Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling.

The physiology and circuitry associated with dorsal cochlear nucleus neurons (DCN) have been well described. The ability to remotely manipulate neuronal activity in these neurons would represent a step forward in the ability to understand the specific function of DCN neurons in hearing. Although, optogenetics has been used to study the function of pathways in other systems for several years, in the auditory system only neurons in the auditory cortex have been studied using this technique. Adeno-associated viral vectors with either channelrhodopsin-2 fused with GFP (ChR2-GFP) or halorhodopsin fused with mCherry (HaloR-mCherry), capable of expressing light sensitive cation channels or chloride pumps, respectively, were delivered into the dorsal cochlear nucleus (DCN). One to 18 months later, expression of ChR2 and HaloR was observed throughout the DCN. Rhodopsin distribution within the DCN was determined to be within several cell types identified based on morphology and location within the DCN. Expression of ChR2-GFP and HaloR-mCherry was found at both the injection site as well as in regions receiving projections from the site. Wavelength appropriate optical stimulation in vivo resulted in neuronal activity that was significantly increased over pre-stimulation levels with no return to baseline levels during the time of the light exposure. We also examined the effects of optically driven neuronal activity on subsequent tone driven responses in the DCN. In the DCN 75% of the 16 electrode sites showed decreased neuronal activity in response to a tone immediately following light stimulation while six percent were decreased following tone stimulation and 19% of the electrode sites showed no change. This is in contrast to tone driven neuronal activity prior to the light exposure in which the majority of electrode sites showed increased neuronal activity. Our results indicate that expression and activation of rhodopsin within neurons involved in auditory processing does not appear to have deleterious effects on hearing even 18 months following expression. In addition, virally targeted rhodopsins may be useful as tract tracers to delineate as well as modulate the activity of pathways and specific neurons. In the future rhodopsins can be targeted to specific subpopulations of auditory neurons. Ultimately, photostimulation may provide a physiologically relevant method for modulating the function of auditory neurons and affecting hearing outcomes. This article is part of a Special Issue entitled Optogenetics (7th BRES).

Vesicular glutamate transporters: spatio-temporal plasticity following hearing loss.

An immunocytochemical comparison of vGluT1 and vGluT3 in the cochlear nucleus (CN) of deafened versus normal hearing rats showed the first example of vGluT3 immunostaining in the dorsal and ventral CN and revealed temporal and spatial changes in vGluT1 localization in the CN after cochlear injury. In normal hearing rats vGluT1 immunostaining was restricted to terminals on CN neurons while vGluT3 immunolabeled the somata of the neurons. This changed in the ventral cochlear nucleus (VCN) 3 days following deafness, where vGluT1 immunostaining was no longer seen in large auditory nerve terminals but was instead found in somata of VCN neurons. In the dorsal cochlear nucleus (DCN), while vGluT1 labeling of terminals decreased, there was no labeling of neuronal somata. Therefore, loss of peripheral excitatory input results in co-localization of vGluT1 and vGluT3 in VCN neuronal somata. Postsynaptic glutamatergic neurons can use retrograde signaling to control their presynaptic inputs and these results suggest vGluTs could play a role in regulating retrograde signaling in the CN under different conditions of excitatory input. Changes in vGluT gene expression in CN neurons were found 3 weeks following deafness using qRT-PCR with significant increases in vGluT1 gene expression in both ventral and dorsal CN while vGluT3 gene expression decreased in VCN but increased in DCN.

A method for evaluating the entrance surface dose from the measurement of exposure and half value layer in intraoral radiography using a radiophotoluminescent dosemeter.

A method for measuring entrance surface dose in intraoral radiography for nationwide survey for the determination of guidance levels was developed using a commercially available radiophotoluminescent dosemeter (RPLD) system. From the ratio of the readings of the RPLD detectors. with and without a 1 mm thick aluminium filter, half value layer (HVL) and the energy dependency correction factor were derived. HVL and exposure for intraoral radiography were obtained with uncertainties of +/- 0.11 mmAl (SD) and +/- 2.1% (CV), respectively. This has been achieved by calibration at various beam qualities used in intraoral radiography. The HVLs and outputs of intraoral radiography units were measured by means of the mailed RPLD holders and also by ionisation chambers in 19 dental schools in Japan. Results obtained from the two methods of measurement agreed well for both HVLs and outputs.

[Indication for surgical and microwave coagulation therapy for multiple (> or = 5) bilobar liver metastases from colorectal cancer].

We analyzed methods and clinicopathological factors for multiple (> or = 5) bilobar liver metastases (H3) from colorectal cancer and evaluated the indication of surgical and microwave coagulation therapy (MCT) for them. Twenty-four patients with H3 underwent surgical therapy and were divided into two groups. Group a: 9 patients with a prognosis of more than 700 days. Group b: the remaining 15 patients. There was no significant difference in prognosis between those receiving MCT and resection + MCT as a surgical therapy. The number and maximum diameter of tumors tended to be smaller in Group a. The number of tumors was less than or equal to 9 and the maximum size of the tumors was 38 mm. Moreover, the tumor could be controlled by MCT alone if the tumor size was less than 30 mm. MCT is a useful therapy for these cases and the indication for surgical therapy may depend on the number and maximum size of tumors.

[Comparison of microwave coagulation using a new type electrode with radiofrequency ablation in the liver of living animals].

In the present study, we compared microwave coagulation using a new type of electrode, a teflon-coated electrode that was developed in order to increase the area of coagulation, with radiofrequency ablation using a Radionics Cool-tip electrode inserted into the pig liver. Two Landrace male pig were put under general anesthesia. A microwave electrode (insulated area: 6 mm, teflon-coated electrode 16 G) and a radiofreqency (RF) electrode (Cool-tip RF single electrode 17 G) were passed through the surface of the livers of the pig. A thermometer was placed 1 cm from the tip of the electrode in order to measure the changes in the temperature of the area surrounding the electrode. In this study, the microwave setting was 80 W, and the RF pulse was set automatically. The coagulated and ablated areas of the liver were measured after 2.5, 5, and 10 minutes of energy delivery (n = 4). The diameter of the coagulated area of the liver following 2.5, 5 and 10 minutes of microwave exposure was 23.5 +/- 4.8 mm, 29.5 +/- 5.2 mm and 32.5 +/- 6.4 mm, respectively. On the other hand, the diameter of the ablated area of the liver following 2.5, 5 and 10 minutes of RF exposure was 18.5 +/- 4.1 mm, 24 +/- 7.8 mm and 28 +/- 4.9 mm, respectively. The mean temperature of the liver 1 cm from the microwave and RF electrodes (measured time: 2 minutes) was 69.6 degrees C and 56.3 degrees C. respectively (n = 12). Thus, the temperature of the area surrounding the microwave electrode was significantly higher than the temperature of the area surrounding the RF electrode (p = 0.0065). The teflon-coated microwave electrode achieved superior results to the Radionics Cool-tip electrode with respect to the diameter of the coagulated area and the temperature of the area in which the electrode was inserted, at the specified times.

Accumulation of beta-catenin in the cytoplasm and the nuclei during the early hepatic tumorigenesis.

Hepatocellular carcinomas (HCCs) are thought to develop as well-differentiated tumors and progress to less-differentiated tumors. However, the genetic changes underlying the development and progression of HCCs are not well understood. Recent studies have shown frequent beta-catenin gene activation in HCCs by somatic alterations involving exon 3, resulting in the activation of the Wnt/Wingless signal transduction pathway. However, the exact process in which activation of Wnt/Wingless signal transduction pathway occurs during hepatic tumorigenesis remains to be elucidated. The aim of the present study was to investigate at what stage of hepatocellular tumorigenesis this pathway was activated. Altered expression of beta-catenin was investigated immunohistochemically with special reference to the grade of histological differentiation in 41 HCCs and eight dysplastic nodules. Mutational analysis of the beta-catenin gene with single-strand conformation polymorphism method and polymerase chain reaction amplification was related with the expression of this protein. beta-Catenin was expressed in the cytoplasm and the nuclei in three cases among eight dysplastic nodules, in four cases among 20 well differentiated HCCs, in five cases among 15 moderately differentiated HCCs, and one case among six poorly differentiated HCCs, respectively. Expression of beta-catenin in the cytoplasm and the nuclei was associated in one case with mutation and two cases without mutation for beta-catenin gene among 11 screened HCCs. It was concluded that beta-catenin was accumulated in the cytoplasm and the nuclei in pre-cancerous lesions of the liver and might contribute, at least in part, to hepatic tumorigenesis.

Dependence on the sample width of signals from a near-field optical microscope.

We numerically investigated signals from a scanning near-field optical microscope (NOM) for samples of various sizes by using the finite-difference time-domain method. Under the usual conditions that apply to detection, the signal intensity depends on the width of the sample, even though the sample will be wider than the wavelength, which is much larger than the aperture, i.e., the lateral resolution of the NOM. This is an obstacle to measuring the local optical constant of samples by means of obtaining the signal intensity. When waves propagating in all directions are collected, this dependence on the sample width is reduced. The whole angle detection is important for observing the distribution of the optical constants.

[Assessment of colorectal cancer patients exhibiting bilobular multiple hepatic metastases].

We assessed 23 patients who underwent surgical therapy of hepatectomy or microwave coagulation therapy (MCT) for bilobular multiple hepatic metastatic foci following curative resection of the primary lesion of colorectal cancer. Hepatectomy was the first surgical therapy for 10 patients, and 6 of them received combined intra-arterial chemotherapy. All 13 patients in the MCT group received intra-arterial chemotherapy, and 8 of them underwent combined hepatectomy. The two-year survival rate of the hepatectomy group, classified according to the first surgical therapy, was 40% against 52% in the MCT group. In comparison with the H2 (2-5 foci) patients in the hepatectomy group, there were 7 H2 patients in the MCT group, and the two-year survival rate of these 7 patients was 50%. No significant difference was observed between hepatectomy and the MCT as the first surgical therapy. The survival rates of the 5 patients who received treatment for recurrence after the first surgery and of the 18 patients without any recurrence treatment were 80% and 40%, respectively. No significant difference existed between the two groups, but a p value of 0.06 was noted. MCT was considered to be useful local therapy for cancer as the first therapy and as a therapy following recurrence.

[A case report of intra-thoracic biliary fistula after percutaneous microwave coagulation therapy].

Among the locoregional therapies for hepatic malignant tumor, percutaneous microwave coagulation therapy (PMCT) has spread widely as a minimally invasive therapy. We performed PMCT on 40 patients under hepatic blood flow interruption with the aim of improving the radical treatment and expanding the coagulation area by single microwave delivery. A patient with a 2.5 cm lesion in S5 of the liver under PMCT transdiaphragmatically, but he developed a postoperative biliary fistula with consequent development of an intra-thoracic abscess through the diaphragm. Biliary fisture and liver abscess disappeared with open drainage under thoracotomy and laparotomy. Liver abscess has occasionally been reported as a PMCT complication, whereas there have been no reports, to the authors' knowledge, about intra-thoracic abscess as a PMCT complication, as in our case. It should be kept in mind that the penetration of the diaphragm as a route for PMCT may result in a biliary fistula flowing into the thorax, leading to a very serious condition.

[Transcatheter arterial chemo-embolization using degradable starch microspheres (DSM) markedly effective for post-hepatectomy intra-hepatic recurrence in a patient with cholangioma].

Transcatheter arterial chemo-embolization (TACE) using degradable starch microspheres (DSM) was performed for multiple recurrence after hepatectomy in a patient with cholangiocarcinoma. The patient was a 68-year-man. He received treatment for hepatitis type C starting in 1996 at a nearby hospital. In November 1997, an increased AFP level was noted and a CT scan of the abdomen revealed an abnormal shadow in the liver. On May 21, 1998, imaging results led to the diagnosis of cholangiocarcinoma or a mixed type of hepatocellular carcinoma with cholangioma. Hepatic S7 sub-sequential resection was performed. The lesion was found to be a tumor-forming type, measuring 2.2 x 2.0 cm in diameter, diagnosed histopathologically as cholangiocarcinoma, tw (-), but Stage III since a nodule suggesting intrahepatic metastasis was noted in the cut surface of the resected liver. CT scan after a month revealed multiple metastatic lesions in the liver. TACE was performed by administering 450 mg of DSM, 10 mg of MMC and 30 mg of FARM, given in three divided doses on October 30, 1998, and February 9, 1999, according to Seldinger's method. A CT scan on January 31, 2000 revealed nearly complete remission of the hepatic SOL. Accordingly, TACE was considered to be useful therapy in combination with DSM, MMC and FARM for intrahepatic recurrence of cholangiocarcinoma.

[Three long surviving patients with gastric cancer metastasizing to the liver under interdisciplinary therapy].

In 30 patients with gastric cancer metastasizing to the liver over the past 15 years at our hospital the primary foci in the stomach could be resected in a curative manner. The authors report herein three long surviving patients in this series. [Case 1] A 49-year-old male. Distal gastrectomy was performed in November 1984. Metastasis to the liver occurred in June 1986. The right lobe of the liver was resected in November 1987 after transcatheter arterial embolization (TAE). Although hepatic arterial infusion chemotherapy was conducted, the cancer metastasized to the whole body, and the patient died in December 1991. [Case 2] A 65-year-old female. Distal gastrectomy was performed in July 1994. The left hepatic lobe and segment 5 in the right lobe were resected in June 1995. Although TAE was performed six times starting in December 1996, the patient died of hepatic failure in July 1999. [Case 3] A 73-year-old male. This patient simultaneously received distal gastrectomy and extended resection of the posterior hepatic segments in September 1997. Cancer recurred in the remaining liver in July 1998. Although microwave coagulation therapy (MCT) and TAE were performed, the patient died of hepatic failure in January 2000. In these patients who survived for a long period, the primary focus was well-differentiated adenocarcinoma under sufficient local control with metastasis limited to the nearest regional lymph nodes (group 1 lymph nodes). The patients could undergo interdisciplinary therapy, including hepatectomy, MCT, TAE, and hepatic arterial infusion chemotherapy.

Activation of the beta-catenin gene by interstitial deletions involving exon 3 as an early event in colorectal tumorigenesis.

beta-Catenin has been identified as an oncogene in several tumors including colorectal cancers. beta-Catenin gene is activated by interstitial deletions involving exon 3 in colorectal carcinomas of Japanese population, in contrast to amino acid substitutions detected among Caucasian population. The aim of this study was to examine the type and frequency of beta-catenin gene mutation during early stages of colorectal tumorigenesis. We screened 100 colorectal adenomas for somatic mutations in the beta-catenin gene by single-strand conformation polymorphism method, as well as polymerase chain reaction amplification. In cases with mutations, sequencing analyses and immunohistochemical staining were also performed. Somatic interstitial deletions of 272-413 bp, each of which included all parts of exon 3, were detected in three tumors. However, no adenoma carried missense mutations. We confirmed accumulation of aberrant beta-catenin protein in cytoplasm and nuclei of adenoma cells by immunohistochemical analysis. Our results suggested that activation of the beta-catenin gene by interstitial deletions involving exon 3 might be less frequent compared with frequent alterations of adenomatous polyposis coli (APC) gene, but could be an early event in colorectal tumorigenesis equivalent to APC gene alterations in the Japanese population.

Molecular and biological analysis of carcinoma of the small intestine: beta-catenin gene mutation by interstitial deletion involving exon 3 and replication error phenotype.

The genetic mechanisms of carcinomas of the small intestine are not well understood. We report the results of analysis of genetic alterations in a case of small intestinal carcinoma. A tumor in the terminal ileum was resected in a 59-yr-old woman. Histologically, the tumor was classified as well-differentiated adenocarcinoma. We screened for genetic alterations in adenomatous polyposis coli (APC), beta-catenin, K-ras, and p53 genes, as well as microsatellite instability, which are known to be involved in colorectal tumorigenesis. The tumor exhibited somatic interstitial deletion of 425-bp, which included the entire exon 3 in beta-catenin gene. Immunohistochemical staining confirmed accumulation of aberrant beta-catenin protein in the cytoplasm and nuclei of the malignant tissue. Furthermore, a frameshift mutation in the transforming growth factor beta receptor type II gene with replication error phenotype was detected in the tumor DNA. In contrast, no genetic alterations were found in the APC, K-ras, and p53 genes. Our results suggested that both beta-catenin gene mutation and replication error phenotype might contribute to carcinogenesis of the small intestinal tumor in our case. This is the first report that activation of beta-catenin gene by somatic gene mutation is involved in the development of carcinoma of the small intestine.

Antibody response to toxic shock syndrome toxin-1 of Staphylococcus aureus in dairy cows.

Antibody response to toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus in dairy cows was examined by enzyme linked immunosorbent assay (ELISA). Serum antibody to TSST-1 was not detected in 39 (76.5%) of 51 calves, which were 1-6 months of age. In contrast, TSST-1 antibody was demonstrated in 1728 (72.6%) of 2380 lactating cows housed on 36 dairy farms. The ELISA values of antibody ranged from 0.2 to 3.0 OD and presented a distribution with the peak at 1.6 OD. The mean ELISA value differed between farms, and it increased slightly along with parturient history. Somatic cell counts of milk from 174 lactating cows was compared with TSST-1 antibody and tst1,000,000 cells per ml. The mean ELISA values in milk were lower than those of sera, but they rose as somatic cells increased. The tst gene of S. aureus detected in 76.0-86.2% of the milk samples containing somatic cells > 500,000 cells per ml, a level which indicates mastitis. The data suggests that many lactating cows may be infected by TSST-1- producing S. aureus.

AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1.

The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.

[Microwave coagulation therapy under laparotomic ischemia for multiple liver metastases of colorectal cancer].

Microwave coagulation therapy (MCT) under laparotomic ischemia induced by partial obstruction of the hepatic artery and portal vein was conducted on patients with multiple liver metastases of colorectal cancer. The patients were then compared with those who underwent non-ischemic MCT. Among the patients with liver metastasis of colorectal cancer we encountered between August 1990 and October 1998, 14 patients who developed multiple cancer (five or more) in the bilateral liver lobes were enrolled in the study. No clear differences were observed in the sex, age, frequency of simultaneousness, therapy other than MCT, number of foci, and number of MCT between the ischemic MCT and non-ischemic MCT group. Postoperative CT revealed residual foci in one of the seven patients in the ischemic MCT group. A comparison of the cumulative survival rate revealed that the ischemic MCT group had a higher one-year survival rate (50%) than the non-ischemic MCT group (14%). A comparison of patients with a residual lesion and those with no residual lesion showed that all six patients with a residual lesion died less than one year after surgery. Eight patients with no residual lesion had a significantly better prognosis (p < 0.05). It is important to eliminate any residual metastatic lesion during surgery in multiple liver metastases of colorectal cancer If the residual lesion is non-resectable, its elimination by ischemic MCT would contribute to the long-term survival of the patients.

A case of Crohn's disease with recurrent massive life-threatening hemorrhage from terminal ileum.

A case of Crohn's disease that underwent bowel resection two times at 3-year intervals due to life-threatening hemorrhage from ileum is presented. The bleeding sites were located in the ulcer lesions of ileum, in the oral side near to the ileum-colon transition. The first bleeding point was at the longitudinal ulcer of the terminal ileum and the secondary bleeding site was at the profound ulcer of ileum appearing in the oral side near to the ileo-colic anastomosis. This is the first case of Crohn's disease with recurrent life-threatening massive hemorrhage in the terminal region of ileum, for which operative bowel resections were necessary. This case suggests that we should search for the bleeding site in ileal lesions developed in the circumference of and especially the oral side near to the anastomosis due to prior resection, when intestinal massive bleeding occurs again after bowel resection, and that the careful follow-up and strict treatment with diet therapy and/or anti-inflammatory drugs are necessary for the protection of recurrence in patients with Crohn's disease.

Production of exfoliative toxin A by Staphylococcus aureus isolated from mastitic cow's milk and farm bulk milk.

The production of exfoliative toxins A and B (ETA and ETB) by Staphylococcus aureus isolated from mastitic cow's milk and farm bulk milk was examined by the reverse passive latex agglutination method (RPLA). ETA was detected in 2 (1.2%) of 162 isolates from mastitic cow's milk and in 1 (0.6%) of 166 isolates from farm bulk milk. RPLA titers of these isolates were much lower than in human isolates. No ETB was detected in any of the isolates tested. These ETA-positive isolates belonged to bovine ecovar. They were non-typable using the international phage set for human strains. When these ETA-positive isolates were subcutaneously inoculated into neonatal mice, general exfoliation of the epidermis accompanied by the so-called Nikolsky sign was not recognized. By the immunoblotting and PCR methods, however, ETA and eta gene were recognized in the ETA-positive isolates from mastitic cow's milk and farm bulk milk. These data suggest that ETA is also produced by bovine isolates of S. aureus, but in smaller quantities.