Indocyanine Green Fluorescence-Guided Laparoscopic Anatomical Segmentectomy of Liver Segment 6: Surgical Strategy and Technical Details.

BACKGROUND: Because of the complex anatomy of the right posterior hepatic pedicle, there have been few reports on standardized laparoscopic portal territory staining-guided anatomical resection of liver segment 6 (LPTAR-S6). This study aimed to elucidate the indocyanine green (ICG) fluorescence staining methods for LPTAR-S6. PATIENTS AND METHODS: LPTAR-S6 can be performed using positive and negative fluorescence staining approaches. We implemented these two approaches for patients with hepatocellular carcinoma. Descriptions of the surgical strategy and technical details are presented. RESULTS: Two patients safely underwent LPTAR-S6 using a preoperative three-dimensional reconstruction plan. The intraoperative ICG fluorescence staining effect was satisfactory, and the anatomical landmarks were fully exposed. CONCLUSIONS: A detailed preoperative three-dimensional reconstruction plan, complete intraoperative application of real-time laparoscopic ultrasound guidance, and ICG fluorescence staining can result in accurate transection of the liver parenchyma during LPTAR-S6.

Indocyanine green fluorescence staining based on the "hepatic pedicle first" approach during laparoscopic anatomic liver resection.

BACKGROUND: Indocyanine green (ICG) fluorescence staining is one of the most challenging procedures for laparoscopic anatomic liver resection (LALR). Here, we introduce a novel method based on the "hepatic pedicle first" approach that can improve the success rate of positive staining. METHOD: The target hepatic pedicle (even for the subsegment) was dissected through the first porta until it became visible. Five milliliters of 0.025 mg/ml ICG was injected after the target hepatic pedicle (extra-Glissonian approach) or portal vein/hepatic artery (intra-Glissonian approach) was punctured successfully using scalp acupuncture under direct vision. Then, the Glissonian pedicle or vessel was clamped immediately to prevent the intrahepatic diffusion of ICG. During the operation, a fluorescence imaging model was used repeatedly to confirm the segmental boundary. RESULTS: Finally, 24 patients underwent LALR with the "hepatic pedicle first" approach for ICG fluorescence-positive staining. In 5 patients, ICG-positive staining failed, representing a 79.17% success rate. The average staining time was 25.92 min ± 14.64 min. There were no complications associated with vessel puncture (bile leakage, hemorrhage, and thrombosis). CONCLUSION: The "hepatic pedicle first" approach is a feasible, convenient, and safe method for ICG-positive staining, with a high success rate.

Super-Selective Intra-Arterial Indocyanine Green Administration for Near-Infrared Fluorescence-Based Positive Staining of Hepatic Segmentation: A Feasibility Study.

BACKGROUND: Despite the increasing number of laparoscopic hepatic procedures for the resection of hepatocellular carcinoma (HCC), intraoperative tumor localization and demarcation remains challenging in comparison to open surgery. In this study, we evaluated the feasibility of positive liver segment staining through the super-selective intra-arterial indocyanine green (ICG) administration. METHODS: Eight patients presenting with a single HCC underwent an interventional vascular procedure followed by laparoscopic surgery. A microcatheter was advanced into the hepatic artery branches perfusing the HCC followed by digital subtraction angiography and angiography computed tomography (angio-CT). Patients were then transferred to the operating room, and a laparoscopic hepatectomy was performed under ultrasound guidance. A 5 mL bolus of ICG with a concentration of .125 mg/mL was injected through the microcatheter, and a near-infrared laparoscope was used to detect the fluorescence signal to assess the correspondence between the fluorescence-based demarcation and the intraoperative ultrasound-based demarcation. RESULTS: The duration for the angiography procedure was 32.7 +/- 5.3 min, and it took 242 +/- 118 min from the end of angiography procedure until the start of the surgical procedure. In all cases, the fluorescent liver segment was corresponding to the angio-CT findings. In 6/8 cases, fluorescence imaging was considered helpful in the identification of the resection line. In 3 patients, the resection line was changed according to the positively stained liver segment. CONCLUSION: We successfully demonstrated the feasibility of the super-selective intra-arterial ICG administration for fluorescence-based positive staining of hepatic segmentation during laparoscopic surgery for HCC (NCT04266548).

Superselective intra-arterial hepatic injection of indocyanine green (ICG) for fluorescence image-guided segmental positive staining: experimental proof of the concept.

BACKGROUND: Intraoperative liver segmentation can be obtained by means of percutaneous intra-portal injection of a fluorophore and illumination with a near-infrared light source. However, the percutaneous approach is challenging in the minimally invasive setting. We aimed to evaluate the feasibility of fluorescence liver segmentation by superselective intra-hepatic arterial injection of indocyanine green (ICG). MATERIALS AND METHODS: Eight pigs (mean weight: 26.01 ± 5.21 kg) were involved. Procedures were performed in a hybrid experimental operative suite equipped with the Artis Zeego®, multiaxis robotic angiography system. A pneumoperitoneum was established and four laparoscopic ports were introduced. The celiac trunk was catheterized, and a microcatheter was advanced into different segmental hepatic artery branches. A near-infrared laparoscope (D-Light P, Karl Storz) was used to detect the fluorescent signal. To assess the correspondence between arterial-based fluorescence demarcation and liver volume, metallic markers were placed along the fluorescent border, followed by a 3D CT-scanning, after injecting intra-arterial radiological contrast (n = 3). To assess the correspondence between arterial and portal supplies, percutaneous intra-portal angiography and intra-arterial angiography were performed simultaneously (n = 1). RESULTS: Bright fluorescence signal enhancing the demarcation of target segments was obtained from 0.1 mg/mL, in matter of seconds. Correspondence between the volume of hepatic segments and arterial territories was confirmed by CT angiography. Higher background fluorescence noise was found after positive staining by intra-portal ICG injection, due to parenchymal accumulation and porto-systemic shunting. CONCLUSIONS: Intra-hepatic arterial ICG injection, rapidly highlights hepatic target segment borders, with a better signal-to-background ratio as compared to portal vein injection, in the experimental setting.

Comparing the accuracy of positive and negative indocyanine green staining in guiding laparoscopic anatomical liver resection: protocol for a randomised controlled trial.

INTRODUCTION: Knowledge of the clinical liver anatomy has evolved with advanced imaging modalities and laparoscopic surgery. Therefore, precise anatomical resection knowledge has become the standard treatment for primary and secondary liver cancer. Segmentectomy, a parenchymal-preserving approach, is regarded as an option for anatomical resections in patients with impaired liver. Indocyanine green (ICG) staining is a promising method for understanding the anatomical borders of the liver segments. There are two methods of ICG staining (positive and negative), and the superiority of either approach has not been determined to date. METHODS AND ANALYSIS: This is a prospective randomised controlled superiority clinical trial performed in a single centre tertiary hospital in Japan. A comparison between the accuracy of positive and negative ICG staining in guiding laparoscopic anatomical liver resection is planned in this study. Possible candidates are patients with liver malignant tumours in whom laparoscopic monosegmentectomy or subsegmentectomy is planned. Fifty patients will be prospectively allocated into the following two groups: group A, ICG-negative staining group, and group B, ICG-positive staining group. The optimal dose of ICG for positive staining will be determined during the preparation phase. To assess the ability of the ICG fluorescence guidance in anatomical resection, the primary endpoint is the success rate of ICG staining, which consists of a SOS based on three components: superficial demarcation in the liver surface, visualisation of the parenchymal borders and consistency with the preoperative three-dimensional simulation. The secondary endpoints are the evaluation of short-term surgical outcomes and recurrence-free survival. ETHICS AND DISSEMINATION: The study was approved by Ageo Central General Hospital Clinical Research Ethical Committee (No: 1044) and it carried out following the Declaration of Helsinki (2013 revision). Informed consent will be taken from the patients before participating. The findings will be disseminated through peer-reviewed publications, scientific meetings and conferences. TRIAL REGISTRATION NUMBER: UMIN000049815.

Optimal Dosage of Indocyanine Green Fluorescence for Intraoperative Positive Staining in Laparoscopic Anatomical Liver Resection.

Introduction Fluorescence imaging technology, specifically utilizing indocyanine green (ICG), has emerged as a valuable tool in laparoscopic hepatectomy. In particular, laparoscopic anatomical liver resection (ALR) has benefited from the implementation of both positive and negative staining methods. A case series study reported a success rate of 53% for the positive staining method, citing potential issues regarding the proper ICG dosage needed for accurate fluorescence. Thus, it is crucial to conduct research to investigate the optimal dosage for ICG-positive staining in clinical practice to maximize the benefits of this technique. Materials and methods This retrospective study was conducted at a single center, Meiwa Hospital, and received approval from the hospital's ethics committee in accordance with the Helsinki Declaration. We reviewed the records of 264 patients who underwent open and laparoscopic hepatectomies for benign and malignant liver diseases from January 2019 to January 2023. Of these, 18 patients who underwent laparoscopic ALR with the ICG-positive staining method were evaluated. Fluorescence-emitting segmental borders were assessed immediately after puncture (first stage) and during parenchymal dissection (second stage). In the first stage, we evaluated the intensity of fluorescence emission, categorizing it as "strong" or "weak." The absence of visible fluorescence emission was considered a puncture failure. During the second stage of evaluation, from parenchymal resection to completion, we assessed the sustainability of fluorescence emission, defining it as "clear" or "contaminated." Both evaluations were subjectively judged by three surgeons at our center. The ICG quantity per targeted portal vein-bearing liver volume (mg/100 mL) was calculated for each patient, and the optimal dosage was determined using receiver operating characteristic (ROC) curve analysis. To ascertain the minimum value for adequate fluorescence emission intensity, ROC curve analysis was performed to discriminate between binary outcomes of "strong" or "weak" emission. Furthermore, to establish the maximum value for maintaining a clear fluorescence border, ROC curve analysis was conducted to discriminate between "clear" and "contaminated" during the second evaluation. Results Among the 18 successful puncture cases, the first-stage evaluation of fluorescence intensity revealed 14 punctures with "strong" intensity and four punctures with "weak" intensity. In the second-stage evaluation, 13 cases demonstrated "clear" borders, while five cases exhibited "contaminated" borders. ROC curve analysis was performed to determine the optimal ICG dose for adequate fluorescence intensity and preservation of clear borders during dissection. The analysis indicated that the appropriate ICG dose for achieving optimal intensity was 0.028 mg/100 mL (area under the curve [AUC]: 0.893), while the dose that prevented contamination of fluorescence in non-target areas until after dissection was 0.083 mg/100 mL (AUC: 0.723). Conclusions Laparoscopic anatomical resection using the positive staining method requires an optimal ICG dosage of 0.028-0.083 mg per 100 mL of liver volume. By employing this methodology, more precise and safer laparoscopic anatomical resections can be conducted, thereby enhancing the safety of the surgical procedure for patients.

A novel method of ultrasound-guided positive staining using indocyanine green fluorescence in laparoscopic anatomical liver resection of segments VII and VIII.

Background: Recently, in many Asian centers, laparoscopic anatomical liver resection (LALR) using the indocyanine green (ICG) fluorescence imaging technique has been increasingly applied in resecting hepatocellular carcinoma, even in colorectal liver metastases. However, LALR techniques have not been fully standardized, especially in right superior segments. Due to the anatomical position, prevailing positive staining using a PTCD (percutaneous transhepatic cholangial drainage) needle was superior to negative staining in right superior segments hepatectomy, while it was difficult to manipulate. Herein, we design a novel method of ICG-positive staining for LALR of right superior segments. Methods: Between April 2021 and October 2022, we retrospectively studied patients in our institute who underwent LALR of right superior segments using a novel method of ICG-positive staining, which comprised a customized puncture needle and an adaptor. Compared to the PTCD needle, the customized needle was not limited by the abdominal wall and could be punctured from the liver dorsal surface, which was more flexible to manipulate. The adapter was attached to the guide hole of the laparoscopic ultrasound (LUS) probe to ensure the precise puncture path of the needle. Guided by preoperative three-dimensional (3D) simulation and intraoperative laparoscopic ultrasound imaging, we punctured the transhepatic needle into the target portal vein through the adaptor and then slowly injected 5-10 ml of 0.025 mg/ml ICG solution into the vessel. LALR can be guided by the demarcation line under fluorescence imaging after injection. Demographic, procedural and postoperative data were collected and analyzed. Results: In this study, 21 patients underwent LALR of the right superior segments with ICG fluorescence-positive staining, and the procedures had a success rate of 71.4%. The average staining time was 13.0 ± 6.4 min, the operative time was 230.4 ± 71.7 min, R0 resection was 100%, the postoperative hospital stay was 7.1 ± 2.4 days, and no severe puncture complications occurred. Conclusions: The novel customized puncture needle approach seems to be feasible and safe for ICG-positive staining in LALR of right superior segments, with a high success rate and a short staining time.

Characterizing the Core-Shell Architecture of Block Copolymer Nanoparticles with Electron Microscopy: A Multi-Technique Approach.

Electron microscopy has proved to be a major tool to study the structure of self-assembled amphiphilic block copolymer particles. These specimens, like supramolecular biological structures, are problematic for electron microscopy because of their poor capacity to scatter electrons and their susceptibility to radiation damage and dehydration. Sub-50 nm core-shell spherical particles made up of poly(hydroxyethyl acrylate)-b-poly(styrene) are prepared via polymerization-induced self-assembly (PISA). For their morphological characterization, we discuss the advantages, limitations, and artefacts of TEM with or without staining, cryo-TEM, and SEM. A number of technical points are addressed such as precisely shaping of particle boundaries, resolving the particle shell, differentiating particle core and shell, and the effect of sample drying and staining. TEM without staining and cryo-TEM largely evaluate the core diameter. Negative staining TEM is more efficient than positive staining TEM to preserve native structure and to visualize the entire particle volume. However, no technique allows for a satisfactory imaging of both core and shell regions. The presence of long protruding chains is manifested by patched structure in cryo-TEM and a significant edge effect in SEM. This manuscript provides a basis for polymer chemists to develop their own specimen preparations and to tackle the interpretation of challenging systems.

Immunological response to porcine reproductive and respiratory syndrome virus in young pigs obtained from a PRRSV-positive exposure status herd in a PRRSV endemic area.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), remains a major economic threat to swine production throughout the world. The aim of this study was to investigate the humoral and cell-mediated immune responses to PRRSV in 10 PRRSV vaccinated and 10 non-vaccinated young pigs obtained from a PRRSV-seropositive herd under field conditions. On day 35 days of post-vaccination (dpv), two PRRSV seropositive mixed-litter pigs were added to each group to co-mingle the animals. Serum and whole blood samples were collected from all pigs on the first day of vaccination, as well as on the 21, 35, 49, and 63 dpv. The PRRSV-specific humoral and cell-mediated immune response was determined by ELISA and flow cytometry analysis. The PRRSV ELISA sample to positive (S/P) ratio was found to be positive at the threshold level until the age of 84 days in both non-vaccinated and vaccinated groups, whereas the IFN-γ positive staining cytotoxic (CD8+) cells were rapidly expressed in the early periods of vaccination and co-mingling, but were not found to be specific to PRRSV. This result might have been due to an unspecific response to stress antigens. Further studies should be conducted to obtain more immune response data over long-term observation periods and to study the effect of PRRSV endemic strain vaccinations in endemically-infected herds.

Application value of laparoscopic ultrosound guided puncture and positive staining tech-nique in anatomical right superior segmentectomy / 中华消化外科杂志

Anatomical right superior segmentectomy has always been a difficult surgical procedure in laparoscopic liver resection. How to accurately judge the interface between liver segments, so as to achieve a true sense of anatomic resection based on portal territory is a new requirement in the era of precision liver surgery. The portal vein puncture and indocyanine green (ICG) fluorescence positive staining technique can effectively solve this problem, which has a series of advantages and certain technical requirements in anatomical right superior segmentectomy. ICG fluorescence staining can intuitively define the three-dimensional range of the liver segment, which is a more accurate navigation mode that helps liver surgeons identify the range of the right superior liver segment, thus conducting surgery more safely and normatively. The authors consult relevant studies and combined with clinical practice to explore the application value of laparoscopic ultrosound guided puncture and positive staining technique in anatomical right superior segmentectomy.

Sad State of Phage Electron Microscopy. Please Shoot the Messenger.

Two hundred and sixty publications from 2007 to 2012 were classified according to the quality of electron micrographs; namely as good (71); mediocre (21); or poor (168). Publications were from 37 countries; appeared in 77 journals; and included micrographs produced with about 60 models of electron microscopes. The quality of the micrographs was not linked to any country; journal; or electron microscope. Main problems were poor contrast; positive staining; low magnification; and small image size. Unsharp images were frequent. Many phage descriptions were silent on virus purification; magnification control; even the type of electron microscope and stain used. The deterioration in phage electron microscopy can be attributed to the absence of working instructions and electron microscopy courses; incompetent authors and reviewers; and lenient journals. All these factors are able to cause a gradual lowering of standards.

Study of the transcytosis of an anti-transferrin receptor antibody with a Fab' cargo across the blood-brain barrier in mice.

One strategy used to transport pharmacologically active substances across the blood-brain barrier (BBB) is to link the substance to a molecule capable of crossing the BBB using a receptor-mediated transcellular transport system in brain capillary endothelial cells. The transferrin receptor (TfR) is related to a transcytosis process in these cells, and the 8D3 antibody, directed against mouse TfR, is able to induce a receptor response. In this work, the potential of 8D3 to carry molecules across the BBB was investigated. This antibody (either unlabeled or FITC-labeled) was intravenously administered to ICR-CD1 mice. Immunocomplexes (ICs) consisting of 8D3 antibody (carrier) and Fab' fragments (simulated cargo), in some cases directly fluorolabeled with FITC, were also administered to ICR-CD1 mice. At different time-points of recirculation, the IC components were studied using immunohistochemical procedures and fluorescence resonance energy transfer (FRET). The results suggested that 8D3 alone or in an IC with Fab' fragments entered the endothelial cells. FRET analysis indicated that the colocalization of their fluorescent signals inside the endothelial cells decreased with time, indicating that ICs can be processed and Fab' fragments probably separated from 8D3. However, neither 8D3 alone nor the IC components crossed the BBB.

High-Resolution Single-Molecule Structure Revealed by Electron Microscopy and Individual Particle Electron Tomography.

The protein is naturally dynamic and heterogeneous in solution. Protein dynamics involves both equilibrium fluctuations that regulate biological function and other non-equilibrium effects of biological motors, which convert chemical energy to mechanical energy. However, a single, unique structure of protein determined from X-ray crystal and conventional single-particle electron microscopy is insufficient to encompass the dynamic nature of proteins in solution. Structure determination of dynamic and heterogeneous protein is essentially required the determination of each individual particle of protein. Recently, Drs. Gang Ren and Lei Zhan published the first single molecule three-dimensional (3D) EM images of individual proteins ever obtained with enough clarity to determine their structure, an IgG antibody (14 Å resolution) and a 17nm HDL (36 Å resolution). These results depended upon four innovations: i) improved cryo-electron microscopy (cryoEM) sample preparation and Electron microscopy (EM) operation conditions resulted in the successful imaging of a 17 nm HDL particle (120-200kDa) by cryo-electron tomography (cryoET); ii) developed an optimized NS (OpNS) protocol that eliminates the rouleau artifact that has plagued EM research for three decades. This OpNS protocol provides high-contrast single lipoprotein images with similar size (<5%) and shape (<5%) to that seen by cryoEM; iii) developed a high-resolution and high contrast sample preparation protocol, cryo-positive-staining (cryoPS) that allows direct visualization of the secondary structure of a small protein, such as the ß-strands in CETP and the helical double belt of apoA-I in spherical HDL; iv) developed a robust tomography reconstruction method, Individual Particle Electron Tomography (IPET) that is a high-resolution, high throughput reconstruction method that, to the best of our knowledge, is the only method for determining an individual protein structure. Remarkably, IPET went against the conventional wisdom that a single protein can NOT be reconstructed by EM and this opens a door for the study of protein dynamics via a particle-by-particle structural comparison.