ObjectiveTo analyze the feasibility, advantages and disadvantages of the fluorescence method and the inflation-deflation method in defining the intersegmental plane during thoracoscopic lung segmental resection.MethodsFrom February to October 2018, 60 patients underwent thoracoscopic anatomical segmentectomy in Thoracic Surgery Department of Nanjing Chest Hospital, with 28 males and 32 females, aged from 25 to 82 years. Three-dimension computed tomography bronchography and angiography was used to reconstruct pulmonary vessels, bronchus and virtual intersegmental plane. Among them, 20 patients used the fluorescence method to define the intersegmental plane, and the other 40 patients used the traditional inflation-deflation method to define the intersegmental plane.ResultsFluorescent injection of indocyanine green (ICG) showed a clear intersegmental line with a duration sufficient to complete the label. With the fluorescence method, the intersegmental plane occurrence time was significantly shortened (10.75±3.78 s vs. 988.00±314.24 s, P<0.001) and had satisfactory repeatability. The lungs did not need to be inflated, which was convenient for the operation. And the operation time was shortened (108.75±31.28 min vs 138.00±32.47 min, P=0.002). No obvious ICG injection-related concurrency symptoms was found.ConclusionCompared with the traditional inflation-deflation method, the fluorescence method can display the intersegmental line quickly, accurately and clearly, reduce the difficulty of surgery, shorten the operation time, and provide reliable technical support for thoracoscopic anatomical segmentectomy. The fluorescence is a safe and effective method that is worthy of clinical application.
Objective Using chemically extracted acellular methods to treat extracranial section of the canine whole facial nerve, to evaluated its effects on nerve structure and the removal extent of Schwann cells and myel in. Methods Twenty whole facial nerves were exposed from 10 canines [weighing (18 ± 3) kg]. The extracranial trunk of canine facial nerve and its branches (temporal branch, zygomatic branch, buccal branch, marginal mandibular branch, and cervical branch) were dissected under l ight microscope. Twenty facial nerves were divided into the experimental group (n=12) and control group (n=8) randomly. In experimental group, the nerve was extracted with the 3%TritonX-100 and 4% sodium deoxycholate. In control group, the nerve was not extracted. HE staining and immunofluorescence histological stainings for Hoechst33258, P75, Zero, and Laminin were performed. Results After histological staining, it was found that myel in and Schwann cells were removed from the facial nerve while the basal lamina tube remained intact. The whole canine facial nerves (one nerve trunk and multiple nerve branches) had the similar result. Conclusion The canine whole facial nerve has natural structure (one nerve trunk and multiple nerve branches) by extracted with chemically extracted acellular methods, so it is an available graft for repairing the defect of the whole facial nerve.
Objective To insure early detection and hence efficient prevention of allograft rejection in transplanted heart, investigate possible applications of NAD(P)H fluorescence components analysis at the level of living cardiac cells to propose new approaches for diagnosis of rejection. Methods NAD(P)H was studied for noninvasive fluorescent probing of the mitochondrial function. Human cardiomyocyte were isolated from one additional endomyocardial biopsy (EMB) of 14 pediatric patients with heart ransplantation. Rat cardiomyocyte (n=5, 13-14 week old) were also isolated by the same approach for human myocytes. Autofluorescence(AF) was recorded in living cardiomyocytes following excitation with 375 nm UVlight and detection by spectrallyresolved time correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and lifetimes. Rat cardiac cells were divided into four groups: normoxic condition, normoxia with Rotenone, ischemic condition and ischemia with Rotenone. Comparison of cardiomyocyte AF between human and rat; compared kinetics of rat cardiomyocytes AF in normoxic conditions to ischemiamimicking ones, induced at physiological temperatures by reducing cell pH and oxygen content; comparison of cardiomyocyte AF dynamic changes in transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Results We have achieved appropriate isolation of living cardiomyocytes from human biopsies, as well as from rat cardiac tissues and determined their AF. At least a 3-exponential decay with 0.5-0.7ns, 1.9-2.4 ns and 9.0-15.0 ns lifetime pools is necessary to describe human cardiomyocyte AF within 420560 nm spectral range. Rat cardiomyocyte steadystate AF in ischemiamimicking condition was significantly increased when compared normoxic ones (Plt;0.05); application of Rotenone induced a significant increase in AF intensity in ischemic and normoxic condition, however no significant difference between the two groups (Plt;0.05).Human cardiomyocyte AF was found significantly lower in comparison to experimental rat model in the same condition(Plt;0.05). A correlation between changes in steadystate NAD(P)H fluorescence and rejection grades was found when comparison of R1 to R0. R1 showed significantly increased fluorescence intensity (Plt;0.05), without change in the spectra shape, results can be comparable to the effect of ischemiamimic conditions. Conclusion Our studies clearly demonstrated that spectrallyresolved fluorescence spectral analysis coupled to fluorescence lifetime are high sensitive approaches to examine mitochondrial metabolic oxidative state directly in living human cardiomyocytes with good reproducibility. Human cardiomyocytes are more metabolically active than the rat ones, while this activity (and thus ATP production) seems lowered during rejection process. In perspective, the advantage of this method is the possibility of its combination to multiphoton confocal microscopy, which can result in the adaptation of this approach directly to tissue biopsy, as well as in vivo directly via cardiac catheterization without the necessity of cell isolation. This approach provides promising new tool for clinical diagnosis and treatment of allograft rejection, and will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level.
ObjectiveTo establish a better immunofluorescence protocol to detect co-localization of p53 and mitochondria which may benefit studies aiming to detect mitochondrial expression of proteins.MethodsHeLa cells were treated with hypoxia and the expression of p53 was detected by immunoblotting. HeLa cells were fixed with methanol, methanol: acetone (1: 1, v/v) mixture, and 4% paraformaldehyde, respectively; the former two groups were not permeable, while the latter was penetrated with 0.1% Triton-X 100 and stained with p53 and mitochondria at the same time. After HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 was reduced to 0.05%, 0.025%, 0.01%, and 0.005%. After the HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 decreased to 0.01% and 0.005% for the first time, then, after staining with p53, the mitochondria were stained with 0.1% Triton-X 100 for the second time.ResultsThe expression of p53 was up-regulated (P<0.01) after hypoxia, which could be used in the following immunofluorescence experiment. The co-localization of p53 and mitochondria was observed in the nucleus and cytoplasm in both the methanol group and the mixed solution group. The co-localization of p53 was the most obvious in the mixed solution group. After using 0.1% Triton-X 100, the p53 signal was mainly in the nucleus, but no co-localization was observed. After fixation with 4% paraformaldehyde, to some extent, the reduced concentration of 0.05% and 0.025% Triton-X 100 weakened the p53 signal in nucleus and enhanced the co-localization signal. However, the signal in nucleus was still stronger than that in cytoplasm. When it was reduced to 0.01% and 0.005%, p53 signal was detected in cytoplasm but not in nucleus, suggesting that the nuclear membrane was not penetrated under this condition, but it also failed to penetrate the mitochondrial membrane, leading to the failure of mitochondrial labeling. The second permeability completely avoided the p53 signal in nucleus, and successfully labeled mitochondria, and the co-localization of p53 and mitochondria was detected.ConclusionsCo-localization of p53 and mitochondria is detectable in cells fixed by methanol or methanol and acetone mixture which brings out better results. Penetrating twice with Triton X-100 of different concentrations following paraformaldehyde fixation help avoid signals in nuclei and falicitate co-localization detection.
This consensus aims to introduce the applications of 4K high-definition technology and fluorescence technology in thoracic surgery, summarize and categorize the technical support for pulmonary segment surgery, and innovatively propose technical support for precise sleeve resection of pulmonary segments. It provides a reference for clinical use, points out the direction for the research and innovation of domestically produced high-end endoscopes, promotes the widespread application of excellent domestically produced medical endoscopes, and facilitates the development of domestically produced medical equipment.
Objective To investigate the relationships between circulating tumor cells (CTCs), circulating tumor endothelial cells (CTECs) and treatment methods in patients with nasopharyngeal carcinoma (NPC) at different stages of treatment. Methods The data of NPC patients at different treatment periods in West China Hospital of Sichuan University from March 2016 to November 2019 were retrospectively collected. The patients received CTCs test and part of those patients received CTECs test, by subtraction enrichment-immunostaining-fluorescence in situ hybridization. The relationships of CTCs and CTECs with radiotherapy and chemotherapy, and the correlations between CTCs and CTECs in NPC patients were analyzed. Results A total of 191 patients were included. Among them, there were 66 cases before initial treatment, 38 cases after induction chemotherapy, and 87 cases after concurrent chemoradiotherapy. A total of 127 patients received CTECs test, including 41 cases before initial treatment, 29 cases after induction chemotherapy, and 57 cases after concurrent chemoradiotherapy. The positive rates of CTCs were 89.4%, 81.6% and 69.0% respectively in the three stages of treatment, and the difference was statistically significant only between the pre-treatment group and the post-concurrent chemoradiotherapy group (P=0.003). The number of CTCs in the post-concurrent chemoradiotherapy group was lower than that in the pre-treatment group and the post-induction chemotherapy group (P<0.001, P=0.002). The number of triploid CTCs in the post-concurrent chemoradiotherapy group was significantly different from that in the pre-treatment group and the post-induction chemotherapy group (P=0.009, P=0.013). The number of tetraploid CTCs in the post-concurrent chemoradiotherapy group was significantly different from that in the post-induction chemotherapy group (P=0.007). The number of polyploidy (pentaploid or > 5 copies of chromosome 8) CTCs in the post-concurrent chemoradiotherapy group was significantly different from that in the pre-treatment group (P<0.001). The positive rates of CTECs were 70.7%, 82.8% and 64.9% respectively in the three stages of treatment, and the difference was not statistically significant (P>0.05). The number of CTECs in the post-concurrent chemoradiotherapy group was only lower than that in the post-induction chemotherapy group (P=0.009). There was no significant difference in the number of triploid or tetraploid CTECs among the three groups (P=0.265, P=0.088). The number of polyploid CTECs was statistically different only between the post-concurrent chemoradiotherapy group and the post-induction chemotherapy group (P=0.007). Spearman correlation analysis showed that there was a significant positive correlation between CTCs and CTECs (rs=0.437, P<0.001). Conclusions Concurrent chemoradiotherapy plays a decisive role in reducing the number of CTCs in the blood of NPC patients, while induction chemotherapy does not appear to directly cause changes in the number of CTCs. In NPC patients, different types of CTCs have different responses to different treatments. There is a significant positive correlation between CTECs level and CTCs level in NPC.
ObjectiveTo investigate the application value of indocyanine green (ICG) fluorescence imaging technology for determining the blood supply of parathyroid in thyroid surgery.MethodsThe patients who underwent total thyroidectomy and bilateral central lymph node dissection for papillary thyroid carcinoma (PTC) from June 1, 2017 to January 1, 2018 were prospectively enrolled and then divided into a study group and control group randomly. The study group used the ICG fluorescence imaging technology to evaluate the blood supply of the parathyroid glands, while the control group assessed the blood supply by naked eyes, then determined that whether the parathyroid glands were retained in situ or autotransplanted. The incidence of hypoparathyroidism, length of hospital stay, and parathyroid hormone (PTH) were compared between the two groups.Results① A total of 60 patients with PTC were included in the study, and 30 patients in each group. There were no significant differences in the baseline informations of the two groups such as the gender, age, comorbidities, and preoperative PTH, Ca2+ levels, etc. (P>0.05). ② The ICG score of type A parathyroid glands (except type A3) was lower than that of type B parathyroid glands (0.99±0.38 versus 1.45±0.58, t=–2.395, P<0.05). ③ The length of postoperative hospital stay was shorter in the study group than in the control group (t=–2.159, P=0.035). ④ The ICG fluorescence imaging could significantly reduce the incidence of temporary hypoparathyroidism (χ2=5.079, P=0.024). The incidence of permanent hypoparathyroidism was not statistically different between the two groups (χ2=1.000, P=0.317), and only 1 case appeared in the control group. ⑤ There were no statistically significant differences in the PTH and serum Ca2+ levels at day 1, month 1, month 3, and month 6 after the surgery between the two groups (P>0.05). ConclusionICG fluorescence imaging technology could be used to determine blood supply of parathyroid in situ in real time during operation. Further studies are needed to confirm this conclusion.
Objective To explore the pathological diagnostic value of optical coherence tomography (OCT) in lung cancer. Methods This study selected patients who underwent general anesthesia and electronic bronchoscope biopsy at the Respiratory Endoscopy Center of Sichuan Provincial People’s Hospital from January 1, 2023, to December 1, 2023. White-light bronchoscopy (WLB), auto-fluorescence bronchoscopy (AFB), and OCT examinations were performed in all patients. Lesions were assessed for benign or malignant characteristics based on AFB and OCT before biopsy. The final pathological results were determined according to pathology report. Results A total of 124 patients were included in the study. The accuracy of OCT in differentiating the nature of lesions was 93.55%, significantly higher than AFB (accuracy 83.06%). The accuracy, sensitivity, and specificity of OCT were all higher than AFB. For squamous carcinoma, adenocarcinoma, and small cell lung cancer, the accuracy rates of OCT imaging characteristics were 91.94%, 94.35%, and 94.35%, respectively. Conclusion OCT can improve the accuracy of pre-bronchoscopic tissue pathology biopsy in determining the nature of lesions and provide rapid pathological typing basis, potentially further promoting the development of non-invasive histological biopsy.
Objective To analyze the variation of intestinal microflora in patients with colorectal cancer by SYBR GreenⅠreal-time fluorescence quantitative PCR and reveal the role and significance of intestinal microflora in the colorectal cancer-associated molecular pathogenesis. Methods A set of 16S rRNA gene group of species-specific primers for Bifidobacterium spp., Lactobacillus group, Escherichia coli, and ddl gene-targeted species-specific primers for Enterococcus faecalis and feces Enterococcus were designed. Patients with colorectal cancer (colorectal cancer group, n=30) and healthy volunteers (normal control group, n=30) were included and whose feces were collected to extract bacterial genome DNA. SYBR GreenⅠ real-time fluorescence quantitative PCR was used to analyze the five mentioned bacterial amounts. Results Level of Bifidobacterium spp. (4.52±0.49) and Lactobacillus group (5.46±0.12) in colorectal cancer group were significantly lower than those （9.25±0.83 and 7.45±0.37） of normal control group (Plt;0.05), whereas levels of Escherichia coli (5.82±0.47), Enterococcus faecalis (10.6±0.30) and feces Enterococcus (5.74±0.16) in colorectal cancer group were significantly higher than those （4.68±0.32, 4.95±0.24, and 5.03±0.43） of normal control group (Plt;0.05). Conclusions The fecal microflora composition of patients with colorectal cancer is significantly decreased in Bifidobacterium spp. and Lactobacillus group, whereas increased in Escherichia coli, Enterococcus faecalis, and feces Enterococcus. These data underline that the occurrence and progress of colorectal cancer may be related to intestinal microflora.
In order to protect the integrity and function of the digestive system, duodenum-preserving total pancreatic head resection is becoming the surgical method which was chosen by more and more doctors for benign lesions or low-grade malignant tumors of the pancreatic head. With the development of minimally invasive concepts and techniques, laparoscopic technology has brought unique advantages to this surgery. In this paper, a series of problems such as the development process and indications of laparoscopic duodenum-preserving total pancreatic head resection were discussed, and the core techniques of surgery and how to reduce the occurrence of complications were emphasized. The aim is to improve the therapeutic effect and quality of life of patients through reasonable surgical methods and treatment strategies.