Assessment of Tissue Viability Following Amputation Surgery Using Near-Infrared Fluorescence Imaging With Indocyanine Green.

BACKGROUND: Patients with chronic limb threatening ischemia have a risk of undergoing a major amputation within 1 year of nearly 30% with a substantial risk of re-amputation since wound healing is often impaired. Quantitative assessment of regional tissue viability following amputation surgery can identify patients at risk for impaired wound healing. In quantification of regional tissue perfusion, near-infrared (NIR) fluorescence imaging using Indocyanine Green (ICG) seems promising. METHODS: This pilot study included adult patients undergoing lower extremity amputation surgery due to peripheral artery disease or diabetes mellitus. ICG NIR fluorescence imaging was performed within 5 days following amputation surgery using the Quest Spectrum PlatformⓇ. Following intravenous administration of ICG, the NIR fluorescence intensity of the amputation wound was recorded for 10 minutes. The NIR fluorescence intensity videos were analyzed and if a fluorescence deficit was observed, this region was marked as "low fluorescence." All other regions were marked as "normal fluorescence." RESULTS: Successful ICG NIR fluorescence imaging was performed in 10 patients undergoing a total of 15 amputations. No "low fluorescence" regions were observed in 11 out of 15 amputation wounds. In 10 out of these 11 amputations, no wound healing problems occurred during follow-up. Regions with "low fluorescence" were observed in 4 amputation wounds. Impaired wound healing corresponding to these regions was observed in all wounds and a re-amputation was necessary in 3 out of 4. When observing time-related parameters, regions with low fluorescence had a significantly longer time to maximum intensity (113 seconds vs. 32 seconds, P = 0.003) and a significantly lesser decline in outflow after five minutes (80.3% vs. 57.0%, P = 0.003). CONCLUSIONS: ICG NIR fluorescence imaging was able to predict postoperative skin necrosis in all four cases. Quantitative assessment of regional perfusion remains challenging due toinfluencing factors on the NIR fluorescence intensity signal, including camera angle, camera distance and ICG dosage. This was also observed in this study, contributing to a large variety in fluorescence intensity parameters among patients. To provide surgeons with reliable NIR fluorescence cut-off values for prediction of wound healing, prospective studies on the intra-operative use of this technique are required. The potential prediction of wound healing using ICG NIR fluorescence imaging will have a huge impact on patient mortality, morbidity as well as the burden of amputation surgery on health care.

Quantitative High-speed Assessment of Droplet and Aerosol From an Eye After Impact With an Air-puff Amid COVID-19 Scenario.

PURPOSE: To quantify aerosol and droplets generated during noncontact tonometry (NCT) and assess the spread distance of the same. METHODOLOGY: This was an experimental study on healthy human volunteers (n=8 eyes). In an experimental setup, NCT was performed on eyes (n=8) of human volunteers under normal settings, with a single and 2 drops of lubricant. High-speed shadowgraphy, frontal lighting technique, and fluorescein analysis were used to detect the possible generation of any droplets and aerosols. Mathematical computation of the spread of the droplets was then performed. RESULTS: In a natural setting, there was no droplet or aerosol production. Minimal splatter along with droplet ejection was observed when 1 drop of lubricant was used before NCT. When 2 drops of lubricant were instilled, a significant amount of fluid ejection in the form of a sheet that broke up into multiple droplets was observed. Some of these droplets traversed back to the tonometer. Droplets ranging from 100 to 500 µm in diameter were measured. CONCLUSIONS: There was no droplet generation during NCT performed in a natural setting. However, NCT should be avoided in conditions with high-tear volume (natural or artificial) as it would lead to droplet spread and tactile contamination.

Assessment of Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles' Biocompatibility with Endothelial Cells in Vitro and Delivery of an Anti-Inflammatory Drug.

Nanoparticles (NPs) produced from amphiphilic derivatives of poly-N-vinylpyrrolidone (Amph-PVP), composed of various molecular weight polymeric hydrophilic fragments linked into hydrophobic n-alkyl chains of varying lengths, were previously shown to exert excellent biocompatibility. Although routes of administration can be different, finally, most nanosystems enter the blood circulation or lymphatic vessels, and by this, they establish direct contact with endothelial cells. In this study, Amph-PVP NPs and fluorescently labeled Amph-PVP-based NPs, namely "PVP" NPs (Amph-PVP-NPs (6000 Da) unloaded) and "F"-NPs (Amph-PVP-NPs (6000 Da) loaded with fluorescent FITC), were synthesized to study Amph-PVP NPs interactions with HMEC-1 endothelial cells. PVP NPs were readily uptaken by HMEC-1 cells in a concentration-dependent manner, as demonstrated by immunofluorescence imaging. Upon uptake, the FITC dye was localized to the perinuclear region and cytoplasm of treated cells. The generation of lipopolysaccharide (LPS)-induced activated endothelium model revealed an increased uptake of PVPNPs, as shown by confocal microscopy. Both unloaded PVP NPs and F-NPs did not affect EC viability in the 0.01 to 0.066 mg/mL range. Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon PVPNPs treatment by assessing the expression of their E-Selectin, ICAM-1, and VCAM-1 adhesion receptors. None of the adhesion molecules were affected by NP treatments of both activated by LPS and nonactivated HMEC-1 cells, at the utilized concentrations (p = NS). In this study, PVP (6000 Da) NPs were used to encapsulate indomethacin, a widely used anti-inflammatory drug. The synthesized drug carrier complex did not affect HMEC-1 cell growth and expression of E-selectin, ICAM-1, and VCAM-1 adhesion receptors. In summary, PVP-based NPs are safe for use on both basal and activated endothelium, which more accurately mimics pathological conditions. Amph-PVP NPs are a promising drug delivery system.

First-in-Human Assessment of cRGD-ZW800-1, a Zwitterionic, Integrin-Targeted, Near-Infrared Fluorescent Peptide in Colon Carcinoma.

PURPOSE: Incomplete oncologic resections and damage to vital structures during colorectal cancer surgery increases morbidity and mortality. Moreover, neoadjuvant chemoradiotherapy has become the standard treatment modality for locally advanced rectal cancer, where subsequent downstaging can make identification of the primary tumor more challenging during surgery. Near-infrared (NIR) fluorescence imaging can aid surgeons by providing real-time visualization of tumors and vital structures during surgery. EXPERIMENTAL DESIGN: We present the first-in-human clinical experience of a novel NIR fluorescent peptide, cRGD-ZW800-1, for the detection of colon cancer. cRGD-ZW800-1 was engineered to have an overall zwitterionic chemical structure and neutral charge to lower nonspecific uptake and thus background fluorescent signal. We performed a phase I study in 11 healthy volunteer as well as a phase II feasibility study in 12 patients undergoing an elective colon resection, assessing 0.005, 0.015, and 0.05 mg/kg cRGD-ZW800-1 for the intraoperative visualization of colon cancer. RESULTS: cRGD-ZW800-1 appears safe, and exhibited rapid elimination into urine after a single low intravenous dose. Minimal invasive intraoperative visualization of colon cancer through full-thickness bowel wall was possible after an intravenous bolus injection of 0.05 mg/kg at least 2 hours prior to surgery. Longer intervals between injection and imaging improved the tumor-to-background ratio. CONCLUSIONS: cRGD-ZW800-1 enabled fluorescence imaging of colon cancer in both open and minimal invasive surgeries. Further development of cRGD-ZW800-1 for widespread use in cancer surgery may be warranted given the ubiquitous overexpression of various integrins on different types of tumors and their vasculature.

Indocyanine green-based fluorescence imaging in visceral and hepatobiliary and pancreatic surgery: State of the art and future directions.

In recent years, the use of fluorescence-guided surgery (FGS) to treat benign and malignant visceral, hepatobiliary and pancreatic neoplasms has significantly increased. FGS relies on the fluorescence signal emitted by injected substances (fluorophores) after being illuminated by ad hoc laser sources to help guide the surgical procedure and provide the surgeon with real-time visualization of the fluorescent structures of interest that would be otherwise invisible. This review surveys and discusses the most common and emerging clinical applications of indocyanine green (ICG)-based fluorescence in visceral, hepatobiliary and pancreatic surgery. The analysis, findings, and discussion presented here rely on the authors' significant experience with this technique in their medical institutions, an up-to-date review of the most relevant articles published on this topic between 2014 and 2018, and lengthy discussions with key opinion leaders in the field during recent conferences and congresses. For each application, the benefits and limitations of this technique, as well as applicable future directions, are described. The imaging of fluorescence emitted by ICG is a simple, fast, relatively inexpensive, and harmless tool with numerous different applications in surgery for both neoplasms and benign pathologies of the visceral and hepatobiliary systems. The ever-increasing availability of visual systems that can utilize this tool will transform some of these applications into the standard of care in the near future. Further studies are needed to evaluate the strengths and weaknesses of each application of ICG-based fluorescence imaging in abdominal surgery.

Quantitative Assessment of Nanoparticle Biodistribution by Fluorescence Imaging, Revisited.

Fluorescence-based whole-body imaging is widely used in the evaluation of nanoparticles (NPs) in small animals, often combined with quantitative analysis to indicate their spatiotemporal distribution following systemic administration. An underlying assumption is that the fluorescence label represents NPs and the intensity increases with the amount of NPs and/or the labeling dyes accumulated in the region of interest. We prepare DiR-loaded poly(lactic- co-glycolic acid) (PLGA) NPs with different surface layers (polyethylene glycol with and without folate terminus) and compare the distribution of fluorescence signals in a mouse model of folate-receptor-expressing tumors by near-infrared fluorescence whole-body imaging. Unexpectedly, we observe that fluorescence distribution patterns differ far more dramatically with DiR loading than with the surface ligand, reaching opposite conclusions with the same type of NPs (tumor-specific delivery vs predominant liver accumulation). Analysis of DiR-loaded PLGA NPs reveals that fluorescence quenching, dequenching, and signal saturation, which occur with the increasing dye content and local NP concentration, are responsible for the conflicting interpretations. This study highlights the critical need for validating fluorescence labeling of NPs in the quantitative analysis of whole-body imaging. In light of our observation, we make suggestions for future whole-body fluorescence imaging in the in vivo evaluation of NP behaviors.

Corneal epithelial permeability to fluorescein in humans by a multi-drop method.

PURPOSE: The permeability of the corneal epithelium to fluorescein Pdc is an indicator of the health of the ocular surface. It can be measured in a clinical setting by determining the accumulation of fluorescein in the stroma following administration of the dye on the ocular surface. Here we demonstrate a new multi-drop method for the measurement of Pdc by a spot fluorometer. METHODS: Twenty-nine healthy participants were recruited for this study. First, a probe-drop of fluorescein (0.35%, 2 µL) was instilled on the conjunctiva. The clearance of the dye from the tears was immediately measured using the fluorometer. Following this, two loading drops (2%; 6 µL each) were administered 10 min apart. Fifteen minutes later, the ocular surface was washed and fluorescence from the stroma Fs was measured. Permeability was calculated using Pdc = (Q x Fs)/ (2 x AUC), where Q is the stromal thickness and AUC is the area under the fluorescence vs. time curve for the loading drops. RESULTS: After the probe drop, the tear fluorescence followed an exponential decay (elimination rate constant; kd = 0.41 ± 0.28 per min; 49 eyes of 29 subjects), but the increase in Fs was negligible. However, after the loading drops, the measured Fs was ~ 20-fold higher than the autofluorescence and could be recorded at a high signal to noise ratio (SNR > 40). The intra-subject variability of kd was insignificant. Since fluorescein undergoes concentration quenching at > 0.5%, the value of AUC for the loading drops was estimated by scaling the AUC of the probe drop. The calculated Pdc was 0.54 ± 0.54 nm/sec (n = 49). A Monte Carlo simulation of the model for the multi-drop protocol confirmed the robustness of the estimated Pdc. CONCLUSIONS: The new multi-drop method can be used in place of the single-drop approach. It can overcome a lack of sensitivity in fluorometers of high axial resolution. The Pdc estimated by the multi-drop method is ~ 11-fold higher than previously reported but closer to the value reported for other drugs with equivalent octanol/water partition coefficient.

Assessment of MMP-2/-9 expression by fluorescence endoscopy for evaluation of anastomotic healing in a murine model of anastomotic leakage.

BACKGROUND: Disturbance of intestinal wound closure leads to insufficient anastomotic healing and is associated with considerable morbidity following colorectal resections. Matrix metalloproteinases (MMPs) play a crucial role in regulation of wound closure. Here fluorescence endoscopy was evaluated for assessment of MMP-2/-9 expression during failed intestinal anastomotic healing. METHODS: Distal colonic anastomoses were performed as a model for disturbed healing in 36 Balb/c mice. Healing was evaluated endoscopically, macroscopically, and histologically after 1, 3 and 5 days. For detection of MMP-2/-9 expression fluorescence endoscopy (FE) was used following i.v.-administration of a Cy5.5-labeled MMP-2/-9 specific tracer. FE was complemented by quantification of the fluorescence signal using the MS-FX PRO Optical Imaging System. An overall leakage score was calculated and correlated with the results of FE. RESULTS: With increasing incidence of anastomotic leakage from POD1 (17%) to POD5 (83%) the uptake of the MMP tracer gradually increased (signal-to-noise ratio (SNR), POD1: 17.91 ± 1.251 vs. POD3: 30.56 ± 3.03 vs. POD5: 44.8 ± 4.473, P<0.0001). Mice with defective anastomotic healing showed significantly higher uptake compared to non-defective (SNR: 37.37± 3.63 vs. 26.16± 3.635, P = 0.0369). White light endoscopy and FE allowed evaluation of anastomotic healing and visualization of mucosal MMPs in vivo. Using FE based detection of MMPs in the anastomosis, an overall positive predictive value of 71.4% and negative predictive value of 66.6% was calculated for detection of anastomotic leakage. CONCLUSION: During disturbed anastomotic healing increased expression of MMP-2/-9 was observed in the anastomotic tissue. Fluorescence endoscopy for detection of MMP-2/-9 during the healing process might be a promising tool for early identification of anastomotic leakage.

Comparative Evaluation of Clinical Methods of Tear Film Stability Assessment: A Randomized Crossover Trial.

Importance: Tear film breakup time assessment is an integral component of dry eye evaluation. To our knowledge, the comparative discriminative ability of the noninvasive Keratograph (Oculus) vs conventional fluorescein method in detecting dry eye is unknown. Objective: To compare tear film stability measurements obtained with an automated noninvasive corneal topographer vs conventional fluorescein methods and evaluate their respective discriminative ability in detecting dry eye. Design, Setting, and Participants: This investigator-masked randomized crossover trial was conducted at a single-center university clinic between May 26, 2016, and October 3, 2016, and included 74 participants 18 years or older. Participants were recruited into 2 equally sized age, sex, and race/ethnicity-matched groups, with and without symptomatic dry eye (Ocular Surface Disease Index ≥13). Interventions: Participants were assigned to receive a noninvasive keratograph evaluation and topical fluorescein instillation in a randomized order. Main Outcomes and Measures: Noninvasive keratograph breakup time (NIKBUT) and fluorescein breakup time (TBUT). Area under the receiver operating characteristic curve, Youden-optimal diagnostic cutoff sensitivity, and specificity of NIKBUT and TBUT in detecting dry eye. Results: Seventy-four participants (74 eyes; 43 women [58.1%]) with a mean (SD) age of 24 (4) years were randomized. Noninvasive keratograph breakup time was significantly longer than TBUT in participants with dry eye (median, 6.3 seconds vs 4.3 seconds [difference, 2.0 seconds]; 95% CI, 1.1-3.4 seconds; P = .003), and healthy participants (median, 11.9 seconds vs 5.0 seconds [difference, 6.9 seconds]; 95% CI, 4.7-7.6 seconds; P < .001). Fluorescein breakup time measurements were more narrowly distributed in both the dry eye (variance, 188 seconds2 vs 27.9 seconds2; P < .001) and control groups (variance, 113 seconds2 vs 13.4 seconds2; P < .001). The discriminative ability of NIKBUT in detecting dry eye (area under the receiver operating characteristic curve, 0.68; 95% CI, 0.56-0.81; P = .007) was greater than that of TBUT (area under the receiver operating characteristic curve, 0.57; 95% CI, 0.44-0.70; P = .31). The optimal diagnostic cutoff for NIKBUT was 9 seconds or less with a sensitivity of 68% (95% CI, 50%-82%), specificity of 70% (95% CI, 53%-84%), positive likelihood ratio of 2.27 (95% CI, 1.32-3.91), and negative likelihood ratio of 0.46 (95% CI, 0.28-0.77). The optimal threshold for TBUT was 5 seconds or less with a sensitivity of 54% (95% CI, 37%-71%), specificity of 68% (95% CI, 50%-82%), positive likelihood ratio of 1.67 (95% CI, 0.96-2.89), and negative likelihood ratio of 0.68 (95% CI, 0.45-1.03). Conclusions and Relevance: Conventional fluorescein tear film breakup time measurements were significantly shorter with narrower distributions, while automated noninvasive keratograph readings displayed superior discriminative ability in detecting dry eye. Trial Registration: anzctr.org.au Identifier: ACTRN12617001428358.

Therapeutic Fluorescent Hybrid Nanoparticles for Traceable Delivery of Glucocorticoids to Inflammatory Sites.

Treatment of inflammatory disorders with glucocorticoids (GCs) is often accompanied by severe adverse effects. Application of GCs via nanoparticles (NPs), especially those using simple formulations, could possibly improve their delivery to sites of inflammation and therefore their efficacy, minimising the required dose and thus reducing side effects. Here, we present the evaluation of NPs composed of GC betamethasone phosphate (BMP) and the fluorescent dye DY-647 (BMP-IOH-NPs) for improved treatment of inflammation with simultaneous in vivo monitoring of NP delivery. Methods: BMP-IOH-NP uptake by MH-S macrophages was analysed by fluorescence and electron microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48 h with BMP-IOH-NPs (1×10-5-1×10-9 M), BMP or dexamethasone (Dexa). Drug efficacy was assessed by measurement of interleukin 6. Mice with Zymosan-A-induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI) were treated intranasally with BMP-IOH-NPs, BMP or Dexa (each 2.5 mg/kg). Efficacy was assessed in vivo by paw volume measurements with µCT and ex vivo by measurement of paw weight for Zymosan-A-treated mice, or in the AAI model by in vivo x-ray-based lung function assessment and by cell counts in the bronchoalveolar lavage (BAL) fluid and histology. Delivery of BMP-IOH-NPs to the lungs of AAI mice was monitored by in vivo optical imaging and by fluorescence microscopy. Results: Uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with the NP load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs in vitro was dose dependent and higher than that of Dexa or free BMP, confirming efficient release of the drug. In vivo, Zymosan-A-induced paw inflammation was significantly reduced in mice treated with BMP-IOH-NPs. AAI mice that received BMP-IOH-NPs or Dexa but not BMP revealed significantly decreased eosinophil numbers in BALs and reduced immune cell infiltration in lungs. Correspondingly, lung function parameters, which were strongly affected in non-treated AAI mice, were unaffected in AAI mice treated with BMP-IOH-NPs and resembled those of healthy animals. Accumulation of BMP-IOH-NPs within the lungs of AAI mice was detectable by optical imaging for at least 4 h in vivo, where they were preferentially taken up by peribronchial and alveolar M2 macrophages. Conclusion: Our results show that BMP-IOH-NPs can effectively be applied in therapy of inflammatory diseases with at least equal efficacy as the gold standard Dexa, while their delivery can be simultaneously tracked in vivo by fluorescence imaging. BMP-IOH-NPs thus have the potential to reach clinical applications.

Qualitative angiographic and quantitative myocardial perfusion assessment using fluorescent cardiac imaging during graded coronary artery bypass stenosis.

Intraoperative graft assessment in coronary artery bypass (CAB) grafting is important to avoid early graft failure. This study aimed to evaluate the accuracy of fluorescent cardiac imaging (FCI) for intraoperative qualitative angiographic and quantitative myocardial perfusion assessment during graded CAB stenosis compared to coronary angiography (CA). After CAB grafting to the left anterior descending coronary artery, graded distal bypass stenoses were created in ten pigs by 25, 50, 75, and 100% flow reduction assessed by transit-time flow measurement (TTFM). Visual angiographic assessment was performed by FCI and CA during baseline and graded bypass stenoses. Altered myocardial perfusion was assessed by quantitative intraoperative fluorescence intensity (QIFI) derived from FCI and correlated to TTFM. Patent bypass grafts and graft occlusion were visualized successfully by FCI and CA, while discrimination between various graded bypass stenosis was possible in 73.3%. The degree of CAB stenosis was overestimated in 16.7% and underestimated in 10.0% by FCI compared to CA. Graded CAB stenosis reduced regional myocardial perfusion quantified by decreased QIFI value (p < 0.001). Mean QIFI value was 76.8 (95% CI 67.2-86.3) during baseline, 55.6 (95% CI 45.3-65.9) during 25% flow-reduction, 30.6 (95% CI 22.3-39.0) during 50% flow-reduction, 20.3 (95% CI 15.4-25.3) during 75% flow-reduction, and 0 during CAB occlusion (p < 0.001) with a significant correlation to TTFM (r = 0.955; p < 0.0001). Solely visual assessment of CAB quality using FCI is limited as compared to CA. Additional QIFI assessment identified graded CAB stenosis and occlusion with a significant correlation to TTFM.

Tear film and ocular surface assessment in psoriasis.

BACKGROUND: Psoriasis is a skin disease with also systemic involvement: its impact on the eye is not well established and often clinically underestimated. Aim of this study was to investigate the presence of ocular discomfort symptoms and of ocular surface changes in a population of patients with psoriasis. METHODS: For this cross-sectional, comparative study, 66 patients with psoriasis were subdivided according to the presence of arthritis and to the use of biological therapy. All patients underwent clinical evaluation with the following tests: Ocular Surface Disease Index Questionnaire, Tearscope examination, meibometry, tear film breakup time, corneal and conjunctival fluorescein staining, Schirmer I test, corneal aesthesiometry, meibomian gland dysfunction (MGD) assessment and conjunctival impression cytology. 28 healthy subjects were also enrolled and treated with the same clinical tests. A statistical analysis of the results was performed. RESULTS: Patients with psoriasis showed a significant deterioration of the ocular surface tests, if compared with healthy subjects, demonstrated by tear film lipid layer alteration, tear film instability, corneal and conjunctival epithelial suffering and mild squamous metaplasia at impression cytology. No differences were found in ocular surface test results of the psoriatic group when patients were divided according to the presence of arthritis, whereas the anti-inflammatory treatment with biological drugs demonstrated a significant improvement of corneal stain and MGD. CONCLUSIONS: Our findings suggest that the ocular surface involvement in patients with psoriasis indicates the need of periodic ophthalmological examinations to diagnose the condition and allow a proper treatment, so contributing to the amelioration of patients' quality of life.

Assessment of dually labelled PEGylated liposomes transplacental passage and placental penetration using a combination of two ex-vivo human models: the dually perfused placenta and the suspended villous explants.

Uptake and passage of nanocarriers through the placenta are critical information to develop new therapeutic approaches during pregnancy. In order to assess nanocarriers transplacental passage and penetration into the placenta, we studied and optimized two ex-vivo human models: the dually perfused placenta and the placenta explants. Doubly labelled PEGylated liposomes were used as models to provide data on the penetration and transplacental passage of drugs and liposomes. A HPLC method was set-up to quantify both carboxyfluorescein and lipid-rhodamine. Transplacental passage was then quantified using HPLC and placental penetration was assessed using spinning disk microscopy. We found a similar transplacental passage rate for both free and encapsulated carboxyfluorescein as well as a homogeneous fluorescence intensity in the outer cell layer of the placental villous, the syncytiotrophoblast, and the mesenchyma. Besides, liposome-rhodamine was not detected in the fetal circulation. The absence of transplacental passage of PEGylated liposomes is also supported by their detection in the sole syncytiotrophoblast. The combination of two ex-vivo models and the monitoring of both the drug and the carrier provided consistent and complementary information. Overall, we suggest combining the perfused human placenta and the human explants villous models to evaluate nanocarriers designed for treatments during pregnancy.

Initial experience with a new quantitative assessment tool for fluorescent imaging in peripheral artery disease.

BACKGROUND: Perioperative evaluation in peripheral artery disease (PAD) by common vascular diagnostic tools is limited by open wounds, medial calcinosis or an altered collateral supply of the foot. Indocyanine green fluorescent imaging (ICG-FI) has recently been introduced as an alternative tool, but so far a standardized quantitative assessment of tissue perfusion in vascular surgery has not been performed for this purpose. The aim of this feasibility study was to investigate a new software for quantitative assessment of tissue perfusion in patients with PAD using indocyanine green fluorescent imaging (ICG-FI) before and after peripheral bypass grafting. PATIENTS AND METHODS: Indocyanine green fluorescent imaging was performed in seven patients using the SPY Elite system before and after peripheral bypass grafting for PAD (Rutherford III-VI). Visual and quantitative evaluation of tissue perfusion was assessed in an area of low perfusion (ALP) and high perfusion (AHP), each by three independent investigators. Data assessment was performed offline using a specially customized software package (Institute for Laser Technology, University Ulm, GmbH). Slope of fluorescent intensity (SFI) was measured as time-intensity curves. Values were compared to ankle-brachial index (ABI), slope of oscillation (SOO), and time to peak (TTP) obtained from photoplethysmography (PPG). RESULTS: All measurements before and after surgery were successfully performed, showing that ABI, TTP, and SOO increased significantly compared to preoperative values, all being statistically significant (P < 0.05), except for TTP (p = 0.061). Further, SFI increased significantly in both ALP and AHP (P < 0.05) and correlated considerably with ABI, TTP, and SOO (P < 0.05). CONCLUSIONS: In addition to ABI and slope of oscillation (SOO), the ICG-FI technique allows visual assessment in combination with quantitative assessment of tissue perfusion in patients with PAD. Ratios related to different perfusion patterns and SFI seem to be useful tools to reduce factors disturbing ICG-FI measurements.

Qualitative assessment of tear dynamics with fluorescein profilometry.

PURPOSE: To describe a new methodology for tear-film dynamics assessment by observing fluorescein decay rate over time and to understand the relationship between the newly defined tear fluorescein washout rate (TFWR) and other measures of the tear film behaviour. METHODS: Forty subjects (24F/16M) aged (mean±standard deviation) 31.8±14.2years volunteered for the study. It consisted of the review of medical history, McMonnies questionnaire (McMQ), slit lamp examination, and TFWR using a newly-developed fluorescein profilometry. The repeatability of TFWR measurements was assessed. TFWR estimates were contrasted against patient age, McMQ score, daytime, fluorescein tear film break-up time (FTBUT), tear meniscus height (TMH) and blink frequency. RESULTS: Mean repeatability of the method was 28.13±9.59%. The group mean TFWR was 39±23% at 30-s mark after the beginning of measurements, ranging from 1.4% to 83%. This indicates that TFWR is highly subject-dependent. Statistically significant correlations were found between the percentage TFWR and McMQ score (r2=0.214, p=0.001) as well as FTBUT (r2=0.136, p=0.009). No statistically significant correlations were found between TFWR and age, daytime, TMH, and blink frequency. CONCLUSIONS: Fluorescein profilometry allows clinicians to follow dynamic changes in the tear film on the entire ocular surface and may be used for qualitative assessment of the tear film dynamics.

Noninvasive Assessment of Elimination and Retention using CT-FMT and Kinetic Whole-body Modeling.

Fluorescence-mediated tomography (FMT) is a quantitative three-dimensional imaging technique for preclinical research applications. The combination with micro-computed tomography (µCT) enables improved reconstruction and analysis. The aim of this study is to assess the potential of µCT-FMT and kinetic modeling to determine elimination and retention of typical model drugs and drug delivery systems. We selected four fluorescent probes with different but well-known biodistribution and elimination routes: Indocyanine green (ICG), hydroxyapatite-binding OsteoSense (OS), biodegradable nanogels (NG) and microbubbles (MB). µCT-FMT scans were performed in twenty BALB/c nude mice (5 per group) at 0.25, 2, 4, 8, 24, 48 and 72 h after intravenous injection. Longitudinal organ curves were determined using interactive organ segmentation software and a pharmacokinetic whole-body model was implemented and applied to compute physiological parameters describing elimination and retention. ICG demonstrated high initial hepatic uptake which decreased rapidly while intestinal accumulation appeared for around 8 hours which is in line with the known direct uptake by hepatocytes followed by hepatobiliary elimination. Complete clearance from the body was observed at 48 h. NG showed similar but slower hepatobiliary elimination because these nanoparticles require degradation before elimination can take place. OS was strongly located in the bones in addition to high signal in the bladder at 0.25 h indicating fast renal excretion. MB showed longest retention in liver and spleen and low signal in the kidneys likely caused by renal elimination or retention of fragments. Furthermore, probe retention was found in liver (MB, NG and OS), spleen (MB) and kidneys (MB and NG) at 72 h which was confirmed by ex vivo data. The kinetic model enabled robust extraction of physiological parameters from the organ curves. In summary, µCT-FMT and kinetic modeling enable differentiation of hepatobiliary and renal elimination routes and allow for the noninvasive assessment of retention sites in relevant organs including liver, kidney, bone and spleen.

Microinjection Technique for Assessment of Gap Junction Function.

Gap junctions are essential for the proper function of many native mammalian tissues including neurons, cardiomyocytes, embryonic tissues, and muscle. Assessing these channels is therefore fundamental to understanding disease pathophysiology, developing therapies for a multitude of acquired and genetic conditions, and providing novel approaches to drug delivery and cellular communication. Microinjection is a robust, albeit difficult, technique, which provides considerable information that is superior to many of the simpler techniques due to its ability to isolate cells, quantify kinetics, and allow cross-comparison of multiple cell lines. Despite its user-dependent nature, the strengths of the technique are considerable and with the advent of new, automation technologies may improve further. This text describes the basic technique of microinjection and briefly discusses modern automation advances that can improve the success rates of this technique.

Liver reserve function assessment by acoustic radiation force impulse imaging.

AIM: To evaluate the utility of liver reserve function by acoustic radiation force impulse (ARFI) imaging in patients with liver tumors. METHODS: Seventy-six patients with liver tumors were enrolled in this study. Serum biochemical indexes, such as aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin (ALB), total bilirubin (T-Bil), and other indicators were observed. Liver stiffness (LS) was measured by ARFI imaging, measurements were repeated 10 times, and the average value of the results was taken as the final LS value. Indocyanine green (ICG) retention was performed, and ICG-K and ICG-R15 were recorded. Child-Pugh (CP) scores were carried out based on patient's preoperative biochemical tests and physical condition. Correlations among CP scores, ICG-R15, ICG-K and LS values were observed and analyzed using either the Pearson correlation coefficient or the Spearman rank correlation coefficient. Kruskal-Wallis test was used to compare LS values of CP scores, and the receiver-operator characteristic (ROC) curve was used to analyze liver reserve function assessment accuracy. RESULTS: LS in the ICG-R15 10%-20% group was significantly higher than in the ICG-R15 < 10% group; and the difference was statistically significant (2.19 ± 0.27 vs 1.59 ± 0.32, P < 0.01). LS in the ICG-R15 > 20% group was significantly higher than in the ICG-R15 < 10% group; and the difference was statistically significant (2.92 ± 0.29 vs 1.59 ± 0.32, P < 0.01). The LS value in patients with CP class A was lower than in patients with CP class B (1.57 ± 0.34 vs 1.86 ± 0.27, P < 0.05), while the LS value in patients with CP class B was lower than in patients with CP class C (1.86 ± 0.27 vs 2.47 ± 0.33, P < 0.01). LS was positively correlated with ICG-R15 (r = 0.617, P < 0.01) and CP score (r = 0.772, P < 0.01). Meanwhile, LS was negatively correlated with ICG-K (r = -0.673, P < 0.01). AST, ALT and T-Bil were positively correlated with LS, while ALB was negatively correlated with LS (P < 0.05). The ROC curve revealed that the when the LS value was 2.34 m/s, the Youden index was at its highest point, sensitivity was 69.2% and specificity was 92.1%. CONCLUSION: For patients with liver tumors, ARFI imaging is a useful tool for assessing liver reserve function.

Use of a fluorescence angiography system in assessment of lower extremity ulcers in patients with peripheral arterial disease: A review and a look forward.

The prevalence of chronic wounds is sharply rising throughout the world due to an aging population and increases in the incidence of obesity, diabetes, and cardiovascular diseases. People with diabetes, hypertension, and hyperlipidemia are at increased risk for developing peripheral arterial disease (PAD). PAD affects 8 to 12 million people over the age of 40 years in the United States and it is a major contributing factor to the development of lower extremity ulcers. Although a number of noninvasive diagnostic tests are available to detect PAD in lower extremities, they have several clinical limitations. In this review, current understanding of the pathophysiology of commonly seen lower extremity ulcers is described and vascular assessments typically used in practice are evaluated. In addition, application of the LUNA Fluorescence Angiography System (Novadaq, Bonita Springs, FL) for the screening and treatment of complex nonhealing wounds in patients with PAD is discussed.

In vivo assessment of HER2 receptor density in HER2-positive tumors by near-infrared imaging, using repeated injections of the fluorescent probe.

HER2 overexpression and amplification of the HER2/neu gene have been found in approximately 25% of invasive breast carcinomas. They are associated with a poor prognosis and resistance to therapy in breast cancer patients. Up to now, clinical evaluation of human epidermal growth factor receptor 2 (HER2) expression is based on ex vivo methods (immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) staining of biopsied tissue). Our goal is to realize "image and treat" paradigm using targeted fluorescent probes to evaluate expression levels of cell biomarkers responsible for cancer progression and to monitor the efficacy of corresponding monoclonal antibody treatments. We used fluorescent Affibody-based probes for in vivo analysis of HER2 receptors using near-infrared optical imaging that do not interfere with binding of the therapeutic agents to these receptors. We have analyzed two types of breast carcinoma xenografts with significant differences in HER2 expression (31 and 21 according to classification) in the mouse model. Using our kinetic model to analyze the temporal variations of the fluorescence intensity in the tumor area after two subsequent injections allowed us to assess quantitatively the difference in HER2 expression levels for two tumor types (BT-474 and MD-MBA-361). This result was substantiated by ELISA ex vivo assays of HER2 expression in the same tumors.