Light transmittance in human atrial tissue and transthoracic illumination in rats support translatability of optogenetic cardioversion of atrial fibrillation.

BACKGROUND: Optogenetics could offer a solution to the current lack of an ambulatory method for the rapid automated cardioversion of atrial fibrillation (AF), but key translational aspects remain to be studied. OBJECTIVE: To investigate whether optogenetic cardioversion of AF is effective in the aged heart and whether sufficient light penetrates the human atrial wall. METHODS: Atria of adult and aged rats were optogenetically modified to express light-gated ion channels (i.e., red-activatable channelrhodopsin), followed by AF induction and atrial illumination to determine the effectivity of optogenetic cardioversion. The irradiance level was determined by light transmittance measurements on human atrial tissue. RESULTS: AF could be effectively terminated in the remodeled atria of aged rats (97%, n = 6). Subsequently, ex vivo experiments using human atrial auricles demonstrated that 565-nm light pulses at an intensity of 25 mW/mm2 achieved the complete penetration of the atrial wall. Applying such irradiation onto the chest of adult rats resulted in transthoracic atrial illumination as evidenced by the optogenetic cardioversion of AF (90%, n = 4). CONCLUSION: Transthoracic optogenetic cardioversion of AF is effective in the aged rat heart using irradiation levels compatible with human atrial transmural light penetration.

The Impact of Reoperations Following Bariatric Surgery on Mid-term Outcomes.

PURPOSE: With the obesity epidemic, the number of bariatric procedures is increasing, and although considered relatively safe, major postoperative complications still occur. In cancer surgery, major complications such as reoperations have been associated with deteriorated mid/long-term outcomes. In obesity surgery, the effects of reoperations on postoperative weight loss and associated comorbidities remain unclear. The aim of this study was to assess mid-term weight loss and comorbidities following early reoperations in obesity surgery. METHODS: A population-based cohort study was performed within the Dutch Audit for Treatment of Obesity (DATO), including all patients that underwent a primary gastric bypass procedure or sleeve gastrectomy. Follow-up data was collected up until 5 years postoperatively on percentage total weight loss (%TWL) and comorbidities. RESULTS: A total of 40,640 patients underwent a gastric bypass procedure or sleeve gastrectomy between 2015 and 2018. Within this cohort, 709 patients (1.7%) suffered a major complication requiring reoperation within 30 days. %TWL at 24 months was 33.1 ± 9.2 in the overall population, versus 32.9 ± 8.7 in the patients who underwent a reoperation (p=0.813). Both analysis per year and Cox regression techniques revealed no differences in long-term follow-up regarding percentage TLW, and weight loss success rates (%TWL>20%) in patients who underwent a reoperation compared to patients without reoperation. At 5 years, the availability of follow-up data was low. No differences were observed in the remission of comorbidities. DISCUSSION: Major complications requiring reoperation within 30 days of gastric bypass surgery or sleeve gastrectomy did not affect long-term outcomes with regard to weight loss or remission of comorbidities.

Risk factors for postoperative bleeding in bariatric surgery.

BACKGROUND: Postoperative bleeding remains a relatively common complication following bariatric surgery and may lead to morbidity and even mortality. OBJECTIVE: To develop a prediction model to identify patients at risk for postoperative bleeding. SETTING: Rode Kruis Ziekenhuis, Beverwijk, the Netherlands. Based on Dutch nationwide obesity audit data. METHODS: Patients undergoing primary bariatric surgery were selected from January 2015 to December 2020 from the Dutch Audit for Treatment of Obesity. The primary outcome was postoperative bleeding within 30 days. Assessed predictors included patient factors and operative data. A prediction model was developed using backward stepwise logistic regression. Internal validation was performed using bootstrapping techniques. RESULTS: A total of 59,055 patients were included; 13,399 underwent a sleeve gastrectomy, and 45,656 underwent a gastric bypass procedure. Postoperative bleeding occurred in 1.5%. The following predictors were identified: male patients (odds ratio [OR] = 1.40; 95% confidence interval [CI]: 1.21-1.63), patients >45 years of age (OR = 1.50; 95% CI: 1.29-1.76), body mass index <40 kg/m2 (OR = 1.22; 95% CI: 1.06-1.41), cardiovascular disease (OR = 1.36; 95% CI: 1.17-1.57), and sleeve gastrectomy (OR = 1.43; 95% CI: 1.24-1.67). Area under the curve for the model was .612. Following bootstrapping for internal validation, a correction of .9817 was applied. CONCLUSION: A clinical decision rule was designed to assess the risk of postoperative bleeding in patients undergoing bariatric surgery. If 3 or more risk factors are present, there is an increased risk for postoperative bleeding. The model can aid in clinical decision-making: implementing extra preventative measures in high-risk patients. External validation is needed to further develop the model.

Conditional immortalization of human atrial myocytes for the generation of in vitro models of atrial fibrillation.

The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6-8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.

Long-term follow-up of thoracoscopic ablation in long-standing persistent atrial fibrillation.

OBJECTIVES: Catheter ablation of long-standing persistent atrial fibrillation (LSPAF) remains challenging, with suboptimal success rates obtained following multiple procedures. Thoracoscopic ablation has shown effective at creating transmural lesions around the pulmonary veins and box; however, long-term rhythm follow-up data are lacking. This study aims, for the first time, to assess the long-term outcomes of thoracoscopic pulmonary vein and box ablation in LSPAF. METHODS: Rhythm follow-up consisted of continuous rhythm monitoring using implanted loop recorders or 24-h Holter recordings. Rhythm status and touch-up interventions were assessed up to 5 years. RESULTS: Seventy-seven patients with symptomatic LSPAF underwent thoracoscopic ablation in 2 centres. Freedom from atrial arrhythmias at 5 years was 50% following a single thoracoscopic procedure and 68% allowing endocardial touch-up procedures (performed in 21% of patients). The mean atrial fibrillation burden in patients with continuous monitoring was reduced from 100% preoperatively to 0.1% at the end of the blanking period and 8.0% during the second year. Antiarrhythmic drug use decreased from 49.4% preoperative to 12.1% and 14.3% at 2 and 5 years, respectively (P < 0.001). Continuous rhythm monitoring resulted in higher recurrence detection rates compared to 24-h Holter monitoring at 2-year follow-up (hazard ratio: 6.5, P = 0.003), with comparable recurrence rates at 5-year follow-up. CONCLUSIONS: Thoracoscopic pulmonary vein and box isolation are effective in long-term restoration of sinus rhythm in LSPAF, especially when complemented by endocardial touch-up procedures, as demonstrated by the 68% freedom rate at 5 years. Continuous rhythm monitoring revealed earlier, but not more numerous documentation of recurrences at 5-year follow-up.

Self-restoration of cardiac excitation rhythm by anti-arrhythmic ion channel gating.

Homeostatic regulation protects organisms against hazardous physiological changes. However, such regulation is limited in certain organs and associated biological processes. For example, the heart fails to self-restore its normal electrical activity once disturbed, as with sustained arrhythmias. Here we present proof-of-concept of a biological self-restoring system that allows automatic detection and correction of such abnormal excitation rhythms. For the heart, its realization involves the integration of ion channels with newly designed gating properties into cardiomyocytes. This allows cardiac tissue to i) discriminate between normal rhythm and arrhythmia based on frequency-dependent gating and ii) generate an ionic current for termination of the detected arrhythmia. We show in silico, that for both human atrial and ventricular arrhythmias, activation of these channels leads to rapid and repeated restoration of normal excitation rhythm. Experimental validation is provided by injecting the designed channel current for arrhythmia termination in human atrial myocytes using dynamic clamp.

Clamping versus nonclamping thoracoscopic box ablation in long-standing persistent atrial fibrillation.

OBJECTIVE: To compare clinical outcomes of clamping devices and linear nonclamping devices for isolation of the posterior left atrium (box) in thoracoscopic ablation of long-standing persistent atrial fibrillation. METHODS: Eighty patients who underwent thoracoscopic pulmonary vein and box isolation using a bipolar clamping device (42 patients) or bipolar nonclamping device (38 patients) to create the roof/inferior lesions for box isolation were included from 2 centers. Follow-up consisted of 24-hour Holter at regular intervals. Freedom from AF during 1-year follow-up and catheter repeat interventions were compared between groups. RESULTS: Acute intraoperative electrical isolation of the box compartment was significantly higher in the clamping group than in the nonclamping group (100% and 79%, respectively, P < .01). At 1-year follow-up, 91% of the clamping group and 79% of the nonclamping group were in sinus rhythm. During 1-year follow-up, recurrence rates did not significantly differ between the 2 groups (P = .08). Repeat catheter interventions were required in 10% of the clamping group and 21% of the nonclamping group (P = .15). Conduction gaps in the roof or inferior lesions were found in 1 patient (2%) in the clamping group versus 4 patients (11%) in the nonclamping group (P = .13). CONCLUSIONS: Thoracoscopic pulmonary vein and box isolation are highly effective in restoring sinus rhythm in long-standing persistent atrial fibrillation on short-term follow-up. Comparison of clamping and nonclamping devices revealed lower rates of intraoperative exit block of the box in the nonclamping group. However, this did not translate into a significant difference in atrial fibrillation freedom at short-term (1-year) follow-up.

Molecular fluorescence-guided surgery of peritoneal carcinomatosis of colorectal origin: a single-centre feasibility study.

BACKGROUND: Optimum cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is essential for the curative treatment of peritoneal carcinomatosis of colorectal origin. At present, surgeons depend on visual inspection and palpation for tumour detection. Improved detection of tumour tissue using molecular fluorescence-guided surgery could not only help attain a complete cytoreduction of metastatic lesions, but might also prevent overtreatment by avoiding resection of benign lesions. METHODS: For this non-randomised, single-centre feasibility study, we enrolled patients with colorectal peritoneal metastases scheduled for cytoreductive surgery and HIPEC. 2 days before surgery, 4·5 mg of the near-infrared fluorescent tracer bevacizumab-IRDye800CW was administered intravenously. The primary objectives were to determine the safety and feasibility of molecular fluorescence-guided surgery using bevacizumab-IRDye800CW. Molecular fluorescence-guided surgery was deemed safe if no allergic or anaphylactic reactions were recorded and no serious adverse events were attributed to bevacizumab-IRDye800CW. The technique was deemed feasible if bevacizumab-IRDye800CW enabled detection of fluorescence signals intraoperatively. Secondary objectives were correlation of fluorescence with histopathology by back-table imaging of the fresh surgical specimen and semi-quantitative ex-vivo analyses of formalin-fixed paraffin embedded (FFPE) tissue on all peritoneal lesions. Additionally, VEGF-α staining and fluorescence microscopy was done. This study is registered with the Netherlands Trial Registry, number NTR4632. FINDINGS: Between July 3, 2014, and March 2, 2015, seven patients were enrolled in the study. One patient developed an abdominal sepsis 5 days postoperatively and another died from an asystole 4 days postoperatively, most probably due to a cardiovascular thromboembolic event. However, both serious adverse events were attributed to the surgical cytoreductive surgery and HIPEC procedure. No serious adverse events related to bevacizumab-IRDye800CW occurred in any of the patients. Intraoperatively, fluorescence was seen in all patients. In two patients, additional tumour tissue was detected by molecular fluorescence-guided surgery that was initially missed by the surgeons. During back-table imaging of fresh surgical specimens, a total of 80 areas were imaged, marked, and analysed. All of the 29 non-fluorescent areas were found to contain only benign tissue, whereas tumour tissue was detected in 27 of 51 fluorescent areas (53%). Ex-vivo semi-quantification of 79 FFPE peritoneal lesions showed a tumour-to-normal ratio of 6·92 (SD 2·47). INTERPRETATION: Molecular fluorescence-guided surgery using the near-infrared fluorescent tracer bevacizumab-IRDye800CW is safe and feasible. This technique might be of added value for the treatment of patients with colorectal peritoneal metastases through improved patient selection and optimisation of cytoreductive surgery. A subsequent multicentre phase 2 trial is needed to make a definitive assessment of the diagnostic accuracy and the effect on clinical decision making of molecular fluorescence-guided surgery. FUNDING: FP-7 Framework Programme BetaCure and SurgVision BV.

Distribution of Matrix Metalloproteinases in Human Atherosclerotic Carotid Plaques and Their Production by Smooth Muscle Cells and Macrophage Subsets.

PURPOSE: In this study, the potential of matrix metalloproteinase (MMP) sense for detection of atherosclerotic plaque instability was explored. Secondly, expression of MMPs by macrophage subtypes and smooth muscle cells (SMCs) was investigated. PROCEDURES: Twenty-three consecutive plaques removed during carotid endarterectomy were incubated in MMPSense™ 680 and imaged with IVIS® Spectrum. mRNA levels of MMPs, macrophage markers, and SMCs were determined in plaque specimens, and in in vitro differentiated M1 and M2 macrophages. RESULTS: There was a significant difference between autofluorescence signals and MMPSense signals, both on the intraluminal and extraluminal sides of plaques. MMP-9 and CD68 messenger RNA (mRNA) expression was higher in hot spots, whereas MMP-2 and αSMA expression was higher in cold spots. In vitro M2 macrophages had higher mRNA expression of MMP-1, MMP-9, MMP-12, and TIMP-1 compared to M1 macrophages. CONCLUSION: MMP-9 is most dominantly MMP present in atherosclerotic plaques and is produced by M2 rather than M1 macrophages.

Multispectral optoacoustic tomography of matrix metalloproteinase activity in vulnerable human carotid plaques.

AIMS: Elevated expression of cathepsins, integrins and matrix metalloproteinases (MMPs) is typically associated with atherosclerotic plaque instability. While fluorescent tagging of such molecules has been amply demonstrated, no imaging method was so far shown capable of resolving these inflammation-associated tags with high fidelity and resolution beyond microscopic depths. This study is aimed at demonstrating a new method with high potential for noninvasive clinical cardiovascular diagnostics of vulnerable plaques using high-resolution deep-tissue multispectral optoacoustic tomography (MSOT) technology. METHODS AND RESULTS: MMP-sensitive activatable fluorescent probe (MMPSense™ 680) was applied to human carotid plaques from symptomatic patients. Atherosclerotic activity was detected by tuning MSOT wavelengths to activation-dependent absorption changes of the molecules, structurally modified in the presence of enzymes. MSOT analysis simultaneously provided morphology along with heterogeneous MMP activity with better than 200 micron resolution throughout the intact plaque tissue. The results corresponded well with epi-fluorescence images made from thin cryosections. Elevated MMP activity was further confirmed by in situ zymography, accompanied by increased macrophage influx. CONCLUSIONS: We demonstrated, for the first time to our knowledge, the ability of MSOT to provide volumetric images of activatable molecular probe distribution deep within optically diffuse tissues. High-resolution mapping of MMP activity was achieved deep in the vulnerable plaque of intact human carotid specimens. This performance directly relates to pre-clinical screening applications in animal models and to clinical decision potential as it might eventually allow for highly specific visualization and staging of plaque vulnerability thus impacting therapeutic clinical decision making.

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results.

The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90-95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α-targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α-targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.

Enhancing surgical vision by using real-time imaging of αvß3-integrin targeted near-infrared fluorescent agent.

BACKGROUND: This study was designed to improve the surgical procedure and outcome of cancer surgery by means of real-time molecular imaging feedback of tumor spread and margin delineation using targeted near-infrared fluorescent probes with specificity to tumor biomarkers. Surgical excision of cancer often is confronted with difficulties in the identification of cancer spread and the accurate delineation of tumor margins. Currently, the assessment of tumor borders is afforded by postoperative pathology or, less reliably, intraoperative frozen sectioning. Fluorescence imaging is a natural modality for intraoperative use by directly relating to the surgeon's vision and offers highly attractive characteristics, such as high-resolution, sensitivity, and portability. Via the use of targeted probes it also becomes highly tumor-specific and can lead to significant improvements in surgical procedures and outcome. METHODS: Mice bearing xenograft human tumors were injected with αvß3-integrin receptor-targeted fluorescent probe and in vivo visualized by using a novel, real-time, multispectral fluorescence imaging system. Confirmatory ex vivo imaging, bioluminescence imaging, and histopathology were used to validate the in vivo findings. RESULTS: Fluorescence images were all in good correspondence with the confirming bioluminescence images in respect to signal colocalization. Fluorescence imaging detected all tumors and successfully guided total tumor excision by effectively detecting small tumor residuals, which occasionally were missed by the surgeon. Tumor tissue exhibited target-to-background ratio of ~4.0, which was significantly higher compared with white-light images representing the visual contrast. Histopathology confirmed the capability of the method to identify tumor negative margins with high specificity and better prediction rate compared with visual inspection. CONCLUSIONS: Real-time multispectral fluorescence imaging using tumor specific molecular probes is a promising modality for tumor excision by offering real time feedback to the surgeon in the operating room.

Intraoperative multispectral fluorescence imaging for the detection of the sentinel lymph node in cervical cancer: a novel concept.

PURPOSE: Real-time intraoperative near-infrared fluorescence (NIRF) imaging is a promising technique for lymphatic mapping and sentinel lymph node (SLN) detection. The purpose of this technical feasibility pilot study was to evaluate the applicability of NIRF imaging with indocyanin green (ICG) for the detection of the SLN in cervical cancer. PROCEDURES: In ten patients with early stage cervical cancer, a mixture of patent blue and ICG was injected into the cervix uteri during surgery. Real-time color and fluorescence videos and images were acquired using a custom-made multispectral fluorescence camera system. RESULTS: Real-time fluorescence lymphatic mapping was observed in vivo in six patients; a total of nine SLNs were detected, of which one (11%) contained metastases. Ex vivo fluorescence imaging revealed the remaining fluorescent signal in 11 of 197 non-sentinel LNs (5%), of which one contained metastatic tumor tissue. None of the non-fluorescent LNs contained metastases. CONCLUSIONS: We conclude that lymphatic mapping and detection of the SLN in cervical cancer using intraoperative NIRF imaging is technically feasible. However, the technique needs to be refined for full applicability in cervical cancer in terms of sensitivity and specificity.

Multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases. Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema. As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor. In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix. The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid. Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion. It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used. The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.