Multi-region calcium imaging in freely behaving mice with ultra-compact head-mounted fluorescence microscopes.

To investigate the circuit-level neural mechanisms of behavior, simultaneous imaging of neuronal activity in multiple cortical and subcortical regions is highly desired. Miniature head-mounted microscopes offer the capability of calcium imaging in freely behaving animals. However, implanting multiple microscopes on a mouse brain remains challenging due to space constraints and the cumbersome weight of the equipment. Here, we present TINIscope, a Tightly Integrated Neuronal Imaging microscope optimized for electronic and opto-mechanical design. With its compact and lightweight design of 0.43 g, TINIscope enables unprecedented simultaneous imaging of behavior-relevant activity in up to four brain regions in mice. Proof-of-concept experiments with TINIscope recorded over 1000 neurons in four hippocampal subregions and revealed concurrent activity patterns spanning across these regions. Moreover, we explored potential multi-modal experimental designs by integrating additional modules for optogenetics, electrical stimulation or local field potential recordings. Overall, TINIscope represents a timely and indispensable tool for studying the brain-wide interregional coordination that underlies unrestrained behaviors.

A new paradigm of learned cooperation reveals extensive social coordination and specific cortical activation in mice.

Cooperation is a social behavior crucial for the survival of many species, including humans. Several experimental paradigms have been established to study cooperative behavior and related neural activity in different animal species. Although mice exhibit limited cooperative capacity in some behavioral paradigms, it is still interesting to explore their cooperative behavior and the underlying neural mechanisms. Here, we developed a new paradigm for training and testing cooperative behavior in mice based on coordinated lever-pressing and analyzed social interactions between the animals during cooperation. We observed extensive social contact and waiting behavior in cooperating animals, with the number of such events positively correlated with the success of cooperation. Using c-Fos immunostaining and a high-speed volumetric imaging with synchronized on-the-fly scan and readout (VISoR) system, we further mapped whole-brain neuronal activity trace following cooperation. Significantly higher levels of c-Fos expression were observed in cortical areas including the frontal pole, motor cortex, anterior cingulate area, and prelimbic area. These observations highlight social interaction and coordination in cooperative behavior and provide clues for further study of the underlying neural circuitry mechanisms.

Preferential subcortical collateral projections of pedunculopontine nucleus-targeting cortical pyramidal neurons revealed by brain-wide single fiber tracing.

The pedunculopontine nucleus (PPN) is a heterogeneous midbrain structure involved in various brain functions, such as motor control, learning, reward, and sleep. Previous studies using conventional tracers have shown that the PPN receives extensive afferent inputs from various cortical areas. To examine how these cortical axons make collateral projections to other subcortical areas, we used a dual-viral injection strategy to sparsely label PPN-targeting cortical pyramidal neurons in CaMKIIα-Cre transgenic mice. Using a high-speed volumetric imaging with on-the-fly-scan and Readout (VISoR) technique, we visualized brain-wide axonal projections of individual PPN-targeting neurons from several cortical areas, including the prelimbic region (PL), anterior cingulate area (ACA) and secondary motor cortex (MOs). We found that each PPN-projecting neuron had a unique profile of collateralization, with some subcortical areas being preferential targets. In particular, PPN-projecting neurons from all three traced cortical areas exhibited common preferential collateralization to several nuclei, with most neurons targeting the striatum (STR), lateral hypothalamic area (LHA) and periaqueductal gray (PAG), and a substantial portion of neurons also targeting the zona incerta (ZI), median raphe nucleus (MRN) and substantia nigra pars reticulata (SNr). Meanwhile, very specific collateralization patterns were found for other nuclei, including the intermediate reticular nucleus (IRN), parvicellular reticular nucleus (PARN) and gigantocellular reticular nucleus (GRN), which receive collateral inputs almost exclusively from the MOs. These observations provide potential anatomical mechanisms for cortical neurons to coordinate the PPN with other subcortical areas in performing different physiological functions.

Efficacy and safety of transcutaneous auricular vagus nerve stimulation combined with conventional rehabilitation training in acute stroke patients: a randomized controlled trial conducted for 1 year involving 60 patients.

Transcutaneous auricular vagus nerve stimulation (ta-VNS) is a novel noninvasive treat-ment for stroke that directly stimulates the peripheral auricular branch of the vagus nerve. There have been recent reports that ta-VNS combined with conventional rehabilitation training promotes the recovery of neurological function of patients with acute stroke. However, these were small-sample-sized studies on the recovery of neurological function in patients after percutaneous vagus nerve stimulation in the subacute and chronic phases after stroke. This double-blinded randomized controlled trial involved 60 acute ischemic or hemorrhagic stroke patients aged 18-80 years who received treatment in the Second Affiliated Hospital of Chongqing Medical University. The subjects were randomly assigned to receive ta-VNS or sham ta-VNS combined with conventional rehabilitation training. The follow-up results over 1 year revealed that ta-VNS combined with conventional rehabilitation training greatly improved the recovery of motor and sensory functions and emotional responses compared with sham ta-VNS combined with conventional rehabilitation training. There were no obvious side effects. These findings suggest that ta-VNS combined with conventional rehabilitation training for the treatment of acute ischemic or hemorrhagic stroke patients is safe and effective.

Prediction of hospital discharge outcome from changes in cerebrospinal fluid/serum albumin quotient and cerebrospinal fluid lactate dehydrogenase in patients with cryptococcal meningitis.

Aim: This study aims to provide reliable prognostic factors for patients with cryptococcal meningitis (CM). Patients & methods: Clinical characteristics and laboratory findings of CM patients were retrospectively reviewed. Results: Sixty-three patients with CM were enrolled and 38/63 were confirmed to be HIV serology positive. Among clinical characteristics, headache, nausea and/or vomiting, and fever were the most common symptoms. Among cerebrospinal fluid (CSF) parameters, changes in leukocyte count, lactate dehydrogenase and chloride were significantly associated with the outcome. An increased CSF/serum albumin quotient (QAlb) was indicative of an unfavorable outcome in HIV-negative patients. Conclusion: CSF lactate dehydrogenase and QAlb may improve the prediction of outcomes in patients with CM.

Sema7A, a brain immune regulator, regulates seizure activity in PTZ-kindled epileptic rats.

AIMS: Semaphorin7A (Sema7A) plays an important role in the immunoregulation of the brain. In our study, we aimed to investigate the expression patterns of Sema7A in epilepsy and further explore the roles of Sema7A in the regulation of seizure activity and the inflammatory response in PTZ-kindled epileptic rats. METHODS: First, we measured the Sema7A expression levels in patients with temporal lobe epilepsy (TLE) and in rats of a PTZ-kindled epilepsy rat model. Second, to explore the role of Sema7A in the regulation of seizure activity, we conducted epilepsy-related behavioral experiments after knockdown and overexpression of Sema7A in the rat hippocampal dentate gyrus (DG). Possible Sema7A-related brain immune regulators (eg, ERK phosphorylation, IL-6, and TNF-α) were also investigated. Additionally, the growth of mossy fibers was visualized by anterograde tracing using injections of biotinylated dextran amine (BDA) into the DG region. RESULTS: Sema7A expression was markedly upregulated in the brain tissues of TLE patients and rats of the epileptic model after PTZ kindling. After knockdown of Sema7A, seizure activity was suppressed based on the latency to the first epileptic seizure, number of seizures, and duration of seizures. Conversely, overexpression of Sema7A promoted seizures. Overexpression of Sema7A increased the expression levels of the inflammatory cytokines, IL-6 and TNF-α, ERK phosphorylation, and growth of mossy fibers in PTZ-kindled epileptic rats. CONCLUSION: Sema7A is upregulated in the epileptic brain and plays a potential role in the regulation of seizure activity in PTZ-kindled epileptic rats, which may be related to neuroinflammation. Sema7A promotes the inflammatory cytokines TNF-α and IL-6 as well as the growth of mossy fibers through the ERK pathway, suggesting that Sema7A may promote seizures by increasing neuroinflammation and activating pathological neural circuits. Sema7A plays a critical role in epilepsy and could be a potential therapeutic target for this neurological disorder.

Long noncoding RNA expression profile from cryptococcal meningitis patients identifies DPY19L1p1 as a new disease marker.

AIMS: LncRNAs play a vital role in the pathological and physiological process. This study aimed to explore the involvement of lncRNAs in cryptococcal meningitis. METHODS: Microarray was performed in cryptococcal meningitis patients, and then, GO and KEGG pathways were analyzed. Coexpression relationship between lncRNA and mRNA was explored. The expressions of the lncRNAs and mRNAs, and their changes after treatment were detected by PCR. RESULTS: A total of 325 mRNAs (201 upregulated and 124 downregulated) and 497 lncRNAs (263 upregulated and 234 downregulated) were identified. The top three enriched GO terms for the mRNAs were arachidonic acid binding, activin receptor binding, and replication fork protection complex. The top three pathways in KEGG were asthma, one carbon pool by folate, and allograft rejection. A total of 305 coexpression relationships were found between 108 lncRNAs and 87 mRNAs. LncRNA-DPY19L1p1 was significantly increased in patients and decreased after treatment. ROC analysis revealed DPY19L1p1 was a potential diagnostic marker (AUCROC  = 0.9389). Furthermore, the target genes of DPY19L1p1 in cis or trans regulation were mainly involved in immune-related pathways like the interleukin signaling pathway. CONCLUSIONS: This study analyzed the differential lncRNA profile in cryptococcal meningitis patients and revealed DPY19L1p1 could be used for treatment evaluation and disease diagnosis.

MTF2 Induces Epithelial-Mesenchymal Transition and Progression of Hepatocellular Carcinoma by Transcriptionally Activating Snail.

BACKGROUND: Metal regulatory transcription factor 2 (MTF2) has been previously reported as a protein binding to the metal response element of the mouse metallothionein promoter, which is involved in chromosome inactivation and pluripotency. However, the function of MTF2 in tumor formation and progression has not yet been completely elucidated. METHODS: The expression of MTF2 and clinicopathological characteristics were evaluated by hepatocellular carcinoma (HCC) tissue microarray of 240 specimens. The role of MTF2 on HCC progression was determined using MTT, crystal violet, and transwell assays. Tumor growth was monitored in a xenograft model, and intrahepatic metastasis models were established. RESULTS: The expression of MTF2 was increased in HCC and strongly associated with the clinical characteristics and prognosis. Forced expression of MTF2 in HCC cells significantly promoted cell growth, migration, and invasion in vitro. In contrast, downregulation of MTF2 inhibited cell growth, migration, and invasion in vitro. Moreover, knock down of MTF2 suppressed tumorigenesis and intrahepatic metastasis of HCC cells in vivo. Mechanistically, MTF2 overexpression may promote growth and epithelial-mesenchymal transition processes of HCC cells by facilitating Snail transcription. CONCLUSION: MTF2 promotes the proliferation, migration, and invasion of HCC cells by regulating Snail transcription, providing a potential therapeutic candidate for patients with HCC.

A comparative study of the therapeutic effect between long and short intramedullary nails in the treatment of intertrochanteric femur fractures in the elderly.

PURPOSE: To compare the clinical effects of long vs. short intramedullary nails in the treatment of intertrochanteric fractures in old patients more than 65 years old. METHODS: A retrospective analysis of 178 cases of intertrochanteric fractures of the femur (AO type A1 and A2) in the elderly was conducted from January 2008 to December 2013. There were 85 males (47.8%) and 93 females (52.2%) with the age of 65e89 (70.2±10.8) years. The patients were treated by closed reduction and long or short intramedullary nail (Gamma 3) fixation. The length of short nail was 180 mm and that for long nail was 320e360 mm. The general data of patients, operation time, intraoperative blood loss, length of hospital stay, preoperative hemoglobin level, blood transfusion rate, postoperative periprosthetic fractures, infections, complications, etc were carefully recorded. RESULTS: There were 76 cases (42.7%) in the long intramedullary nail group and 102 cases (57.3%) in the short nail group. All the cases were followed up for 12e48 (21.3±6.8) months, during which there were 21 deaths (11.8%), mean (13.8±6.9) months after operation. The intraoperative blood loss was (90.7±50.6) ml in short nail group, greatly less than that in long nail group (127.8±85.9) ml (p=0.004). The short nail group also had a significantly shorter operation time (43.5 min±12.3 min vs. 58.5 min±20.3 min, p=0.002) and lower rate of postoperative transfusion (42.3% vs. 56.7%, p=0.041). But the length of hospital stay showed no big differences. After operation, in each group there was 1 case of periprosthetic fracture with a total incidence of 1.1%, 1.3% in long nail group and 0.9% in short nail group. At the end of the follow-up, all patients achieved bony union. The average healing time of the long nail group was (6.5±3.1) months, and the short nail group was (6.8±3.7) months, revealing no significant differences (p=0.09). Postoperative complications showed no great differences either. CONCLUSION: Both the intramedullary long and short nail fixation has a good clinical effect in treating intertrochanteric femur fractures in the elderly. They showed no significant difference in terms of therapeutic effect, hospital stay and postoperative complications. The incidence of periprosthetic fractures treated by either length of nails was low. But short intramedullary nailing can obviously decrease the intraoperative blood loss, operation time and postoperative blood transfusion.

Inhibition of KL-6/MUC1 glycosylation limits aggressive progression of pancreatic cancer.

AIM: To evaluate the significance of KL-6/MUC1 (a type of MUC1) glycosylation in pancreatic cancer progression. METHODS: KL-6/MUC1 expression was detected by immunohistochemistry in 48 patients with pancreatic duct cell carcinoma. The N-/O-glycosylation inhibitors (tunicamycin and benzyl-N-acetyl-α-galactosaminide) were then used to interfere with KL-6/MUC1 glycosylation in two pancreatic carcinoma cell lines, and the effects on KL-6/MUC1 expression, and cell adhesion and invasion were determined. In addition, protein expression of epithelial-mesenchymal transition markers, E-cadherin and vimentin, were evaluated in cells after treatment with glycosylation inhibitors. RESULTS: Overexpression of KL-6/MUC1 was found in all pancreatic cancer tissues, but not in the surrounding normal pancreatic tissues. The expression profile of KL-6/MUC1 was significantly decreased after treatment with the inhibitors. The adhesion and invasive ability of cancer cells were significantly decreased after drug treatment, and increased E-cadherin and decreased vimentin expression were found. CONCLUSION: KL-6/MUC1 glycosylation is involved in pancreatic cancer metastasis and invasion. Therapeutic strategies which target this may help control the aggressive behavior of pancreatic cancer cells.

Clinical implication of TMPRSS4 expression in human gallbladder cancer.

Altered expression of transmembrane protease/serine 4 (TMPRSS4) is observed in various types of human cancers. However, the clinical significance of TMPRSS4 expression in gallbladder cancer (GBC) remains largely unknown. The present study aims to explore the clinicopathological significance and prognostic value of TMPRSS4 in GBC. The levels of TMPRSS4 mRNA and protein in GBC tissues and adjacent noncancerous tissues were evaluated by quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry. To investigate the correlations between TMPRSS4 and the clinicopathological features of GBC, the expression of TMPRSS4 in 97 patients with GBC were detected by immunohistochemistry. The correlation of TMPRSS4 expression with patients' survival rate was assessed by Kaplan-Meier and Cox regression. Our results showed that the expression levels of TMPRSS4 mRNA and protein in GBC tissues were both significantly higher than those in adjacent noncancerous tissues. Immunohistochemical staining revealed that high TMPRSS4 expression was closely correlated with tumor size (P=0.032), histological grade (P=0.002), pathologic T stage (P=0.005), clinical stage (P=0.013), and lymph node metastasis (P=0.003). Moreover, the results of Kaplan-Meier analysis indicated that a high expression level of TMPRSS4 resulted in a significantly poor prognosis of GBC patients. Multivariate analysis showed that the status of TMPRSS4 expression was an independent prognostic factor for GBC patients. Our results showed that TMPRSS4 plays a key role in GBC and therefore may provide an opportunity for developing a novel therapeutic target as well as a prognostic marker in GBC.

Ex-situ liver surgery without veno-venous bypass.

AIM: To evaluate the results of hepatic resection with ex-situ hypothermic perfusion and without veno-venous bypass. METHODS: In 3 patients with liver tumor, the degree of the inferior vena cava and/or main hepatic vein involvement was verified when the liver was dissociated in the operation. It was impossible to resect the tumors by the routine hepatectomy, so the patients underwent ex-situ liver surgery, vein cava replacement and hepatic autotransplantation without veno-venous bypass. All surgical procedures were carried out or supervised by a senior surgeon. A retrospective analysis was performed for the prospectively collected data from patients with liver tumor undergoing ex-situ liver surgery, vein cava replacement and hepatic autotransplantation without veno-venous bypass. We also compared our data with the 9 cases of Pichlmayr's group. RESULTS: Three patients with liver tumor were analysed. The first case was a 60-year-old female with a huge haemangioma located in S1, S4, S5, S6, S7 and S8 of liver; the second was a 64-year-old man with cholangiocarcinoma in S1, S2, S3 and S4 and the third one was a 55-year-old man with a huge cholangiocarcinoma in S1, S5, S7 and S8. The operation time for the three patients were 6.6, 6.4 and 7.3 h, respectively. The anhepatic phases were 3.8, 2.8 and 4.0 h. The volume of blood loss during operation were 1200, 3100, 2000 mL in the three patients, respectively. The survival periods without recurrence were 22 and 17 mo in the first two cases. As for the third case complicated with postoperative hepatic vein outflow obstruction, emergency hepatic vein outflow extending operation and assistant living donor liver transplantation were performed the next day, and finally died of liver and renal failure on the third day. Operation time (6.7 ± 0.47 h vs 13.7 ± 2.6 h) and anhepatic phase (3.5 ± 0.64 h vs 5.7 ± 1.7 h) were compared between Pichlmayr's group and our series (P = 0.78). CONCLUSION: Ex-situ liver resection and liver autotransplantation has shown a potential for treatment of complicated hepatic neoplasms that are unresectable by traditional procedures.

[A meta-analysis of clinical outcomes after laparoscopic operation for rectal cancer].

OBJECTIVE: To evaluate the clinical outcomes after laparoscopic surgery for rectal cancer. METHODS: A systematic literature search (Medline, Embase, Cochrane Library) as of March 2010 was performed to identify all eligible studies. Two reviewers independently screened and extracted the data. Differences in short-term and long-term clinical outcomes after laparoscopic resection (LR) and open resection (OR) were analyzed using RevMan 5. RESULTS: A total of 1042 abstracts were retrieved and 16 clinical controlled studies finally included. The total number of patients was 2850. There were 1145 patients received LR and 1705 received OR. The analyses showed that LR had longer operative time (WMD=42.50, 95%CI: 29.27 to 55.74, P<0.05), less harvested lymph nodes (WMD=-0.94, 95%CI: -1.47 to -0.41, P<0.05), and less blood loss (WMD=-158.46, 95%CI: -221.08 to -95.84, P<0.05) as compared to OR. LR was superior to OR in terms of surgical mortality (OR=0.40, 95%CI: 0.18 to 0.92, P=0.03), postoperative complications (OR=0.73, 95%CI: 0.61 to 0.87, P<0.05), and 5-year overall survival rate (OR=1.56, 95%CI: 1.21 to 2.02, P<0.05). There was no significant difference in positive rate of circumferential resection margin between the two groups (OR=1.00, 95%CI: 0.45 to 2.20, P=1.00). CONCLUSION: Compared to open surgery, short-term and long-term clinical outcomes after laparoscopic surgery are favorable.

Measurement of serum and tissue des-gamma-carboxyprothrombin in resectable hepatocellular carcinoma.

BACKGROUND: The total des-gamma-carboxyprothrombin (DCP) produced in hepatocellular carcinoma (HCC) and the surrounding non-cancer liver tissue was estimated and the correlation between tissue DCP and serum DCP levels was examined. PATIENTS AND METHODS: Forty patients with a single primary HCC nodule served as the subjects. The DCP was quantified by electrochemiluminescence immunoassay and the tissue volumes were estimated by computed tomography (CT). RESULTS: The mean tissue DCP in the cancer tissue, non-cancer tissue and the whole HCC specimen was 147,400, 20,200 and 190,000 mAU, respectively, which correlated positively with the serum DCP level (r2 = 0.657, 0.452, and 0.684, respectively). The DCP expression levels in the cancer and non-cancer liver tissue were also confirmed by immunohistochemical detection of DCP. CONCLUSION: The elevation of serum DCP levels in HCC patients is influenced by DCP production not only in the cancer tissue, but also in the surrounding non-cancer liver tissue.

Demographic and clinical characteristics of resectable hepatocellular carcinoma in China and Japan.

BACKGROUND/AIMS: To describe and compare the demographic and clinical characteristics of hepatocellular carcinoma (HCC) patients subject to surgical operation at two referral centers in China and Japan. METHODOLOGY: Information on surgical HCC patients was retrieved from medical records. All patients were subjected to regular follow-up. The characteristics of HCC cases from two countries were described and compared. RESULTS: A total of 353 newly diagnosed cases subject to surgical resection for HCC from 1995 to 1999 were analyzed in China and Japan. Differences were noted in the demographic and clinical characteristics of HCC patients between the two countries, such as average age of incidence, history of blood transfusion, serum examination, and history of hepatic viral infection. Statistical analysis also indicated differences in surgical systematic anatomical resection (p < 0.001), liver cirrhosis (p < 0.05), tumor diameter, and distant metastases (p < 0.001). The Chinese patients in early TNM stage I showed poorer outcomes in terms of cumulative survival in comparison with Japanese patients (p < 0.001). CONCLUSIONS: HCC patients in Japan were diagnosed earlier, subject to more standard treatment, and had better prognosis. Examination with a larger population and more clinical data, including repeated treatment for recurrent HCC, will be required to provide a more accurate assessment of HCC in the two countries.