1304 Fig. 1. Illustration of gas exposure apparatus. Korean J Otolaryngol 2001;44:1303-9

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1 KISEP Rhinology Korean J Otolaryngol 2001;44: 공업용본드가스에노출된생쥐후각점막의형태학적변화 안희영 강기훈 한동열 김동욱 이병돈 오천환 장혁순 Morphological Changes of the Olfactory Mucosa in Mice Exposed to Industrial ond Vapor Hee-Young hn, MD, Ki-Hoon Kang, MD, Dong-Yul Han, MD, Dong-Wook Kim, MD, yung-don Lee, MD, Cheon-Hwan Oh, MD and Hyuck-Soon Chang, MD Department of Otolaryngology, College of Medicine, Soonchunhyang University, Seoul, Korea STRCT ackground and ObjectivesRecently, accidents have been reported of young adults who inhale industrial bond vapor. cquiring industrial bond is easy for young adults, and glue sniffing has thus become a serious social problem. However, there have been few reports concerning the changes in the olfactory epithelium after exposure of industrial bond vapor. The aim of this study is to describe the morphological changes of the olfactory mucosa after being exposed to industrial bond vapor, using HE,, -PS stains, immunohistochemistry and the electron microscopy. Materials and MethodHealthy ICR mice were exposed to industrial bond vapor acetone ppm, c-hexane ppm, methylcyclopentane ppm, n-hexane ppm and toluene ppm. The vapor was given to each animal with duration of 20 min/day for 3 days, 5 days, 7 days or 14 days in a specially designed box for this study. fter exposure to industrial bond vapor, mice olfactory mucosae were excised and prepared for HE,, -PS stains, immunohistochemistry and electron microscopy. ResultsLoss of cells and disarrangements of olfactory epithelium were prominent on the third day of exposure and the epithelium recovered to the normal state after the day 7 in the HE stain. nd there were also definite ultrastructural changes in the epitheliumloss of microvilli in supporting cells, exposure of olfactory vesicles to the luminal surface and apoptotic bodies in electron microscopic study. Study with the and -PS stains demonstrated an evidence of decreased mucin secretion on the day 3 and the content of mucin was normalized after the day 7. In the PCN study, cells were unable to proliferate until day 3. The proliferation potential were increased 4 fold on day 5 and returned to normal on day 14. ConclusionThis result indicated that industrial bond vapor can damage the olfactory epithelium of mice which can recover rapidly. poptosis and active proliferation could be the factors that are involved in the recovery of the epithelium that is open to regular industrial bond vapor exposure on daily basis. Korean J Otolaryngol 2001;44: KEY WORDSond Olfactory mucosa poptosis. 1303

2 1304 Fig. 1. Illustration of gas exposure apparatus. Korean J Otolaryngol 2001;44:1303-9

안희영 외 Fig. 2. The olfactory mucosa of mice (HE stain 100).

Note tall olfactory epithelial cells and the underlying bundles (n). The 3 days group.

C Fig. 3. owman s glands are stained with purple (-PS stain 100). The control group. The 3 days group.

PCN immunohistochemistry of olfactory epithelia ( 200). The nuclei of proliferating basal cells are seen. The control group.

PCN항체를 4 에서 24시간 반응시킨 뒤 PS(phosphate buffered saline)로 수세한 다음 biotinated anti mouse IgG와 streptoavidine peroxidase를 처리한 후에

전자현미경의 표본 제작 주사전자현미경 마취된 생쥐의 머리를 반쪽으로 절개한 후 코 안을 PS로 조심스럽게 씻어 점액을 제거한 다음, 2.5% glutaraldehyde 에 고정하였다. 3시간 고정한 조직을 다시 0.

The sustentacular cells with oval nuclei (N) are occupied most of upper portion of olfactory epithelium.

Immediately below the surface is a terminal web area, where is relatively free of cytoplasmic organelles.

supranuclear cytoplasm. The olfactory cells (OC) are shown between the sustentacular cells.

3 안희영 외 Fig. 2. The olfactory mucosa of mice (HE stain 100). The control lamina propria containing owman s glands and olfactory nerve group. Note tall olfactory epithelial cells and the underlying bundles (n). The 3 days group. Loss of mucus substance and exfoliated cell clump in the lumen are found. C The 5 days group. Note hyperplasia of olfactory cells. C Fig. 3. owman s glands are stained with purple (-PS stain 100). The control group. The 3 days group. The mucus substance of owman s gland is decreased. C The 5 days group. The mucus substance of owman s gland is restored. C Fig. 4. PCN immunohistochemistry of olfactory epithelia ( 200). The nuclei of proliferating basal cells are seen. The control group. The 5 days group. methanol로 내인성 peroxidase를 파괴한 후 20배 희석된 normal swine serum으로 비특이성 반응을 억제시켰다. PCN항체를 4 에서 24시간 반응시킨 뒤 PS(phosphate buffered saline)로 수세한 다음 biotinated anti mouse IgG와 streptoavidine peroxidase를 처리한 후에 diaminobenxidine(d)으로 발색시켜 단위길이(1 mm)에서 양성을 보 이는 기저세포의 수를 비교하였다. 전자현미경의 표본 제작 주사전자현미경 마취된 생쥐의 머리를 반쪽으로 절개한 후 코 안을 PS로 조심스럽게 씻어 점액을 제거한 다음, 2.5% glutaraldehyde 에 고정하였다. 3시간 고정한 조직을 다시 0.1 M phosphate buffer, ph 7.4로 씻어낸 후에, 2% osmium tetroxide Fig. 6. The olfactory epithelium of a control mouse (TEM. 8960). The sustentacular cells with oval nuclei (N) are occupied most of upper portion of olfactory epithelium. The surfaces of the sustentacular cells are provided with long microvilli (mv). Immediately below the surface is a terminal web area, where is relatively free of cytoplasmic organelles. bundant agranular endoplasmic reticula (ser), mitochondria (M), granular endoplasmic reticula (rer) are densely packed in the supranuclear cytoplasm. The olfactory cells (OC) are shown between the sustentacular cells. distal process (arrow) extend upward from olfactory cell body and form the dilated olfactory vesicles (OV). The cilia with tapering ends (arrowheads), project in all direction, from olfactory vesicles, and mingle with microvilli of sustentacular cells. 로 다시 1시간 고정하였다. 고정된 조직은 알콜의 농도를 높 여가면서 조직을 탈수한 다음 hexamethyldisilazane(hm- 투과전자현미경 DS)으로 건조시킨 후 nm 두께로 금코팅하여 후각 절취한 조직을 2.5% glutaraldehyde-1.5% paraform- 세포 표면의 변화를 JSM 5410 LV 주사전자현미경(JEOL, aldehyde액에 고정한 뒤, 7일간 탈회한 조직을 1% osmium Tokyo, Japan)으로 관찰하였다. tetroxide액에서 다시 3시간 동안 2차 고정하고, 알콜로 탈 수한 다음에 araldite mixture에 포매하였다. 포매된 조직을 1305

공업용 본드가스에 노출된 생쥐 후각점막의 형태학적 변화 1 μm 두께의 절편으로 만들어 toluidine blue로 염색하여 후각점막부위를 선택한 후에 LK-V ultratome으로 60 70 nm 두께의

The olfactory epithelium of a mouse exposed to bond vapor for 3 days (TEM. 5400).

round lysosome (asterisk) is seen within the cytoplasm. C Fig. 5. The olfactory epithelia of control mice.

The luminal surface is covered by a tangled web of cilia and microvilli ( 1500). The 5 day group ( 5500).

Micrograph showing crowded olfactory vesicles. Most of sustentacular cells are disappeared 1306 Fig. 8.

4 공업용 본드가스에 노출된 생쥐 후각점막의 형태학적 변화 1 μm 두께의 절편으로 만들어 toluidine blue로 염색하여 후각점막부위를 선택한 후에 LK-V ultratome으로 nm 두께의 투과전자현미경용 절편을 작성하였고, uranyl acetate와 lead citrate로 대조염색하여 후각세포의 미세 구조적 변화를 JEM 100CXII 투과전자현미경(JEOL, To- Fig. 7. The olfactory epithelium of a mouse exposed to bond vapor for 3 days (TEM. 5400). poptotic bodies (arrows) of various sizes and shapes lie in the intercellular space. round lysosome (asterisk) is seen within the cytoplasm. C Fig. 5. The olfactory epithelia of control mice. Scanning electron micrographs (SEM). The control group. The luminal surface is covered by a tangled web of cilia and microvilli ( 1500). The 5 day group ( 5500). Numerous olfactory cell bodies (asterisks) and distal processes are seen. C The 5 day group ( 2000). Micrograph showing crowded olfactory vesicles. Most of sustentacular cells are disappeared 1306 Fig. 8. Free border of the olfactory epithelium exposed to bond vapor for 5days (TEM. 6160). The bleb-like cytoplasms (asterisk) of sustentacular cells filled with vesicular agranular endoplasmic reticula are protruded. Note the reduction of microvilli (mv). OV olfactory vesicle. Korean J Otolaryngol 2001 ;44 :1303-9

5 HE염색결과 와 -PS염색결과 PCN에대한면역조직화학염색결과 주사전자현미경관찰 투과전자현미경관찰 1307

6 1308 Korean J Otolaryngol 2001;44:1303-9

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KISEP Rhinology Korean J Otolaryngol 2000;43: 오존노출에의한백서비점막의형태학적변화 정진욱 김용민 나기상 김병국 박찬일 Morphologic Changes in Nasal Mucosa of the Rat after Ozon

KISEP Rhinology Korean J Otolaryngol 000;:9500 오존노출에의한백서비점막의형태학적변화 정진욱 김용민 나기상 김병국 박찬일 Morphologic Changes in Nasal Mucosa of the Rat after Ozone Exposure Jin Uk Jeong, MD, Yong Min Kim, MD, Ki Sang Rha,