Sugata Takahashi

Transcranial fluorescence imaging of auditory cortical plasticity regulated by acoustic environments in mice

Functional brain imaging using endogenous fluorescence of mitochondrial flavoprotein is useful for investigating mouse cortical activities via the intact skull, which is thin and sufficiently transparent in mice. We applied this method to investigate auditory cortical plasticity regulated by acoustic environments. Normal mice of the C57BL/6 strain, reared in various acoustic environments for at least 4 weeks after birth, were anaesthetized with urethane (1.7 g/kg, i.p.). Auditory cortical images of endogenous green fluorescence in blue light were recorded by a cooled CCD camera via the intact skull. Cortical responses elicited by tonal stimuli (5, 10 and 20 kHz) exhibited mirror‐symmetrical tonotopic maps in the primary auditory cortex (AI) and anterior auditory field (AAF). Depression of auditory cortical responses regarding response duration was observed in sound‐deprived mice compared with naïve mice reared in a normal acoustic environment. When mice were exposed to an environmental tonal stimulus at 10 kHz for more than 4 weeks after birth, the cortical responses were potentiated in a frequency‐specific manner in respect to peak amplitude of the responses in AI, but not for the size of the responsive areas. Changes in AAF were less clear than those in AI. To determine the modified synapses by acoustic environments, neural responses in cortical slices were investigated with endogenous fluorescence imaging. The vertical thickness of responsive areas after supragranular electrical stimulation was significantly reduced in the slices obtained from sound‐deprived mice. These results suggest that acoustic environments regulate the development of vertical intracortical circuits in the mouse auditory cortex.

Effects of sinus surgery on asthma in aspirin triad patients.

The aspirin triad (nasal polyposis, asthma and sensitivity to aspirin) is a well-recognized clinical entity, also known as aspirin-induced asthma (AIA). The sinusitis associated with AIA is often difficult to treat and aggravates the asthmatic symptoms. In order to evaluate the surgical treatment of sinusitis in AIA, 22 patients who underwent sinus surgery were studied. Twenty patients (90.9%) got any relief of their asthma symptoms from sinus surgery. Postoperative pulmonary function test 1 year after surgery showed statistically significant improvement over the preoperative one. Three of 5 patients (60%) who used systemic steroids were able to eliminate or reduce their dosages. Also, 8 of 17 patients (47.1%) who were using inhaled topical steroids reduced their dosages and statistical analysis showed a significant difference in the doses of topical steroid used before and after surgery. Subjective evaluation of 20 patients (90.9%) indicated that the sinus surgery was effective for their asthma condition; showing from mild to marked improvement. For AIA patients aggravated by sinus disease, we recommend sinus surgery to improve the quality of life.

The olfactory route for cerebrospinal fluid drainage into the peripheral lymphatic system

Drainage of the cerebrospinal fluid through the olfactory nerves into the nasal lymphatics has been suggested repeatedly. To investigate precisely the morphology of this pathway, India ink was injected into the subarachnoidal space of the rat brain, and samples including the olfactory bulbs, olfactory tracts and the nasal mucosa were observed by light and electron microscopy. Under the dissecting microscope, ink particles were found within the subarachnoid space and along the olfactory nerves. At the nasal mucosa, a lymphatic network stained in black was identified near the olfactory nerves, which finally emptied into the superficial and deep cervical lymph nodes. Light microscopically, ink particles were found in the subarachnoid space, partially distributed around the olfactory nerves and within the lymphatic vessels. By electron microscopy, the subarachnoid space often formed a pocket‐like space in the entrance of the fila olfactoria. The olfactory nerves were partially surrounded by ink particles within the space between perineurial cells and epineurial fibroblasts. At the nasal mucosa, the lymphatics were frequently located close to the nerves. These results indicate that the cerebrospinal fluid drains from the subarachnoid space along the olfactory nerves to the nasal lymphatics, which in turn, empties into the cervical lymph nodes. This anatomical communication, thus, allows the central nervous system to connect with the lymphatic system. The presence of this route may play an important role in the movement of antigens from the subarachnoidal space to the extracranial lymphatic vessels, resulting in inducement of an immune response of the central nervous system.

Immunohistochemical analysis of the olfactory mucosa by use of antibodies to brain proteins and cytokeratin.

The present study deals with the immunohistochemical detection of four brain-derived proteins and cytokeratin in the normal olfactory mucosa of humans and guinea pigs. Neurofilament protein immunoreactivity was found in the olfactory vesicles, dendrites, and perikaryon of receptor cells, and in thick nerve bundles located deep in the lamina propria. The antiserum to neuron-specific enolase (NSE) selectively stained olfactory receptor cells throughout the length of the bundles. The NSE immunoreactivity also was recognized in nerve bundles of various sizes throughout the lamina propria. Glia-specific S-100 protein immunoreactivity was present in Bowmans glands as well as in all nerve bundles in the lamina propria, but not in any cellular elements constituting the olfactory epithelium. Immunoreactivity for spot-35 protein, which was considered to be specific for cerebellar Purkinje cells, was found in flasklike cells (microvillar cells) occurring near the free surface of the epithelium. The basal cells in the olfactory and respiratory epithelium were stained positively with a cytokeratin antiserum.

Neuron-specific enolase, neurofilament protein and S-100 protein in the olfactory mucosa of human fetuses

SummaryImmunohistochemical examination for neuronspecific enolase (NSE), neurofilament protein (NFP), and S-100 protein was performed in the olfactory mucosa of human fetuses. NSE and NFP immunoreactivities were found in the olfactory receptor cells, while no S-100 immunoreactive cells were recognized within the olfactory epithelium. The anti-NSE serum stained various types of nerve bundles in the lamina propria mucosae; a population of the NSE-positive nerve bundles was also immunoreactive for NFP. The anti-S-100 serum clearly demonstrated Schwann cells associated with the nerve fibers in the lamina propria mucosae. These findings 1) suggest a possibility of NSE and NFP as new marker substances for olfactory cells and 2) indicate that immunohistochemistry is a useful tool to analyse the cellular components of the olfactory organs in normal and pathological conditions.

Transcranial photo-inactivation of neural activities in the mouse auditory cortex.

Flavoprotein fluorescence in the brain is intimately coupled with neuronal aerobic energy metabolism. If flavoproteins are photobleached, neural activities may be affected owing to dysfunction in aerobic energy metabolism in mitochondria. We tested this possibility in cortical slices from mice, and found that exposure to blue light (lambda = 475 nm) derived from a 20 mW diode laser for 50 min suppresses trans-synaptic components of field potentials. This finding formed the basis of a transcranial photo-inactivation technique, that was used to investigate auditory signal transmission between the anterior auditory field (AAF) and the primary auditory cortex (AI) in anesthetized mice. Cortical responses in AAF and AI, elicited by 5 kHz tonal stimuli, were visualized using transcranial flavoprotein fluorescence imaging. After determining responsive areas in AAF and AI, the auditory cortex was exposed to the blue diode laser via the intact skull, while either AAF or AI was protected with a piece of carbon paper. Although the photo-inactivation of AI had no significant effect on the fluorescence responses in AAF, the photo-inactivation of AAF significantly reduced the fluorescence responses in AI, indicating the presence of auditory signal transmission from AAF to AI.

Inactivation of tumor suppressor Dlg1 augments transformation of a T-cell line induced by human T-cell leukemia virus type 1 Tax protein

BackgroundThe interaction of human T-cell leukemia virus type 1 (HTLV-1) Tax1 protein with the tumor suppressor Dlg1 is correlated with cellular transformation.ResultsHere, we show that Dlg1 knockdown by RNA interference increases the ability of Tax1 to transform a mouse T-cell line (CTLL-2), as measured interleukin (IL)-2-independent growth. A Tax1 mutant defective for the Dlg1 interaction showed reduced transformation of CTLL-2 compared to wild type Tax1, but the transformation was minimally affected by Dlg1 reduction. The few Tax1ΔC-transduced CTLL-2 cells that became transformed expressed less Dlg1 than parental cells, suggesting that Dlg1-low cells were selectively transformed by Tax1ΔC. Moreover, all human T-cell lines immortalized by HTLV-1, including the recombinant HTLV-1-containing Tax1ΔC, expressed less Dlg1 than control T-cell lines.ConclusionThese results suggest that inactivation of Dlg1 augments Tax1-mediated transformation of CTLL-2, and PDZ protein(s) other than Dlg1 are critically involved in the transformation.

Prevalence of human papillomavirus in mobile tongue cancer with particular reference to young patients

The carcinogenetic role of human papillomavirus (HPV) in mobile tongue cancer remains unclear because of conflicting results reported in the literature. This disparity is likely to be due to variations in the samples and methods used. Furthermore, despite a tendency for increased prevalence of mobile tongue cancer in young adults, only a few reports specifically in young patients have been published. In the present study on 32 patients, including six in their 20s, we genotyped the prevalence of HPV using a highly sensitive detection tool in fresh‐frozen samples from surgical specimens and a novel detection device with electrochemical DNA chip and loop‐mediated isothermal amplification. In addition, we confirmed HPV prevalence by in situ hybridization and immunohistochemistry for the p16INK4a protein, regarded as a biomarker of HPV‐associated cancers. The frequency of 13 genotypes of high‐risk HPV was 0/32 (0%), which was further confirmed by in situ hybridization. Overexpression of p16INK4a protein was observed in six of the 32 patients (19%), with four (67%) also overexpressing p53. Because there is usually a lack of p53 overexpression in HPV‐associated cancer, it is unlikely that p16INK4a protein overexpression is correlated with HPV infection. Consequently, it is unlikely that HPV infection plays an important role in mobile tongue carcinogenesis, in particular in young adults. In addition, our data suggest that the overexpression of p16INK4a protein is not an appropriate biomarker for HPV association in mobile tongue carcinogenesis. (Cancer Sci 2012; 103: 161–168)

Surgical treatment of 52 cases of auditory ossicular malformations

OBJECTIVE The aim of this study was to determine the relationship between hearing improvements and the pathological conditions of auditory ossicular malformations. METHODS Fifty-two ears (49 patients) with auditory ossicular malformations without congenital aural atresia were studied. The classification of the pathological conditions was based on surgical findings. Group 1 showed defects in the incudo-stapedial (I-S) joint, Group 2, fixation of the stapes, Group 3, fixation of the malleus and incus and Group 4, defects in the I-S joint with fixation of the stapes. Hearing improvements at the final examination were designated as successful when both of the following were satisfied. (1) Air-bone gap was reduced to 20 dB or less. (2) Postoperative hearing gain exceeded 15 dB. RESULTS Successful hearing improvements after operations were achieved in 20 ears (95%) in Group 1, 21 ears (91%) in Group 2, three ears (75%) in Group 3 and two ears (50%) in Group 4. They were observed in 88% of all cases. CONCLUSIONS Postoperative hearing improvements of auditory ossicular malformations yielded good results, particularly in Groups 1 and 2. In retrospect, the unsuccessful cases with fixation of the stapes would have been improved if stapedectomy were chosen rather than mobilization. In defects to the long process of Group 4, we wished to perform a reconstruction using the malleus attachment piston after small-fenestra stapedectomy with regard to the long-term hearing results.

Caspase-3, caspase-8, and nuclear factor-kappaB expression in human cholesteatoma.

Background: Cholesteatoma is characterized by the accumulation of keratinizing epithelium resulting from the proliferation and differentiation of epithelium. Researchers are presently unraveling the role that apoptosis plays in the disease seen in cholesteatoma epithelium. Caspases play a key role in apoptosis. Caspase-8, which is activated by the induction of tumor necrosis factor-α, leads to activation of caspase-3, which activates apoptotic nucleases. Nuclear factor-κB is a transcription factor known to inhibit apoptosis induced by tumor necrosis factor-α. Hypothesis: In this study, we hypothesized that expression of caspase-3, caspase-8, and nuclear factor-κB is uniquely connected to the proliferation, differentiation, and programmed cell death of keratinocytes during the growth and development of cholesteatoma. Methods: We obtained 41 cholesteatoma specimens for this study. The presence of these proteins in cholesteatoma was examined using immunohistochemistry and a colorimetric assay system. We also examined the patterns of apoptosis by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Results: Using the immunoperoxidase staining method, caspase-3 was found to be densely localized in the spinous and granular layers of cholesteatoma epithelium; caspase-8 was also found in the granular layer. Nuclear factor-κB was localized densely in the perinuclear region of the epithelium. The results obtained with immunoperoxidase staining agreed with those obtained with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. In addition, the colorimetric assay method was used to measure the activity of caspase-3. Conclusion: These findings suggest that caspase-3 and caspase-8 play important roles in programmed cell death, which results in the accumulation of keratin debris during the growth of cholesteatoma. Nuclear factor-κB was found in cholesteatoma epithelium, but the transcription factor appeared to be inactivated.