Faculty Profiles - NISHIDA Keiichiro

Papers - NISHIDA Keiichiro

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New approach for assessing vascular distribution within bone tumors using dynamic contrast-enhanced MRI

Yukio Kawakami, Toshiyuki Kunisada, Shinsuke Sugihara, Atsushi Ono, Keiichiro Nishida, Nobuhiro Abe, Akira Kawai, Kazuo Fujiwara, Yuki Morimoto, Toshifumi Ozaki

JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY 133 ( 10 ) 697 - 703 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Purpose To differentiate benign from malignant bone tumors by analyzing the vascular distribution within bone tumors with dynamic contrast-enhanced MRI.
Methods We studied dynamic contrast-enhanced MRI for 49 bone tumors (22 malignant and 27 benign tumors). Seven small regions of interest (ROI) were set inside the largest portion of each tumor. Four ROI were placed evenly on the periphery and three ROI were placed evenly on the line of the longest breadth within the tumor. The slope of the curve (%Slope) was calculated on the time-intensity curves of the whole tumor and of each ROI. The variance values for the %Slope of the ROI were calculated to assess the dispersion of the intensity change at each ROI within the tumor.
Results Mean value of the %Slopes of whole tumor regions for malignant bone tumors (70.4 +/- 60.3%) was significantly higher than that for benign bone tumors (37.6 +/- 52.9%) (P = 0.015), although giant cell tumor (GCT), a locally aggressive tumor, had a relatively higher %Slope. Mean value of the variance of %Slopes for malignant bone tumors (3485.9 +/- 5942.5) was significantly higher than that for all benign tumors (470.4 +/- 583.9) (P = 0.012), indicating that the %Slope values of seven ROI within malignant bone tumors varied more widely compared with the ROI inside benign bone tumors. GCT also demonstrated a lower value.
Conclusion Our method of analyzing the signal intensity change at seven separate regions that evaluates the vascular distribution within a tumor could be a useful tool for differentiating between benign and malignant bone tumors.

DOI： 10.1007/s00432-006-0190-9

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A novel dominant-negative mutation in Gdf5 generated by ENU mutagenesis impairs joint formation and causes osteoarthritis in mice

Hiroshi Masuya, Keiichiro Nishida, Tatsuya Furuichi, Hideaki Toki, Gen Nishimura, Hidehiko Kawabata, Haruka Yokoyama, Aki Yoshida, Sayaka Tominaga, Junko Nagano, Aya Shimizu, Shigeharu Wakana, Yoichi Gondo, Tetsuo Noda, Toshihiko Shiroishi, Shiro Ikegawa

HUMAN MOLECULAR GENETICS 16 ( 19 ) 2366 - 2375 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Growth and differentiation factor 5 (GDF5) has been implicated in chondrogenesis and joint formation, and an association of GDF5 and osteoarthritis (OA) has been reported recently. However, the in vivo function of GDF5 remains mostly unclarified. Although various human GDF5 mutations and their phenotypic consequences have been described, only loss-of-function mutations that cause brachypodism (shortening and joint ankylosis of the digits) have been reported in mice. Here, we report a new Gdf5 allele derived from a large-scale N-ethyl-N-nitrosourea mutagenesis screen. This allele carries an amino acid substitution (W408R) in a highly conserved region of the active signaling domain of the GDF5 protein. The mutation is semi-dominant, showing brachypodism and ankylosis in heterozygotes and much more severe brachypodism, ankylosis of the knee joint and malformation with early-onset OA of the elbow joint in homozygotes. The mutant GDF5 protein is secreted and dimerizes normally, but inhibits the function of the wild-type GDF5 protein in a dominant-negative fashion. This study further highlights a critical role of GDF5 in joint formation and the development of OA, and this mouse should serve as a good model for OA.

DOI： 10.1093/hmg/ddm195

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Comparative biomechanical analysis of an improved novel pedicle screw with sheath and bone cement

Tomoyuki Takigawa, Masato Tanaka, Hitoshi Konishi, Hisanori Ikuma, Haruo Misawa, Yoshihisa Sugimoto, Kazuo Nakanishi, Koichi Kuramoto, Keiichiro Nishida, Toshifumi Ozaki

JOURNAL OF SPINAL DISORDERS & TECHNIQUES 20 ( 6 ) 462 - 467 2007.8

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Study Design: A human cadaveric biomechanical study of fixation strength of an improved novel pedicle screw (NPS) with cement and a conventional screw.
Objective: To clarify whether the NPS has adequate fixation strength without leakage in vertebrae with low bone quality.
Summary of Background Data: The fixation strength of pedicle screws decreases in frail spines of elderly osteoporotic patients. Augmentation of screw fixation with bone cement must be balanced against increased difficulty of screw removal and risk of cement leakage. We developed the NPS consisting of an internal screw and an outer sheath to mitigate the disadvantages of cement augmentation.
Methods: The T12 and L1 vertebrae obtained from 18 formalin preserved cadavers (11 males and 7 females; mean age, 82.7y) were used. The mean bone mineral density was 0.39 +/- 0.14 g/cm(2). The NPS was inserted into one pedicle of each vertebra and the control screw, a Compact CD2 screw, was inserted into the contralateral pedicle. Both screws were 6 mm in diameter and 40 mm in length. Pull-out tests were performed at a crosshead speed of 10 mm/min. Cyclic loading tests were performed with a maximum 250 N load at 2 Hz until 30,000 cycles.
Results: Cement leakage did not occur in any of the specimens tested. The mean maximum force at pull-out was 760 +/- 344 N for the NPS and 346 172 N for the control screw (P < 0.01). Loosening of 50% of the screws was observed after 17,000 cycles of the NPS and after 30 cycles of the control screw. The hazard ratio of loosening was 19.6 (95% confidence interval 19.3-19.9) (P < 0.001).
Conclusions: The NPS showed a significantly higher mechanical strength than the control screw in both pull-out tests and cyclic loading test than the control screw in both pull-out tests and cyclic loading test. The NPS showed more than adequate strength without cement leakage.

DOI： 10.1097/BSD.0b013e318030d2d6

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The association of C-reactive protein with an oxidative metabolite of LDL and its implication in atherosclerosis

Masako Tabuchi, Katsumi Inoue, Hitomi Usui-Kataoka, Kazuko Kobayashi, Misako Teramoto, Koji Takasugi, Kenichi Shikata, Masahiro Yamamura, Kenji Ando, Keiichiro Nishida, Junko Kasahara, Noriaki Kume, Luis R. Lopez, Kazuaki Mitsudo, Masakiyo Nobuyoshi, Tatsuji Yasuda, Toru Kita, Hirofumi Makino, Eiji Matsuura

JOURNAL OF LIPID RESEARCH 48 ( 4 ) 768 - 781 2007.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

C-reactive protein (CRP) is one of the strongest independent predictors of cardiovascular disease. We have previously reported that oxidized LDL (oxLDL) interacts with beta 2-glycoprotein I (beta 2GPI), implicating oxLDL/P2GPI complexes as putative autoantigens in autoimmune-mediated atherosclerotic vascular disease. In this study, we investigated the interaction of CRP with oxLDL/beta 2GPI complexes and its association with atherosclerosis in patients with diabetes mellitus (DM). CRP/oxLDL/R2GPI complexes were predominantly found in sera of DM patients with atherosclerosis. In contrast, noncomplexed CRP isoforms were present in sera of patients with acute/chronic inflammation, i.e., various pyrogenic diseases, rheumatoid arthritis (RA), and DM. Immunohistochemistry staining colocalized CRP and beta 2GPI together with oxLDL in carotid artery plaques but not in synovial tissue from RA patients, strongly suggesting that complex formation occurs during the development of adierosclerosis. Serum levels of CRP correlated with soluble forms of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and oxLDL/beta 2GPI complexes correlated with total cholesterol and hemoglobin Al c. Thus, the generation of CRP/oxLDL/beta 2GPI complexes seems to be associated with arterial inflammation, hyperglycemia, and hypercholesterolemia. CRP/oxLDL/R2GPI complexes can be distinguished from pyrogenic noncomplexed CRP isoforms and may represent a more specific and predictive marker for atherosclerosis.

DOI： 10.1194/jlr.M600414-JLR200

マウス成長軟骨におけるADAMTS-9発現の検討

熊岸加苗, 西田圭一郎, 百田龍輔, 山合友一朗, 広畑聡, Demircan Kadir, 内藤一郎, 二宮善文, 大塚愛二

解剖学雑誌 82 ( Suppl. ) 150 - 150 2007.3

A new Gdf5 allele with fusion of knee joint Reviewed

Masuya Hiroshi, Furuichi Tatsuya, Nishida Keiichirou, Nagano Junko, Yokoyama Haruka, Miura Ikuo, Wakana Shigeharu, Ikegawa Shirou, Shiroishi Toshihiko

GENES & GENETIC SYSTEMS 81 ( 6 ) 447 2006.12

IV型コラーゲンα鎖特異抗体を用いた血管基底膜の三次元観察

森本尊雅, 内藤一郎, 田口勇仁, 芳原成美, 長谷川治子, 中野和代, 佐渡義一, 西田圭一郎, 二宮善文, 大塚愛二

解剖学雑誌 81 ( 3 ) 96 - 96 2006.9

Decreased levels of insulin-like growth factor-1 and vascular endothelial growth factor relevant to the ossification disturbance in femoral heads spontaneous hypertensive rats

Takamitsu Komiyama, Keiichiro Nishida, Masanori Yorimitsu, Hideyuki Doi, Shinichi Miyazawa, Ai Kitamura, Aki Yoshida, Yoshihisa Nasu, Nobuhiro Abe, Toshifumi Ozaki

ACTA MEDICA OKAYAMA 60 ( 3 ) 141 - 148 2006.6

Language：English Publishing type：Research paper (scientific journal) Publisher：OKAYAMA UNIV MED SCHOOL

Ossification disturbance in femoral head reportedly is seen in the Spontaneously Hypertensive rats (SHR) between ages of 10 and 20 weeks. We investigated serum and tissue levels of insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VIEGF) in SHR relevant to the ossification disturbance and osteonecrosis of the femoral head. Serum levels of IGF-1 and VEGF were significantly lower in SHR than in Wistar Kyoto rats (WKY) at weeks 5, 10, 15 and 20 (P < 0.005). The incidence of histological ossification disturbance of the femoral head was higher in SHR (59%) than in WKY (40%) at week 20. Lower serum and local levels of VEGF in SHR appeared to be related to the incomplete ossification of the femoral heads. Immunohistochemical study showed significantly lower numbers of IGF-1 and VEGF positive chondrocytes in the femoral epiphyseal cartilage of SHR than in those of WKY at weeks 10, 15 and 20. Our results suggest that local and/or systemic levels of IGF-1 and VEGF between ages of 5 and 20 weeks might play roles in the pathogenesis of ossification disturbance of the femoral head in SHR.

DOI： 10.18926/AMO/30749

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血管基底膜IV型コラーゲンの免疫組織学的解析共焦点レーザー顕微鏡を用いた三次元観察

森本尊雅, 内藤一郎, 百田龍輔, 芳原成美, 長谷川治子, 田口勇仁, 西田圭一郎, 佐渡義一, 二宮善文, 大塚愛二

解剖学雑誌 81 ( Suppl. ) 155 - 155 2006.3

In situ preparation of colloidal iron by microwave irradiation for transmission electron microscopy

S Nakatani, Naito, I, R Momota, N Hinenoya, K Horiuchi, K Nishida, A Ohtsuka

ACTA MEDICA OKAYAMA 60 ( 1 ) 59 - 64 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：OKAYAMA UNIV MED SCHOOL

We attempted to prepare colloidal iron within tissues by means of microwave irradiation. Mouse tissue blocks were fixed with a mixture of paraformaldehyde and ferric chloride in a cacodylate buffer, immersed in a cacodylate buffered ferric chloride solution, and irradiated in a microwave processor. Colloidal iron was prepared within tissues or cells, and was observed in the form of electron dense fine granules (1-2 nm in diameter) by transmission electron microscopy. Collagen fibrils in the connective tissue showed colloidal iron deposition at regular periodical intervals. Cells in the splenic tissue showed that fine colloidal granules were deposited on the ribosomes but not on the nuclear chromatin. This finding suggests that ferric ions could not diffuse into the nucleus, which was surrounded by the nuclear envelope. The podocyte processes of the renal glomerulus were stained diffusedly. Though this microwave in situ colloidal iron preparation method has some limitations, it is convenient for use in biomedical specimen preparation in transmission electron microscopy.

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Novel transdermal photodynamic therapy using ATX-S10·Na(II) induces apoptosis of synovial fibroblasts and ameliorates collagen antibody-induced arthritis in mice

Miyazawa, S., Nishida, K., Komiyama, T., Nakae, Y., Takeda, K., Yorimitsu, M., Kitamura, A., Kunisada, T., Ohtsuka, A., Inoue, H.

Rheumatology International 26 ( 8 ) 2006

Expression of WISP-1 (ccn4), WISP-2 (ccn5) and WISP-3 (ccn6) in Rheumatoid Arthritic Synovium Evaluated by DNA Microarrays

Mori Hiroki, Nishida Keiichiro, Ozaki Toshifumi, Inoue Hajime, Setsu Kojun, Tsujigiwa Hidetsugu, Nagatsuka Hitoshi, Gunduz Mehmet, Nakanishi Tohru

Journal of hard tissue biology 15 ( 3 ) 89 - 95 2006

Language：English Publisher：The Society of Hard Tissue Biology

Rheumatoid disease (RA) is one of the complex diseases that showed multiple progression stages. At first inflammatory response is occurred by self antibodies, and then the proliferation of synoviocytes is abnormally promoted. These synoviocytes invade into articular structures and finally articular cartilage is destroyed. In order to analyze growth-promoting factors in synovial cells from articular tissues of RA, we performed DNA microarray analysis and compared the expression patterns of their mRNA to that of synoviocytes from osteoarthritis (OA). The results showed that the expression of many genes was up-regulated in RA synoviocytes comparing with OA synoviocytes. Among these genes, RT-PCR analysis revealed that Wnt-1-induced secreted protein 2 (WISP-2, CCN5) and protein 3 (WISP-3, CCN6) which are belong to the novel gene family, CCN, were highly up-regulated in RA synoviocytes. In addition, one truncated isoform of WISP-3 which may be related to the ongoing of some type of RA, was detected in RA synoviocytes. Furthermore, immunohistochemical analysis showed that the products of both CCN genes were highly distributed in active synoviocytes of articular tissues of RA. These results indicate that both CCN genes and their products play some roles in the progression of RA.

DOI： 10.2485/jhtb.15.89

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Other Link： http://search.jamas.or.jp/link/ui/2007191489

Accuracy of a portable electroneurometer for measuring distal motor latency

H Tanaka, H Hashizume, C McCown, M Senda, K Nishida, H Inoue

JOURNAL OF ORTHOPAEDIC SCIENCE 10 ( 5 ) 445 - 450 2005.9

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER TOKYO

Background A facile electromyographic test in the outpatient clinic is needed to evaluate the outcome of screening for carpal tunnel syndrome. The purpose of the current prospective study was to compare the accuracy of Nervepace digital electroneurometer (Nervepace) and Dantec Neuromatic 2000M (Dantec) measurements of a wide range of distal motor latency (DML) of the median nerve.
Methods. The DML values for 112 of 126 median nerves examined with Nervepace and conventional electromyography, Dantec were statistically analyzed in this prospective study. The 30 hands of 15 clinically healthy adults 20-24 years old served as the reference group. Sixty consecutive patients (96 hands) with idiopathic carpal tunnel syndrome provided a wider range of DML values. All of the patients had standard clinical tests and standard motor (Dantec and Nervepace) and sensory (Dantec) electrodiagostic tests before endoscopic carpal tunnel release.
Results. The DML was measurable in all 30 hands of the reference group, in all 96 hands of the patients by Dantec, but in only 82 hands (52 patients) by Nervepace. Nervepace and Dantec DML values of 6.0 ms or less had high correlation and agreement.
Conclusions. Correlation and agreement decreased significantly among values greater than 6.0ms.

DOI： 10.1007/s00776-005-0928-2

ADAMTS-9 is synergistically induced by interleukin-1 beta and tumor necrosis factor alpha in OUMS-27 chondrosarcoma cells and in human chondrocytes

K Demircan, S Hirohata, K Nishida, OF Hatipoglu, T Oohashi, T Yonezawa, SS Apte, Y Ninomiya

ARTHRITIS AND RHEUMATISM 52 ( 5 ) 1451 - 1460 2005.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Objective. To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1β (IL-1β) and tumor necrosis factor a (TNFα) in chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.
Methods. OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1β and/or TNFa. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1β and/or TNFα. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1β-stimulated OUMS-27 cells was investigated.
Results. IL-1β increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1β stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1β and TNFα had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1β-stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 upregulation in OUMS-27 cells.
Conclusion. ADAMTS9 is an IL-1β- and TNFα-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis.

DOI： 10.1002/art.21010

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Calpain inhibitors prevent neuronal cell death and ameliorate motor disturbances after compression-induced spinal cord injury in rats

S Arataki, K Tomizawa, A Moriwaki, K Nishida, M Matsushita, T Ozaki, T Kunisada, A Yoshida, H Inoue, H Matsui

JOURNAL OF NEUROTRAUMA 22 ( 3 ) 398 - 406 2005.3

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT INC

Traumatic spinal cord injury (SCI) results in widespread neuronal cell death. Recent studies have suggested that activated calpain mediates neuronal cell death in the central nervous system. We conducted a study to determine whether calpain mediates neuronal cell death in the motor neurons of the spinal cord after SCI, and whether postinjury administration of the calpain inhibitors N-acetyl-Leu-Leu-Met-CHO (ALLM) and calpain inhibitor III (CI III) (MDL28170) reduces the motor disturbances in rats with a model of SCI. Adult male Wistar rats were subjected to SCI by application of a 20-g weight impactor probe to the spinal cord at T12 for 20 min. The rats were divided into three groups according to whether they were injected intravenously with 0.05-2.5 mg/kg ALLM, 10 mg/kg CI III, or 0.1% DMSO as a control every 24 h for I week after SCI. Calpain was activated in the spinal cord at 8 h, 24 h, and 5 days after SCI, and administration of ALLM inhibited its activation. ALLM, as compared to the DMSO vehicle alone, also significantly reduced the number of motor neurons in spinal-cord lesions that were positively labeled at 24 h after SCI with the terminal deoxynucleotidyl transferase-uridine nucleotide end-labeling (TUNEL) technique. Additionally, both the inclined plane test and footprint analysis showed markedly better motor activity after 4 weeks in rats injected with ALLM or CI III than in rats given vehicle only. These results suggest that activation of calpain plays a critical role in the neuronal cell death that follows SCI, and that calpain inhibitors may have benefit in treating the motor disturbances that follow SCI.

DOI： 10.1089/neu.2005.22.398

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Suppression of chondrosarcoma cells by 15-deoxy-Delta (12,14)-prostaglandin J(2) is associated with altered expression of Bax/Bcl-xL and p21

ZN Shen, K Nishida, H Doi, T Oohashi, S Hirohata, T Ozaki, A Yoshida, Y Ninomiya, H Inoue

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 328 ( 2 ) 375 - 382 2005.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

We previously reported that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent agonist for peroxisome proliferator-activated receptor gamma (PPARgamma), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ(2)-induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ2-induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ2, but p 16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ2 was partly, but not completely, blocked by PPARgamma antagonist (GW9662) suggesting the 15d-PGJ2 exerted its effect by PPARgamma-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ2 in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma. (C) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.12.186

ヒト皮膚とその付属器の基底膜を構成するIV型コラーゲン分子のα鎖構成

長谷川治子, 内藤一郎, 中野和代, 米澤朋子, 西田圭一郎, 田口勇仁, 佐渡義一, 二宮善文, 大塚愛二

解剖学雑誌 80 ( Suppl. ) 151 - 151 2005.3

ヒト皮膚および付属器の基底膜IV型コラーゲンα鎖構成

長谷川治子, 内藤一郎, 中野和代, 米澤朋子, 西田圭一郎, 田口勇仁, 佐渡義一, 二宮善文, 大塚愛二

解剖学雑誌 80 ( 1 ) 20 - 20 2005.3

Hoechst 33342は関節軟骨修復の長期研究において細胞標識物質として有用である

川崎啓介, 杉原進介, 大塚愛二, 西田圭一郎, 尾崎敏文, 吉田晶, 井上一

移植 40 ( 1 ) 78 - 79 2005.2

Three-dimensional ultrastructure of the brush border glycocalyx in the mouse small intestine: a high resolution scanning electron microscopic study

K Horiuchi, Naito, I, K Nakano, S Nakatani, K Nishida, T Taguchi, A Ohtsuka

ARCHIVES OF HISTOLOGY AND CYTOLOGY 68 ( 1 ) 51 - 56 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：INT SOC HISTOLOGY & CYTOLOGY

The three-dimensional ultrastructure of the filamentous glycocalyx of the brush border in the mouse small intestine was successfully demonstrated by high resolution scanning electron microscopy (SEM). The specimens were fixed with 2% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4), and rinsed with buffered solutions with differently adjusted pH values (pH 3.0, 7.0 or 11.0). They were then osmicated, dried, spatter-coated with gold (1.0-1.5 nm), and observed under a high resolution SEM. The glycocalyx on the luminal surface of the intestinal villi covered the top of the microvilli of the epithelial cells and were well preserved in the specimens treated with an alkaline buffer (pH 11.0). The glycocalyx was observed as filamentous structures, 7 to 15 nm thick in diameter. These filaments repeatedly branched and anastomosed with neighboring ones to form an actual network or plexus as a whole, in contrast with superimposed images in transmission electron microscopy (TEM) which suggested that such anastomoses were pseudo-networks. The filaments thickened globularly at the sites of the filament bifurcation or branching. On the other hand, specimens rinsed with an acid or neutral buffer showed no glycocalyx on their microvilli, whose naked top had knob-like structures. Thus, the pH values of the washing buffer solutions were considered to affect the preservation of the surface coat due to molecular characteristics.

DOI： 10.1679/aohc.68.51

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