Nerve growth factor-induced hyperexcitability of rat sensory neuron in culture

Abnormal spontaneous firing of primary sensory neurons is considered to be a cause of neuropathic pain. However, pathogenic mechanisms of hyperexcitable sensory neurons in neuropathic model animals are unclear. We examined effects of chronic treatment of nerve growth factor (NGF), one of candidate mediators for the pathogenesis, on excitability of sensory neurons by voltage-clamped recording in a cell-attached configuration. From rat dorsal root ganglion (DRG) neurons cultured without NGF, only stable holding currents without spontaneous firing activity were recorded. On the other hand, more than 20% neurons cultured in the presence of NGF for more than 3 days showed spontaneous current spikes at frequencies between 0.1 and 5 Hz. Each spikes had an initial inward phase followed by the outward phase, resulted from spontaneous transient depolarization followed by transient hyperpolarization. These spontaneous spikes were abolished by tetrodotoxin, lidocaine and reduction of extracellular concentration of Na+ from 154 mM to 100 mM, in all-or-none fashion, suggesting that spontaneous current spikes reflected spontaneous action potentials. From these results, it became evident that DRG neurons of adult rats had a nature to respond to NGF and obtained the abnormal hyperexcitability to fire spontaneously.     

The expression of vascular endothelial growth factor-A and -C, and receptors 1 and 3: correlation with lymph node metastasis and prognosis in tongue squamous cell carcinoma

We determined whether the expression of VEGF-A and -C and their receptors, Flt-1 and Flt-4, are associated with primary tumor size, regional lymph node metastasis, distant metastasis and prognosis in patients with tongue carcinoma. The expression of VEGF-A and -C, and their receptors, Flt-1 and Flt-4, in biopsy specimens taken from 73 patients with tongue carcinoma were examined by immunohistochemical staining. VEGF-A expression was associated with distant failure and VEGF-C expression correlated with locoregional recurrence and distant failure. Furthermore, VEGF-C expression was associated with lymph node recurrence in N0 cases. Multivariate analysis revealed that VEGF-C expression was an exclusively independent factor influencing lymph node metastasis. In terms of the overall 5-year survival rate, there was no significance correlation between the overall 5-year survival rate and expression of VEGF-A, Flt-1 and Flt-4 expression, whereas there was a significant difference between VEGF-C-positive and VEGF-C-negative cases (VEGF-C-positive, 51.7% vs VEGF-C-negative, 94.2%). Furthermore, there was a significant difference between positive and negative expression for both VEGF-A and VEGF-C. Multivariate analysis revealed that lymph node metastasis and VEGF-C expression were exclusive, independent factors influencing the overall survival rate. VEGF-C expression may be a predictive factor of regional lymph node recurrence and prognosis in patients with tongue carcinoma.     

Direct attachment of fibronectin to tresyl chloride-activated titanium

The aim of this study was to bind fibronectin directly to a titanium surface treated with tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride) for the development of a strong connection of a dental implant to subepithelial connective tissues and/or peri-implant epithelia. Basic terminal OH groups of mirror polished titanium were allowed to react with tresyl chloride at 37 degrees C for 2 days. The tresylated titanium disk was then immersed into a fibronectin/phosphate-buffered saline solution for 24 h at 37 degrees C. The activation reaction of the basic OH of titanium with tresyl chloride was confirmed by S2p, F1s, and O1s spectra using X-ray photoelectron spectroscopy and -O-S-O2- bonds using Fourier transform infrared reflection-absorption spectroscopy. After the reaction of fibronectin with titanium, the X-ray photoelectron spectroscopy revealed the remarkable effect of the activation of terminal OH groups with the tresyl chloride treatment. The N1s peak derived from the attached fibronectin still remained after 60 s of argon-ion sputtering after tresyl chloride treatment. In contrast, the N1s peak of the specimen not treated with tresyl chloride almost disappeared after only 10 s of argon-ion etching. Fibronectin, a well-known cell-adhesive protein, could easily be attached to the titanium surface by use of the tresyl chloride activation technique.     

Wear properties of alumina/zirconia composite ceramics for joint prostheses measured with an end-face apparatus

While only alumina is applied to all-ceramic joint prostheses at present, a stronger ceramic is required to prevent fracture and chipping due to impingement and stress concentration. Zirconia could be a potential substitute for alumina because it has high strength and fracture toughness. However, the wear of zirconia/zirconia combination is too high for clinical use. Although some investigations on composite ceramics revealed that mixing of different ceramics was able to improve the mechanical properties of ceramics, there are few reports about wear properties of composite ceramics for joint prosthesis. Since acetabular cup and femoral head of artificial hip joint are finished precisely, they indicate high geometric conformity. Therefore, wear test under flat contact was carried out with an end-face wear testing apparatus for four kinds of ceramics: alumina monolith, zirconia monolith, alumina-based composite ceramic, and zirconia based composite ceramic. Mean contact pressure was 10 MPa and sliding velocity was 40 mm/s. The wear test continued for 72 hours and total sliding distance was 10 km. After the test, the wear factor was calculated. Worn surfaces were observed with a scanning electron micrograph (SEM). The results of this wear test show that the wear factors of the both composite ceramics are similarly low and their mechanical properties are much better than those of the alumina monolith and the zirconia monolith. According to these results, it is predicted that joint prostheses of the composite ceramics are safer against break down and have longer lifetime compared with alumina/alumina joint prostheses.     

Novel TSC2 mutation in a patient with pulmonary tuberous sclerosis: lack of loss of heterozygosity in a lung cyst

A Japanese patient with tuberous sclerosis (TSC), who manifested with multiple lung cysts and pneumothorax, is described. All exons of two TSC genes, TSC1 and TSC2, in peripheral blood leukocytes from the patient were analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). A novel T-to-G transition was found in exon 19 of TSC2 at nucleotide position 2168. This mutation caused an amino acid change, L717R. There was no such mutation in any other family members or in 100 normal Japanese. An automated sequencer-assisted quantitative analysis of normal and mutated SSCP-bands revealed no loss of heterozygosity (LOH) in the lung cyst tissue of the patient.     

Rare glomerular capillary regeneration and subsequent capillary regression with endothelial cell apoptosis in progressive glomerulonephritis.

Glomerulonephritis (GN) leading to glomerular sclerosis remains an important cause of renal failure. The glomerulus is a capillary network, but endothelial and vascular reactions during progressive GN are not well understood. We have, therefore, examined the morphological alterations of glomerular capillary network and endothelial cells during the progression of damaged glomeruli to glomerular sclerosis. A progressive model of anti-glomerular basement membrane (GBM) GN was induced in Wistar-Kyoto (WKY) rats with a single injection of anti-rat GBM antibody. Severe necrotizing glomerular injuries were observed between day 5 and week 3 with a reduction in the number of total glomerular endothelial cells and total glomerular capillary lumina per glomerular cross sections. In necrotizing lesions, the glomerular endothelial cells were lost with the destruction of the glomerular capillary network. Moreover, angiogenic capillary repair with proliferation of endothelial cells was rare in severely damaged regions of glomeruli. Subsequently, mesangial hypercellularity and marked mesangial matrix accumulation occurred with absence of the development of a capillary network, and the necrotizing lesions progressed to sclerotic scars until 8 weeks. Although active necrotizing lesions could not be seen in damaged glomeruli between week 4 and week 8, the number of apoptotic endothelial cells gradually increased in the glomerular capillaries (0.10 +/- 0.01 apoptotic endothelial cells/glomerular cross section at week 8 versus 0.00 +/- 0.00 control cells (mean +/- SEM; P < 0.05) with the progression of glomerular sclerosis. Whereas the number of apoptotic endothelial cells increased in the damaged glomeruli, the number of total glomerular endothelial cells decreased (9.3 +/- 3.0 cells/glomerular cross section at week 8 versus 24.8 +/- 3.0 cells in control (mean +/- SD); P < 0.001) with regression of glomerular capillaries (3.6 +/- 2.5 capillary lumina/glomerular cross section at week 8 versus 35.0 +/- 5.0 capillary lumina in control (mean +/- SD); P < 0.001). Finally, glomerular endothelial cells could not be detected in the sclerotic lesions in progressive anti-GBM GN in WKY rats. These data indicate that the destruction of the capillary network of glomeruli and subsequent incomplete angiogenic capillary repair leads to glomerular sclerosis in progressive GN. Endothelial cell apoptosis with glomerular capillary regression may also contribute to the development of glomerular sclerosis. Injury of the glomerular capillary network with endothelial cell damage, including apoptosis and subsequent incomplete capillary repair, plays an important role in the progression of glomerular sclerosis during anti-GBM GN in WKY rats.     

A girl with 1p36 deletion syndrome and congenital fiber type disproportion myopathy

Chromosome 1p36 deletion syndrome is characterized by hypotonia, moderate to severe developmental and growth retardation, and characteristic craniofacial dysmorphism. Muscle hypotonia and delayed motor development are almost constant features of the syndrome. We report a 4-year-old Japanese girl with 1p36 deletion syndrome whose muscle pathology showed congenital fiber type disproportion (CFTD) myopathy. This is the first case report of 1p36 deletion associated with CFTD. This association may indicate that one of the CFTD loci is located at 1p36. Ski proto-oncogene −/− mice have phenotypes that resemble some of the features observed in patients with 1p36 deletion syndrome. Because fluorescent in situ hybridization analysis revealed that the human SKI gene is deleted in our patient, some genes in 1p36, including SKI proto-oncogene, may be involved in muscle hypotonia and delayed motor development in this syndrome. Received: March 4, 2002 / Accepted: July 7, 2002     

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.     

Liver metastasis from rectal cancer with prominent intrabile duct growth

Intrabiliary growth of liver metastases from colorectal cancer has rarely been studied. A surgically resected case of a metastatic liver tumor with prominent intrabiliary growth derived from rectal cancer is reported. The patient was a 62-year-old man who had received a low anterior resection for rectal cancer in March 2000. He was re-admitted due to obstructive jaundice in January 2003, and was diagnosed with hepatic malignancy in segment II of the liver with an intrabiliary tumor extending from the intrahepatic bile duct of segment II to the common hepatic duct. He underwent a left hepatectomy, a partial resection of segment VI, and an extrahepatic bile duct resection with reconstruction of the biliary tract. In the resected specimen, there were whitish tumors of 3 cm and 1.5 cm in diameter in segments II and VI, respectively, and an intrabiliary tumor originating from the main tumor in segment II extended to the common hepatic duct. Both the liver tumors and the intrabiliary tumor consisted of a well- to moderately differentiated adenocarcinoma, which showed the same histological features as the rectal cancer. The immunohistochemical findings strongly supported that these tumors, including the intrabiliary growth, were liver metastasis from the rectal cancer. The intrabiliary invasion and growth of metastatic liver tumors has generally been overlooked, notwithstanding their frequently observed biological behavior. The present case is informative, and further investigation into this type of metastatic liver tumor may be warranted.