[Clinical value of routine contrast esophagram in the diagnosis of anastomotic leakage for three-incision esophagectomy with cervical anastomosis].

Objective: To examine the clinical value of routine contrast esophagram (RCE) for the diagnosis of anastomotic leakage (AL) after three-incision esophagectomy with cervical anastomosis. Methods: Clinical data of 1 022 patients with esophageal cancer who underwent McKeown three-incision esophagectomy with cervical anastomosis from January 2015 to December 2019 at Department of Minimally Invasive Esophageal Surgery, Tianjin Medical University Cancer Hospital and Institute were analyzed retrospectively. There were 876 males and 146 females, aging(M(IQR)) 48(16) years (range: 36 to 84 years). There were 253 patients (24.8%) with neoadjuvant therapy, and 817 patients (79.9%) with minimally invasive esophagectomy. According to the diagnosis and treatment habits of the attending surgeons, 333 patients were included in the RCE group, and RCE was performed on the 7th day postoperative, while 689 patients were included in the non-RCE group, and RCE was performed when the patients had suspicious symptoms. Taking clinical symptoms, RCE, CT, endoscopy and other methods as reference to the diagnosis of AL, the sensitivity and specificity were used to analyze and evaluate the efficacy of RCE for the diagnosis of AL. The data were compared by U test or χ² test between groups. Results: The incidence rate of AL after three-incision esophagectomy was 7.34% (75/1 022), including 30 cases in the RCE group and 45 cases in the non-RCE group (9.0%(30/333) vs. 6.5%(45/689), χ²=2.027, P=0.155). The diagnostic time of AL was 9(5) days postoperative (range: 4 to 30 days). Among them, 23 cases showed cervical leakages, 50 cases showed intro-thoracic leakages, and 2 cases both cervical and intro-thoracic leakages. The diagnostic time of patients with intro-thoracic leakages was longer than that of cervical leakages (10(4) days vs. 6(3) days, Z=-2.517, P=0.012). Among the 333 patients in the RCE group, 16 cases of RCE indicated leakages including 11 cases of true positive and 5 cases determined to be false positive, while 317 cases indicated no abnormalities including 19 cases developed leakages. The sensitivity and specificity of RCE to detect AL were 36.7%(11/30) and 98.3%(298/333), respectively. The Youden-index was 0.35, and the diagnostic accuracy was 92.8%(309/333). The positive and negative predictive value were 11/16 and 94.0%(298/317), respectively. Conclusions: Routine contrast esophagram after three-incision esophagectomy with cervical anastomosis has low sensitivity and high specificity in the diagnosis of AL. The diagnostic time of AL is the 9th day after surgery. It is necessary to prolong the observation time clinically, and combine RCE with CT, endoscopy and other inspection methods for diagnosis.

Ultrasonic detection and developmental changes in calcification of the placenta during normal pregnancy in mice.

High resolution ultrasound imaging of the mouse placenta during development revealed highly echogenic foci localized near the materno-placental interface in early gestation and, near term, in the placental labyrinth (the exchange region of the placenta). Echogenic foci and calcium deposits identified in histological sections using Alizarin red staining showed similar localization and changes with gestation. Calcium deposits caused the echogenic foci because incubating uteri in a decalcifying solution eliminated both the deposits and echogenic foci. Transmission electron microscopy, X-ray microanalysis, and electron diffraction were used to show that deposits were calcium hydroxyapatite crystals. Calcium deposits were extensive and densely packed at days 7.5-9.5 of gestation at the border between the maternal decidua and the fetal trophoblast giant cells of ectoplacental cone. After the formation of the chorio-allantoic placenta (approximately day 10.5), calcification deposits appeared larger and more rarefied but were still localized at the border between the maternal decidua and the fetal trophoblast giant cells of the placenta. Calcification deposits were not observed in the labyrinthine region of the mouse placenta until > or = day 15.5 (day 18.5 is full term). We conclude that deposits of calcium hydroxyapatite crystals in the mouse placenta are detectable by high resolution ultrasound imaging. These deposits provide an ultrasound detectable marker of the maternal-placental interface that is particularly prominent during the establishment of the chorio-allantoic placenta between days 7.5 and 9.5 of gestation.

Glial cell line-derived neurotrophic factor promotes proliferation of neuroglioma cells by up-regulation of cyclins PCNA and Ki-67.

OBJECTIVE: We wished to test whether glial cell line-derived neurotrophic factor (GDNF) stimulates proliferation of gliomas by up-regulating expression of nuclear cyclins PCNA and Ki37. MATERIALS AND METHODS: As a model, we tested rat C6 glioma cell line exposed to basal conditions, vehicle control, or exogenous GDNF at different concentrations (0-90 µg/L) or different times (0-72 hours). Cell proliferation was quantified by MTT test, cell cycle by flow cytometry and propidium iodide staining, expression of PCNA and Ki67 by intracellular antibody staining and flow cytometry. RESULTS: We first observed that cell proliferation was most stimulated by GDNF at concentration of 70 µg/L and incubation time of 48 hours. Using this concentration and incubation time, we next documented that GDNF increased the percentage of cells in the S phase (47.98% vs. 32.57% in basal cells; p < 0.05), while not affecting the percentage of cells in G0/G1 or G2/M phases. Finally, we demonstrated that expression of both PCNA and Ki67 was significantly increased in cells exposed to GDNF. CONCLUSIONS: We demonstrate that GDNF stimulates proliferation of glioma cells by up-regulating expression of cyclins PCNA and Ki-67.

Applications for multifrequency ultrasound biomicroscopy in mice from implantation to adulthood.

A new multifrequency (19-55 MHz) ultrasound biomicroscope with two-dimensional imaging and integrated Doppler ultrasound was evaluated using phantoms and isoflurane-anesthetized mice. Phantoms revealed the biomicroscope's lateral resolution was between 50 and 100 microm, whereas that of a conventional 13 MHz ultrasound system was 200-500 microm. This difference was apparent in the markedly higher resolution images achieved using the biomicroscope in vivo. Transcutaneous images of embryos in pregnant mice from approximately 2 days after implantation (7 days gestation) to near term (17.5 days) were obtained using frequencies from 25 to 40 MHz. The ectoplacental cone and early embryonic cavities were visible as were the placenta and embryonic organs throughout development to term. We also evaluated the ability of the biomicroscope to detect important features of heart development by examining embryos from 8.5 to 17.5 day gestation in exteriorized uteri using 55 MHz ultrasound. Cardiac looping, division of the outflow tract, and ventricular septation were visible. In postnatal imaging, we observed the heart and kidney of neonatal mice at 55 MHz, the carotid artery in juveniles (approximately 8 g body wt) and adults (approximately 25 g body wt) at 40 MHz, and the adult heart, aorta, and kidney at 19 MHz. The coefficient of variation of carotid and aortic diameter measurements was 1-3%. In addition, blisters in GRIP1 -/- embryos and aortic valvular stenosis in two adults were readily visualized. Using image-guided Doppler function, low blood velocities in vessels as small as 100 microm in diameter including the primitive heart tube at day 8.5 were measurable, but high blood velocities (>37.5 cm/s) such as in the heart and large arteries in late gestation and postnatal life were off-scale. Accurate cardiac dimension measurements were impeded by poor temporal resolution (4 frames/s). In summary, the multifrequency ultrasound biomicroscope is a versatile tool well suited to detailed study of the morphology of various organ systems throughout development in mice and for hemodynamic measurements in the low velocity range.

Expression of rat oligodendrocyte transcription factor 2 in COS-7 cells following its eukaryotic expression vector construction.

OBJECTIVES: The basic HLH transcription factor Olig is a key regulator for differentiating the oligodendrocyte lineage cells during development. Oligodendrocyte transcription factor 2 (Olig2) plays a crucial role in differentiating the oligodendrocytes in the spinal cord. We aimed to construct and investigate the eukaryotic expression recombinant plasmid in the rat Olig2. DESIGN, TIME AND SETTING: The experiment was performed at the Laboratory of Neurobiology, Xuzhou Medical College from October 2011 to March 2012. MATERIALS AND METHODS: The pEGFP-N1 vector was purchased from Invitrogen. JM101 competent cells and COS-7 cells were preserved at the Laboratory of Neurobiology, Xuzhou Medical College, China. The Olig2 cDNA fragment was cloned by RT-PCR with the total RNA from the neonatal rat spinal cord, and subsequently cloned into pGEM-T vector. The confirmed Olig2 fragment was then cloned into the pEGFP-N1 vector. The right recombinant was transfected into COS-7 cells by lipofectamine 2000. The expression of the Olig2 in COS-7 cells was detected by RT-PCR and immunoblot analysis. Enzyme digestion and sequencing of the recombinant plasmid; and expression of the Olig2 were analyzed by fluorescence microscope and western blot. RESULTS: The correct pEGFP-N1-Olig2 cloning was verified by restriction endonuclease digestion and sequencing. The western blot analysis indicated that the Olig2-GFP fusion protein was expressed in the COS-7/pEGFP-N1-Olig2 cells at 72 h. CONCLUSIONS: The pEGFP-N1-Olig2 vector was constructed successfully. The Olig2-GFP fusion protein was expressed in the COS-7/pEGFP-N1-Olig2 cells. This study lays the foundation for further research in gene therapy for central nervous system demyelinating diseases.

Expression of cyclinD1 and Ki-67 proteins in gliomas and its clinical significance.

OBJECTIVES: To investigate the expression of cyclinD1 and Ki-67 proteins in gliomas and its significance. PATIENTS AND METHODS: The immunohistochemistry was used to detect the expression of cyclinD1 and Ki-67 proteins in 18 cases of normal brain tissues, 32 cases of low-grade gliomas, and 24 cases of high-grade gliomas. RESULTS: The cyclinD1 positive ratio in normal brain tissues, low-grade gliomas, and high-grade gliomas were 4/18, 15/32, and 18/24, respectively, with statistically significant difference (p < 0.05). Differences were significant by pairwise comparison between normal brain tissue with high-grade gliomas and low-grade gliomas with high-grade glioma groups (p < 0.01). However, there was no significant differences between normal brain tissue with low-grade gliomas. The Ki-67 positive ratio in normal brain tissues, low-grade gliomas, and high-grade gliomas were 5/18, 21/32, and 20/24, respectively. The difference among three tissues was statistically significant (p < 0.05). Differences were significant by pairwise comparison between normal brain tissue with low-grade gliomas and normal brain tissue with high-grade glioma group (p < 0.01). There is no difference between low-grade gliomas and high-grade gliomas (p > 0.05). Spearman's rank correlation confirmed that cyclinD1 and Ki-67 was positively correlated in low-grade gliomas and high-level brain tumor (p < 0.05), but no correlation in the normal brain tissue (p > 0.05). CONCLUSIONS: The expression of CyclinD1 and Ki-67 increased in gliomas, suggesting that both may play an important role in the occurrence of gliomas.