflow facility is therefore important when investigating the pathophysiology of glaucoma and testing candidate treatments for lowering IOP. Due to similar anatomy and response to pharmacological treatments, mouse eyes are a common model of human aqueous humour dynamics. The ex vivo preparation, in which an enucleated mouse eye is mounted in a temperature controlled bath and cannulated, has been well characterised and is widely used. The postmortem in situ model, in which the eyes are perfused within the cadaver, has received relatively little attention. In this study, we investigate the postmortem in situ model using the iPerfusion system, with a particular focus on i) the presence or absence of pressure-independent flow, ii) the effect of evaporation on measured flow rates and iii) the magnitude and pressure dependence of outflow facility and how these properties are affected by postmortem changes. Measurements immediately after cannulation and following multi-pressure facility measurement demonstrated negligible pressure-independent flow in postmortem eyes, in contrast to assumptions made in previous studies. Using a humidity chamber, we investigated whether the humidity of the surrounding air would influence measured flow rates. We found that at room levels of humidity, evaporation of saline droplets on the eye resulted in artefactual flow rates with a magnitude comparable to outflow, which were eliminated by a high relative humidity (>85%) environment. Average postmortem outflow facility was ∼4 nl/min/mmHg, similar to values observed ex vivo, irrespective of whether a postmortem delay was introduced prior to cannulation. The intra-animal variability of measured outflow facility values was also reduced relative to previous ex vivo data. The pressure-dependence of outflow facility was reduced in the postmortem relative to ex vivo model, and practically eliminated when eyes were cannulated >40 min after euthanisation. Overall, our results indicate that the moderately increased technical complexity associated with postmortem perfusion provides reduced variability and reduced pressure-dependence in outflow facility, when experimental conditions are properly controlled.
Topics: Animals; Aqueous Humor; Glaucoma; Intraocular Pressure; Mice; Perfusion; Tonometry, Ocular; Trabecular Meshwork
PubMed: 35525299
DOI: 10.1016/j.exer.2022.109103 -
Frontiers in Immunology 2023In response to the increasing demand for lung transplantation, lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs....
In response to the increasing demand for lung transplantation, lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs. However despite an expanding use in clinical practice, the responses of the different lung cell types to EVLP are not known. In order to advance our mechanistic understanding and establish a refine tool for improvement of EVLP, we conducted a pioneer study involving single cell RNA-seq on human lungs declined for transplantation. Functional enrichment analyses were performed upon integration of data sets generated at 4 h (clinical duration) and 10 h (prolonged duration) from two human lungs processed to EVLP. Pathways related to inflammation were predicted activated in epithelial and blood endothelial cells, in monocyte-derived macrophages and temporally at 4 h in alveolar macrophages. Pathways related to cytoskeleton signaling/organization were predicted reduced in most cell types mainly at 10 h. We identified a division of labor between cell types for the selected expression of cytokine and chemokine genes that varied according to time. Immune cells including CD4 and CD8 T cells, NK cells, mast cells and conventional dendritic cells displayed gene expression patterns indicating blunted activation, already at 4 h in several instances and further more at 10 h. Therefore despite inducing inflammatory responses, EVLP appears to dampen the activation of major lung immune cell types, what may be beneficial to the outcome of transplantation. Our results also support that therapeutics approaches aiming at reducing inflammation upon EVLP should target both the alveolar and vascular compartments.
Topics: Humans; Perfusion; CD8-Positive T-Lymphocytes; Endothelial Cells; Lung Transplantation; Lung; Inflammation
PubMed: 37465668
DOI: 10.3389/fimmu.2023.1142228 -
Journal of Visualized Experiments : JoVE Sep 2023This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas...
This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 °C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material's enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.
Topics: Mice; Animals; Eosine Yellowish-(YS); Hematoxylin; Polypropylenes; Polyurethanes; Liver Transplantation; Organ Preservation; Mice, Inbred C57BL; Liver; Perfusion
PubMed: 37811934
DOI: 10.3791/65363 -
Frontiers in Immunology 2022Normothermic machine perfusion (NMP) allows viability assessment and potential resuscitation of donor livers prior to transplantation. The immunological effect of NMP on...
BACKGROUND
Normothermic machine perfusion (NMP) allows viability assessment and potential resuscitation of donor livers prior to transplantation. The immunological effect of NMP on liver allografts is undetermined, with potential implications on allograft function, rejection outcomes and overall survival. In this study we define the changes in immune profile of human livers during NMP.
METHODS
Six human livers were placed on a NMP device. Tissue and perfusate samples were obtained during cold storage prior to perfusion and at 1, 3, and 6 hours of perfusion. Flow cytometry, immunohistochemistry, and bead-based immunoassays were used to measure leukocyte composition and cytokines in the perfusate and within the liver tissue. Mean values between baseline and time points were compared by Student's t-test.
RESULTS
Within circulating perfusate, significantly increased frequencies of CD4 T cells, B cells and eosinophils were detectable by 1 hour of NMP and continued to increase at 6 hours of perfusion. On the other hand, NK cell frequency significantly decreased by 1 hour of NMP and remained decreased for the duration of perfusion. Within the liver tissue there was significantly increased B cell frequency but decreased neutrophils detectable at 6 hours of NMP. A transient decrease in intermediate monocyte frequency was detectable in liver tissue during the middle of the perfusion run. Overall, no significant differences were detectable in tissue resident T regulatory cells during NMP. Significantly increased levels of pro-inflammatory and anti-inflammatory cytokines were seen following initiation of NMP that continued to rise throughout duration of perfusion.
CONCLUSIONS
Time-dependent dynamic changes are seen in individual leukocyte cell-types within both perfusate and tissue compartments of donor livers during NMP. This suggests a potential role of NMP in altering the immunogenicity of donor livers prior to transplant. These data also provide insights for future work to recondition the intrinsic immune profile of donor livers during NMP prior to transplantation.
Topics: Cytokines; Humans; Liver; Liver Transplantation; Living Donors; Organ Preservation; Perfusion
PubMed: 35720395
DOI: 10.3389/fimmu.2022.788935 -
Acta Neuropathologica Communications Sep 2019Perfusing fixatives through the cerebrovascular system is the gold standard approach in animals to prepare brain tissue for spatial biomolecular profiling, circuit... (Meta-Analysis)
Meta-Analysis
BACKGROUND
Perfusing fixatives through the cerebrovascular system is the gold standard approach in animals to prepare brain tissue for spatial biomolecular profiling, circuit tracing, and ultrastructural studies such as connectomics. Translating these discoveries to humans requires examination of postmortem autopsy brain tissue. Yet banked brain tissue is routinely prepared using immersion fixation, which is a significant barrier to optimal preservation of tissue architecture. The challenges involved in adopting perfusion fixation in brain banks and the extent to which it improves histology quality are not well defined.
METHODOLOGY
We searched four databases to identify studies that have performed perfusion fixation in human brain tissue and screened the references of the eligible studies to identify further studies. From the included studies, we extracted data about the methods that they used, as well as any data comparing perfusion fixation to immersion fixation. The protocol was preregistered at the Open Science Framework: https://osf.io/cv3ys/ .
RESULTS
We screened 4489 abstracts, 214 full-text publications, and identified 35 studies that met our inclusion criteria, which collectively reported on the perfusion fixation of 558 human brains. We identified a wide variety of approaches to perfusion fixation, including perfusion fixation of the brain in situ and ex situ, perfusion fixation through different sets of blood vessels, and perfusion fixation with different washout solutions, fixatives, perfusion pressures, and postfixation tissue processing methods. Through a qualitative synthesis of data comparing the outcomes of perfusion and immersion fixation, we found moderate confidence evidence showing that perfusion fixation results in equal or greater subjective histology quality compared to immersion fixation of relatively large volumes of brain tissue, in an equal or shorter amount of time.
CONCLUSIONS
This manuscript serves as a resource for investigators interested in building upon the methods and results of previous research in designing their own perfusion fixation studies in human brains or other large animal brains. We also suggest several future research directions, such as comparing the in situ and ex situ approaches to perfusion fixation, studying the efficacy of different washout solutions, and elucidating the types of brain donors in which perfusion fixation is likely to result in higher fixation quality than immersion fixation.
Topics: Brain; Humans; Perfusion; Tissue Banks; Tissue Fixation
PubMed: 31488214
DOI: 10.1186/s40478-019-0799-y -
Transplant International : Official... 2023Pancreas transplantation is the only curative treatment for patients with complicated diabetes, and organ shortage is a common and increasing problem. Strategies to...
Pancreas transplantation is the only curative treatment for patients with complicated diabetes, and organ shortage is a common and increasing problem. Strategies to expand the donor pool are needed, and normothermic perfusion of the pancreas has the potential to test and repair grafts before implantation. Between January 2021 and April 2022, six human pancreases, declined for transplantation or islet isolation, were perfused using a previously established method by our group. All 6 cases were successfully perfused for 4 h, with minimal edema. The mean age of the donors was 44.16 ± 13.8 years. Five grafts were obtained from neurological death donors, and one was obtained from a donation after cardiac death. The mean glucose and lactate levels decreased throughout perfusion and insulin levels increased. All 6 grafts were metabolically active during perfusion and histopathology showed minimal tissue injury and no edema. Human normothermic perfusion of the pancreas is feasible and safe and has the potential to expand the donor pool. Future studies will focus on tests and biomarkers for the assessment of grafts.
Topics: Humans; Adult; Middle Aged; Organ Preservation; Feasibility Studies; Perfusion; Tissue Donors; Pancreas; Allografts
PubMed: 37252614
DOI: 10.3389/ti.2023.10936 -
Critical Care (London, England) Jun 2021In acute respiratory distress syndrome (ARDS), non-ventilated perfused regions coexist with non-perfused ventilated regions within lungs. The number of unmatched regions... (Observational Study)
Observational Study
BACKGROUND
In acute respiratory distress syndrome (ARDS), non-ventilated perfused regions coexist with non-perfused ventilated regions within lungs. The number of unmatched regions might reflect ARDS severity and affect the risk of ventilation-induced lung injury. Despite pathophysiological relevance, unmatched ventilation and perfusion are not routinely assessed at the bedside. The aims of this study were to quantify unmatched ventilation and perfusion at the bedside by electrical impedance tomography (EIT) investigating their association with mortality in patients with ARDS and to explore the effects of positive end-expiratory pressure (PEEP) on unmatched ventilation and perfusion in subgroups of patients with different ARDS severity based on PaO/FiO and compliance.
METHODS
Prospective observational study in 50 patients with mild (36%), moderate (46%), and severe (18%) ARDS under clinical ventilation settings. EIT was applied to measure the regional distribution of ventilation and perfusion using central venous bolus of saline 5% during end-inspiratory pause. We defined unmatched units as the percentage of only ventilated units plus the percentage of only perfused units.
RESULTS
Percentage of unmatched units was significantly higher in non-survivors compared to survivors (32[27-47]% vs. 21[17-27]%, p < 0.001). Percentage of unmatched units was an independent predictor of mortality (OR 1.22, 95% CI 1.07-1.39, p = 0.004) with an area under the ROC curve of 0.88 (95% CI 0.79-0.97, p < 0.001). The percentage of ventilation to the ventral region of the lung was higher than the percentage of ventilation to the dorsal region (32 [27-38]% vs. 18 [13-21]%, p < 0.001), while the opposite was true for perfusion (28 [22-38]% vs. 36 [32-44]%, p < 0.001). Higher percentage of only perfused units was correlated with lower dorsal ventilation (r = - 0.486, p < 0.001) and with lower PaO/FiO ratio (r = - 0.293, p = 0.039).
CONCLUSIONS
EIT allows bedside assessment of unmatched ventilation and perfusion in mechanically ventilated patients with ARDS. Measurement of unmatched units could identify patients at higher risk of death and could guide personalized treatment.
Topics: Adult; Aged; Electric Impedance; Female; Humans; Italy; Male; Middle Aged; Perfusion; Prognosis; Prospective Studies; Respiration, Artificial; Respiratory Distress Syndrome; Simplified Acute Physiology Score
PubMed: 34082795
DOI: 10.1186/s13054-021-03615-4 -
Annals of Surgery Nov 2023To provide mechanistic insight into key biological alterations in donation after circulatory death kidneys during continuous pefusion we performed mass spectrometry... (Randomized Controlled Trial)
Randomized Controlled Trial
OBJECTIVE
To provide mechanistic insight into key biological alterations in donation after circulatory death kidneys during continuous pefusion we performed mass spectrometry profiling of perfusate samples collected during a phase 3 randomized double-blind paired clinical trial of hypothermic machine perfusion with and without oxygen (COMPARE).
BACKGROUND
Despite the clinical benefits of novel perfusion technologies aiming to better preserve donor organs, biological processes that may be altered during perfusion have remained largely unexplored. The collection of serial perfusate samples during the COMPARE clinical trial provided a unique resource to study perfusate proteomic profiles, with the hypothesis that in-depth profiling may reveal biologically meaningful information on how donor kidneys benefit from this intervention.
METHODS
Multiplexed liquid chromatography-tandem mass spectrometry was used to obtain a proteome profile of 210 perfusate samples. Partial least squares discriminant analysis and multivariate analysis involving clinical and perfusion parameters were used to identify associations between profiles and clinical outcomes.
RESULTS
Identification and quantitation of 1716 proteins indicated that proteins released during perfusion originate from the kidney tissue and blood, with blood-based proteins being the majority. Data show that the overall hypothermic machine perfusion duration is associated with increasing levels of a subgroup of proteins. Notably, high-density lipoprotein and complement cascade proteins are associated with 12-month outcomes, and blood-derived proteins are enriched in the perfusate of kidneys that developed acute rejection.
CONCLUSIONS
Perfusate profiling by mass spectrometry was informative and revealed proteomic changes that are biologically meaningful and, in part, explain the clinical observations of the COMPARE trial.
Topics: Humans; Kidney Transplantation; Proteome; Proteomics; Organ Preservation; Kidney; Perfusion; Tissue Donors
PubMed: 37503631
DOI: 10.1097/SLA.0000000000006046