Effect of Intrauterine Growth Restriction on Cell Proliferation in the Fetal Cerebellum of the Guinea Pig

young-Sig Hyun(현영식),Yoon-Young Chung(정윤영),Sandra Rees 대한체질인류학회 2008 해부·생물인류학 (Anat Biol Anthropol) Vol.21 No.3

자궁속성장지연은 유아의 신경학적 결함 및 행동장애를 일으키는 원인이 될 수 있지만 이와 관련한 생물학적 원인은 정확히 밝혀져 있지 않다. 또한 뇌발생시 출생전 손상에 영향 받기 쉬운 소뇌의 세포 증식에 미치는 자궁속성장지연의 영향은 잘 알려져 있지 않다. 본 연구는 자궁속성장지연이 태생기 소뇌의 세포 증식에 미치는 영향을 알아보고 세포증식의 변화와 소뇌 성장과의 관계를 규명하고자 시행되었다. 임신 30일 된 기니픽의 한쪽 자궁동맥을 묶어 만성적인 태반 부전으로 인한 성장지연 기니픽 태자를 만들였으며 태생 60일에 희생시켜 태자의 체중과 뇌의 무게를 측정하였다. Ki67 면역조직화학염색을 이용하여 세포 증식의 변화를 관찰하였고 태생 60일의 소뇌 바깥과립층의 폭을 측정하였다. 본 연구 결과, 바깥과립층의 전체 세포 수에 대한 중식세포의 비율의 차이는 정상군과 성장지연군 간에 통계학척 의의는 없었으나 바깥과립층의 폭은 성장지연군에서 유의하게 크게 나타났다. 이상의 연구 결과는 자궁속성장지연이 발생 중인 기니픽 태자의 소뇌 성장을 감소시키지만 이는 태생기 소뇌의 세포 종식과는 연관성이 없음을 나타내 주었다. 또한 태생 60일에 정상문과 성장지연군 간에 바깥과립층 전체 세포 수에 대한 증식세포 수의 비율의 차이는 없었음에도 불구하고 성장지연군의 바깥과립층의 폭은 정상군에 비해 증가되었는데 이는 세포 발생 및 이동 과정의 지연 때문일 것으로 생각된다. Intrauterine growth restriction (IUGR) is a risk factor for neurological and behavioral deficits in children although the precise underlying biological mechanisms are unclear. It is also unclear whether IUGR affects cell proliferation in the cerebellum, which is vulnerable to prenatal insults during brain development. The aim of this study is to determine whether IUGR affects the alteration of cell proliferation in the fetal cerebellum and whether this change correlate with reduction in the growth of cerebellum. IUGR was induced via unilateral ligation of the uterine artery in pregnant guinea pigs at 30 days of gestation (dg; term - 67 dg) to produce growth restricted (GR) fetuses. Fetal (control, n=7 and GR, n=7) body and organ weights at 60 dg were measured and Ki67 immunohistochemistry was performed to detect cell proliferation. The width of the external granular layer (EGL) was also measured at 60 dg. The mean body and organ weights of GR fetuses at 60 dg were significantly reduced. The proportion of proliferating cells to the total cell number in the EGL was not different in GR compared with control animals but the width of the EGL was significantly increased in GR animals. These results demonstrate that significant reductions in the growth of the cerebellum do not have a well-defined relationship to cell proliferation in IUGR guinea pig fetuses. In addition, despite there was no difference in the proportion of proliferating to post-mitotic cells between control and GR fetuses in the EGL at 60 dg, the width of the EGL was in￢creased in GR fetuses compared to controls. This could be interpreted as a delay in the process of cell production or migration.

척수를 손상시킨 후 꼬리정맥에 주입한 사람탯줄혈액세포가 뇌줄기에 미치는 영향

김종중 · 정윤영 · 박영란 · 문영민 · 현영식 · 정영욱 · 문정석 조선대학교 의학연구원 2007 The Medical Journal of Chosun University Vol.32 No.1

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모체 thyroxine 투여가 태아알코올효과를 가진 흰쥐 대뇌겉질 및 해마에서 NPY함유 신경세포의 생후 발달에 미치는 영향

김복 · 박상기 · 박영란 · 김종중 · 문정석 · 김주수 · 문영민 · 현영식 · 천관영 · 정윤영 조선대학교 의학연구원 2005 The Medical Journal of Chosun University Vol.30 No.2

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척수손상 후 숨뇌와 척수회색질기둥 앞뿔에 분포하는 calbindin D-28K와 parvalbumin면역반응세포에 관한 형태학적 연구

정윤영,김종중,현영식 대한체질인류학회 2004 해부·생물인류학 (Anat Biol Anthropol) Vol.17 No.3

This study was examined and compared the immunocytochemical distribution of the two calcium-binding proteins calbindin D-28K and parvalbumin immunreactive neurons in the medulla oblongata and spinal cord after transection of spinal cord in rats. In this experiment, calbindin D-28K immnunoreactive neurons were mainly found in many pyramidal cells distributed medulla oblongata and spina1 cord of rats. Parvalbumin immunoreactive cells were demonstrated in all lamina of the gray matter of the spinal cord. These immunoreactive cells had the most high density in the severa1 nuclei of the ventra1 horn of the all segments of the spina1 cord. Calbindin D-28K neuropil labeling was strongly noted in spina1 all segments of the spinal cord. In contrast parva1bumin immunoreactive, little differences were found in distribution, size and morphology of calbindin D-28K cell body or neuropil staining in the spinal cord. The number of parvalbumin immunoreactive cells were more than twice in the medulla oblongata and spinal cord compared to the calbindin D-28K immunoreactive cells. Calbindin D-28K and parvalbumin-immmoreactive somata were round, ova1, spind1e and polygona1 in shape, and the immunoreactive neurons were unipolar, bipolar, multipolar and horizontal in shape. The diameters of the somata of the two immunoreactive neurons were 40~50 µm, respectively. Also dendrites of two immunoreactive neurons were densely arrayed in network. These results suggest that CB-IR and PV-IR most high density in the of the VII~X layers in the ventra1 horn of the all segments of the spina1 cord. 척수를 손상시키지 않은 정상 군과 완전히 손상시킨 군 그리고 각각 오른쪽과 왼쪽을 반씩 손상시킨 후 5, 10일동안 사육한 흰쥐의 숨뇌와 각 척수 분절의 회색질기둥 앞뿔에서 칼슘결합단백질인 calbindin D-28K (CB) 와 parvalbumin (PV)에 단세포군 항체를 이용하여 면역조직화학적 방법으로 염색하여 CB-IR세포와 PV-IR세포 의 분포양상과 세포의 크기 및 형태에 대하여 관찰하여 다음과 같은 결과를 얻었다. 1. 숨뇌의 그물핵에서 CB-IR세포와 PV-IR세포를 관찰할 수 있었다. 2. CB-IR세포와 PV-IR세포는 척수 각 분절의 회색질기둥 앞뿔의 앞가쪽핵, 뒤안쪽핵, 뒤가쪽핵, 뒤안쪽핵, 중앙핵 등에서 관찰할 수 있었다. 특히 허리분절에서 강한 양성 반응을 나타내었다. 3. 척수 손상 후 5일간 사육한 실험군의 경우 완전히 손상 받은 가슴분절 아래부위에서는 CB-IR세포와 PV-IR세 포는 드물게 관찰되었다. 4. 척수 손상 후 10일간 사육한 실험군에서 왼쪽 손상부위와 오른쪽 손상부위에서 CB-IR세포와 PV-IR세포의 수는 거의 비슷하였다. 5. CB-IR세포와 PV-IR세포는 방추형, 타원형, 원형, 삼각형이었으며 크기는 40~50 µm 정도였고, 모양은 홑극, 두 극, 뭇극, 수평형이었다. 이상의 실험 결과를 요약하면 척수 손상 후 5, 10일간 사육한 실험군에서 CB-IR세포와 PV-IR세포는 각 척수분 절의 회색질기둥 앞뿔의 VII~X층에서 가장 많이 관찰 할 수 있었다.

척수를 손상시킨 후 꼬리정맥에 주입한 사람탯줄혈액세포가 뇌줄기에 미치는 영향

김종중,정윤영,박영란,문영민,현영식,정영욱,문정석 朝鮮大學校 附設 醫學硏究所 2007 The Medical Journal of Chosun University Vol.32 No.1

Background and Objectives: Stem cells are a valuable resource for treatment of many disease, but limited access to stem cells in some organs such as brain restricts their utility. Many approaches have been attempted to restore the function following brain stem injury (BSI) and spinal cord injury (SCI). The use of the human umbilical cord blood cells (hUCB) - a rich source of nonembryonic or adult stem cells - has recently been reported to ameliorate the behavioral consequences of stroke. Mateiials and Methods: Forty rats were divided into 8 groups: (1) SCI l+hUCB (infused 1 day post injury); (2) SCI 2+hUCB (infused 2 days post injury); (3) SCI 3+hUCB (infused 3 days post injury); (4) SCI 4+hUCB (infused 4 days post injury); (5) SCI 5+hUCB (mfusedt 5 days post injury); (6) SCI 6+hUCB (infused 6 days post injury); (7) LO+hUCB (laminectomy+hUCB); and (8) LO (laminectomy only). SCI was produced by compressing the spinal cord for one minute with an aneurysm clip calibrated to a closing pressure of 50 g. We report here that immunhistotochemical identification of fluorescent hUCB positive cells in the brain stem after compressed spinal cord injury using mouse anti-human mitochondria monoclonal antibody (MAB1273). Results: All SCI+hUCB (1-8) groups contained fluorescent hUCB positive cells in the all area of the brain stem. But especially a large number of fluorescent hUCB positive cells were observed in the whole area of the brain stem of the experimental 5 (SCI 5+hUCB) and 6 (SCI 6+hUCB)groups. No hUCB positive cells were found in the brain stem of group with non-injured spinal cord of these animals and group with laminectomy only. Conclusion: These results suggest that hUCB are potentially useful as a vector for treating a variety of the central nervous system disorders, and we are sure that continuous of stem cell study will give an best opportunity to treat the uncurable disorders in the future.

모체 Thyroxine 투여가 태아알코올효과를 가진 흰쥐 대뇌겉질 및 해마에서 NPY함유 신경세포의 생후 발달에 미치는 영향

김복,박상기,박영란,김종중,문정석,김주수,문영민,현영식,천관영,정윤영 朝鮮大學校 附設 醫學硏究所 2005 The Medical Journal of Chosun University Vol.30 No.2

Background and Objectives: Maternal alcohol abuse is considered to be one of the most prominent cause of neurobiological malformations in the postnatal and adult life of the offspring. In this study, we investigated the effects of maternal alcohol drinking on the postnatal development of NPY-containing neuron, and, the influence of thyroxine treatment on the cerebral cortex and hippocampus of pups of alcohol abused mother. Materials and Method: Time-pregnant rats were divided into three groups. Alcohol-fed group A received 35 calories of liquid alcohol diet daily from gestation day 6; control pair-fed group B was fed a liquid diet in dextrin replaced alcohol isocalorically: alcohol + T4 group C received 35 calories liquid alcohol diet and exogenous thyroxine subcutaneously. Results: Group C showed prominent NPY immunoreactivity in the cerebral cortex compared to group A and B at P7. In group C, NPY-containing neurons were widely distributed in the all layers of cerebral cortex after P14. Besides, numerical decrease of NPY-containing neuron as age increases was not found in group C. However, the decrease of NPY-containing neuron was clearly observed in group A compared to group C after P14. In hippocampus, group Band C were appeared similar patterns after P7. Additionally, in group C, NPY immunoreactivity was prominently appeared in CA2 and CA3 at P14 as compared to group B. Conclusions: The present results showed the increase of intensity and number of NPY-containing neurons in cerebral cortex and hippocampus of pups of exogenous T₄ supplemented alcohol-exposed dams as compared to control pair-fed and alcohol-exposed pups at P7. It presumably suggest that NPY-containing neurons might be regulated by the early postnatal growth stimulatory effect of the exogenously supplemented T₄. Therefore, the increase of NPY synthesis caused by maternal administration of exogenous thyroxine may ameliorate fetal alcohol effect, one of the ill effects as a result of the dysthyroid state following maternal alcohol abuse.

모체 Thyroxine 투여가 태아알코올효과를 가진 흰쥐 뇌의 BDNF함유 신경세포 생후 발달에 미치는 영향

강양수(Yang Soo Kang),정윤영(Yoon Young Chung),박영란(Young Lan Park),현영식(Young Sig Hyun),김종중(Jong Joong Kim),문정석(Jeong Seok Moon),문영민(Young Min Mun),오재욱(Jae Wook Oh),신성희(Sung Heui Shin),배춘상(Choon Sang Bae) 대한해부학회 2006 Anatomy & Cell Biology Vol.39 No.4

임부의 알코올 남용은 태아 정신발육지연의 흔한 원인이며 특히 태아의 뇌 발생에 예민한 결정적 기간 (critical period) 동안에 지속적으로 음주하는 경우 태아알코올효과를 나타내기 쉽다. 본 연구에서는 임신 기간중 지속적으로 알코올을 섭취한 모체에 thyroxine을 투여하여 알코올의 유해한 영향으로 인한 태아알코올효과를 개선시킬 수 있는지를 관찰하기 위하여 흰쥐 뇌의 생후 연령에 따라 brain-derived neurotrophic factor(BDNF)함유 신경세포의 성숙 양상을 면역조직화학염색을 이용하여 관찰하였고 BDNF 함량을 측정하였다. 실험동물은 매일 35 칼로리 정도의 알코올을 섭취한 알코올군, 알코올 대신 dextrin이 첨가된 유동액을 섭취한 정상군 및 알코올을 섭취하고 thyroxine을 매일 5 μg/kg 피하 주사한 알코올+T4군으로 나누었으며 생후 0, 7, 14, 21, 28일에 희생시켰다. 본 연구 결과 BDNF 단백 양은 알코올+T4군에서 알코올군에 비해 생후 7, 14, 21일에 증가하였으며 특히 생후 7일의 알코올+T4군에서 가장 높았다. 알코올군은 모든 연령에서 정상군보다 감소하였다. 소뇌에서는 알코올+T4군에서 생후 14일부터 정상군과 유사한 BDNF함유 신경세포들이 조롱박세포층에 분포하였다. 알코올+T4군에서는 알코올군에 비해 대뇌겉질, 시상하부 및 해마에서 생후 7일에 성숙되고 두드러진 양성반응을 나타냈으며 해마에서는 생후 28일까지도 뚜렷한 양성반응을 나타냈다. 이상의 결과는 임신 중 알코올을 음용한 흰쥐 모체에 thyroxine을 투여하면 태아알코올효과를 가지고 태어날 수 있는 후손들의 뇌에 분포하는 BDNF함유 신경세포들이 생후 발달동안 정상군과 유사하거나 더 빠르게 발달시킬 수 있을 것으로 생각된다. 이는 모체에 투여하는 지속적인 thyroxine 처치가 출생 초기에 BDNF 합성을 증가시켜 모체의 알코올 남용으로 야기되는 태아알코올효과와 같은 출생결함을 개선시킬 수 있음을 시사한다. Maternal alcohol abuse is thought to be the common cause of mental retardation. Especially, continuous alcohol consumption during critical period of brain development induce fetal alcohol effects. In this study, the authors investigated the effects of maternal alcohol drinking on the postnatal changes of BDNF contents and patterns of BDNF-containing neuron in neonatal rat brain, and, the influence of maternal thyroxine treatment on the brain of pups of alcohol abused mother. Pregnant rats were divided into three groups. Alcohol-fed group (n=4) received 35 calories of liquid alcohol diet daily from gestation day 6; control pair-fed group (n=4) was fed a liquid diet in dextrin replaced alcohol isocalorically; alcohol+T4 group (n=4) received 35 calories liquid alcohol diet and exogenous thyroxine (5 μg/kg/day) subcutaneously. The amount of BDNF was significantly higher in the alcohol+T4 group as compared to the alcohol group at P7, P14 and P21, especially, alcohol+T4-exposed pups showed a significant increase of BDNF at P7. The decrease in BDNF was found in alcohol group compared to control pair-fed group at all ages. In alcohol+T4 group, BDNF-containing Purkinje cells exhibited mature pattern and monolayer arrangement at P14. Alcohol+T4 group showed mature pattern and numerical increase of BDNF-containing cells in cerebral cortex, hypothalamus and hippocampus at P7. The BDNF immunoreactivity of hippocampus continued to show prominent configuration in alcohol+T4 group at P28. These results indicate that the increase of the BDNF-containing neurons and BDNF amount in pups of thyroxinesupplemented alcohol-exposed dams as compared to control pair-fed and alcohol-exposed pups at P7, presumably suggest the early postnatal growth stimulatory effect of the exogenously supplemented thyroxine. Therefore, the increase of BDNF synthesis caused by maternal administration of exogenous thyroxine may ameliorate fetal alcohol effects, one of the ill effects as a result of the dysthyroid state following maternal alcohol abuse.