Organ transplantation is a life-saving procedure affecting over 100,000 people on the transplant waitlist. Ischemia reperfusion injury (IRI) is a major challenge in the field as it can cause ...post-transplantation complications and limit the use of organs from extended criteria donors. Machine perfusion technology has the potential to mitigate IRI; however, it currently fails to achieve its full potential due to a lack of highly sensitive and specific assays to assess organ quality during perfusion. We developed a real-time and non-invasive method of assessing organs during perfusion based on mitochondrial function and injury using resonance Raman spectroscopy. It uses a 441 nm laser and a high-resolution spectrometer to quantify the oxidation state of mitochondrial cytochromes during perfusion. This index of mitochondrial oxidation, or 3RMR, was used to understand differences in mitochondrial recovery of cold ischemic rodent livers during machine perfusion at normothermic temperatures with an acellular versus cellular perfusate. Measurement of the mitochondrial oxidation revealed that there was no difference in 3RMR of fresh livers as a function of normothermic perfusion when comparing acellular versus cellular-based perfusates. However, following 24 h of static cold storage, 3RMR returned to baseline faster with a cellular-based perfusate, yet 3RMR progressively increased during perfusion, indicating injury may develop over time. Thus, this study emphasizes the need for further refinement of a reoxygenation strategy during normothermic machine perfusion that considers cold ischemia durations, gradual recovery/rewarming, and risk of hemolysis.
Ischemia reperfusion injury (IRI) is a critical problem in liver transplantation that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. ...However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria play a central role in IRI and are an ideal diagnostic target. During ischemia, changes in the mitochondrial redox state form the first link in the chain of events that lead to IRI. In this study we used resonance Raman spectroscopy to provide a rapid, non-invasive, and label-free diagnostic for quantification of the hepatic mitochondrial redox status. We show this diagnostic can be used to significantly distinguish transplantable versus non-transplantable ischemically injured rat livers during oxygenated machine perfusion and demonstrate spatial differences in the response of mitochondrial redox to ischemia reperfusion. This novel diagnostic may be used in the future to predict the viability of human livers for transplantation and as a tool to better understand the mechanisms of hepatic IRI.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background: Normothermic ex situ perfusion of vascularized composite allografts (VCAs) necessitates high oxygen demand and, thus, increased metabolic activity, which, in turn, requires the use of ...blood-based perfusion solutions. However, blood-derived perfusates, in turn, constitute an antigenic load. To circumvent this immunogenic problem, we used a perfusate enriched with acellular dextrane oxygen microcarriers to perfuse rat hindlimbs. Methods: Rat hindlimbs (n = 11) were perfused with either (non-), oxygenated dextrane-enriched Phoxilium, or Phoxilium enriched with dextrane oxygen microcarriers (MO2) for 12 h at 37 °C or stored on ice. Oxygenation of the skeletal muscle was assessed with Raman spectroscopy, tissue pO2-probes, and analysis of the perfusate. Transmission electronic microscopy was utilized to assess the ultrastructure of mitochondria of the skeletal muscle. Results: For all evaluated conditions, ischemia time until perfusion was comparable (22.91 ± 1.64 min; p = 0.1559). After 12 h, limb weight increased significantly by at least 81%, up to 124% in the perfusion groups, and by 27% in the static cold storage (SCS) group. Raman spectroscopy signals of skeletal muscle did not differ substantially among the groups during either perfusion or static cold storage across the duration of the experiment. While the total number of skeletal muscle mitochondria decreased significantly compared to baseline, mitochondrial diameter increased in the perfusion groups and the static cold storage group. Conclusion: The use of oxygen microcarriers in ex situ perfusion of VCA with acellular perfusates under normothermic conditions for 12 h facilitates the maintenance of mitochondrial structure, as well as a subsequent recovery of mitochondrial redox status over time, while markers of muscle injury were lower compared to conventional oxygenated acellular perfusates.
Abstract Background The knowledge of hemoglobin oxygen saturation (SO2 ) and tissue oxygenation is critical to identify the presence of shock and therapeutic options. The resonance vibrational ...enhancement of hemoglobin allows measurement of oxy- and deoxy species of hemoglobin and resonance Raman spectroscopy (RRS-StO2 ) has been successfully used to measure aggregate microvascular oxygenation. We tested the hypothesis that noninvasive oxygen saturation measured by RRS-StO2 could serve as surrogate of systemic central venous SO2. Methods In anesthetized rats, measurements of RRS-StO2 made in oral mucosa, skin, muscle, and liver were compared with measurements of central venous SO2 using traditional multi-wavelength oximetry. Various oxygenation levels were obtained using a stepwise hemorrhage while over 100 paired blood samples and Raman-based measurements were performed. The relationships between RRS-StO2 and clinically important systemic blood parameters were also evaluated. RRS-StO2 measurements were made in 3-mm diameter tissue areas using a microvascular oximeter and a handheld probe. Results Significant correlations were found between venous SO2 and RRS-StO2 measurements made in the oral mucosa ( r = 0.913, P < 0.001), skin ( r = 0.499, P < 0.01), and liver ( r = 0.611, P < 0.05). The mean difference between sublingual RRS-StO2 and blood sample SO2 values was 5.4 ± 1.6%. Sublingual RRS-StO2 also correlated with lactate ( r = 0.909, P < 0.01), potassium ( r = 0.757, P < 0.01), and pH ( r = 0.703, P < 0.05). Conclusions Raman-based oxygen saturation is a promising technique for the noninvasive evaluation of oxygenation in skin, thin tissues, and solid organs. Under certain conditions, sublingual RRS-StO2 measurements correlate with central venous SO2.
Abstract only
Introduction:
Balance between oxygen delivery and demand is typically assessed based on venous oxyhemoglobin saturation (SvO2), which requires repeated blood sampling from a central ...location. Alternative, non-invasive methods such as near-infrared spectroscopy (NIRS) demonstrate poor sensitivity to tissue hypoxia.
Methods:
We explored the possibility of measuring regional tissue oxyhemoglobin saturation in the esophagus (EsSO2) using a new, linear probe using resonance Raman spectroscopy (RRS, 420 nm excitatory light, A) to quantify the fraction of oxyhemoglobin. The RRS probe was passed from the mouth into the mid-esophagus in Yorkshire swine (n=7, weight 11.2-13.4 kg). Cerebral (cNIRS) and splanchnic NIRS (sNIRS, Somanetics) were continuously recorded. Swine underwent complete asphyxia by endotracheal tube occlusion for 12 minutes, during which time they were resuscitated using CPR and drugs. EsSO2, cNIRS, sNIRS were compared with SO2 based on arterial blood gas co-oximetry (SaO2). For the purpose of analysis, SaO2 was considered functionally equivalent to SvO2 due to the obligate intrapulmonary (veno-arterial) shunting in this physiology.
Results:
During asphyxia, SO2 rapidly declined to nearly 0%. This was reflected in EsSO2, but cNIRS and sNIRS decreased to a lesser extent (B). EsSO2 exhibited a strong correlation with SO2 (r
2
=0.63, p<0.001) compared to sNIRS (r
2
=0.53, p<0.001) and cNIRS (r
2
=0.06, p=0.106, C). Defining SO2 <30% as the presence of critical hypoxia, EsSO2 had a higher sensitivity than both the sNIRS and cNIRS (D). EsSO2 was <30 in 100% (33/33) of timepoints in which critical hypoxia was present, significantly more sensitive than rSO2 based on cNIRS (which was <30 in 58% (19/33) of timepoints) and sNIRS (<30 in 24% (8/33) of timepoints).
Conclusion:
EsSO2 accurately detects critical hypoxic states, more so than cNIRS or sNIRS, and may represent an alternative, continuous, less invasive approach to central venous monitoring.
Abstract Introduction Assessment of thermal burns in the acute phase is critical since healing may require surgical intervention depending on the depth of injury. Early depth estimation guides ...debridement and planning care strategy. Current assessment standard has limitations, and biopsies are invasive and painful. There is an urgent need for a rapid, non-invasive, and objective metric of burn depth. Methods We created a model to study intermediate burn depth in Yucatan miniature pigs, which is clinically relevant. Brass blocks of 4cm radius create contact burns by preheating to 63°C for 2nd-degree (intermediate), and 95°C for 3rd-degree (full thickness) burns. Contact duration varied between 15 to 45 seconds for 2-degree burns to create different depths. Burns are evenly distributed and randomized on the dorsal paravertebral region of the pigs. Full-thickness burns were debrided on POD 3. Each wound was assessed using a custom bench-top Resonance Raman Spectroscopy (RRS) device to measure oxygen saturation, hemoglobin content, and autofluorescence spectrum. Measurements with RRS were performed immediately ( < 2 hours), 2 days, and 3 days after injury. Animals received punch biopsies and dressing changes every week for 9 wks and photographic documentation of healing. Post hoc analysis was performed in GraphPad Prism software to observe correlation between RRS readings on POD 0 to 3 and wound healing duration. Results We successfully created intermediate and full-thickness burns, as confirmed by visual observation and histological analysis. We also created different depths of intermediate burns that vary in healing potential (>95% re-epithelialization). Of 9 wounds on 2 pigs, 4 healed in 5 wks, 3 in 7 wks, and 2 did not heal by 9 wks. RRS measurements had 4 technical replicates for each wound on POD 0, 2, and 3, and were correlated with wound type (2nd vs. 3rd) and duration of healing for 2nd-degree burns. We defined Burn Index (BI) as a marker of burn severity- Burn Index B.I. = Oxygen Saturation / (Normalized Laser Induced Fluorescence * Normalized Hemoglobin Content) BI distinguished between 2nd-degree (n=9) and 3rd-degree (n=4) burns on POD 0 with an area under the curve (AUC) for a receiver operating characteristic (ROC) of 79.17%. It could also distinguish wounds taking more than 9 wks to heal (n=2) from those that healed earlier (n=7) by POD 2, with AUC 97.77%, indicating high sensitivity and specificity. By POD 3, BI distinguished healing in less than 5 wks (n=4) from others (n=5) with AUC 79.38%. Conclusions We established a model of intermediate burns with Yucatan miniature pigs. Resonance Raman Spectroscopy (RRS) based Burn index (BI) could distinguish full and intermediate thickness burns by POD 0 and predict healing time by POD 2 and POD 3 with high specificity and sensitivity. Applicability of Research to Practice Rapid, early, non-invasive quantification of burn depth using our portable device can aid decision-making regarding intermediate-depth wound care, such as skin grafting.
Current oxygen monitoring by pulse oximetry has limitations and cannot provide estimates of the oxygen content in the microvasculature, where oxygen is used. Resonance Raman spectroscopy (RRS) ...provides noninvasive microvascular oxygen measurement. The objectives of this study were to (i) measure the correlation between preductal RRS microvascular oxygen saturations (RRS-StO2) and central venous oxygen saturation (SCVO2), (ii) develop normative data for RRS-StO2 measurements in healthy preterm infants, and (iii) determine the effect of blood transfusion on RRS-StO2.
Thirty-three buccal and thenar RRS-StO2 measurements were performed in 26 subjects to correlate RRS-StO2 with SCVO2. Thirty-one measurements were performed in 28 subjects to develop normative RRS-StO2 values, and eight subjects were enrolled in the transfusion group to assess changes in RRS-StO2 with blood transfusion.
There were good correlations for buccal (r = 0.692) and thenar (r = 0.768) RRS-StO2 versus SCVO2. The median RRS-StO2 in healthy subjects was 76% (IQR 68.7-80.8). There was a significant increase of 7.8 ± 4.6% in the thenar RRS-StO2 after blood transfusion.
RRS appears to be a safe and noninvasive means of monitoring microvascular oxygenation. Thenar RRS-StO2 measurements are more feasible and practical to use than buccal. In healthy preterm infants, the median RRS-StO2 was calculated based on measurements across various gestational age and gender. More studies evaluating the effects of gestational age of RRS-StO2 in various critical clinical settings are needed to confirm the findings.
BACKGROUND:In this study, we examined the ability of resonance Raman spectroscopy to measure tissue hemoglobin oxygenation (R-StO2) noninvasively in critically ill patients and compared its ...performance with conventional central venous hemoglobin oxygen saturation (ScvO2).
METHODS:Critically ill patients (n = 138) with an indwelling central venous or pulmonary artery catheter in place were consented and recruited. R-StO2 measurements were obtained by placing a sensor inside the mouth on the buccal mucosa. R-StO2 was measured continuously for 5 minutes. Blood samples were drawn from the distal port of the indwelling central venous catheter or proximal port of the pulmonary artery catheter at the end of the test period to measure ScvO2 using standard co-oximetry analyzer. A regression algorithm was used to calculate the R-StO2 based on the observed spectra.
RESULTS:Mean(SD) of pooled R-StO2 and ScvO2 were 64(7.6) % and 65(9.2) % respectively. A paired t-test showed no significant difference between R-StO2 and ScvO2 with a mean(SD) difference of -1(7.5) % (95% CI-2.2, 0.3%) with a Clarke Error Grid demonstrating 84.8% of the data residing within the accurate and acceptable grids. Area under the receiver operator curve for R-StO2ʼs was 0.8(0.029) (95% CI0.7, 0.9 p < 0.0001) at different thresholds of ScvO2 (≤60%, ≤65%, and ≤70%). Clinical adjudication by five clinicians to assess the utility of R-StO2 and ScvO2 yielded Fleiss’ Kappa agreement of 0.45 (p < 0.00001).
CONCLUSIONS:R-StO2 has the potential to predict ScvO2 with high precision and might serve as a faster, safer, and non-invasive surrogate to these measures.
Assessing the adequacy of oxygen delivery to tissues is vital, particularly in the fields of intensive care medicine and surgery. As oxygen delivery to a cell becomes deficient, changes in ...mitochondrial redox state precede changes in cellular function. We describe a technique for the continuous monitoring of the mitochondrial redox state on the epicardial surface using resonance Raman spectroscopy. We quantify the reduced fraction of specific electron transport chain cytochromes, a metric we name the resonance Raman reduced mitochondrial ratio (3RMR). As oxygen deficiency worsens, heme moieties within the electron transport chain become progressively more reduced, leading to an increase in 3RMR. Myocardial 3RMR increased from baseline values of 18.1 ± 5.9 to 44.0 ± 16.9% (
= 0.0039) after inferior vena cava occlusion in rodents (
= 8). To demonstrate the diagnostic power of this measurement, 3RMR was continuously measured in rodents (
= 31) ventilated with 5 to 8% inspired oxygen for 30 min. A 3RMR value exceeding 40% at 10 min predicted subsequent cardiac arrest with 95% sensitivity and 100% specificity area under the curve (AUC), 0.98, outperforming all current measures, including contractility (AUC, 0.51) and ejection fraction (AUC, 0.39). 3RMR correlated with indices of intracellular redox state and energy production. This technique may permit the real-time identification of critical defects in organ-specific oxygen delivery.
PDF
