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� � ��������� Vol. 33, pp. 373�381, 2005
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� �������� � Aquaporin-4 �AQP4�!"#$%&'��'�()*+,� ��-��-�.�-/0'1�2�3456*789:'� ��;-�<=->�?@ABC*78� DEF�!��GHI- ICP -JK?��;-LM4�N)*7897O�'P�)�ICP QR'S,��;-�TUV4WX)� ICP 9 AQP4 �(->��9� GHI- AQP4�(9Y-Z['\7* controlled cortical impactor'S8�]^-��GH_`a4�7*b )c� AQP4 �('! Western blotting !4� AQP -Z['\7*!de"fgh!4�7*b )c� �i�j!��'�i�j_kl��mno�^`pqrq4s#)*QR)c� AQP4�(!GHt�$u'+7*GHIvw)� GHI 48xyz{v|4@)c�}%� �i�j!GHI~�'JK)� GHI 48xyz{��|4@)c� "f����b z!� ��H���'+7*GHI 48xy'gh�vw!&�2�6c?� ��H���z!GHI 48xy'{� AQP4 -gh�?vw)*7c� AQP4 �(9�i�j'�?�()*78���� GHI-��;-�T' AQP4 ?>�)*789���Cc� �cGHt�$u'+�8 AQP4 �(!})z!2�6c?� �C!Z�'+�8T���-�74�N)c�-9���Cc�
���Aquaporin4, brain edema, controlled cortical impactor, traumatic brain injury,
intracranial pressure
�
���BCc Aquaporin �AQP� !��-T��* '�[�8������� �z�,� �*��az�-�.�/0'>�)*789���C*781�� ([��BC*78 13�-r l¡¢-O£"#$%&'+7*! Aquaporin-4 �AQP4�?{���'�89BC1, 2�� m���* �glial lim-iting membrane, pericapillary astrocyte foot pro-
ceses�� J¤�*� ¥�¢a¦�§�*� tanycyte'¨©�()*78� �-SO2Z[�+��AQP4!��-���.�-/0'1�2�34ªc)*789���C*783, 4����;!«¬®¯GH� ;,� °g� v�n�]±«²� ³�´-�� vµ¶U·2¸)�2.·'/6*�T)� ����'! astrocyte -0�41¹9)c cytotoxic edema 9� blood-brain bar-rier -2º'S8�*y»=-��¼3'S8 va-
sogenic edema '4BC85�� ��;-�T'S, intracranial pressure �ICP� ?5½)���'-�������� �$%G
373
17
����������� ������ ����������������� ������� � ���������� !"#$����� %&'�� ��(��� �)*���+�,-.����� �./�����01����2������34.�����5�����67� AQP4 89:;<=>< � �2 cyto-toxic edema, vasogenic edema ?.@.��ABC�DE� AQP4 !5�F��"G��4.���� Manley/6�� 2F� cytotoxic edemaABC�D��� #$H>< �%I� AQP4 89:;<=>< ��!$�&'4.2�J/�cytotoxic edema �D�� AQP4 ���(K4L�"G����� M)� Papadopoulos /7���3 F� vasogenic edema ABC�D��� AQP489:;<=>< � ICP *+N,-�.�O/0�2�P�J/� vasogenic edema �D�� AQP4 ��!$�&'��Q ��"G����� ��RP�� AQP489:;<=>< � �2S1J/� AQP4 ��!$T�U��KO2V4.����34)���5W)X�678���J�9Y��Z[�:O� 34)�� AQP4 !5�U���"G�\]4.��� ;^�M<�2]_�=/.����� ?��>���)�ABC�?`�ab�� )��K@�Ac�`����db/.�� ?�2e7fg��34)�� ICP �/0���(K�Bh�����PC�DE��ICP �Wi��F<������!$�j�Gk�� ICP AQP4 !5�UHI�F���l234)�� AQP4 !5?�mn�F��controlled cortical impactor �CCI� �R�>< �34)�ABC� ��S1�2�
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JoKL�M�p��U���N>q;rstXAuKLJo�vO�wx�,x2�� CCI model ���34)�ABC�� 6�8 PQ� �4 28�36 g �CD1 RI>< �6Syz{C|�q}{HT[U� ~V�� ��QW�2� 2.6� 2,2,2 -
tribromoethanol �Sigma-Aldrich, St. Louis, MO��20 ml�kg �X����� ��YZ�,�x2�>< �34� stereotactic frame ��<�� �[
��\����\��]��� �^������RP_� 4 mm��3�,�x2�Dixon/8��ABC��d� CCI device �VirginiaCommonwealth Biomedical Engineering Facility�� ��� _� 3 mm�y9���^�`��a_���RP 60@��b�FE�� velocity 3.2 m�s�c4 2 mm�34)�ABC�QW�2� )�QW�3��\��2� ICP F< � �2>< �� )�QW��P� �����4�d��2� 3��\��2� �3���,�x2����r="{C�2� 34)�QW 3� 24� 48 i�� 7� 146�>< ��ef�r="{C>< ��ef�Jo� �2�g� ��� Western blotting �� ��h�ij�� ICP F<��[email protected]��>< �� �2�� Western blotting)�Ql�>< �n5� �����r="{
C>< �n5� R��ef�M�m�� n����4�o��2)�[�p)�[�A�q��+E2� ?.@.� 0.6 ml ��A�s#�r�}9� {�ab�r/��A�s#|�� 4�C �1,000 rpm� 10+�¡s�� /t�o��2���>#:"y¢{£�u.vb� wxy@� 0.5 mg�ml�Gz�2� 12� Tris-Glycine Gels �Invitrogen,Carlsbad, CA� ��L�{¤¥K�2� polyvinilidene difluoride �PVDF�^ �Invitrogen��|}�� 4�C]�M~ 5� ¦�§C:�PBS �1 mM �¨©��ª*� ��£"9¦r�B«�,�x2�|}�2 PVDF ^�� 1��7¬®t�PBS ��500�¯��2M°±���� Rabbit anti- aqua-porin 4 polyclonal antibody �Chemicon, Temecula,CA� �ab�� �(� 2i�B«4L2� TBS-T Bu#er �0.1� �v�v� TritonN, 20 mM=q ©�²� pH 7.6�����2� °�� 1��7¬®t�PBS �� 5000�¯��2�{ �B³9´¢µC¶¦´�{·���°±� �Amersham Biosci-ences, Little Chalfont, Buckinghamshire� ���(� 1i�B«4L� TBS-T Bu#er ����2�?� ECL plus Western blotting detection system�Amersham Biosciences� � ��!�4L� ImageReader LAS-3000 version 1.11 �FUJIFILM� �¸���¹�_��,�x2� =/.2º»� NIHimage 1.62 soft ware �Ver. 1.62, NIH, Bethesda,
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18
MJ, USA� ��������� ��� ����� AQP4��� 1���� ���� AQP4 ������������������ ICP ����� �� �n�5�� ����
�n�5� �� stereotactic frame �!��� "#$%�&' �(��)��*�+,��*-.��/ 3 mm�0�12�� ICP monitoring trans-ducer �Codman, Raynham, MA��3��"4��5��6�� ICP 7��� �89:-�;�*�+,<=)�<>,�������� ?#�@�����0�1�� ICP �7���� ICP 7��A ICP ExpressTM �Codman� ����� �.B�ICP ��C.��� ����D���EF���1*2���� �������� �n�7� G��A��� �� �n�7�� 4� paraformaldehyde�PBSHI�!����� �JK��;�� ����L�MN�� O� 4� paraformaldehyde�PBS ���!��1�� P�� 30� sucrose H�Q��� Tissue-Tec
�O.C.T. Compound �SAKURA, Tokyo� ��
!��RS 80�CH"TUV� microtome ��WU 14 mm�X#�YZ��� "TX#� PBS ��[\�� 5� ]^_`ab�PBS ����� cdH 1�ef�ghi�1*2�� j�H 1�]^_`ab�PBS�� 500$kl��Ojmn Rabbitanti-aquaporin 4 polyclonal antibody �Chemicon���V� 4�C HO�Rhop���� PBS ��[\�� 1� ]^_`ab�PBS �� 500 $kl��qr%sm�t`mn �Vector Laboratories,Burlingame, CA� ��V� 2 �ecdH&uUV�� PBS ��[\�� VECTASHIELD�
Hard
SetTM �Vector Laboratories� ��'�!���� �
�#4��5�v��A� �w����YZ�(+xy� z{ � 2 mm |�� �w�})�w��~����� �w�����w��C.*B�#4�+�� ,��#4�+�PB�B������ AQP4 ��������������������� ���-����EF���A Student � t ���1*2�� -��A-�%��FH��� p�0.05��E����
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�� AQP4 ����� ICPAQP4 A 30kDa .~�������/UB��Fig. 1�� ��#4��5����A� ��� 3 �eC.��� AQP4 ���0{�/�� ��� 48�e��@��-0{����� ��� 712|��A�3��-������� O ICP A��� H 4�5 mmHg HG2��-� ��� 3 �e+x��-/�.B� ��� 48�eH�����40 mmHg���� ��� 712|��*��{��� ��� 141�A��� ���4�p �*2� �Fig. 2a��,��#4��5����A� ��#������� 48�e�HA AQP4 ��A0{�� ��� 71|��A�3��-������� ICP ��5@��#������HG2� �Fig. 2b���� AQP4 �����#�w�HA� ��� 3�e+x6^� ¡�A¢*2�7£%� AQP4 -¤.B� ���24�eH�@¥¦/�.B�� ��� 48�e�*�����A§§¨©��7£%� AQP4 @¨ª�� ��� 7 12�A«¬C�7£%� AQP4 �/���¤HG2�� ��� 1412�A7£%�AQP4A®�JK-=)����� =)�)¯�¯°�+,� AQP4 -±%���UB����Fig. 3���w�})HA� �w��¢*x��� 24
�eC. 48�e� AQP4 �����8.C*¨©A/�.B¬� ²���� ���4�������� �Fig. 4���w���HA� ��� 3 �e+x AQP4 ����-¨©�³�� ��� 48�eH�@���-¨©��� ��� 712�*���3���-�´�� ��� 1412HAU.����-µ�2��� �Fig. 5����,��#HA� AQP4�����0{-4��5¶n�/�.B�� P����A��� 48�e��@0{��-� ��� 712|��*�����A�´�� ��� 1412HAU.����-µ�2��x� ��#��������������� �Fig. 6�� 9·¸HA�:�*¹ºA1,»�-H�*C2�-� ¼���¶�� �������T½-�.B�����G�»�-�/UB��
������ AQP4 �� 375
19
Fig. 1. Expression of AQP4 after head injury by Western blotting. At 48 hours after
the head injury AQP4 expression decreased in both hemispheres.
Fig. 2. Variation of ICP and AQP4 expression after the head injury. Bar graphs
show AQP4 expression �n�5 in each point�. There was a significant dif-ference between AQP4 expression in the control and injured mice �p�0.05�.Line graph demonstrates ICP �n�5 in each point�. ICP increased after thehead injury, at 48 hours significantly.
a. Traumatized hemisphere
b. Non-traumatized hemisphere
� �� ���� 376
20
Fig. 3. Variation of AQP 4 expression after the head injury at the contusional area by
immunofluorescence. AQP4 found as small particles after the head injury, at 24 hours
significantly. At 14days after the head injury, damaged tissues disappeared, �10.�; the defective area of the tissue
Fig. 4. Variation of AQP 4 expression after the head injury at the peripheral area of the
contusion by immunofluorescence. Compared with the distant area of the contusion,
AQP4 expression was maintained at 48 hours after the head injury. The micrograph
showed linear staining of AQP4 surrounding the defective area of the tissue at 14days
after the head injury, �10.�; the defective area of the tissue
������� AQP4 � 377
21
Fig. 5. Variation of AQP4 expression after the head injury at the distant area of the contusion
by immunofluorescence. Compared with the control, AQP4 expression began to decrease
at 3 hours after head injury, and the microphotograph showed the minimum expression
at 48 hours after the head injury. The expression recovered at 14 days after head injury,
�10.
Fig. 6. Variation of AQP4 expression after the head injury at the non traumatized hemisphere
by immunofluorescence. As well as at the distant area of the contusion, AQP4 expression
began to decrease at 3 hours after head injury, and the microphotograph showed the
minimum expression at 48 hours after the head injury. The expression recovered at 14
days after head injury, �10.
� �� ���� 378
22
� �
������������� cytotoxic
edema � vasogenic edema ������������9�� �������������������������� ����� ��������������� � ����� AQP4 !�"#�$ ����%&�'�� AQP4���(�)�*�&+,-./��0��1���234��� AQP4 �5������Kiening�10� �67����89:� CCI234&����;���� Kiening ��� ��< 48=>�������?��� AQP4 ���5 @A�B��� ��C����� D��67EF�G��A�.H� � I� Sun�11���� JK���L��234.���< 24=>����.� AQP4 mRNA �5� M����� ���N!.�O��B�� ���%�"� �'P.�#Q 0%� ��C�����67������<� AQP4 ���5�R=$��B&%S� ��< 48=>.@A�&&T� � '�� ICP �-U� �0�V��� W�=>RX�I���� �'Y(Z[$\)�EF.�� ��������*]�^0+_`� AQP4 %S�� � '�� AQP4 a,-�.��-��.H��#'�&+/��� ���"�0bc� a,-�12 ��d�ef4&gh�� ��a,-12�.�� AQP4 ijc�� +_`�%S�� A��+,�� � I�� 3���k���lm.� Western blotting 4�EF������<48=>� AQP4 �5��B&%S � �%�� ���N!�����nJ��� AQP4�5�5�%0�B�%So� pq:rs4��6.H� �t-�"#�������?���� AQP4 �5�I�.0%� � �67t��(Z[$\)ku7$0\)12�14�����A�������N!����lm.�� ��d�^0� 8* v'�T����'�"#0Western blotting4�'Y(Z[$\w�"EF��xy9z�� Western blotting 4.�����3�����#���?>.�9{&|��� S� (Z[$\).%S�� "#0
JK.� AQP4 �5�}��8~c����o��(Z����:.v'��� AQP4 �5��B��#g.I�$�;5c� S��� �+,��� Western blotting 4.A���&�������N!� ���lm��:���;&|0#'�&�� � D� �� ���%�<���(Z�89:%�<��A�"��c1� ��� �(Z&:��JK.� AQP4 �5��;&|#'���=.H� ���D� �� CCI 234�JK����234.H � 67�EF� 3������?����A�������� ICP� ���5� 'Y(Z[$0#Q&%S� � ICP 40 mmHg >��.-U����� ���?�@�� A�)���&+/��� JK��B�������B� M�� /���234��,� 3������?� ICP -U� ��� ������? ������ n�� �>&�C��� S�������?����|��# ICP -U���H�������B��"�(Z�D�E�"���+,�� � �%�0 � Sun ��234����L������234.H'�%�� ���.��� 234����W? ^0����� ��}� AQP4 �5�F}���� �+,�� ����67EF&��� �����#G�B��CH�$��o����3������?�A�� ��< ICP �-U����?�D.�AQP4�5��B IJ��%S�� � 'Y(Z[$K�.�� ���N!.�JK$� AQP4��5 W���"#�� c� � K¡�.�(Z�0¢�£¤1L¥0¦_ M�G��� SI�$0�;��=.H� �%�IN$���<���� W� ICP �-U&§v��+,������ �67���������|� AQP4 �5��$I� H�+,�� �
� �
����� O�B¨P©ª«�¬*®¯���°h±�"�|�� �����²ªQR³�� �´R�µ[¶·��·STUMc�0����´R�µ¯J¸¹�� �µ[67·�º�� ��V²»WR� º��A�1²X¼�-½���
����� AQP4 �5 379
23
� �
1� King LS, Agre P. Pathophysiology of the aqua-porin water channels. Ann Rev Physiol 1996;
58: 619�648.2� Manley GT, Binder DK, Papadopoulos MCand Verkman AS. New insights into water
transport and edema in the central nervous
system from phenotype analysis of aquaporin-4
null mice. Neuroscience 2004; 129: 983�991.3� Badaut J, Lasbennes F, Magistretti PJ andRegli L. Aquaporins in brain: Distribution,
Physiology, and Pathophysiology. J Cereb
Blood Flow Metab 2002; 22: 367�378.4� Papadopoulos MC, Krishna S and VerkmanAS. Aquqporin water channels and brain
edema. Mt Sinai J Med 2002; 69: 242�248.5� Klatzo I. Neuropathological aspects of brainedema. J Neuropathol Exp Neurol 1967; 26: 1�14.
6� Manley TG, Fujimura M, Ma T, Noshita N,Filiz F, Bollen AW, Chan P and Verkman AS.
Aquaporin-4 deletion in mice reduces brain
edema after acute water intoxication and
ischemic stroke. Nat Med 2000; 6: 159�163.7� Papadopoulos MC, Manley GT, Krishna Sand Verkman AS. Aquaporin-4 facilitates re-
absorption of excess fluid in vasogenic brain
edema. FASEB J 2004; 11: 1291�1293.8� Dixon CE, Clifton GL, Lighthall JW, Yagh-mai AA and Hayes LR. A controlled cortical
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rat. J Neurosci Methods1991; 39: 253�262.9� Go KG. The normal and pathological physiol-ogy of brain water. Adv Tech Stand Neurosurg
1997; 23: 47�142.10� Kiening KL, van Landeghem HFK, SchreiberS, Thomale UW, von Deimling A, Unterberg
AW and Stover JF. Decreased hemispheric
aquqporin-4 is linked to evolving brain edema
following controlled cortical impact injury in
rats. Neurosci Lett 2002; 324: 105�108.11� Sun M-C, M.B., Honey RC, M.D., D.Phil.,Berk C, M.D., Wong LM N, Ph.D., and Tsui
KC J, M.D. Regulation of aquaporin-4 in a
traumatic brain injury model in rats. J Neuro-
surg 2003; 98: 565�569.12� Katayama Y and Kawamata T. Edema fluid
accumulation within necrotic brain tissue as a
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2003; 86: 323�327.13� Marmarou A. Pathophysiology of traumaticbrain edema: current concepts. Acta Neuro-
chir Suppl 2003; 86: 7�10.14� Stoop R, Thomale UW, Pauser S, BernardingJ, Vollmann W, Wolf JK Lanksch WR and
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� �� ���� 380
24
Abstract
Variation of Selective Water Transport Protein Aquaporin-4
Expression in Traumatic Brain Injury Model
Akiko Hoshi, Kotaro Oshio, Hiroyuki Watanabe, and Takuo Hashimoto
The brain contains abundant aquaporin-4 �AQP4� selective water transporting membrane protein, andthis protein plays a significant role in regulations of water homeostasis.
It is suggested that AQP4 is involved in the development of brain edema. Since we found that
intracranial pressure �ICP� reflected the developing brain edema, we investigated the developing brain edemaby measuring ICP. We also examined expression and localization of AQP4 after head injury in mice. Head
injury was produced in male CD-1 mice using a controlled cortical impactor device. We examined protein
expression of AQP4 by Western blot analysis and localization by immunofluorescence. ICP monitoring
transducer was put in the brain to measure ICP. Expression of AQP4 by Western blot analysis significantly
decreased in the traumatized hemisphere at 48 hours after the head injury. Whereas, ICP markedly increased
at 48 hours after the injury. Expression of AQP4 by immunofluorescence was maintained at the peripheral
areas of the contusion at 48 hours after the injury, however, it decreased at the distant area of contusion after
the injury, the minimum expression appearing at that time. Our experiment showed a decrease of AQP4
expression and increase of ICP concurrently. Therefore, it was suggested that AQP4 was involved in
development of brain edema following the head injury. It was indicated that di#erentiation of AQP4expression at each area of the traumatized hemisphere reflected the di#erent vital reactions for the injury.
Department of Neurosurgery, St. Marianna University School of Medicine
������� AQP4 � 381
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