Clinicopathologic analysis of intraductal papillary neoplasm of bile duct: Korean multicenter cohort study.

BACKGROUND: IPNB is very rare disease and most previous studies on IPNB were case series with a small number due to low incidence. The aim of this study is to validate previously known clinicopathologic features of intraductal papillary neoplasm of bile duct (IPNB) based on the first largest multicenter cohort. METHODS: Among 587 patients previously diagnosed with IPNB and similar diseases from each center in Korea, 387 were included in this study after central pathologic review. We also reviewed all preoperative image data. RESULTS: Of 387 patients, 176 (45.5%) had invasive carcinoma and 21 (6.0%) lymph node metastasis. The 5-year overall survival was 80.9% for all patients, 88.8% for IPNB with mucosal dysplasia, and 70.5% for IPNB with invasive carcinoma. According to the "Jang & Kim's modified anatomical classification," 265 (68.5%) were intrahepatic, 103 (26.6%) extrahepatic, and 16 (4.1%) diffuse type. Multivariate analysis revealed that tumor invasiveness was a unique predictor for survival analysis. (p = 0.047 [hazard ratio = 2.116, 95% confidence interval 1.010-4.433]). CONCLUSIONS: This is the first Korean multicenter study on IPNB through central pathologic and radiologic review process. Although IPNB showed good long-term prognosis, relatively aggressive features were also found in invasive carcinoma and extrahepatic/diffuse type.

Predictive value of sarcopenia and visceral obesity for postoperative pancreatic fistula after pancreaticoduodenectomy analyzed on clinically acquired CT and MRI.

OBJECTIVE: To evaluate predictive values of sarcopenia and visceral obesity measured from preoperative CT/MRIs for postoperative pancreatic fistula (POPF) after pancreaticoduodenectomy in patients with periampullary malignancies. METHODS: From the prospectively constructed surgical registry, we included adult patients treated with pancreaticoduodenectomy. Based on CT/MRIs, body morphometric analysis was performed to evaluate the visceral obesity and sarcopenia, based on the areas of visceral fat and skeletal muscle measured at the L3 vertebrae level. We retrieved various perioperative factors from registry. As outcomes of postoperative complications, we evaluated POPF and major complications based on the Clavien-Dindo classification. Multivariate logistic regression analyses were performed. RESULTS: From a total of 284 patients (163 males, 121 females) who met the inclusion/exclusion criteria, POPF, major complications, and 60-day mortality occurred in 52 (18.3%), 34 (12.0%), and 6 (2.1%), respectively. Sarcopenia and visceral obesity were noted in 123 (75.5%) and 66 (40.5%) of men and 68 (56.2%) and 53 (43.8%) of women, respectively. Combination of sarcopenia and obesity (sarcopenic obesity) was noted in 31.9% (52/163) of men and in 26.4% (32/121) of women. In multivariate logistic regression analyses, sarcopenic obesity was the only independent predictor for POPF (OR 2.65, 95% CI 1.43-4.93), and the vascular resection during pancreaticoduodenectomy was the only independent predictor for severe complications (OR 3.75, 95% CI 1.61-8.70). CONCLUSION: Sarcopenic obesity might be highly predictive for POPF. Body morphometric analysis in preoperative CT/MRI combined with assessment of perioperative clinical features may help to identify high-risk patients and determine perioperative management strategies. KEY POINTS: • Sarcopenic obesity might be predictive for postoperative pancreatic fistula after pancreaticoduodenectomy. • The vascular resection during pancreaticoduodenectomy might be predictive of major complications. • Body morphometric analysis might be helpful for identifying high-risk patients.

Revisiting the Topographic Anatomy of the Marginal Mandibular Branch of Facial Nerve Relating to the Surgical Approach.

BACKGROUND: The marginal mandibular branch (Mbr) of the facial nerve is vulnerable to damage during rhytidoplasty, surgical reduction of the mandibular angle, parotidectomy, and excision of the submandibular gland. OBJECTIVES: The authors sought to map the Mbr and determine the relationship between the number of Mbr offshoots and the course of the Mbr. METHODS: The Mbr was examined in 29 hemifaces from 12 embalmed and 4 fresh cadavers (10 males, 6 females; mean age, 73.7 years). RESULTS: The Mbr was located ≤5 mm from the gonion (Go) in 24 of 29 hemifaces (82.8%) and ≤10 mm from the intersection of the facial artery and mandible (ie, FM) in 26 hemifaces (89.7%). In 16 hemifaces (55.2%), offshoots arose from the Mbr inferior to the mandible. The Mbr ran below the Go in 14 hemifaces (48.3%) and ran below FM in 13 hemifaces (44.8%). Except for minute offshoots deep to the platysma, the Mbr was not found to pass >2 cm below the mandible. The mean (± standard deviation) quantity of Mbr offshoots was 1.5 (± 0.6). A greater number of offshoots was associated with a higher likelihood of an inferiorly located nerve. The Mbr proceeded under the lower border of the mandible in 13 hemifaces (44.8%) and reached the mandible at a mean distance of 33.1±5.2 mm anterior to the Go. CONCLUSIONS: To avoid damaging the Mbr, surgical maneuvers should be positioned 4.5 cm anterior to the Go and 2 cm below the mandible.

Human umbilical cord blood-derived mesenchymal stem cells improve functional recovery through thrombospondin1, pantraxin3, and vascular endothelial growth factor in the ischemic rat brain.

Cell therapy is a potential therapeutic method for cerebral ischemia, which remains a serious problem. In the search for more effective therapeutic methods, many kinds of stem cells from various tissues have been developed and tested as candidate therapeutic agents. Among them, human umbilical cord blood (hUCB)-derived mesenchymal stem cells (MSCs) are widely used for cell therapy because of their genetic flexibility. To confirm that they are effective and understand how they affect ischemic neural cells, hUCB-MSCs were directly administered ipsilaterally into an ischemic zone induced by middle cerebral artery occlusion (MCAO). We found that the neurobehavioral performance of the hUCB-MSC group was significantly improved compared with that of the vehicle-injected control group. The infarct was also remarkably smaller in the hUCB-MSC group. Additionally, hUCB-MSC transplantation resulted in a greater number of newly generated cells and angiogenic and tissue repair factors and a lower number of inflammatory events in the penumbra zone. To determine why these events occurred, hUCB-MSCs were assayed under hypoxic and normoxic conditions in vitro. The results showed that hUCB-MSCs exhibit higher expression levels of thrombospondin1, pantraxin3, and vascular endothelial growth factor under hypoxic conditions than under normoxic conditions. These results were found to be correlated with our in vivo immunofluorescent staining results. On the basis of these findings, we suggest that hUCB-MSCs may have a beneficial effect on cerebral ischemia, especially through angiogenesis, neurogenesis, and anti-inflammatory effects, and thus could be used as a therapeutic agent to treat neurological disorders such as cerebral ischemia.

Restoration of portal flow using a pericholedochal varix in adult living donor liver transplantation for patients with total portosplenomesenteric thrombosis.

In total portosplenomesenteric thrombosis patients, cavoportal hemitransposition (CPHT) is indicated but rarely applicable for adult-to-adult (A-to-A) living donor liver transplantation (LDLT) because partial liver graft requires splanchno-portal inflow for liver graft regeneration. If intra- & peri-pancreatic collaterals draining into pericholedochal varix were present, pericholedochal varix may provide splanchnic blood flow to the transplanted liver and also relieve recipient's portal hypertension. To date, however, there is no successful report using pericholedochal varix in liver transplantation (LT). We successfully performed A-to-A LDLTs using pericholedochal varix for those 2 patients. The surgical strategies are followings: (a) dissection of hepatic hilum to isolate left hepatic artery using for arterial reconstruction of implanted right lobe graft, (b) en-mass clamping of the undissected remaining hilum if we can leave adequate length of stump from the clamping site, and then hilum is divided, (c) delay the donor hepatectomy until the feasibility of the recipient operation is confirmed. Portal flow was established between the sizable pericholedochal varix (caliber > 1cm) and graft portal vein, but the individually designed approaches were used for each patients. Currently, they have been enjoying normal life on posttransplant 92 and 44 months respectively. In conclusion, enlarged pericholedochal varix in patients with totally obliterated splanchnic veins might be an useful inflow to restore portal flow and secure good outcome in A-to-A LDLT. AASLD.

Survival differences between Milan criteria after down-staging and De novo Milan in living donor liver transplantation for hepatocellular carcinoma.

BACKGROUND/AIMS: This study reports our experiences of adult living donor liver transplantation (LDLT) corresponding to downstaging. METHODOLOGY: Between July 1992 and April 2008, we performed 553 adult LDLTs (35.1%, 553/1575) for HCC. Sixty-five patients was not treated before LDLT and belonged to Milan criteria, classified as De novo Milan group (De novo-M); 22 HCC patients did not meet Milan criteria initially, but subsequently met the criteria after downstaging, classified as artificial Milan group (Artificial-M). The evaluation of downstaging was based on preoperative CT scan and explanted liver biopsy, and excluded the patients having unclear treatment history on analysis. RESULTS: Artificial-M showed significantly less Child C patients (25%) than De novo-M (64.5%) (0.037). Artificial-M had greater tumor burden than De novo-M in maximal tumor size (2.5 +/- 1.2 versus 2.2 +/- 0.95 cm), sum of tumor diameter (3.4 +/- 1.4 versus 2.4 +/- 1.0 cm), number of nodules (1.8 +/- 0.9 versus 1.2 +/- 0.5), respectively. Five-year cumulative survival was not different between Artificial-M and De novo-M (83.9% versus 93.9%), but 5-year disease free survival were significantly different (71.1% versus 96.5%) (p = 0.0016). CONCLUSIONS: Five year overall survival rates after LDLT were good in both groups. However, stricter follow-up is necessary in Artificial-M considering greater tumor burden and higher recurrence rate compared to De novo-M.

Improvement of voice quality and prevention of deafness by a bone-conduction device.

In modern society, people are involuntarily being exposed to various noises in their everyday-life environments. The increasing use of mobile phones and other portable devices as a primary means of communication outside of homes makes the current noise condition even worse. During the exchange of information on these devices, the volume is usually set 15 dB higher than the surrounding noise in order for the sound to be perceived more clearly. Hence, the sum of noise on these devices is usually estimated to be around 110 dB. This level of noise can cause noise-induced hearing impairment or even hearing loss to users when continued for a long time. A bone-conduction system can be a possible solution to reducing the noise while enhancing the quality of voice signals in mobile phones. In this study, we suggest that the implementation of the bone-conduction feedback system in mobile phones will raise the ratio of signal to noise with about 17 dB, enhancing the quality of voice signals.

3D printed multi-growth factor delivery patches fabricated using dual-crosslinked decellularized extracellular matrix-based hybrid inks to promote cerebral angiogenesis.

Generally, brain angiogenesis is a tightly regulated process, which scarcely occurred in the absence of specific pathological conditions. Delivery of exogenous angiogenic factors enables the induction of desired angiogenesis by stimulating neovasculature formation. However, effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. Herein, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs), using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink for printing patches through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition reaction with combining methacrylated hyaluronic acid (HAMA) and vascular-tissue-derived decellularized extracellular matrix (VdECM), and thermal crosslinking of VdECM. 3D printing technology, a useful approach with fabrication versatility with customizable systems and multiple biomaterials, is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by label-free photoacoustic microscopy in vivo. The developed multi-GFs releasing patch may offer a promising therapeutic approach of spatiotemporal drugs releasing such as cerebral ischemia, ischemic heart diseases, diabetes, and even use as vaccines. STATEMENT OF SIGNIFICANCE: Effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. In this study, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs) using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition, and thermal crosslinking. 3D printing technology is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by photoacoustic microscopy in vivo.

Determination of the Unilaterally Damaged Region May Depend on the Asymmetry of Carotid Blood Flow Velocity in Hemiparkinsonian Monkey: A Pilot Study.

The hemiparkinsonian nonhuman primate model induced by unilateral injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the carotid artery is used to study Parkinson's disease. However, there have been no studies that the contralateral distribution of MPTP via the cerebral collateral circulation is provided by both the circle of Willis (CoW) and connections of the carotid artery. To investigate whether MPTP-induced unilaterally damaged regions were determined by asymmetrical cerebral blood flow, the differential asymmetric damage of striatal subregions, and examined structural asymmetries in a circle of Willis, and blood flow velocity of the common carotid artery were observed in three monkeys that were infused with MPTP through the left internal carotid artery. Lower flow velocity in the ipsilateral common carotid artery and a higher ratio of ipsilateral middle cerebral artery diameter to anterior cerebral artery diameter resulted in unilateral damage. Additionally, the unilateral damaged monkey observed the apomorphine-induced contralateral rotation behavior and the temporary increase of plasma RANTES. Contrastively, higher flow velocity in the ipsilateral common carotid artery was observed in the bilateral damaged monkey. It is suggested that asymmetry of blood flow velocity and structural asymmetry of the circle of Willis should be taken into consideration when establishing more efficient hemiparkinsonian nonhuman primate models.

Role of apoptosis in retinoic acid-induced cleft palate.

PURPOSE: Although the mechanism by which retinoic acid (RA) induces cleft palate has been intensely investigated, some controversies remain. Some researchers argue that RA inhibits apoptosis, resulting in a failure of palatal shelves to fuse, whereas others propose that RA disrupts elevation or retards the growth of palatal shelves. This study investigated the mechanism underlying RA-induced formation of cleft palate in the rat, focusing mainly on the role of apoptosis. METHODS: Using an RA-induced cleft palate model of Sprague-Dawley rats described in our previous study, we examined a total of 92 embryos. Retinoic acid was injected intraperitoneally in experimental group animals on embryonic day 11 (D11), a time when our previous study indicated that RA-induced cleft palate was maximally developed. Control animals were treated with normal saline mixed with sesame oil. Timed pregnant rats were killed by an overdose of ether on D13, D14, D15, D16, and D17, and 8 sections were prepared from the anterior to the posterior of the palate. Growth of palatal shelves was evaluated histologically by examining sections stained with hematoxylin-eosin, periodic acid-Schiff, trichrome, and cresyl violet. Differences in apoptosis were monitored using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays. RESULTS: Histologic examinations revealed underdevelopment of palatal shelves in the experimental group compared with the control group. In the RA-treated group, the overall process of palatal shelf development was delayed 1 day, and palatal elevation was observed. In hard-palate areas of both groups, apoptosis was maximal immediately after the fusion of palatal shelves. In the soft-palate areas, the saline-treated group showed fusion of palatal shelves, whereas the RA-treated group showed retarded growth of palatal shelves that resulted in failure of palatal fusion. Moreover, apoptosis occurred before palatal contact. CONCLUSIONS: Apoptotic manifestations did not differ between RA-induced cleft palates and control palates, suggesting that apoptosis makes a minimal contribution to the cleft palate formed in response to RA. Instead, growth retardation of the palatal shelves appears to play a major role in RA-induced cleft palate.

Tumor Spheroids of an Aggressive Form of Central Neurocytoma Have Transit-Amplifying Progenitor Characteristics with Enhanced EGFR and Tumor Stem Cell Signaling.

Central neurocytoma (CN) has been known as a benign neuronal tumor. In rare cases, CN undergoes malignant transformation to glioblastomas (GBM). Here we examined its cellular origin by characterizing differentiation potential and gene expression of CN-spheroids. First, we demonstrate that both CN tissue and cultured primary cells recapitulate the hierarchal cellular composition of subventricular zone (SVZ), which is comprised of neural stem cells (NSCs), transit amplifying progenitors (TAPs), and neuroblasts. We then derived spheroids from CN which displayed EGFR+/ MASH+ TAP and BLBP+ radial glial cell (RGC) characteristic, and mitotic neurogenesis and gliogenesis by single spheroids were observed with cycling multipotential cells. CN-spheroids expressed increased levels of pluripotency and tumor stem cell genes such as KLF4 and TPD5L1, when compared to their differentiated cells and human NSCs. Importantly, Gene Set Enrichment Analysis showed that gene sets of GBM-Spheroids, EGFR Signaling, and Packaging of Telomere Ends are enriched in CN-spheroids in comparison with their differentiated cells. We speculate that CN tumor stem cells have TAP and RGC characteristics, and upregulation of EGFR signaling as well as downregulation of eph-ephrin signaling have critical roles in tumorigenesis of CN. And their ephemeral nature of TAPs destined to neuroblasts, might reflect benign nature of CN.

Brain tumor diagnostic model and dietary effect based on extracellular vesicle microbiome data in serum.

The human microbiome has been recently associated with human health and disease. Brain tumors (BTs) are a particularly difficult condition to directly link to the microbiome, as microorganisms cannot generally cross the blood-brain barrier (BBB). However, some nanosized extracellular vesicles (EVs) released from microorganisms can cross the BBB and enter the brain. Therefore, we conducted metagenomic analysis of microbial EVs in both serum (152 BT patients and 198 healthy controls (HC)) and brain tissue (5 BT patients and 5 HC) samples based on the V3-V4 regions of 16S rDNA. We then developed diagnostic models through logistic regression and machine learning algorithms using serum EV metagenomic data to assess the ability of various dietary supplements to reduce BT risk in vivo. Models incorporating the stepwise method and the linear discriminant analysis effect size (LEfSe) method yielded 12 and 29 significant genera as potential biomarkers, respectively. Models using the selected biomarkers yielded areas under the curves (AUCs) >0.93, and the model using machine learning resulted in an AUC of 0.99. In addition, Dialister and [Eubacterium] rectale were significantly lower in both blood and tissue samples of BT patients than in those of HCs. In vivo tests showed that BT risk was decreased through the addition of sorghum, brown rice oil, and garlic but conversely increased by the addition of bellflower and pear. In conclusion, serum EV metagenomics shows promise as a rich data source for highly accurate detection of BT risk, and several foods have potential for mitigating BT risk.

Intrastriatal administration of coenzyme Q10 enhances neuroprotection in a Parkinson's disease rat model.

Parkinson's disease is a neurodegenerative disorder, and no treatment has been yet established to prevent disease progression. Coenzyme Q10, an antioxidant, has been considered a promising neuroprotective agent; however, conventional oral administration provides limited efficacy due to its very low bioavailability. In this study, we hypothesised that continuous, intrastriatal administration of a low dose of Coenzyme Q10 could effectively prevent dopaminergic neuron degeneration. To this end, a Parkinson's disease rat model induced by 6-hydroxydopamine was established, and the treatment was applied a week before the full establishment of this disease model. Behavioural tests showed a dramatically decreased number of asymmetric rotations in the intrastriatal Coenzyme Q10 group compared with the no treatment group. Rats with intrastriatal Coenzyme Q10 exposure also exhibited a larger number of dopaminergic neurons, higher expression of neurogenetic and angiogenetic factors, and less inflammation, and the effects were more prominent than those of orally administered Coenzyme Q10, although the dose of intrastriatal Coenzyme Q10 was 17,000-times lower than that of orally-administered Coenzyme Q10. Therefore, continuous, intrastriatal delivery of Coenzyme Q10, especially when combined with implantable devices for convection-enhanced delivery or deep brain stimulation, can be an effective strategy to prevent neurodegeneration in Parkinson's disease.

Totally Laparoscopic Distal Gastrectomy in Post Liver Transplant Patient.

The risk of malignancy after transplantation is higher than that of general population. Laparoscopic surgery has become a standard treatment of gastric cancer. However, there are no case reports evaluating totally laparoscopic gastrectomy in patients with previous liver transplantation. Herein we report our experience with a liver transplant recipient who underwent totally laparoscopic distal gastrectomy (TLDG) for gastric cancer. A 63 year-old man underwent orthotopic liver transplantation (OLT) for cryptogenic liver cirrhosis. 8 years later, gastric cancer was diagnosed during the follow-up. Endoscopic submucosal dissection was performed and additional surgical resection was needed. TLDG and D1+ lymph node dissection was performed, and the patient was discharged on the 8th post-operative day without any complications. To the best of our knowledge, this is the first case of de novo gastric cancer treated with TLDG after OLT. This suggests that TLDG is a feasible for patients after OLT.

Clinical impact of fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography in patients with resectable pancreatic cancer: diagnosing lymph node metastasis and predicting survival.

PURPOSE: To evaluate the diagnostic accuracy of fluorine-18-fluorodeoxyglucose PET/computed tomography (F-FDG PET/CT) for lymph node (LN) metastasis and the prognostic significance of F-FDG PET/CT LN parameters in patients with resectable pancreatic cancer. PATIENTS AND METHODS: Patients with resectable pancreatic cancer who underwent staging F-FDG PET/CT between May 2007 and September 2016 were retrospectively enrolled and analyzed through medical record and image re-evaluation. The diagnostic accuracy of F-FDG PET/CT in predicting LN metastasis was evaluated and compared with that of contrast-enhanced abdominal computed tomography (CECT). Prognostic variables, including LN parameters assessed by F-FDG PET/CT [standardized uptake value (SUV)LN and LN/tumor SUV ratio], that affect disease-free survival (DFS) and overall survival (OS) were evaluated by regression analysis. RESULTS: When predicting LN metastasis, F-FDG PET/CT showed greater sensitivity, positive predictive value, negative predictive value, and accuracy than CECT. Among prognostic factors affecting DFS, PET-positive LN (P=0.008), and LN/tumor SUV ratio (P=0.003) were found to be significant by regression analysis. Among the variables affecting OS, lymphovascular invasion (P=0.018) and the LN/tumor SUV ratio (P=0.046) were found to be significant. CONCLUSION: F-FDG PET/CT showed higher diagnostic accuracy in predicting LN metastasis than CECT in patients with resectable pancreatic cancer. Only the LN/tumor SUV ratio of F-FDG PET/CT was an independent prognostic variable in both DFS and OS.

Effect of Single and Double Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Following Focal Cerebral Ischemia in Rats.

Stem cell therapies are administered during the acute phase of stroke to preserve the penumbral tissues from ischemic injury. However, the effect of repeated cell therapy during the acute phase remains unclear. In this study, we investigated and compared the functional outcome of single (two days post-injury) and repeated (two and nine days post-injury) treatment with human umbilical cord derived mesenchymal stem cells (hUCB-MSCs) after middle cerebral artery occlusion (MCAO). The rotarod and limb placement tests were utilized to investigate functional outcomes, while infarct volume and tissue damage were measured by immunofluorescent staining for neovascularization, neurogenesis, apoptosis, and inflammation in the penumbral zones. We observed notable motor dysfunction and a significant decrease in infarcted brain volume, as well as increases in neurons and vessels in both single and repeated hUCB-MSC treatments compared to the control group. Interestingly, repeated administration of hUCB-MSCs was not found to elicit additional or synergistic improvements over monotherapy. This study suggests that a clearer understanding of the therapeutic window after stroke will facilitate the development of more efficient treatment protocols in the clinical application of stem cell therapy.

Inhibition of HIF1α and PDK Induces Cell Death of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors. GBMs, like other tumors, rely relatively less on mitochondrial oxidative phosphorylation (OXPHOS) and utilize more aerobic glycolysis, and this metabolic shift becomes augmented under hypoxia. In the present study, we investigated the physiological significance of altered glucose metabolism and hypoxic adaptation in the GBM cell line U251 and two newly established primary GBMs (GBM28 and GBM37). We found that these three GBMs exhibited differential growth rates under hypoxia compared to those under normoxia. Under normoxia, the basal expressions of HIF1α and the glycolysis-associated genes, PDK1, PDK3, and GLUT1, were relatively low in U251 and GBM28, while their basal expressions were high in GBM37. Under hypoxia, the expressions of these genes were enhanced further in all three GBMs. Treatment with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), induced cell death in GBM28 and GBM37 maintained under normoxia, whereas DCA effects disappeared under hypoxia, suggesting that hypoxic adaptation dominated DCA effects in these GBMs. In contrast, the inhibition of HIF1α with chrysin suppressed the expression of PDK1, PDK3, and GLUT1 and markedly promoted cell death of all GBMs under both normoxia and hypoxia. Interestingly, however, GBMs treated with chrysin under hypoxia still sustained higher viability than those under normoxia, and chrysin and DCA co-treatment was unable to eliminate this hypoxia-dependent resistance. Together, these results suggest that hypoxic adaptation is critical for maintaining viability of GBMs, and targeting hypoxic adaptation can be an important treatment option for GBMs.

Behavior, PET and histology in novel regimen of MPTP marmoset model of Parkinson's disease for long-term stem cell therapy.

Stem cell technologies are particularly attractive in Parkinson's disease (PD) research although they occasionally need long-term treatment for anti-parkinsonian activity. Unfortunately, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) widely used as a model for PD has several limitations, including the risk of dose-dependent mortality and the difficulty of maintenance of PD symptoms during the whole experiment period. Therefore, we tested if our novel MPTP regimen protocol (2 mg/kg for 2 consecutive days and 1 mg/kg for next 3 consecutive days) can be maintained stable parkinsonism without mortality for long-term stem cell therapy. For this, we used small-bodied common marmoset monkeys (Callithrix jacchus) among several nonhuman primates showing high anatomical, functional, and behavioral similarities to humans. Along with no mortality, the behavioral changes involved in PD symptoms were maintained for 32 weeks. Also, the loss of jumping ability of the MPTP-treated marmosets in the Tower test was not recovered by 32 weeks. Positron emission tomography (PET) analysis revealed that remarkable decreases of bindings of 18F-FP-CIT were observed at the striatum of the brains of the marmosets received MPTP during the full period of the experiment for 32 weeks. In the substantia nigra of the marmosets, the loss of tyrosine hydroxylase (TH) immunoreactivity was also observed at 32 weeks following the MPTP treatment. In conclusion, our low-dose MPTP regimen protocol was found to be stable parkinsonism without mortality as evidenced by behavior, PET, and TH immunohistochemistry. This result will be useful for evaluation of possible long-term stem cell therapy for anti-parkinsonian activity.

The Unreliability of MTT Assay in the Cytotoxic Test of Primary Cultured Glioblastoma Cells.

MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.