9
臟腶腻腦腾腱膄膍臮膗膼膿 Vol. 33, pp. 373381, 2005 腔腗腉腍腆腅腇腂腀腁腄腏腑腓腎腙腐腒腕 Aquaporin-4 腖腋腃腊腌腓腘腈 腒腋 腅腈 腇腇 腋腇 腊腆 腕腆 腖腌 腎腑 腐腕 腔腈 腏腋 腓腍 腌腉 : 17 8 19 臡臰臼臙膏臛Aquaporin-4 AQP4腚臶臚臕膧膦腘腣腗膮腌腓腅腢腍腇腙臙 腙膲臑臛腙膁臁腘臇膘腥臲腑腓腂腣腕膠腘腍腙臄腙臱腛腙膞腇臃膶腋腤腓腂 膵膑膐腄腚膓臊膰腙 ICP 腙臐臌腇腍腙臄腙臦膡腥膆腌腓腂腣腕腂腃腘臵ICP 臨臻腘腠腢腍腙臄腙臞臒膣腥臹膷腌ICP AQP4 膮腙膞臛腕膓臊膰腙 AQP4 膮腕腎腙膤膻腘腒腂腓 controlled cortical impactor 腘腠腣腶腨腭腙腄腚膓臊腸腯腼腥腂腓 腌腏AQP4 膮腘腚 Western blotting AQP 腙膤膻腘腒腂腓腚膨膱膇臠臔腂腓膬腌腏膀腚腍腇膀腸腲腮腻腾腫腰腺腾腭腯腹腀腬腀腥臥腌腓臨臻 腌腏AQP4 膮腚膓臊臧臮腍腒膟腘腅腂腓膓臊膰臺膋腌膓臊膰 48 臂膝腔膹臺致腥臃腌腏膀腚膓臊膰臉腘臐臌腌膓臊膰 48 臂膝腔膹腞膴致腥臃腌腏膇臤臓膗臼膬 腔腚膸臊臆腟腚腘腅腂腓膓臊膰 48 臂膝腘臠臔臛臺膋腚腝腗腆腑腏腇膸臊膈膖腔腚膓臊膰 48 臂膝腘膹腞 AQP4 腙臠臔臛腇臺膋腌腓腂腏AQP4 膮腕膌腇腰腅 腌腓腂腣臀腆腡膓臊膰腙腍腙臄腙臞腘 AQP4 腇膞腌腓腂腣腕膳腄腡腤腏腜腏膓臊臧 腍腒膟腘腅腉腣 AQP4 膮腚膅腔腚腗腆腑腏腇腊腤腚膤臈腘腅腉腣臞臬膊腙膃腂腥 膆腌腏腞腙腕膳腄腡腤腏ῐῌ῏ Aquaporin4, brain edema, controlled cortical impactor, traumatic brain injury, intracranial pressure 腋腐膭腋腤腏 Aquaporin AQP腚臢臖腙臞臬 腢腦腘臩膻腍腣臡臰臼臙膏臛腔腁腢腽腵腼腔臙腙膲臑臛膁臁腘膞腌腓腂腣腕膳腄腡 腤腓腂腣 1膮膻膭腋腤腓腂腣 13 膯腙腬腳腮腧腴 腙腃腐臶臚臕膧膦腘腅腂腓腚 Aquaporin-4 AQP4腇膹腞腣腗腘腁腣腕腋腤 1, 2腫腻腦膺glial lim- iting membrane, pericapillary astrocyte foot pro- ceses臐膂膺腴腼腪腾腩膺tanycyte 腘自腈膮腌腓腂腣腊腙腠腃腗膤膻腛腘腆腡 AQP4 腍腇腙臙膲臑臛腙膁臁腘臇膘腥膎 腏腌腓腂腣腕膳腄腡腤腓腂腣 3, 4腍腙臄腚膪膛臏膔腟膓臊膚臠臺腱腰腻 腨腷膪臎臺膽臣臒臭腗腖腑腓臞腌臤臓膗臼腘腚 astrocyte 膌腥 臸腕腌腏 cytotoxic edema blood-brain bar- rier 膒腘腠腣膺膝膩腛腙臙腘腠腣 va- sogenic edema 腘臮腋腤腣 5腍腙臄腙臞腘腠 intracranial pressure ICP臗腌臤臓腘臟腶腻腦腾腱膄膍臮膗 臕膧膓膍 373 17

Aquaporin-4 ˘ˇ ˆ˙ - Marianna Uigakukai.marianna-u.ac.jp/idaishi/www/335/04-33-5Akiko... · 2006-01-11 · iting membrane, pericapillary ... 1 King LS, Agre P ... Aquaporin-4 facilitates

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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

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����������� ������ ����������������� ������� � ���������� !"#$����� %&'�� ��(��� �)*���+�,-.����� �./�����01����2������34.�����5�����67� AQP4 89:;<=>< � �2 cyto-toxic edema, vasogenic edema ?.@.��ABC�DE� AQP4 !5�F��"G��4.���� Manley/6�� 2F� cytotoxic edemaABC�D��� #$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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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����

�� 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��

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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���

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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

impact model of traumatic brain injury in the

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

cause of the mass e#ect of cerebral contusion in

head trauma patients. Acta Neurochir Suppl

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

Unterberg AW. Magnetic resonance imaging

studies with cluster algorithm for characteriza-

tion of brain edema after controlled cortical

impact injury �CCII�. Acta Neurochir Suppl1998; 71: 303�305.

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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

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