Department of Plastic Surgery
Helsinki University Hospital
University of Helsinki
Finland
THE DISAPPEARENCE AND REGROWTH
OF ADRENERGIC INNERVATION IN
ARTERIAL GRAFTS IN RATS
A MICROSURGICAL EXPERIMENTAL STUDY
by
Heikki Penttilä
To be publicly discussed with the permission of the Faculty of Medicine of The University
of Helsinki, in the main auditorium of Töölö Hospital, Helsinki University Hospital,
Topeliuksenkatu 5, Helsinki, on 10th February 2012 at 12 noon
Helsinki 2012
6XSHUYLVHGE\
'RFHQW7LPR:DULV
&KLHIRI3ODVWLF6XUJHU\UHW
2XOX8QLYHUVLW\+RVSLWDO
2XOX)LQODQG
5HYLHZHGE\
'RFHQW2XWL.DDUHOD
'HSDUWPHQWRI6XUJHU\
2XOX8QLYHUVLW\+RVSLWDO
2XOX)LQODQG
DQG
3URIHVVRU6HSSR6RLQLOD
'HSDUWPHQWRI1HXURORJ\
7XUNX8QLYHUVLW\+RVSLWDO
7XUNX)LQODQG
2SSRQHQW
'RFHQW.DOHUYR:HUNNDOD
'HSDUWPHQWRI7KRUDFLF6XUJHU\
+HOVLQNL8QLYHUVLW\+RVSLWDO
+HOVLQNL)LQODQG
,6%1QLG
,6%13')
Omistettu pojalleni Kaarlolle
,1752'8&7,21
CONTENTS
/,672)25,*,1$/38%/,&$7,216
$EEUHYLDWLRQV
$%675$&7
,1752'8&7,21
5(9,(:2)7+(/,7(5$785( $XWRQRPLFQHUYHV 6\PSDWKHWLFQHUYHV 3DUDV\PSDWKHWLFQHUYHV 7\SHVRIWUDQVPLVVLRQ &ODVVLFDOWUDQVPLVVLRQ 9ROXPHWUDQVPLVVLRQ 1RQH[RF\WRWLFWUDQVPLVVLRQ &RORFDOL]DWLRQDQGFRWUDQVPLVVLRQ
)DOVHWUDQVPLVVLRQ $GUHQHUJLFLQQHUYDWLRQRIWKHYDVFXODUZDOO ,QWURGXFWLRQ /RFDOL]DWLRQRIQHUYHVLQWKHYDVFXODUZDOO 75$160,77(568%67$1&(6
2WKHUQHXURQDOHOHPHQWV
1HXUR¿ODPHQWV
9DVFXODUJUDIWVLQDFOLQLFDOFRQWH[W
9HQRXVJUDIWV
$UWHULDOJUDIWV $,062)7+(678'< 0$7(5,$/6$1'0(7+2'6 0DWHULDOV 0HWKRGV 6XUJLFDOPHWKRG,,,,,,DQG,9
$QHVWKHVLD,,,,,,DQG,9
*O\R[\OLFDFLGLQGXFHGÀXRUHVFHQFH,
+LJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\,,DQG,,,
0LFURVFRSLFHYDOXDWLRQ,DQG,9
,PPXQRKLVWRFKHPLVWU\,9
6WDWLVWLFDODQDO\VLV,,DQG,,,
5(68/76
*HQHUDO
&RQWUROV
*UDIWV ',6&866,21
*HQHUDOPHWKRGRORJ\
*O\R[\OLFDFLGLQGXFHGÀXRUHVFHQFH +LJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\
,PPXQRKLVWRFKHPLVWU\
([SHULPHQWDOPRGHO
'HJHQHUDWLRQ
5HJHQHUDWLRQ
&OLQLFDOFRQVLGHUDWLRQV
&21&/86,216 $&.12:/('*(0(176
5()(5(1&(6
,1752'8&7,21
LIST OF ORIGINAL PUBLICATIONS
7KLVWKHVLVLVEDVHGRQWKHIROORZLQJRULJLQDOSXEOLFDWLRQVZKLFKZLOOEHUHIHUUHG
WRE\WKHLU5RPDQQXPHUDOV
, ',6$33($5$1&( 2) &$7(&+2/$0,1( )/825(6&(1&( )520 7+(
$'5(1(5*,&1(59(6,1$57(5,$/*5$)76,15$76$1(;3(5,0(17$/
)/825(6&(1&(+,672&+(0,&$/678'<+HLNNL.73HQWWLOl.DUO$-YRQ
6PLWWHQDQG7LPR+:DULV6FDQG-3ODVW5HFRQVWU+DQG6XUJ±
,, 125$'5(1$/,1( /(9(/6 5,6( 6,*1,),&$17/< ,1 5$76¶ $57(5,$/
*5$)76$7+2856326723(5$7,9(/<$1'7$.(+285672&20(
'2:1+HLNNL3HQWWLOl0'3LUNNR+XWWXQHQ3K'.DUOYRQ6PLWWHQ0'
3K'7LPR:DULV0'3K'1XUHGGLQ$VKDPPDNKL0'3K')5&6(G
0LFURVXUJHU\±
,,, 5(,11(59$7,21 2) $57(5,$/ *5$)76 %< $'5(1(5*,&
1(59(6 2&&856 ,1 5$76 $6 ,1',&$7(' %< ,1&5($6(' /(9(/6 2)
125$'5(1$/,1( +HLNNL 3HQWWLOl 0' 3LUNNR +XWWXQHQ 06F .DUO YRQ
6PLWWHQ0'3K'1XUHGGLQ$VKDPPDNKL0'3K')5&6(G7LPR:DULV
0'3K'3ODVW5HFRQVWU6XUJ±
,9'(*(1(5$7,21$1'5(*(1(5$7,212)3(5,9$6&8/$5,11(59$7,21
,1$57(5,$/*5$)763HQWWLOl+HLNNL0':DULV7LPR3URIHVVRU$VKDPPDNKL
1XUHGGLQ )5&6(G 9DQKDWDOR 6DPSVD 0' 3K' 7KH -RXUQDO RI &UDQLRIDFLDO
6XUJHU\±-XO\
,1752'8&7,21
ABBREVIATIONS
$&K
$5
&K$7 &16 *$%$ *$3
+3/& ,5
1$
1)
12
126 H126 L126 Q126
WD
7+
WP
9,3
DFHW\OFKROLQH
DGUHQDOLQH
FKROLQHDFHW\OWUDQVIHUDVH
FHQWUDOQHUYRXVV\VWHP
JDPPDDPLQREXW\ULFDFLG
JURZWKDVVRFLDWHGSURWHLQ
KLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\
LPPXQRUHDFWLYLW\
QRUDGUHQDOLQH
QHXUR¿ODPHQW
QLWULFR[LGH
QLWULFR[LGHV\QWKDVH
HQGRWKHOLDO126
LQGXFLEOH126
QHXURQDO126
WXQLFDDGYHQWLWLD
W\URVLQHK\GUR[\ODVH
WXQLFDPHGLD
YDVRDFWLYHLQWHVWLQDOSHSWLGH
ABSTRACT
,QPDQ\¿HOGVRIVXUJHU\VXFKDVFRURQDU\E\SDVVPLFURYDVFXODUIUHHÀDSRUJDQ
WUDQVSODQWDWLRQYDVFXODUDQGKDQGVXUJHU\WKHIXQFWLRQRIWUDQVSODQWHGDUWHULHV
LVRIFUXFLDOLPSRUWDQFH,QDOOWKHVH¿HOGVRIVXUJHU\YDVRVSDVPLVUHJDUGHGDV
DVHULRXVWKUHDWWRDVXFFHVVIXORXWFRPH,WFDQEHFDXVHGE\PDQ\IDFWRUVVXFK
DVPHFKDQLFDOKDQGOLQJRIWKHUHFLSLHQWDUWHU\GUXJVSHULRSHUDWLYHWUDXPDDQG
FKDQJHVLQWHPSHUDWXUH:HVWLOOGRQRWIXOO\XQGHUVWDQGWKHH[DFWPHFKDQLVPV
EHKLQGYDVRVSDVPDQGWKHUHLVJUHDWYDULHW\LQWKHH[SUHVVLRQRIWKLVSKHQRPHQRQ
EHWZHHQGLIIHUHQWLQGLYLGXDOVIURPDOPRVWQLOWRYHU\SURIRXQGDVLQ5D\QDXG¶V
GLVHDVH 7KH IDFWRUV EHKLQG YDVRVSDVP KDYH EHHQ VWXGLHG LQ FRQQHFWLRQ ZLWK
RSHUDWLYHWUDXPDLQYHQRXVJUDIWVEXWQRWLQDUWHULDOJUDIWV
:KHQDQDUWHU\LVGLVVHFWHGIURPLWVVXUURXQGLQJWLVVXHVLWORVHVLWVV\PSDWKHWLF
LQQHUYDWLRQ7KHHDUO\GLVDSSHDUDQFHRIFDWHFKRODPLQHVIURPYHLQJUDIWVKDVEHHQ
VKRZQSUHYLRXVO\+RZHYHUWKHIDWHRIDGUHQHUJLFQHUYHVLQDUWHULDOJUDIWVKDVQRW
EHHQVWXGLHG
7RLQYHVWLJDWHWKHFKDQJHVLQDGUHQHUJLFQHUYHVLQWUDQVSODQWHGDUWHULDOJUDIWV
LQ UDWV IHPDOH ODERUDWRU\ UDWV RI WR ZHHNV ROG ZHUH RSHUDWHG RQ )HPRUDO
DUWHULDOJUDIWVZHUHDQDVWRPRVHGWRFDURWLGDUWHULHVDQGFRPSDUHGZLWKFRQWURO
QRQRSHUDWHGIHPRUDODUWHU\VHJPHQWV
,QRXU¿UVWH[SHULPHQWWKHGLVDSSHDUDQFHRIFDWHFKRODPLQHÀXRUHVFHQFHIURP
QRUDGUHQDOLQFRQWDLQLQJ V\PSDWKHWLF QHUYH ¿EHUV DIWHU DUWHULDO WUDQVSODQWDWLRQ
ZDVVWXGLHGXVLQJIHPRUDODUWHU\JUDIWV,QWKH¿UVWVWXG\JO\R[\OLFDFLGLQGXFHG
ÀXRUHVFHQFHZDVXVHGWRGHPRQVWUDWHDGUHQHUJLFQHUYHVKLVWRFKHPLFDOO\,QWKH
VHFRQGDQGWKLUGVWXGLHVKLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\+3/&ZLWK
HOHFWURFKHPLFDOGHWHFWLRQZDVXVHGWRGHWHUPLQHQRUDGUHQDOLQHFRQWHQWLQJUDIWV
DQG FRQWUROV 7KH IRXUWK H[SHULPHQW ZDV SHUIRUPHG WR FKDUDFWHUL]H QHXURQDO
GHJHQHUDWLRQDQGUHJHQHUDWLRQHYHQWVTXDOLWDWLYHO\XVLQJLPPXQRKLVWRFKHPLVWU\
6SHFLPHQVZHUHDVVHVVHGIRUYDVRDFWLYHLQWHVWLQDOSHSWLGHQHXUR¿ODPHQWVJURZWK
DVVRFLDWHGSURWHLQW\URVLQHK\GUR[\ODVHDQGQLWULFR[LGHV\QWKDVHLVRHQ]\PHV
$FFRUGLQJWRRXUH[SHULPHQWDOVWXGLHVDWVL[KRXUVSRVWRSHUDWLYHO\WKHQHWZRUNRI
¿EHUVKDGVWDUWHGWRGHJHQHUDWHDQGFDWHFKRODPLQHÀXRUHVFHQFHIURPWKHDGUHQHUJLF
QHUYHVKDG DOPRVW FRPSOHWHO\ GLVDSSHDUHGZLWKLQKRXUVRIJUDIWLQJ&RQWURO
VSHFLPHQVIURPQRUPDOIHPRUDODUWHULHVVKRZHGDGHQVHQHWZRUNRIÀXRUHVFHQW
DGUHQHUJLFQHUYHV,PPHGLDWHO\DIWHUJUDIWLQJWKHQRUDGUHQDOLQHFRQWHQWZDV
RIWKHFRQWUROYDOXHDWKDQGDWKRIWKHFRQWUROYDOXH7KHGURS
LQ JUDIW QRUDGUHQDOLQH FRQWHQW DW K ZDV VWDWLVWLFDOO\ VLJQL¿FDQW S 7KHGLIIHUHQFHEHWZHHQKKDQGKJURXSVZDVVWDWLVWLFDOO\LQVLJQL¿FDQW
,1752'8&7,21
&RPSDUHGZLWKFRQWUROVSHFLPHQVJUDIWVFRQWDLQHGOHVVQRUDGUHQDOLQHDIWHUK
DQGLWGLVDSSHDUHGDOPRVWFRPSOHWHO\IURPIHPRUDODUWHULDOJUDIWVLQUDWVZLWKLQ
K7KHRSHUDWLRQLWVHOIUHVXOWHGLQGHFUHDVHGQRUDGUHQDOLQHFRQFHQWUDWLRQVLQWKH
JUDIWVWRRIWKDWLQWKHFRQWUROVHJPHQWV
2QHZHHNDIWHUWKHRSHUDWLRQWKHQRUDGUHQDOLQHFRQFHQWUDWLRQKDGIDOOHQWROHVV
WKDQRIFRQWUROYDOXHVDQGLWVWDUWHGWRUHFRYHUWKHUHDIWHU2QHPRQWKDIWHUWKH
RSHUDWLRQLWZDVDWPRQWKVLWZDVDQGDWZHHNVLWZDVRIFRQWURO
YDOXHV7KHGHFUHDVHIURPWLPHWRZHHNZDVVLJQL¿FDQWSDVZDVWKH
LQFUHDVHIURPZHHNWRZHHNVS&DWHFKRODPLQHOHYHOVLQFUHDVHGRYHUWKH
IROORZXSSHULRGRIZHHNVDVDUHVXOWRIUHLQQHUYDWLRQRIWKHJUDIWV,QUDWIHPRUDO
DUWHULDOJUDIWVWUDQVSODQWHGLQWRFDURWLGDUWHULHV1$FRQFHQWUDWLRQVKDGULVHQWR
OHYHOVWKDWGLGQRWVLJQL¿FDQWO\GLIIHUIURPWKRVHLQFRQWUROVRUWKHGD\JURXS
'XULQJ QHXURQDO GHJHQHUDWLRQ YDVRDFWLYH LQWHVWLQDO SHSWLGH GLVDSSHDUHG
ZLWKLQGD\WUDQVPLWWHUV\QWKHVL]LQJHQ]\PHVQLWULFR[LGHV\QWKDVHDQGW\URVLQH
K\GUR[\ODVHKDGYDQLVKHGE\GD\DQGQHXUR¿ODPHQWVF\WRVNHOHWDOPDUNHUVKDG
HVVHQWLDOO\GLVDSSHDUHGDIWHUZHHN
,Q WKH UHJHQHUDWLRQ SKDVH WKH PRVW UREXVW D[RQDO JURZWK DV YLVXDOL]HG E\
JURZWKDVVRFLDWHG SURWHLQ ZDV REVHUYHG DW PRQWK IROORZHG E\ D JUDGXDO
LQFUHDVHLQQHXURWUDQVPLWWHUPDUNHUVDWDQGPRQWKVZKHUHDVWKHQHXUR¿ODPHQWV
LQFUHDVHGJUDGXDOO\XSWRWKHHQGRIWKHPRQWKREVHUYDWLRQSHULRG5HLQQHUYDWLRQ
VWDUWHGIURPWKHSUR[LPDOFDURWLGKRVWWUXQNDQGSURFHHGHGGLVWDOO\WRWKHJUDIW
$[RQDO UHJURZWK RFFXUUHG PDLQO\ LQ DUWHULDO DGYHQWLWLD ,QQHUYDWLRQ GHQVLW\ DV
YLVXDOO\DVVHVVHGZDVGHQVHULQWKHJUDIWWKDQLQWKHKRVWZKLFKLVFRQVLVWHQWZLWK
WKH GHQVLW\ RI QHXURQDO QHWZRUNV LQ WKH FRUUHVSRQGLQJ DUWHULHV LQ WKHLU QDWXUDO
VXUURXQGLQJV
,QFRQFOXVLRQZHVXJJHVWWKDWWKHUHODWLYHO\UDSLGOLEHUDWLRQRIFDWHFKRODPLQHV
IURPGHJHQHUDWLQJDGUHQHUJLFQHUYHVPD\KDYHDQLPSRUWDQWUROHLQHDUO\YDVRVSDVP
LQPLFURYDVFXODUDQGFRURQDU\E\SDVVVXUJHU\2XU¿QGLQJVDOVRVXJJHVWWKDWWKH
PDLQVHTXHQFH RI GHJHQHUDWLRQ DQG UHJHQHUDWLRQIROORZVWKDWUHSRUWHGLQRWKHU
PRGHOV RI QHXURQDO GHJHQHUDWLRQ 2XU ¿QGLQJV LQGLFDWH WKDW UHLQQHUYDWLRQ RI
DUWHULDOJUDIWVFRPHVPDLQO\IURPKRVWDUWHULHVDQGLQQHUYDWLRQGHQVLW\LQWKHJUDIW
PD\GLIIHUIURPWKDWLQWKHKRVWZKLFKPD\VXJJHVWWDUJHWGHULYHGUHJXODWLRQRI
LQQHUYDWLRQ$QXQH[SHFWHG¿QGLQJZDVWKDWWKHUHLVDULVHLQQRUDGUHQDOLQHOHYHOVLQ
JUDIWVVL[KRXUVDIWHUJUDIWLQJ7KLVPD\EHEHFDXVHRIWKHIDFWWKDWWKHQHUYH¿EHUV
GRQRWUHFHLYHVWLPXOLZKLFKFDXVHVWKHPWRUHOHDVHQRUDGUHQDOLQHWRWKHV\QDSWLF
YRLG$WWKHVDPHWLPHWKHDFWLYHHQHUJ\DQGVRGLXPLRQGHSHQGHQWUHXSWDNHRI
QRUDGUHQDOLQHRFFXUVLQWKHWHUPLQDOHQGVRIWKH¿EHUV
1. INTRODUCTION
9DVRVSDVP LV D PDMRU WKUHDW WR VXFFHVVIXO RXWFRPH RI VXUJHU\ DQG LW KDV EHHQ
VWXGLHGIRUGHFDGHV%ORRGÀRZWKURXJKDYHVVHOLVGRPLQDWHGE\VHYHUDOIDFWRUV
SUHVVXUHJUDGLHQWRYHUWKHREVHUYHGYHVVHOYHVVHOGLDPHWHUYHVVHOOHQJWKDQGÀXLG
G\QDPLFYLVFRVLW\3RLVHXLOOHVODZ)LJGHPRQVWUDWHVWKHUHODWLRQVKLSVEHWZHHQ
WKHGLIIHUHQWIDFWRUVLQRQHHTXDWLRQ6XWHUDDQG6NDODN*R\WRQ
Fig. 1. Poiseuille´s law, R=flow rate (ml/s), r=radius of the pipe, Ș=dynamic fluid viscosity
(Pascal × seconds), p=pressure gradient, l=length of the pipe.
)URPWKHHTXDWLRQLWFDQHDVLO\EHVHHQWKDWDQLQFUHDVHLQSLSHYHVVHOOHQJWK
DQGLQFUHDVHGYLVFRVLW\WKLFNQHVVRIWKHÀXLGGHFUHDVHWKHÀRZUDWHDQGLQFUHDVLQJ
WKHGLDPHWHUUDGLXVRIWKHSLSHYHVVHOLQFUHDVHVWKHÀRZUDWHWRWKHSRZHURI
IRXU7KXVGRXEOLQJWKHYHVVHOGLDPHWHUZLOOLQFUHDVHWKHÀRZIROGDQGHYHQ
DPRGHVWLQFUHDVHE\DIDFWRURIZLOOGRXEOHWKHÀRZ3RLVHXLOOHVODZDVVXFK
LV DSSOLFDEOH WR QRQFRPSUHVVLEOH KRPRJHQHRXV 1HZWRQLDQ ÀXLGV %ORRG LV D
VKHDUWKLQQLQJQRQ1HZWRQLDQÀXLGDQGLWVÀRZLQWKHYHVVHOVLVDPRUHFRPSOH[
SKHQRPHQRQ,QWKLQYHVVHOVZLWKDGLDPHWHURIDERXW—PEORRGYLVFRVLW\IDOOV
JUHDWO\7KLVLVWKRXJKWWRKDSSHQEHFDXVHEORRGFHOOVZLOODJJUHJDWHWRWKHFHQWHU
RIWKHYHVVHODQGFUHDWHDFHOOIUHHORZYLVFRVLW\OD\HURISODVPDDJDLQVWWKHYHVVHO
ZDOO)RUEHVDQG/RZH
&DWHFKRODPLQHVIUHHGIURPQHUYHHQGLQJVGXULQJDQGDIWHUDQRSHUDWLRQDUH
SRVVLEO\DPDMRUFRQWULEXWRUWRVPRRWKPXVFOHFRQWUDFWLRQDQGYDVRVSDVP7KH
XVHRIDUWHULHVDVJUDIWPDWHULDOKDVLQFUHDVHGFRQVLVWHQWO\RYHUWKH\HDUV7RGD\
DUWHULHV DUH XVHG LQ IUHHÀDS VXUJHU\ RUJDQ WUDQVSODQWDWLRQ YDVFXODU VXUJHU\
UHSODQWDWLRQ VXUJHU\ DQG FRURQDU\ E\SDVV VXUJHU\ 9HQRXV JUDIWV KDYH EHHQ
VWXGLHGLQUHJDUGWRYLDELOLW\RIWKHJUDIWZDOOSHULRSHUDWLYHGDPDJHIDWHRIWKH
HQGRWKHOLXPDQGLQQHUYDWLRQ,QFRURQDU\E\SDVVVXUJHU\ERWKWKHUDGLDODUWHU\
DQGWKHULJKWJDVWURHSLSORLFDUWHU\DUHZHOONQRZQIRUWKHLUWHQGHQF\WRJRLQWR
VSDVPGXULQJWKHRSHUDWLRQ&KHQHWDO*UDQGMHDQHWDO,QYHQRXV
JUDIWVQRUDGUHQDOLQHFRQFHQWUDWLRQVLQFUHDVHDQGUHDFKDVWHDG\VWDWHZLWKLQ
ZHHNV,QPHVHQWHULFDUWHULHVUHLQQHUYDWLRQKDVEHHQIRXQGWREHFRPSOHWHE\
ZHHNVZKHUHDVLQFDURWLGDUWHULHVLWKDVEHHQWKRXJKWWRWDNHORQJHU&RZHQHW
DO7KHDXWRQRPLFQHUYRXVV\VWHPLVDSRZHUIXOFRQWUROOHURIDUWHULDOEORRG
SUHVVXUHJDVWURLQWHVWLQDOVHFUHWLRQDQGPRWLOLW\VZHDWLQJEODGGHUHPSW\LQJDQG
,1752'8&7,21
RWKHUYLWDOIXQFWLRQVRIWKHERG\&OLQLFDODQGKLVWRFKHPLFDOVWXGLHVKDYHLQGLFDWHG
WKDWZKHQRSHQKHDUWVXUJHU\LVSHUIRUPHGFDWHFKRODPLQHVIUHHGIURPKHDUWWLVVXH
FDQEHDULVNIDFWRU,IWKHKHDUWWLVVXHFRQWDLQVODUJHDGUHQHUJLFQHUYHVZLWK³GURSOHW
¿EHUV³RU³DGUHQDOLQHERPEV³FRQWDLQLQJKXJHORFDOLQWUDD[RQDOFRQFHQWUDWLRQVRI
FDWHFKRODPLQHVPRVWO\QRUDGUHQDOLQHWKHUHLVDQLQFUHDVHGULVNRIDUUK\WKPLDDQG
HYHQGHDWK.\|VRODHWDO5HPHVDQGFRZRUNHUV5HPHVHWDO5HPHVHW
HOKDYHGHPRQVWUDWHGWKDWLQVWUXPHQWDWLRQFDXVHVFUXVKLQMXU\WRWKHYHVVHO
ZDOOZLWKORVVRIPHWDEROLFDFWLYLW\DQGLQÀDPPDWRU\FHOOLQ¿OWUDWLRQ
2. REVIEW OF THE LITERATURE
2.1. AUTONOMIC NERVES
2.1.1. SYMPATHETIC NERVES
6\PSDWKHWLF FRQWURO RI WKH FLUFXODWLRQ ZDV ¿UVW GHVFULEHG LQ E\ %HUQDUG
%HUQDUGDQGE\%URZQ6HTXDUG%URZQ6HTXDUGDQG*DVNHOO
DQG/DQJOH\ERWKGHVFULEHGWKHDQDWRP\RIWKHDXWRQRPLFQHUYRXVV\VWHPDQGLWV
HIIHFWVRQHQGRUJDQV*DVNHOODDQGE/DQJOH\DDQGE
6\PSDWKHWLFLQQHUYDWLRQLVPDLQO\FRQWUROOHGE\FHQWHUVLQWKHVSLQDOFRUGEUDLQ
VWHPDQGK\SRWKDODPXV7KHOLPELFFRUWH[ZKLFKLVSDUWRIWKHFHUHEUDOFRUWH[FDQ
DOVRWUDQVPLWVLJQDOVWRORZHUFHQWHUVDQGWKHUHIRUHFRQWUROWKHDXWRQRPLFQHUYRXV
V\VWHP7KHSDUDYHQWULFXODUQXFOHXV391KDVEHHQVKRZQWREHDQLPSRUWDQW
FHQWUDOQXFOHXVDVUHJDUGVFRQWURORIWKHV\PSDWKHWLFV\VWHP391PLFURLQMHFWLRQV
RIH[FLWDWRU\DPLQRDFLGVKDYHEHHQVKRZQWRSURGXFHQRQXQLIRUPFKDQJHVLQ
WKHOHYHORIV\PSDWKHWLFQHUYHDFWLYLW\7KHVHUHVXOWVVXSSRUWWKHFRQFHSWWKDWWKH
391LVDQLPSRUWDQWFRPSRQHQWRIWKHFHQWUDOQHXURFLUFXLWU\UHJXODWLQJIXQFWLRQDO
FKDUDFWHULVWLFVRIHIIHUHQWV\PSDWKHWLFQHUYHRXWÀRZ.HQQH\HWDO/HHHWDO
+LJD7DQLJXFKLHWDO
7KHDXWRQRPLFQHUYRXVV\VWHPFRQWUROVDUWHULDOEORRGSUHVVXUHJDVWURLQWHVWLQDO
VHFUHWLRQDQGPRWLOLW\VZHDWLQJEODGGHUHPSW\LQJDQGRWKHUYLWDOERG\IXQFWLRQV
7KHV\PSDWKHWLFV\VWHPLVDYHU\SRZHUIXOUHJXODWRURIWKHVHIXQFWLRQVLWFDQGRXEOH
DUWHULDOSUHVVXUHZLWKLQWRVHFRQGVLWFDQLQFUHDVHKHDUWUDWHWRGRXEOHRI
QRUPDOZLWKLQWKUHHWR¿YHVHFRQGVDQGVZHDWLQJFDQVWDUWZLWKLQVHFRQGV:UD\
HWDO/HHHWDO
7KH V\PSDWKHWLF QHUYRXV V\VWHP FRQVLVWV RI WZR SDUDYHUWHEUDO V\PSDWKHWLF
FKDLQVRIJDQJOLDWKDWDUHORFDWHGRQERWKVLGHVRIWKHYHUWHEUDOFROXPQDQGWZR
SDUDYHUWHEUDO JDQJOLD WKH K\SRJDVWULF DQG FRHOLDF DQG WKH QHUYHV IURP WKHVH
JDQJOLDWRWKHWDUJHWRUJDQV7KHV\PSDWKHWLFQHUYHVDULVHIURPWKHVSLQDOFROXPQ
EHWZHHQVHJPHQWV7DQG/7KH\WKHQSDVVWKURXJKWKHV\PSDWKHWLFFKDLQWR
WKH HQG RUJDQV WKDW DUH UHJXODWHG E\ WKH QHUYHV 7KH V\PSDWKHWLF QHXURQV ULVH
IURPWKHLQWHUPHGLDWHODWHUDOKRUQRIWKHVSLQDOFROXPQ7KHDIIHUHQW¿EHUVSDVV
WKURXJKWKHSRVWHULRUURRWWRWKHJDQJOLDRIWKHV\PSDWKHWLFFKDLQDQGV\QDSVHWR
VHFRQGDU\QHXURQV$QDOWHUQDWLYHURXWHLVWKDWWKH\SDVVWKURXJKWKHV\PSDWKHWLF
FKDLQDQGV\QDSVHLQDSHULSKHUDOJDQJOLRQDQGDQRWKHUDOWHUQDWHURXWHLVWKDW
WKH\SDVVXSZDUGVRUGRZQZDUGVLQWKHV\PSDWKHWLFFKDLQDQGWKHQV\QDSVHZLWK
WKHSHULSKHUDOQHXURQV7KHSULPDU\QHXURQVXVHDFHW\OFKROLQHDVWUDQVPLWWHUDQG
5(9,(:2)7+(/,7(5$785(
QRUDGUHQDOLQHLVXVHGLQVHFRQGDU\QHXURQVH[FHSWLQWKRVHOHDGLQJWRVZHDWJODQGV
DQG SLORPRWRU PXVFOHV ZKLFK DUH DOZD\V FKROLQHUJLF 6RPH RI WKH V\PSDWKHWLF
QHXURQV SDVV EDFN IURP WKH V\PSDWKHWLF FKDLQ WKURXJK WKH JUD\ UDPL LQWR WKH
VSLQDOQHUYHV7KHVHSDWKZD\VFRPSULVHW\SH&¿EHUVDQGWKH\SDVVWRDOOSDUWVRI
WKHERG\ZLWKWKHVSLQDOQHUYHV7KH¿EHUVFRQWUROSLORHUHFWRUPXVFOHVVZHDWJODQGV
DQGEORRGYHVVHOV$ERXWHLJKWSHUFHQWRIQHUYH¿EHUVRIDQ\SHULSKHUDOQHUYHDUH
V\PSDWKHWLFDIDFWWKDWXQGHUOLQHVWKHLULPSRUWDQFH*X\WRQDQG+DOOHG7H[WERRN
RI0HGLFDO3K\VLRORJ\WKHGLWLRQ3KLODGHOSKLD3D86$6DXQGHUV
6\PSDWKHWLFLQQHUYDWLRQRIEORRGYHVVHOVLQGLIIHUHQWRUJDQVYDULHVJUHDWO\LQGHQVLW\
DVVKRZQE\+LOODUSDQGE\0RUJDQDQGLWDOVRYDULHVEHWZHHQLQGLYLGXDOV+LOODUS
0RUJDQHWDO&KHQHWDO/DUJHQXPEHUVRIV\PSDWKHWLFQHUYHV
LQQHUYDWHWKHDUWHULROHVRIWKHVNLQDQGWKHVSODQFKQLFYHVVHOVEXWWKHV\PSDWKHWLF
QHWZRUNLQYHLQVLQWKHVHDUHDVLVOHVVGHQVH7KHHIIHFWVRIDGUHQHUJLFLQQHUYDWLRQ
KDYHEHHQWKRURXJKO\VWXGLHGDQGDUHVXPPDUL]HGLQ7DEOH
Table 1. Effects of sympathetic stimulation on different receptors, organs and tissues (Zaugg and Shaubb
2005).
Adrenergic
receptor
subtype
Main signaling pathways
Effects on neuronal
and cardiac tissue
Effects on
vascular tissue
ȕ1
G-protein signaling component-adenyl Positive on heart rate
cyclase-cAMP-protein kinase A
inotropy, lusitropy,
metabolism, growth,
myocyte toxicity
_
ȕ2
G-protein signaling componentadenyl cyclase- cAMP-target-specific
serine/theorine protein kinase;
G-protein signaling component
inhibiting adenyl cyclase; G-protein
signaling component- phospholipase
C-diacylglycerol-target-specific
serine/threonine protein kinase and
G-protein signaling components−
phosphatidylinositol 3-kinase-PI3Kdependent kinase-target-specific
serine/threonine protein kinase
Positive on inotropy,
lusitropy, metabolism,
growth, myocyte
survival
Relaxation of
vascular smooth
muscle cells
ȕ3
G-protein signaling component
inhibiting adenylyl cyclase; G-protein
signaling components-phospholipaseC-diacylglycerol-target-specific
serine/threonine protein kinase and
G-protein signaling component-NOSNO-guanylyl cyclase- cyclic guanosine
monophosphate-target-specific
serine/threonine protein kinase
Negative on inotropy
and myocyte survival
_
Į1A
G-protein signaling component
- phospholipase-C- inositol
trisphosphate-Ca2++
Positive on heart rate,
inotropy, and growth in
myocytes
Vasoconstriction
Į1B
G-protein signaling component
- phospholipase-C- inositol
trisphosphate-Ca2++
Positive on heart rate,
inotropy, and growth in
myocytes
_
Į1D
G-protein signaling component
- phospholipase-C- inositol
trisphosphate-Ca2++
Contraction of smooth
muscle cells
Vasoconstriction
Į2A
G-protein signaling componentinhibiting adenyl cyclase
Presynaptically reduces
sympathetic outflow
Lowers RR
Į2B
G-protein signaling componentinhibiting AC, also G-protein signaling
component-stimulating adenyl cyclase
Postsynaptically
counteracts the effects
of Į2 A
Peripheral
vasoconstriction
Į2C
G-protein signaling componentinhibiting adenyl cyclase
Presynaptically reduces
sympathetic outflow,
postsynaptically lowers
cAMP
Lowers RR and
participates in
vasoconstriction
after exposure
to low
temperatures
5(9,(:2)7+(/,7(5$785(
2.1.2. PARASYMPATHETIC NERVES
7KHSDUDV\PSDWKHWLFQHUYRXVV\VWHP3616LVWKHRWKHURIWKHWZRPDLQGLYLVLRQV
RIWKHDXWRQRPLFQHUYRXVV\VWHP$166\PSDWKHWLFDQGSDUDV\PSDWKHWLFGLYLVLRQV
W\SLFDOO\ IXQFWLRQ LQ RSSRVLWLRQ WR HDFK RWKHU 7KLV QDWXUDO RSSRVLWLRQ LV EHWWHU
XQGHUVWRRG DV FRPSOHPHQWDU\ LQ QDWXUH UDWKHU WKDQ DQWDJRQLVWLF 0RRUH DQG
$JXU7KHWUDQVPLWWHUVXEVWDQFHLV$&KDQGWKHUHDUH¿YHGLIIHUHQWW\SHVRI
UHFHSWRUV0LVLQWKH&16DQG0LQWKHKHDUWZKHUHWKH\VORZGRZQWKHKHDUWUDWH
DQGGHFUHDVHWKHFRQWUDFWLOHIRUFH0UHFHSWRUVDUHORFDWHGLQPDQ\SODFHVLQWKH
ERG\VXFKDVWKHLQWHVWLQDOV\VWHPOXQJVDQGEORRGYHVVHOV0DQG0UHFHSWRUV
KDYHSRVVLEOH&16HIIHFWV
8QGHUVWDQGLQJWKHHIIHFWRI0UHFHSWRUVRQEORRGYHVVHOVLVLPSRUWDQWLQWKLV
ZRUN$FHW\OFKROLQHFDXVHVWKHSURGXFWLRQRI12DQGDVDUHVXOWRILWVDFWLRQVPRRWK
PXVFOHFHOOVUHOD[DQGYDVRGLODWDWLRQRFFXUV
$VLQWKHV\PSDWKHWLFQHUYRXVV\VWHPSDUDV\PSDWKHWLFQHUYHVLJQDOVDUHFDUULHG
WRWKHLUHQGRUJDQVIURPWKHFHQWUDOQHUYRXVV\VWHPE\DV\VWHPRIWZRQHXURQV
7KH¿UVWQHXURQLQWKLVSDWKZD\LVUHIHUUHGWRDVWKHSUHJDQJOLRQLFQHXURQ,WVFHOO
ERG\LVORFDWHGLQWKHFHQWUDOQHUYRXVV\VWHPDQGLWVD[RQXVXDOO\H[WHQGVWRD
JDQJOLRQVRPHZKHUHHOVHLQWKHERG\ZKHUHLWV\QDSVHVZLWKWKHGHQGULWHVRIWKH
VHFRQGQHXURQLQWKHFKDLQ7KLVVHFRQGQHXURQLVUHIHUUHGWRDVWKHSRVWJDQJOLRQLF
QHXURQ,QWKH&16WKHSUHV\QDSWLFQHXURQVDUHORFDWHGLQVSHFL¿FQXFOHLLQWKH
SHOYLFGLYLVLRQLQWKHODWHUDOJUD\KRUQRIWKHVSLQDOFRUG7KHD[RQVRISUHV\QDSWLF
SDUDV\PSDWKHWLF QHXURQV DUH XVXDOO\ ORQJ WKH\ H[WHQG IURP WKH &16 LQWR D
JDQJOLRQWKDWLVHLWKHUYHU\FORVHWRRUHPEHGGHGLQWKHWDUJHWRUJDQ$VDUHVXOWWKH
SRVWV\QDSWLFSDUDV\PSDWKHWLFQHUYH¿EHUVDUHYHU\VKRUW0RRUHDQG$JXU
$OWKRXJKWKHSDUDV\PSDWKHWLFQHUYHVFDXVHYDVRGLODWDWLRQLQYLVFHUDORUJDQVLW
KDVEHHQUHSRUWHGWKDWWKHQHUYHVUHVSRQVLEOHIRUYDVRGLODWDWLRQLQWKHSHULSKHUDO
YHVVHOVDUHV\PSDWKHWLFYDVRGLODWRU¿EHUV&KDUFRXGLDQ7KLVZRXOGLQGLFDWH
WKDWWKHSDUDV\PSDWKHWLFV\VWHPZRXOGWKHQEHUHVSRQVLEOHIRUYDVRGLODWDWLRQRQO\
LQ WKH YLVFHUDO RUJDQV +RZHYHU WKH DXWKRU RI WKH UHSRUW VHHPV WR KDYH GUDZQ
IDOVHFRQFOXVLRQV+HVWDWHVWKDWWKHWUDQVPLWWHULVXQNQRZQZKLOHUHSRUWLQJWKDW
ERWXOLQWR[LQDEROLVKHVWKHDFWLRQ+HDOVRVWDWHVWKDWWKHPXVFDULQHUHFHSWRUEORFNHU
DWURSLQHGRHVQRWDEROLVKWKHHIIHFW)URPWKLVGDWDLWZRXOGEHPRUHORJLFDOWR
DVVXPHWKDWWKHWUDQVPLWWHULV$&KERWXOLQWR[LQHIIHFWEXWWKHUHFHSWRULVQRWD
PXVFDULQHUHFHSWRUODFNRIDWURSLQHHIIHFW
2.2. TYPES OF TRANSMISSION
2.2.1. CLASSICAL TRANSMISSION
,QFODVVLFDOWUDQVPLVVLRQDWUDQVPLWWHUVXEVWDQFHLVV\QWKHVL]HGLQDQHXURQDQG
LWLVWKHQVWRUHGLQJUDQXOHVLQWKHSUHV\QDSWLFUHJLRQ1RUDGUHQDOLQHOLNHRWKHU
WUDQVPLWWHUVJRHVWKURXJKWKHVDPHSDWKZD\LQWKHV\PSDWKHWLFQHXURQV3HSWLGH
WUDQVPLWWHUVDUHW\SLFDOO\V\QWKHVL]HGLQWKHVRPDRIWKHQHXURQDQGWKHQWUDQVSRUWHG
WRWKHD[RQE\D[RQDOWUDQVSRUW7KHWUDQVPLWWHULVUHOHDVHGIURPWKHJUDQXOHVDIWHU
DQD[RQSRWHQWLDOHQWHUVWKHV\QDSWLFUHJLRQDQGFDXVHVRSHQLQJRIWKH&DFKDQQHOV
7KLVHYHQWXDOO\OHDGVWRIXVLQJRIWKHVWRUDJHJUDQXOHZLWKWKHFHOOPHPEUDQHDQGWKH
FRQWHQWVRIWKHJUDQXOHLHWKHWUDQVPLWWHUVXEVWDQFHVDUHUHOHDVHGWRWKHV\QDSWLF
FOHIW7KHWUDQVPLWWHUVXEVWDQFHVWKHQELQGWRFRUUHVSRQGLQJUHFHSWRUVFDXVLQJ
HLWKHUSHUPHDELOLW\FKDQJHVLQWKHWDUJHWFHOOPHPEUDQHRUDFWLYDWLRQRIDVHFRQGDU\
PHVVHQJHU V\VWHP 7KH ¿UVW RI WKHVH LV UDSLG DQG KDSSHQV ZLWKLQ PLOOLVHFRQGV
ZKHUHDVWKHVHFRQGHYHQWODVWVVHFRQGV,QWKHODVWSKDVHWKHWUDQVPLWWHUPROHFXOH
QRUDGUHQDOLQHLQWKHV\PSDWKHWLFV\VWHPLQWKHHQGRUJDQV\QDSVHLVHOLPLQDWHG
IURPWKHV\QDSWLFFOHIWHLWKHUE\VSHFL¿FHQ]\PHV&RRSHUHWDOLQWKHV\QDSWLF
FOHIWRUWUDQVSRUWHGLQWRSUHSRVWV\QDSWLFRUJOLDOFHOOV/HVWHUHWDO7KHUHLW
ZLOOEHUHXVHGLQSUHV\QDSWLFFHOOVRULQDFWLYDWHGLQWKHSRVWV\QDSWLFRUJOLDOFHOOV
2.2.2. VOLUME TRANSMISSION
,QFODVVLFDOWUDQVPLVVLRQVLJQDOVDUHSDVVHGIURPRQHFHOOWRDQRWKHUWKURXJKD
VSHFLDOL]HGVWUXFWXUHWKHV\QDSVHLQDTXDQWDOPDQQHU7KLVFRQFHSWZDVFKDOOHQJHG
LQWKHODWHVDQGLQWKHVE\QHZ¿QGLQJV%XUQVWRFN.XSIHUPDQQ
7KHUHZDVDPLVPDWFKLQGLVWULEXWLRQRIUHFHSWRUVDQGWUDQVPLWWHUV7KLV
VXJJHVWHGWKDWWKHVHUHFHSWRUVHLWKHUKDGQRUROHLQVLJQDOWUDQVPLVVLRQRUWKDW
WKHUHLVDQDOWHUQDWLYHIRUPRIWUDQVPLVVLRQRXWVLGHV\QDSVHV$IWHUWKDWWKHUHZHUH
¿QGLQJVRIQHXURQVZLWKV\QDSWLFJUDQXOHVLQDUHDVZLWKQRV\QDSWLFPHPEUDQH
VSHFLDOL]DWLRQ7KLVNLQGRIWUDQVPLVVLRQKDVEHHQIRXQGLQERWKWKHFHQWUDODQG
SHULSKHUDOQHUYRXVV\VWHPV,QYROXPHWUDQVPLVVLRQWKHWUDQVPLWWHUVXEVWDQFHV
PD\GLIIXVHORQJGLVWDQFHVIURPWKHLUVRXUFHVWRWKHLUWDUJHWV9ROXPHWUDQVPLVVLRQ
JLYHV D WRWDOO\ QHZ FRQFHSW WR QHXURORJLFDO WUDQVPLVVLRQ LQ DGGLWLRQ WR TXDQWDO
WUDQVPLVVLRQ :LWK YROXPH WUDQVPLVVLRQ ODUJH SRSXODWLRQV RI QHXURQV FDQ EH
FRQWUROOHGLQDQHYHQWRQLFPDQQHUE\DWUDQVPLWWHUVXEVWDQFH7KHPRVWLPSRUWDQW
IDFWRUVDIIHFWLQJWKHH[WHQWRIYROXPHWUDQVPLVVLRQDUHWKHDPRXQWRIWUDQVPLWWHU
UHOHDVHGWKHYROXPHDQGWRUWXRVLW\RIWKHH[WUDFHOOXODUVSDFHDQGWKHWLPHVSDQRI
DFWLYLW\RIWKHWUDQVPLWWHUWKLVEHLQJGHWHUPLQHGE\WKHDFWLYLW\RIWKHWUDQVPLWWHU
5(9,(:2)7+(/,7(5$785(
GHJUDGLQJPHFKDQLVPV)X[HDQG$JQDWL6\NRYi9ROXPHWUDQVPLVVLRQ
LVSUHVHQWLQDOOSDUWVRIWKHQHUYRXVV\VWHPDQGFRQGLWLRQVIDYRULQJLWWKHGLIIXVLRQ
RI WUDQVPLWWHU DERXW WKH V\QDSWLF FOHIW DQG WR DQRWKHU QHXURQ DUH WKH VDPH DV
FRQGLWLRQVIDYRULQJIDOVHWUDQVPLVVLRQ*OLDOFHOOVSOD\DQLPSRUWDQWUROHLQYROXPH
WUDQVPLVVLRQE\UHJXODWLQJWKHYROXPHWUDQVPLVVLRQFRQGLWLRQV
9ROXPHWUDQVPLVVLRQLVLQYROYHGLQPDQ\SK\VLRORJLFDOSKHQRPHQDVXFKDVSDLQ
PRGXODWLRQVHQVRU\SHUFHSWLRQYLVLRQUHJHQHUDWLRQH[FLWDELOLW\PRWRUUHJXODWLRQ
DURXVDODQGDWWHQWLRQDVZHOODVPDQLFGHSUHVVLYHGLVHDVHDQGH[LWRWR[LFLW\
2.2.3. NONEXOCYTOTIC TRANSMISSION
4XDQWDO UHOHDVH RI WUDQVPLWWHUV ZKLFK LV DOVR NQRZQ DV H[RF\WRWLF UHOHDVH RI
WUDQVPLWWHUV ZDV ORQJ UHJDUGHG DV WKH ³ULJKW´ PHWKRG RI WUDQVPLVVLRQ ,Q
QRQH[RF\WRWLF WUDQVPLVVLRQ QHXURQV UHOHDVH WUDQVPLWWHU VXEVWDQFHV LQ D QRQ
TXDQWDOPDQQHUWKURXJKUHYHUVDORIWKHWUDQVPLWWHUWUDQVSRUWPHFKDQLVP7KLV
UHOHDVHLVLQGHSHQGHQWRI&DLRQV$WWZHOOHWDO6LQFHWKLVUHOHDVHLVFDUULHG
RXWE\WKHWUDQVSRUWPHFKDQLVPLWLVDOVRFDOOHGFDUULHUPHGLDWHGUHOHDVH*DVHRXV
WUDQVPLWWHUVVXFKDVQLWULFR[LGH12DQGFDUERQPRQR[LGH&2DUHUHOHDVHG
VROHO\E\QRQH[RF\WRWLFWUDQVPLVVLRQZKLFKLVDOVRQRQFDUULHUPHGLDWHG&OLQLFDOO\
QRQH[RF\WRWLFWUDQVPLVVLRQLVLQYROYHGLQPDQ\SDWKRORJLFDOVWDWHVLQWKHFHQWUDO
QHUYRXVV\VWHP&16DQGKHDUW,QWKH&16DOOHQGRWKHOLDODQGJOLDOWUDQVPLVVLRQ
LVFDUULHGRXWYLDQRQH[RF\WRWLFWUDQVPLVVLRQ$WWZHOOHWDO+XV]WLHWDO
2.2.4. COLOCALIZATION AND COTRANSMISSION
7KHUHDUHVHYHUDOFULWHULDWKDWQHHGWREH¿OOHGWRHVWDEOLVKWKDWWZRFRPSRXQGV
UHDOO\DUHFRWUDQVPLWWHUVDQGQRWRQO\FRORFDOL]HGLQWKHQHUYHV7KHXQLYHUVDOO\
DFFHSWHGRQHVDUH
7KHVXEVWDQFHPXVWEHV\QWKHVL]HGDQGVWRUHGLQWKHQHUYH
7KHUHOHDVHRIWKHVXEVWDQFHLVFDXVHGE\QHUYHVWLPXODWLRQ
7KHUHDUHVSHFL¿FUHFHSWRUVIRUWKHVXEVWDQFHRQSRVWMXQFWLRQDOVLWHVWKDWZKHQ
VWLPXODWHGZLWKWKHVXEVWDQFHFDXVHVFKDQJHVLQSRVWMXQFWLRQDODFWLYLW\
7KHUHLVDQH[LVWLQJWUDQVSRUWV\VWHPIRUWKHVXEVWDQFHLWVHOIRULWVEUHDNGRZQ
SURGXFWV WKDW WDNHV LWWKHP XS DQG FDXVHV UHQHZDO RI PHVVHQJHU VWRUDJH LQ
QHUYHWHUPLQDOV
'LIIHUHQWQHXURWUDQVPLWWHUVDUHUHOHDVHGE\GLIIHUHQWSXOVHVHTXHQFHVPXOWLSOHRU
VLQJOHGHSHQGLQJRQWKHLUPROHFXODUVL]H%UDGOH\HWDO9HQWXUDHWDO
1HJDWLYH FURVVWDON PHDQV WKDW WKH FRWUDQVPLWWHUV LQKLELW WKH UHVSRQVHV WKDW
DUH HYRNHG E\ WKH VDPH WUDQVPLWWHUV LQ WKH SRVWV\QDSWLF UHFHSWRUV 6RPHWLPHV
WKH FRWUDQVPLWWHU VXEVWDQFHV PD\ DFW DV ORQJWHUP WURSKLF IDFWRUV ZKLOH EHLQJ
QHXURWUDQVPLWWHUV)RUH[DPSOH$73DIWHUHFWRHQ]\PDWLFEUHDNGRZQFDQDFWRQ
3UHFHSWRUVRU3DGHQRVLQHUHFHSWRUVWRSURPRWHFHOOSUROLIHUDWLRQPRWLOLW\
GLIIHUHQWLDWLRQRUGHDWKDQGQHXURSHSWLGH<UHOHDVHGIURPV\PSDWKHWLFQHUYHVKDV
FDUGLRYDVFXODUWURSKLFHIIHFWVLQHQGVWDJHUHQDOGLVHDVH(VOHU
$OWKRXJK FRWUDQVPLWWHUV XVXDOO\ KDYH VLPLODU DFWLRQV RQ SRVWV\QDSWLF FHOOV
WKHUHDUHVRPHH[DPSOHVRIFRWUDQVPLWWHUVKDYLQJRSSRVLWHDFWLRQV<DQJHWDO
/DQGLV &RWUDQVPLWWHU SODVWLFLW\ RFFXUV GXULQJ GHYHORSPHQW DQG
DJLQJIROORZLQJWUDXPDRUVXUJHU\DQGDIWHUFKURQLFH[SRVXUHWRGUXJVDVZHOO
DVLQGLVHDVHDQGLWKDVEHHQUHYLHZHGE\%XUQVWRFN%XUQVWRFND
(YLGHQFHZDVSUHVHQWHGLQDUHFHQWVWXG\LQYROYLQJSULPDU\FXOWXUHVRIQHRQDWDO
UDWVSLQDOQHXURQVIRUWKHUHJXODWLRQRIVXEVWDQFH3631.UHFHSWRUH[SUHVVLRQ
E\FDOFLWRQLQJHQHUHODWHGSHSWLGH&*536H\EROGHWDO7KHUHKDYHEHHQ
VRPHRXWVWDQGLQJHDUO\VWXGLHVRIWKHIDFWRUVLQÀXHQFLQJFRWUDQVPLWWHUH[SUHVVLRQ
LQV\PSDWKHWLFQHUYHVQHXURQV*UDQWDQG/DQGLV$UHFHQWVWXG\SURYLGHG
HYLGHQFHWKDWQHXURWURSKLFIDFWRUVIURPWKUHHGLIIHUHQWSURWHLQIDPLOLHVJOLDOFHOO
OLQHGHULYHGQHXURWURSKLFIDFWRUQHXURWURSKLQDQGFLOLDU\QHXURWURSKLFIDFWRU
SURPRWHFKROLQHUJLFGLIIHUHQWLDWLRQLQV\PSDWKHWLFQHXURQVZKHUHDVQRUDGUHQHUJLF
GLIIHUHQWLDWLRQLVSURPRWHGE\QHUYHJURZWKIDFWRU%URGVNLHWDO
2.2.5. FALSE TRANSMISSION
7KHFRQFHSWRIIDOVHWUDQVPLVVLRQFDPHDERXWZKHQLWZDVQRWHGWKDWDQHXURQPD\
WDNHXSVWRUHDQGUHOHDVHWUDQVPLWWHUVXEVWDQFHWKDWLVQRWWKHDFWXDOWUDQVPLWWHU
VXEVWDQFHXWLOL]HGE\WKHQHXURQ%XUQVWRFN.XSIHUPDQ7KLVRWKHU
VXEVWDQFHRIWHQVWUXFWXUDOO\UHVHPEOHVWKHDFWXDOWUDQVPLWWHUVXEVWDQFH2ULJLQDOO\
DQDORJVDQGPHWDEROLWHVRIQRUDGUHQDOLQHZHUHLGHQWL¿HGDVIDOVHWUDQVPLWWHUVEXW
ODWHUFKROLQHUJLFDQG*$%$HUJLFIDOVHWUDQVPLWWHUVZHUHLGHQWL¿HGDQGIRXQGWREH
XVHIXOUHVHDUFKWRROVIRUVWXG\RIWKHQHUYRXVV\VWHP)DOVHWUDQVPLVVLRQJDLQHG
D QHZ GLPHQVLRQ ZKHQ LW ZDV QRWHG WKDW D JHQXLQH WUDQVPLWWHU VXEVWDQFH PD\
DFWDVDIDOVHWUDQVPLWWHU LQ DQRWKHU SRSXODWLRQRIQHXURQV7KLVLVEHFDXVHWKH
WUDQVPLWWHU WUDQVSRUW V\VWHPV DUH QRW DEVROXWHO\ VSHFL¿F WR WKHLU WUDQVPLWWHUV
UDWKHUWKH\KDYHUHODWLYHVSHFL¿FLWLHVIRUWKHLUWUDQVPLWWHUV7KHFRQFHQWUDWLRQRI
WUDQVPLWWHUVXEVWDQFHLQH[WUDFHOOXODUÀXLGLVDQLPSRUWDQWIDFWRULQGHWHUPLQLQJLI
WKHWUDQVPLWWHULVWDNHQXSE\DQHXURQ6LQFHGLIIHUHQWFRQFHQWUDWLRQVRIYDULRXV
WUDQVPLWWHUV DUH SUHVHQW DOO WKH WLPH LQ WKH QHUYRXV V\VWHP IDOVH WUDQVPLVVLRQ
SUHVXPDEO\RFFXUVDOOWKHWLPH7KHHIIHFWVRIDIDOVHWUDQVPLWWHURQDWDUJHWQHXURQ
UHVHPEOHWKRVHRIFRWUDQVPLVVLRQ$WWKHSUHV\QDSWLFQHXURQOHYHODIDOVHWUDQVPLWWHU
PD\HLWKHULQWHUIHUHZLWKRUDLGDXWKHQWLFWUDQVPLVVLRQ
5(9,(:2)7+(/,7(5$785(
$FODVVLFH[DPSOHRIIDOVHWUDQVPLVVLRQLVWKHHIIHFWRIVHURWRQLQRQYDVFXODUWRQH
LQSHULYDVFXODUV\PSDWKHWLFQRQDGUHQHUJLFQHXURQV1DWXUDOO\WKHVHQHXURQVGR
QRWV\QWKHVL]HVHURWRQLQEXWLWKDVEHHQIRXQGWREHSUHVHQWLQWKHP6HURWRQLQLV
WDNHQXSE\WKHFRFDLQHVHQVLWLYHWUDQVSRUWV\VWHPDQGFDQEHUHOHDVHGE\HOHFWULFDO
VWLPXODWLRQRIWKHQHUYH7KHUHOHDVHRIVHURWRQLQKDVEHHQUHSRUWHGWRHQKDQFH
WKHYDVRFRQVWULFWLRQFDXVHGE\QRUDGUHQDOLQHVXJJHVWLQJV\QHUJLVWLFDFWLRQRIWKHVH
VXEVWDQFHV&RKHQ
2.3. ADRENERGIC INNERVATION OF THE VASCULAR WALL
2.3.1. INTRODUCTION
$GUHQHUJLF QHXURQV FDQ EH VWXGLHG E\ XVLQJ PDQ\ GLIIHUHQW PHWKRGV
LPPXQRKLVWRFKHPLVWU\FKHPLFDODQDO\VLVOLJKWPLFURVFRS\HOHFWURQPLFURFRS\
DQGIXQFWLRQDOVWXGLHVLQOLYLQJRUJDQLVPV(DFKRIWKHVHPHWKRGVRIIHUVVSHFL¿F
DGYDQWDJHVDQGFDUULHVFHUWDLQGLVDGYDQWDJHV,IDQDWRPLFLQIRUPDWLRQLVVRXJKW
WKHQHXURQVRUWLVVXHPXVWEH¿[HGLQIRUPDOGHK\GHRURWKHU¿[DWLYHLWPXVWE\
G\HGIRUH[DPLQDWLRQVOLFHGWKLQO\DQGPRXQWHGRQVOLGHV6LQFHWKHQHXURQVDUH
GHDGDOOIXQFWLRQDOGDWDLVORVW,IIXQFWLRQDOGDWDLVVRXJKWWKHQHXURQVWLVVXHRU
OLYLQJRUJDQLVPLVVXEMHFWHGWRVWLPXOLDQGWKHUHVSRQVHIRUH[DPSOHKHDUWUDWH
LVPHDVXUHG8QGHUVXFKFRQGLWLRQVDQDWRPLFGDWDLVQRWDYDLODEOH8QGHUFHUWDLQ
FLUFXPVWDQFHVOLYLQJQHXURQVFDQEHVWXGLHGXQGHUWKHPLFURVFRSHEXWRQO\OLPLWHG
GDWDFDQEHREWDLQHG
2.3.2. LOCALIZATION OF NERVES IN THE VASCULAR WALL
%ORRGÀRZGLVWULEXWLRQZLWKLQDQRUJDQLVFRQWUROOHGWKURXJKFRPSOH[LQWHUDFWLRQV
WKDWUHJXODWHDUWHULRODUUHVLVWDQFHDQGGH¿QHWKHSDWKVRIEORRGÀRZ7KHUHVLVWDQFHV
RIWKHHQWLUHDUWHULRODUQHWZRUNFXOPLQDWHWRGH¿QHZKDWLVFRPPRQO\NQRZQDV
RUJDQUHVLVWDQFH7KHVSHFL¿FUHVLVWDQFHRIDQLQGLYLGXDODUWHULROHLVFRQWUROOHGE\
DQXPEHURIORFDOYDULDEOHVLQFOXGLQJÀRZSUHVVXUHPHWDEROLWHVDQGQHXUDOLQSXW
ZKLOHDFRRUGLQDWHGQHWZRUNUHVSRQVHUHTXLUHVYDVFXODUFRPPXQLFDWLRQ9DVFXODU
FRPPXQLFDWLRQLVWKHSURFHVVZKHUHE\DUHDVRIWKHYDVFXODUQHWZRUNDGMXVWWRWKH
YDVRPRWRUVLJQDOVWKDWDUHJHQHUDWHGHLWKHUZLWKLQWKHWLVVXHRUJHQHUDWHGDWRWKHU
UHPRWHORFDWLRQVDORQJWKHYDVFXODUWUHH,WSRWHQWLDOO\SOD\VDUROHLQWKHSUHFLVH
FRQWURORILQWUDRUJDQEORRGGLVWULEXWLRQ$OWKRXJKYDULRXVHIIRUWVKDYHEHHQPDGH
WKHXQGHUO\LQJPHFKDQLVPRIWKLVYDVFXODUFRPPXQLFDWLRQLVQRWIXOO\XQGHUVWRRG
7KHQJFKDLVULDQG5LYHUV
7KHUH KDYH EHHQ PDQ\ KLVWRFKHPLFDO VWXGLHV RI WKH UHODWLRQVKLS EHWZHHQ WKH
DGUHQHUJLF QHUYHV DQG WKH EORRG YHVVHOV $GUHQHUJLF ¿EHUV QRUPDOO\ UXQ WR WKH
SHULSKHU\LQWKHVHQVRU\QHUYHV6\PSDWKHWLFQHUYHV¿EHUVDUHORFDWHGLQWKHWXQLFD
DGYHQWLWLD7KH\WHUPLQDWHLQWKHPXVFXODUOD\HUZKHUHWKH\SHUIRUPWKHLUUHJXODWRU\
IXQFWLRQRQWKHYDVFXODUZDOO7KLVRFFXUVWKURXJKĮDGUHQHUJLFUHFHSWRUVORFDWHGLQ
WKHPXVFXODULVOD\HURIWKHYDVFXODUZDOOWKDWFDXVHYDVRFRQVWULFWLRQDQGǃDGUHQHUJLF
UHFHSWRUVWKDWFDXVHYDVRGLODWDWLRQ7KLVUHJXODWRU\IXQFWLRQLVVLPLODULQDOOYDVFXODU
ZDOOVPRRWKPXVFOHV7KH\KDYHDOVREHHQIRXQGDORQJWKHYDVDYDVRUXP(KULQJHU
HWDO$VDUXOHDGUHQHUJLFLQQHUYDWLRQLVOHVVGHQVHLQYHLQVWKDQLQDUWHULHV
(KULQJHUHWDODQGWKHUHGRHVQRWVHHPWREHDQ\LQWKHFDSLOODULHV(KULQJHU
HWDO)XUQHVVDQG0DUVKDOO:DULVDQG3DUWDQHQ
7UDGLWLRQDOO\ FDWHFKRODPLQHV ZHUH VSHFL¿FDOO\ GHPRQVWUDWHG E\ PHDQV RI
IRUPDOGHK\GHLQGXFHGÀXRUHVFHQFH(UlQN|(UlQN|)DOFNDQG
WKHPHWKRGZDVUH¿QHGE\WKHLQWURGXFWLRQRIJO\R[\OLFDFLGLQGXFHGÀXRUHVFHQFH
$[HOVVRQHWDO/LQGYDOO:DULVDQG3DUWDQHQ7KLVPHWKRGKDV
WKHDGYDQWDJHRIGLUHFWO\GHPRQVWUDWLQJWKHWUDQVPLWWHUVXEVWDQFHLWVHOIZKHUHDV
LPPXQRKLVWRFKHPLFDOGHPRQVWUDWLRQRIDGUHQHUJLFQHUYHVLVEDVHGRQUHYHDOLQJWKH
HQ]\PHW\URVLQHK\GUR[\ODVH7+,WLVWKHUDWHOLPLWLQJHQ]\PHIRUFDWHFKRODPLQH
SURGXFWLRQ LQ FHOOV EXW LW RQO\ VKRZV WKH ORFDWLRQ RI SRWHQWLDO FDWHFKRODPLQH
SURGXFWLRQ QRW WKH WUDQVPLWWHU VXEVWDQFH 7KHUHIRUH GHVSLWH WKH DJH RI WKH
PHWKRGRORJ\JO\R[\OLFDFLGLQGXFHGÀXRUHVFHQFHFDQEHUHJDUGHGDVDYDOXDEOH
UHVHDUFKWRRODQGLWVWLOOKROGVLWVSODFHLQKLVWRFKHPLVWU\,WLVQRWDTXDQWLWDWLYH
PHWKRGKRZHYHU,QSUHFLVHPHDVXUHPHQWVRIWKHDPRXQWRIFDWHFKRODPLQHVLQ
WLVVXHVKLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\+3/&KDVSURYHQWREHDQ
LQYDOXDEOHWRRO7D\ORU&KHQJDQG.XR7D\ORU
5(9,(:2)7+(/,7(5$785(
Figure 2: Schematic representation of adrenergic innervation of the vascular wall. These two
differ from each other significantly. Both fibers leave the spinal column through the posterior
root. The fibers that are destined to go to the blood vessel leave the spinal column and traverse
the sympathetic ganglia and enter the spinal nerves that take them to their end organ. Red =
cholinergic, green = VIPergic, blue = adrenergic fibers (posterior root sensory fibers). Modified
from Guyton and Hall, Textbook of Medical Physiology, 11th edition, Saunders 2006.
2.3.3. TRANSMITTER SUBSTANCES
1RUDGUHQDOLQHLVV\QWKHVL]HGIURPW\URVLQHLQDWKUHHHQ]\PHSDWKZD\WKURXJKWKH
LQWHUPHGLDWHVGLK\GUR[\SKHQ\ODODQLQH'23$DQGGRSDPLQH7KHVHUHDFWLRQV
DUHFDWDO\]HGE\WKHHQ]\PHVW\URVLQHK\GUR[\ODVHDPLQRDFLGGHFDUER[\ODVHDQG
GRSDPLQHǃK\GUR[\ODVH7\URVLQHK\GUR[\ODVHZDV¿UVWFKDUDFWHUL]HGE\1DJD]X
/HYLWWDQG8GHQIULHQG1DJD]XHWDO,WLVWKHUDWHOLPLWLQJHQ]\PHLQWKH
SDWKZD\DQGZDVWKHODVWWREHFKDUDFWHUL]HG7KHVHFRQGHQ]\PHLQWKHSDWKZD\
DPLQRDFLGGHFDUER[\ODVHZDVLQLWLDOO\GHVFULEHGDVGLK\GUR[\SKHQ\ODODQLQH
GHFDUER[\ODVH,WUHPRYHVWKHFDUER[\OJURXSIURPWKHVLGHFKDLQRIWKHPROHFXOH
WRSURGXFHGRSDPLQHIURP'23$7KHVHUHDFWLRQVWDNHSODFHLQWKHF\WRSODVPRI
WKHQHUYHHQGLQJV7KHWKLUGHQ]\PHGRSDPLQHǃK\GUR[\ODVHFRQYHUWVGRSDPLQH
WRQRUDGUHQDOLQHE\DGGLQJDK\GUR[\OJURXSWRLWVVLGH
Fig. 3: Synthesis of noradrenaline (and adrenaline) from their common precursor tyrosine through
enzymatic steps where tyrosine hydroxylase is the rate-limiting enzyme. Tyrosine hydroxylase
also needs oxygen, tetrahydrobiopterin and NADPH for the conversion of tyrosine to DOPA
(Kanagy 2005).
5(9,(:2)7+(/,7(5$785(
FKDLQ7KLVODWWHUV\QWKHVLVWDNHVSODFHLQHOHFWURQGHQVHYHVLFOHVWKDWDUHWR
NjPLQGLDPHWHU7KHVHYHVLFOHVDUHIRXQGLQWKHQHUYHHQGLQJVRIWKHDGUHQHUJLF
QHUYHV7KH\DOVRFRQWDLQ$73DVFRUELFDFLGDQGHQFHSKDOLQSUHFXUVRUSHSWLGHV
2QO\DERXWKDOIRIWKHGRSDPLQHIRUPHGLQWKHF\WRSODVPLVFDSWXUHGE\WKHYHVLFOHV
WKHUHVWLVGHDPLQDWHGWRGLK\GUR[\SKHQ\ODFHWLFDFLGDQGWKHQPHWK\ODWHG
WRKRPRYDQLOOLFDFLG
9DVRDFWLYHLQWHVWLQDOSHSWLGH9,3LVDDPLQRDFLGSHSWLGH¿UVWLVRODWHGE\
6DLGDQG0XWW6DLGDQG0XWW,WEHORQJVWRDIDPLO\RISHSWLGHVFDOOHG
WKHJOXFDJRQVHFUHWLQVXSHUIDPLO\ZLWKLWVKRPRORJRXVSHSWLGHV3$&$3SLWXLWDU\
DGHQ\ODWHF\FODVHDFWLYDWLQJSHSWLGHDQG3+,SHSWLGHZLWK1WHUPLQDOKLVWLGLQHDQG
&WHUPLQDOLVROHXFLQHDPLGH)DKUHQNUXJ3+,VKDUHVWKHVDPHSUHFXUVRU
SHSWLGHZLWK9,3WKHSUHSUR9,3IURPZKLFKERWKFDQEHFOHDYHG$ORQJHUYDULDQW
RI3+,3+9LVELRORJLFDOO\DOPRVWDVDFWLYHDV9,3DVDVPRRWKPXVFOHUHOD[LQJ
PROHFXOHZKHUHDV3+,LVOHVVSRWHQW)DKUHQNUXJDQG+DQQLEDO7KHQRUPDO
DFWLRQVRI9,3LQFOXGHYDVRGLODWLRQEURQFKRGLODWLRQDQWLLQÀDPPDWRU\DFWLRQV
LPPXQRVXSSUHVVLRQ KRUPRQH VHFUHWLRQ FHOO SUROLIHUDWLRQ DQG VPRRWK PXVFOH
UHOD[DWLRQ*R]HVDQG)XUPDQ,WVUHFHSWRUVFDQEHIRXQGLQDOPRVWDOOKXPDQ
FHOOVLQFOXGLQJFDQFHUFHOOVDQGWKH93$& UHFHSWRULVIRXQGLQVPRRWKPXVFOHRI
YDULRXVRUJDQVLQFOXGLQJWKHYDVFXODUZDOO9,3DQG$73DUHFRWUDQVPLWWHUVZLWK
DFHW\OFKROLQHLQSDUDV\PSDWKHWLFQHUYHV%XUQVWRFN9,3LPPXQRUHDFWLYLW\
KDV EHHQ IRXQG WR EH FRORFDOL]HG ZLWK WKH DFHW\OFKROLQHV\QWKHVL]LQJ HQ]\PH
FKROLQHDFHW\OWUDQVIHUDVH&K$7/XQGEHUJHWDO2WKHUVWXGLHVRQWKHUDW
63* VSKHQRSDODWLQH JDQJOLRQ KDYH GHPRQVWUDWHG WKDW &K$7LPPXQRUHDFWLYH
¿EHUVLQWKHPLGGOHFHUHEUDODUWHULHVRULJLQDWHLQWKH63*6X]XNLHWDO
6WXGLHVRIWKH63*RIWKHUDWKDYHSURYLGHGIXUWKHUHYLGHQFHWKDW9,3DQG$&KDUH
FRORFDOL]HGLQWKHVDPHFHOOVLQWKH63*DQGWKDWWKH63*LVDOVRDPDMRUVRXUFH
RI9,3HUJLFLQQHUYDWLRQ<XHWDO,QWKHPLGGOHFHUHEUDODUWHULHVRIWKHSLJ
9,3,DQG&K$7,¿EHUVKDYHDOVREHHQIRXQGWREHFRLQFLGHQWZLWK126,DQG
1$'3+G¿EHUVVXJJHVWLQJWKDW9,3QLWULFR[LGHDQG$&KDUHFRORFDOL]HGLQWKH
VDPHD[RQVLQWKHSHULYDVFXODUQHUYHVRIWKHVHEORRGYHVVHOV<XHWDO
Nitric oxide and nitric oxide synthase (NOS) isoenzymes
1LWULFR[LGH12LVDPDMRUVHFUHWRU\SURGXFWRIPDPPDOLDQFHOOVZLWKFULWLFDO
IXQFWLRQVLQKRPHRVWDVLVDQGKRVWGHIHQVH,WLVVXUSULVLQJWKDWVRVLPSOHÀHHWLQJ
DQGLQGLVFULPLQDWHDUHDFWDQWFDQFRQYH\HQRXJKLQIRUPDWLRQLQDUHJXODWHGPDQQHU
WRKHOSFRQWUROYLWDOVHUYRPHFKDQLVPVVXFKDVYDVFXODUWRQHDQGQHXURWUDQVPLVVLRQ
1DWKDQ
7KHUHDUHWKUHHQLWULFR[LGHV\QWKDVH126LVRIRUPVQHXURQDO126Q126
W\SH,LQGXFLEOH126L126W\SH,,DQGHQGRWKHOLDO126H126W\SH,,,7KHVH
LVRIRUPVGLIIHUIURPHDFKRWKHUQRWRQO\LQORFDWLRQEXWDOVRLQUHJXODWLRQFDWDO\WLF
SURSHUWLHVLQKLELWRUVHQVLWLYLW\VWUXFWXUHDQGORFDWLRQVLQWKHJHQRPH$OOLVRIRUPV
DUH GLPHULF LQ WKHLU DFWLYH IRUP EXW UHTXLUH WZR FDOPRGXOLQV FDOFLXPELQGLQJ
SURWHLQVWKDWSOD\DQLPSRUWDQWUROHLQHXNDU\RWLFLQWUDFHOOXODUVLJQDOLQJIRUPLQJLQ
HIIHFWDWHWUDPHU7KLVSURWHLQFRPSOH[WKHQELQGVÀDYLQDGHQLQHGLQXFOHRWLGH)$'
DQGÀDYLQPRQRQXFOHRWLGHULERÀDYLQSKRVSKDWH)01DQGLURQSURWRSKRUSK\ULQ
,;KDHP,QWKHUHDFWLRQLQ)LJ1$'3+GRQDWHVWZRHOHFWURQVDQGEHFRPHV
1$'3DQG/DUJLQLQHLVFRQYHUWHGWRFLWUXOOLQHDQG12
Fig. 4: Nitric oxide (NO) is synthesized by five-electron oxidation of a guanidino nitrogen of
L-arginine to yield the gaseous radical (N0) via an Nȧ-hydroxyl-L-arginine intermediate. Two
electrons are donated by NADPH for the formation of this intermediate, plus one electron for
its oxidation. Both steps are catalyzed by nitric oxide synthase (NOS). Both NO and the ureido
group of citrulline incorporate molecular oxygen in the process. Sub-stoichiometric amounts
of tetrahydrobiopterin are also needed with respect to NO generated, if tetrahydrobiopterin
can be regenerated from its oxidized form, quinonoid dihydrobiopterin. Regeneration of
tetrahydrobiopterin proceeds through two enzymes, the methotrexate-sensitive enzyme
dihydrofolate reductase (DHFR) and the methotrexate-resistant enzyme, dihydropteridine
reductase (DHPR) (Xie and Nathan 1992).
1LWULFR[LGHFDQDOVREHJHQHUDWHGE\GLUHFWQRQHQ]\PDWLFUHGXFWLRQXQGHUWKH
DFLGLFDQGKLJKO\UHGXFHGFRQGLWLRQVWKDWPD\RFFXULQLVFKHPLD
5HFHSWRUDFWLYDWLRQE\EUDG\NLQLQRUDFHW\OFKROLQHRUVKHDUVWUHVVLQWKHYDVFXODU
HQGRWKHOLXP FDXVHV DQ LQÀX[ RI FDOFLXP 7KH LQFUHDVH LQ LQWUDFHOOXODU FDOFLXP
VWLPXODWHVWKHQLWULFR[LGHV\QWKDVHHQ]\PHFRPSOH[7KHQLWULFR[LGHIRUPHGE\
WKLVHQ]\PHGLIIXVHVWRQHDUE\VPRRWKPXVFOHFHOOVUHVXOWLQJLQHQKDQFHGV\QWKHVLV
RIF\FOLFJXDQRVLQHPRQRSKRVSKDWHF*037KLVLQFUHDVHLQF*03LQWKHVPRRWK
PXVFOHFHOOVOHDGVWRWKHLUUHOD[DWLRQDQGYDVRGLODWDWLRQ0RQFDGDDQG+LJJV
5(9,(:2)7+(/,7(5$785(
1LWULFR[LGHDOVRLQKLELWVSODWHOHWDJJUHJDWLRQE\ZD\RIDF*03GHSHQGHQW
PHFKDQLVPDQGV\QHUJL]HVZLWKSURVWDF\FOLQZKLFKE\LQFUHDVLQJWKHFRQFHQWUDWLRQV
RIF\FOLF$03LQKLELWVWKHDJJUHJDWLRQRISODWHOHWVEXWXQOLNHSURVWDF\FOLQQLWULF
R[LGHDOVRLQKLELWVSODWHOHWDGKHVLRQ3ODWHOHWVWKHPVHOYHVDOVRJHQHUDWHQLWULFR[LGH
ZKLFKDFWVYLDDQHJDWLYHIHHGEDFNPHFKDQLVPWRLQKLELWSODWHOHWDFWLYDWLRQ7KXV
SODWHOHWDJJUHJDWLRQLQYLYRPD\EHUHJXODWHGE\SODWHOHWGHULYHGQLWULFR[LGHDV
ZHOODVE\QLWULFR[LGHDQGSURVWDF\FOLQUHOHDVHGIURPWKHYDVFXODUHQGRWKHOLXP
7KHUHIRUHQLWURYDVRGLODWRUVLQFRPELQDWLRQZLWKSURVWDF\FOLQDQDORJVPD\SURYLGH
DXVHIXOIRUPRIDQWLWKURPERWLFWKHUDS\0RQFDGDDQG+LJJV
2.4. OTHER NEURONAL ELEMENTS
2.4.1. NEUROFILAMENTS
1HXUR¿ODPHQWV 1)V DUH VWUXFWXUDO SURWHLQV IRXQG LQ DOO QHXURQV 7KH\ ZHUH
GHVFULEHGDVHDUO\DVLQWKHODWHV$SDWK\&DMDODIWHUWKHVLOYHU
VWDLQLQJ PHWKRG ZDV GLVFRYHUHG $IWHU WKH LQYHQWLRQ RI HOHFWURQ PLFURVFRS\ LQ
*HUPDQ\E\(UQVW5XVNDDQG0D[.QROOLQQHXUR¿ODPHQWVZHUHGHVFULEHG
DV¿ODPHQWVRIURXJKO\QPLQGLDPHWHUDQGSUHVHQWRQO\LQQHXURQDOFHOOV7KHUH
DUHWKUHHJURXSVRIF\WRVNHOHWDOSURWHLQVPLFUR¿ODPHQWV0)VURXJKO\±QP
LQWHUPHGLDWH¿ODPHQWV,)VURXJKO\QPDQGPLFURWXEXOHV07VURXJKO\
QPLQGLDPHWHU0LFUR¿ODPHQWVEHORQJWRWKHW\SH¿YH,)JURXS1HXUR¿ODPHQWV
VKDUHFRPPRQVWUXFWXUHVZLWKSHULSKHULQĮLQWHUQH[LQDQGQHVWLQ7KH\FRQVLVW
RI DQ ĮKHOLFDO DPLQR DFLG FHQWUDO URG WKDW LV ÀDQNHG E\ D QRQĮKHOLFDO
FDUER[\WHUPLQDOVLGHDUPHQGLQJDQGDJOREXODU1WHUPLQDOUHJLRQ7KHVHVXEXQLWV
DUH DVVHPEOHG LQ SRO\PHUV ZKHUH OLJKW VXEXQLWV 1)/V DUH SDLUHG ZLWK HLWKHU
PHGLXP1)0VRUKHDY\1)+VVXEXQLWVVRWKDWLQHYHU\SDLUWKHUHLVRQH1)/
DQGRQHRIWKHRWKHUV/LXHWDO7KHVHSDLUVDUHIRUPHGLQDKHDGWRHQG
PDQQHUVRWKDWWKH1HQGRIWKH1)/LVDJDLQVWWKH&HQGRIWKHRWKHUVXEXQLW7KH
ÀDQNLQJ&DQG1WHUPLQDOVLGHDUPVVWLFNRXWRIWKLVDVVHPEO\DQGWKHDPLQR
DFLGKHOLFDOSRUWLRQVRIWKHVXEXQLWVIRUPWKHSDLUVZKHUHWKHURGVVWD\SDUDOOHOWR
HDFKRWKHU3HUURQH&DSDQRHWDO7KHSDLUVDUHWKHQJURXSHGLQWRELJJHU
XQLWVFRPSULVHGRIVXFKSDLUVWKDWIRUPWKH¿QDOQHXUR¿ODPHQW7KHIRUPDWLRQ
RIWKHW\SLFDO,)RIQPGLDPHWHULVGHWHUPLQHGE\WKHFRUUHFWDVVHPEO\RIWKH
1)/1)0DQG1)+VXEXQLWV$VVHPEO\RIWKHWKUHH1)VXEXQLWVLVGHSHQGHQWRQ
WKH1WHUPLQDOKHDGUHJLRQ1)/LVNQRZQWRSRO\PHUL]HRQLWVRZQLQYLWURZKLOVW
1)0DQG1)+FDQQRW3HW]ROG7KHH[DFWDVVHPEO\PHFKDQLVPLVQRW\HWIXOO\
XQGHUVWRRG/LXHWDO+RZ1)VDUHWUDQVSRUWHGLQWRGLIIHUHQWSDUWVRIWKH
QHXURQLVXQFOHDU/LXHWDOEXWWKHUHKDYHEHHQVRPHUHFHQWDGYDQFHV%DDV
DQG%XVWHULQFODULI\LQJWKHFRQFHSWRI³VORZD[RQDOWUDQVSRUW´±IRUPHUO\
WKRXJKWWREHUHVSRQVLEOHIRUWUDQVSRUWRIWKHVHSURWHLQVHLWKHUDVSRO\PHUVRUDV
VXEXQLWVWRWKHSHULSKHU\RIWKHQHXURQ0LFURWXEXOHVDQGDFWLQ¿ODPHQWVDOVR
DFWDVUDLOZD\VIRUWKHPRWRUEDVHGWUDQVSRUWRIVXEFHOOXODURUJDQHOOHV7KLV³VORZ
WUDQVSRUW´DFWXDOO\VHHPVWRWDNHSODFHLQVKRUWUDSLGEXUVWVIROORZHGE\SHULRGV
RILPPRELOLW\'XULQJWKHSDVW\HDUVGDWDKDVDFFXPXODWHGVKRZLQJDGGLWLRQDO
PHFKDQLFDODQGQRQPHFKDQLFDOUROHVRIQXFOHDUDQGF\WRSODVPLF,)VLQSRVLWLRQLQJ
RUJDQHOOHVDQGFRQWULEXWLQJWRWKHLUVKDSHDQGIXQFWLRQDQGWDUJHWLQJSURWHLQVWR
WKHLUSURSHUVXEFHOOXODUFRPSDUWPHQWV7RLYRODHWDO
2.5 VASCULAR GRAFTS IN A CLINICAL CONTEXT
2.5.1. VENOUS GRAFTS
9HQRXV JUDIWV KDYH EHHQ VWXGLHG IRU H[DPSOH WR DVVHVV WKH YLDELOLW\ RI JUDIW
ZDOOVSHULRSHUDWLYHGDPDJHDQGFROODJHQPHWDEROLVP5HPHVHWDO7KHVH
LQYHVWLJDWRUVVWXGLHGF\WRFKURPHR[LGDVHDQGVXFFLQDWHGHK\GURJHQDVHLQRUGHU
WR DVVHVV DHURELF DFWLYLW\ RI WKH JUDIWV DQG ODFWDWH GHK\GURJHQDVH WR PHDVXUH
DQDHURELF DFWLYLW\ *OXFRVHSKRVSKDWH GHK\GRJHQDVH ZDV PHDVXUHG WR DVVHVV
WKHKH[RVHPRQRSKRVSKDWHVKXQW,QWKHVWXG\LQQHUYDWLRQZDVQRWLQYHVWLJDWHGEXW
LWZDVGLVFRYHUHGWKDWDYHLQWUDQVSODQWHGWRDQDUWHU\VXIIHUHGWUDQVLHQWPHWDEROLF
GHSUHVVLRQIRUWKUHHGD\VDQGWKHQUHFRYHUHG5HPHVHWDO
:DULVHWDOVWXGLHGDRUWLFSDWFKJUDIWVLQUDWVXVLQJWKUHHNLQGVRIPDWHULDO
DV JUDIWV 7KHVH PDWHULDOV ZHUH V\QJHQHLF ULJKW DWULXP RI WKH KHDUW V\QJHQHLF
VXSUDGLDSKUDJPDWLFYHQDFDYDLQIHULRUDQGVXSUDGLDSKUDJPDWLFYHQDFDYDLQIHULRU
ZKLFKKDGEHHQWUHDWHGZLWKJOXWDUDOGHK\GHIRUWZRKRXUV7KH¿QGLQJVLQWKLV
VWXG\ZHUHWKDWGH¿FLHQWDQGSDWFK\LQQHUYDWLRQZDVSUHVHQWLQWKHYHQRXVJUDIWVDQG
ULJKWDWULDOJUDIWVDIWHUWKHREVHUYDWLRQSHULRGRIZHHNVEXWWKHJOXWDUDOGHK\GH
WUHDWHGYHQRXVJUDIWVUHPDLQHGZLWKRXWDQ\DGUHQHUJLFLQQHUYDWLRQ
+XNNLHWDOVWXGLHGWKHQRUDGUHQDOLQHFRQWHQWRIYHQRXVJUDIWV7KH\XVHG
LQIHULRUYHQDFDYDWUDQVSODQWHGLQWRWKHDEGRPLQDODRUWDRIUDWV7KH\GLVFRYHUHG
WKDW QRUDGUHQDOLQH FRQFHQWUDWLRQV LQ WKH JUDIWV IHOO VLJQL¿FDQWO\ FRPSDUHG ZLWK
FRQWUROYHLQVDQGWKH\UHPDLQHGORZ+RZHYHUWKH\KDGUHDFKHGOHYHOVFRPSDUDEOH
WRWKRVHLQWKHDRUWDZHHNVDIWHUWKHRSHUDWLRQ
5(9,(:2)7+(/,7(5$785(
2.5.2. ARTERIAL GRAFTS
9DVRVSDVPPD\EHFDXVHGE\YDULRXVIDFWRUVVXFKDVPHFKDQLFDOKDQGOLQJRIWKH
UHFLSLHQWDUWHU\GUXJVRUSHULRSHUDWLYHWUDXPD%HQPLOORXGHWDO2¶%ULHQHW
DO.UROOHWDO7KHXOWLPDWHPHFKDQLVPVEHKLQGSRVWDQGSHULRSHUDWLYH
YDVRVSDVPLQDUWHULDOJUDIWVUHPDLQHGXQH[SORUHG$UWHULDOGLDPHWHUDQGKHQFH
DUWHULDOEORRGÀRZLVUHJXODWHGE\ERWKQHXUDODQGHQGRWKHOLDOIDFWRUV7KXVWKH
SRWHQWLDOFDXVHRIYDVRVSDVPPD\EHFRQQHFWHGZLWKGHJHQHUDWLRQRIWKHSHULYDVFXODU
LQQHUYDWLRQDQGRUDOWHUHGHQGRWKHOLDOIXQFWLRQ
$UWHULHVKDYHEHHQVWXGLHGLQFRURQDU\E\SDVVVXUJHU\ZKHUHWKH\DUHNQRZQIRU
WKHLUWHQGHQF\WRJRLQWRVSDVP&DWHHWDO$NDUHWDO*LUHUGHWDO
7DXROLVHWDO2WKHUZRUNHUVKDYHVKRZQWKDWV\PSDWKHWLFQHUYHVGLVDSSHDU
IURPWUDQVSODQWHGLULGHVZLWKLQ±KRXUV6HLJHUHWDO9DVRFRQVWULFWLRQ
UHVXOWVIURPWKHVWLPXODWLRQRIĮDGUHQHUJLFUHFHSWRUVLQDUWHULDOVPRRWKPXVFOH
E\QRUDGUHQDOLQH+HDQG<DQJ*XVWDIVVRQHWDO6HJDOHWDO,Q
FRURQDU\E\SDVVVXUJHU\ERWKWKHUDGLDODUWHU\DQGULJKWJDVWURHSLSORLFDUWHU\DUH
ZHOONQRZQIRUWKHLUWHQGHQF\WRJRLQWRVSDVPGXULQJWKHRSHUDWLRQ&DWHHWDO
,QWUDQVSODQWHGWLVVXHVWKHUHLVLQLWLDOGHJHQHUDWLRQRIQHXUDOD[RQVZLWKLQWKH
JUDIWIROORZHGE\UHJHQHUDWLRQRIQHUYH¿EHUVIURPWKHVXUURXQGLQJWLVVXHV&RZHQ
HWDOVWXGLHGWKHHIIHFWRIGHQHUYDWLRQLQFDURWLGDQGPHVHQWHULFDUWHULHV&RZHQHW
DO7KH\VWXGLHGFUXVKLQMXU\DQGH[SHULPHQWDOGHQHUYDWLRQDQGGLVFRYHUHG
WKDWGHQHUYDWLRQRFFXUUHGUDSLGO\LQWKUHHGD\V7KLVZDVFRPSOHWHLQWKHFDURWLG
DUWHU\DQGSDUWLDOLQWKHPHVHQWHULFDUWHU\5HLQQHUYDWLRQRIWKHPHVHQWHULFDUWHU\
ZDVIRXQGWREHFRPSOHWHE\HLJKWZHHNVZKLOHLQFDURWLGDUWHULHVLWZDVWKRXJKW
WRWDNHORQJHU
&UXVK,QMXU\DQGRSHUDWLYHWUDXPDKDYHEHHQVWXGLHGE\5HPHVDQGFRZRUNHUV
7KH\XVHGUDWVLQZKLFKWKHDRUWDZDVFODPSHGZLWKWZRW\SHVRIFODPSIRUDQG
PLQXWHV7KHLQYHVWLJDWRUVPHDVXUHGODFWDWHGHK\GURJHQDVH/'DQGVXFFLQDWH
GHK\GURJHQDVHHQ]\PHDFWLYLWLHVDVLQGLFDWRUVRIPHWDEROLFDFWLYLW\RIWKHDUWHULDO
ZDOO7KH\IRXQGWKDWDIWHUPLQXWHVRIFODPSLQJHQ]\PHDFWLYLWLHVZHUHGHFUHDVHG
IRU GD\V SRVWRSHUDWLYHO\ DQG DIWHU PLQXWHV RI FODPSLQJ HQ]\PH DFWLYLWLHV
ZHUHGLPLQLVKHGIRURQHDQGWKUHHGD\VWKXVGHPRQVWUDWLQJLQMXU\WRWKHDRUWLF
ZDOO+RZHYHUWKLVLQMXU\ZDVQRWIRXQGLQDOOVSHFLPHQV7KLVZDVWKRXJKWWREH
EHFDXVHWKHFODPSVWKH\XVHGFDXVHGRQO\PLOGLQMXU\WRWKHDRUWLFZDOO7KLVZDV
IXUWKHUHPSKDVL]HGE\WKHIDFWWKDWRQHZHHNDIWHULQMXU\HQ]\PHDFWLYLWLHVKDG
UHWXUQHGWRQRUPDO5HPHVHWDO
*RGGHQ DQG FRZRUNHUV VWXGLHG WKH HIIHFW RI PLFURYDVFXODU DQDVWRPRVLV
RQ V\PSDWKHWLF LQQHUYDWLRQ 7KH\ UDLVHG JURLQ ÀDSV LQ UDWV DQG WZR WR GD\V
SRVWRSHUDWLYHO\ VDFUL¿FHG WKH DQLPDOV 7KH\ PHDVXUHG YDVFXODU WRQH ZKHQ WKH
VSHFLPHQ DUWHULHV ZHUH H[SRVHG WR LQFUHDVLQJ FRQFHQWUDWLRQV RI SKHQ\OHSKULQH
D V\PSDWKHWLF DJHQW 7KLV VHQVLWLYLW\ ZDV EORFNHG E\ SKHQWRODPLQH DQG WKH
LQYHVWLJDWRUVLQWHUSUHWHGWKLVDVDVLJQRIV\PSDWKHWLFGHQHUYDWLRQ*RGGHQHWDO
.DDUHODDQGFRZRUNHUVVWXGLHGUDEELWIRUHSDZV.DDUHODD3HULYDVFXODU
V\PSDWKHFWRP\ZDVFDUULHGRXWRQRQHRUWZRIRUHSDZDUWHULHVLQUDEELWV%ORRG
P
ÀRZZDVPHDVXUHGE\PHDQVRI7F
DQJLRJUDSK\EHIRUHWKHLQLWLDORSHUDWLRQ
DQG¿YHGD\VDIWHULW1RVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHZDVIRXQGEHWZHHQWKH
RSHUDWHGOHIWVLGHDQGWKHULJKWVLGHZKLFKVHUYHGDVFRQWURO.DDUHODHWDO
.DDUHODDQGFRZRUNHUVDOVRVWXGLHGWKHHIIHFWRISHULYDVFXODUV\PSDWKHFWRP\LQ
PRQNH\KDQGV.DDUHODHWDOE7KH\SHUIRUPHGSHULYDVFXODUV\PSDWKHFWRP\
RQFRPPRQGLJLWDODUWHULHVRIPDFDFDDUFURLGHVPRQNH\VDQGVWXGLHGJO\R[\OLF
DFLGLQGXFHGÀXRUHVFHQFHLQWKHV\PSDWKHFWRPL]HGDQGRSSRVLWHFRQWUROKDQG
GLJLWDODUWHULHV7KHUHZDVQRGLIIHUHQFHLQWKHGLVWDODGUHQHUJLFQHUYHQHWZRUNDIWHU
WKHSURFHGXUHDQGWKHLQYHVWLJDWRUVVXJJHVWHGWKDWWKHUHIRUHWKHRSHUDWLRQVKRXOG
EHFDOOHGDGYHQWLWHFWRP\LQVWHDGRIV\PSDWKHFWRP\7KHVDPHNLQGRIUHVXOWZDV
REWDLQHGLQFRQQHFWLRQZLWKWKHUDEELWHDUFHQWUDODUWHU\ZKHUHV\PSDWKHFWRP\
ORZHUHGQRUDGUHQDOLQHFRQFHQWUDWLRQVEXWGLGQRWDEROLVKLWIURPWKHWLVVXH.DDUHOD
HWDOF
6LPLODUSKHQRPHQDDUHSUHVHQWLQWUDQVSODQWHGRUJDQV7KH\DUHFRPSOHWHO\
GHWDFKHGIURPWKHLUVXUURXQGLQJVDQGUHDQDVWRPRVHGWRDQHZVLWH8QGHUWKHVH
FLUFXPVWDQFHVQHZO\IRUPHGQHUYHWLVVXHFRQWDFWVPD\EHIXQFWLRQDODQGFRQWULEXWH
WRQHXURQDOFRQWURORIWKHRUJDQ/DVVPDQQDQG3L]D.DZDJXFKLHWDO
8EHUIXKUHWDOD8EHUIXKUHWDOE+RZHYHURIWHQWKHJUDIWHGWLVVXH
RQO\VWLPXODWHVUREXVWVSURXWLQJRIQHUYHWHUPLQDOVIURPWKHKRVWWLVVXHLQWRWKH
JUDIWDQGRQO\OLPLWHGRUQRIXQFWLRQDOLQQHUYDWLRQPD\EHIRXQG6FKURHGHU
7DNDFKLHWDO'RHULQJHWDO(QKDQFLQJIXQFWLRQDOUHLQQHUYDWLRQRI
GHQHUYDWHGWLVVXHVHLWKHUDIWHUQHXURQDOWUDXPDRUDIWHUWLVVXHWUDQVSODQWDWLRQ
FRQVWLWXWHVDPDMRUFKDOOHQJHLQPDQ\¿HOGVRIVXUJHU\.L\RFKLHWDO)DQVD
HWDO.MDHUDQG6HFKHU
$,062)7+(678'<
3. AIMS OF THE STUDY
1HXURQDO UHJXODWLRQ RI YDVFXODU WRQH LV LPSRUWDQW IRU WKH FRQWURO RI EORRG ÀRZ
LQ YDVFXODU JUDIWV \HW WKHUH KDYH EHHQ IHZ VWXGLHV RQ WKH EDVLF IHDWXUHV RI WKH
FKDQJHVRFFXUULQJLQSRVWRSHUDWLYHLQQHUYDWLRQSDWWHUQVLQDUWHULDOJUDIWV7KHUHIRUH
ZHVHWRXWWRVWXG\GHJHQHUDWLRQDQGUHJHQHUDWLRQRISHULYDVFXODULQQHUYDWLRQLQ
WUDQVSODQWHGIHPRUDODUWHULHVXVLQJDQH[SHULPHQWDOUDWPRGHO:HXVHGDQXPEHURI
GLIIHUHQWKLVWRFKHPLFDOPDUNHUVWRUHYHDOGLVWLQFWDVSHFWVRISHULYDVFXODULQQHUYDWLRQ
$LPVRIWKHFXUUHQWVWXG\
7RTXDOLWDWLYHO\H[DPLQHWKHGLVDSSHDUDQFHRIQRUDGUHQDOLQHIURPDUWHULDOJUDIWV
7R TXDQWLWDWLYHO\ PHDVXUH WKH UDWH RI GLVDSSHDUDQFH RI QRUDGUHQDOLQH IURP
DUWHULDOJUDIWV
7RTXDQWLWDWLYHO\PHDVXUHWKHUHJHQHUDWLRQRIDGUHQHUJLFQHUYHVLQDUWHULDOJUDIWV
7R VWXG\ KLVWRFKHPLFDO FKDQJHV LQ ERWK GHJHQHUDWLRQ DQG UHJHQHUDWLRQ RI
DGUHQHUJLFQHUYHVLQDUWHULDOJUDIWV
4. MATERIALS AND METHODS
4.1. MATERIALS
Experimental animals (I, II, III and IV)
,QEUHG$2.V2&ZHHNWRPRQWKROGIHPDOHUDWVZHUHXVHG7KH\ZHUHNHSWLQ
WKHYLYDULXPRIWKH8QLYHUVLW\RI+HOVLQNL6XUJLFDO+RVSLWDO7KH\KDGIUHHDFFHVVWR
FRPPHUFLDOURGHQWIRGGHUDQGZDWHUZDVVXSSOLHGE\DGULSERWWOH7KH\ZHUHKRXVHG
LQSODVWLFDQLPDOER[HVZLWKZLUHFRYHUVLQURRPVPDLQWDLQHGDWDWHPSHUDWXUHRI
R&7KHZHLJKWRIWKHDQLPDOVUDQJHGIURPWRJUDPV7KUHHUDWVZHUH
XVHGLQHDFKJURXSIRURXU¿UVWVHWRIH[SHULPHQWV¿YHWRVHYHQLQHDFKJURXSIRU
WKHVHFRQGDQGWKLUGVHWVRIH[SHULPHQWVDQGWZRIRUHDFKREVHUYDWLRQSHULRGLQ
WKHIRXUWKVHWRIH[SHULPHQWV$WRWDORIDQLPDOVZDVXVHGIRUWKLVVWXG\7KH
QXPEHURIDQLPDOVXVHGLQHDFKRIWKHRULJLQDOVWXGLHVLVVXPPDUL]HGLQ7DEOH
Table 2.
EXPERIMENTAL ANIMALS
Follow-up
time
Rats/group
330–351
0h, 6h, 24h
3
6
STUDY
N
Age
Weight, g
I
9
6 months
II
30
3–10 months
220–373
0h, 6h, 12h,
24h, 48h
III
31
2–10 months
254–373
0h, 1w, 4w,
12w, 20w
5 to 7
IV
12
2–3 months
180–284
1d, 3d, 7d, 1m,
3m, 5m
2
4.2. METHODS
$YDULHW\RIPHWKRGVZDVXVHGWRVWXG\WKHJUDIWVDWGLIIHUHQWWLPHSRLQWVDQGWKH\
DUHVXPPDUL]HGLQ7DEOH
0$7(5,$/6$1'0(7+2'6
Table 3. Methods and time points used in the original studies
Methods and
time points
Glyoxylic acid
fluorescence
HPLC
HPLC
Immunohistochemistry
Article
I
II
III
IV
0h
x
x
x
x
6h
x
x
12h
x
24h/1d
x
x
x
48h/2d
x
72h/3d
x
1w
x
x
4w
x
x
12w
x
x
20w
x
x
Time
'LIIHUHQW PHWKRGV ZHUH HPSOR\HG DW GLIIHUHQW WLPH SRLQWV GHSHQGLQJ RQ WKHLU
VXLWDELOLW\
4.2.1. SURGICAL METHOD (I, II, III AND IV)
%HIRUHWKHRSHUDWLRQWKHUDWVZHUHZHLJKHGDQGDSSURSULDWHDUHDVZHUHVKDYHG
7KHOHIWIHPRUDODUWHU\ZDVH[SRVHGLQWKHLQJXLQDOUHJLRQWKURXJKDORQJLWXGLQDO
LQFLVLRQWKHVXEFXWDQHRXVIDWZDVOLIWHGFUDQLDOO\DQGWKHDUWHU\ZDVIUHHGIURPWKH
IHPRUDOYHLQDQGQHUYH&DUHZDVWDNHQQRWWRGDPDJHWKHVHVWUXFWXUHV7KHDUWHU\
ZDVWKHQOLJDWHGMXVWEHORZWKHLQJXLQDOOLJDPHQWDQGMXVWEHORZWKHEUDQFKLQJ
SRLQW RI WKH LQIHULRU HSLJDVWULF YHVVHOV 7KH GHHS EUDQFK RI WKH IHPRUDO DUWHU\
ZDVFUXVKHGZLWKDFODPSWRVWRSLWIURPEOHHGLQJDQGGLYLGHG7KHVHJPHQWRI
IHPRUDODUWHU\EHWZHHQWKHOLJDWXUHVDERXWPPLQOHQJWKZDVWKHQUHPRYHG
ZLWKVFLVVRUVULQVHGZLWKKHSDULQVROXWLRQPORIKHSDULQVROXWLRQ>/RHYHQV
.HPLVNH)DEULN%DOOHQUXS'HQPDUN@DQGPORIVWHULOH1D&ODQGWKH
HQGVVOLJKWO\GLODWHGZLWKPLFURVXUJLFDOIRUFHSVXQGHUDQRSHUDWLQJPLFURVFRSH
7KHDGYHQWLWLDZDVFDUHIXOO\UHWUDFWHGIURPWKHHQGVRIWKHDUWHU\7KHVHJPHQWZDV
VWRUHGLQVWHULOHVDOLQH7KHLQJXLQDOLQFLVLRQZDVWKHQVXWXUHGLQRQHOD\HU
ZLWKSRO\JO\FROLFDFLG'H[RQ
$WUDQVYHUVHLQFLVLRQZDVWKHQPDGHMXVWFUDQLDOO\RIWKHMXJXOXP7KHVDOLYDU\
JODQGVZHUHGLVVHFWHGIUHHFDXGDOO\DQGOLIWHGFUDQLDOO\7KHOHIWFDURWLGDUWHU\ZDV
H[SRVHGMXVWODWHUDOWRWKHWUDFKHDDQGZDVFDUHIXOO\IUHHGIURPWKHVXUURXQGLQJ
WLVVXHV$PLFURVXUJLFDODSSUR[LPDWRUFOLSZDVSODFHGRQWKHFDURWLGDUWHU\DQG
WKHDUWHU\ZDVWKHQFXWEHWZHHQWKHFOLSMDZV7KHFDURWLGDUWHU\ZDVQRWUHVHFWHG
7KHDGYHQWLWLDZDVSXOOHGZLWKPLFURVXUJLFDOIRUFHSVDQGFXWZLWKVFLVVRUVDWERWK
HQGVLQRUGHUWRNHHSLWIURPJHWWLQJEHWZHHQWKHHQGRIWKHDUWHU\DQGWKHJUDIW
7KHJUDIWZDVSODFHGEHWZHHQWKHHQGVRIWKHDUWHU\DQGVXWXUHGLQSODFHZLWK
LQWHUUXSWHGQ\ORQPRQR¿ODPHQWVXWXUHV)LUVWWKHSUR[LPDODQDVWRPRVLVZDV
FRPSOHWHGDQGFKHFNHGE\LQMHFWLQJKHSDULQVROXWLRQLQWRWKHJUDIWWKURXJKWKH
RSHQHQG7KHQWKHGLVWDODQDVWRPRVLVZDVFRPSOHWHG$IWHUFRPSOHWLQJWKHGLVWDO
DQDVWRPRVLVUHWURJUDGHÀRZZDVEURXJKWDERXWE\HDVLQJWKHGLVWDOFOLSRIWKH
DSSUR[LPDWRUGHYLFH+HQFHSDWHQF\DQGKHPRVWDVLVZHUHLQYHVWLJDWHG,IQHFHVVDU\
KHPRVWDWLFVXWXUHVZHUHSODFHGDWWKDWSRLQWWKHSUR[LPDOFOLSZDVHDVHGDQGD
SDWHQF\WHVWSHUIRUPHG:KHQIUHHÀRZFRXOGEHGHPRQVWUDWHGDQGWKHUHZDVQR
EOHHGLQJWKHMXJXODUZRXQGZDVFORVHGZLWKFXWLFXODUSRO\JO\FROLFDFLGVXWXUHV
4.2.2. ANESTHESIA (I, II, III AND IV)
7KHUDWVZHUHDQHVWKHWL]HGZLWKDPL[WXUHRI'RUPLFXPPLGD]RODPPJPO
)+RIIPDQ/D5RFKH%DVOH6ZLW]HUODQGDQG+\SQRUPPL[WXUHRISKHQWDQ\O
FLWUDWHPJPODQGÀXQLVROHPJPO-DQVHQ3KDUPDFHXWLFDO/WG*URYH
2[IRUG8.)RUDQHVWKHVLDDVROXWLRQFRQVLVWLQJRIHTXDOSDUWVRIERWKDJHQWVDQG
WZRSDUWVRIZDWHUVWHULOHGLVWLOOHGSKDUPDFHXWLFDOZDWHUZDVSUHSDUHGDQGWKHUDWV
ZHUHJLYHQ±PORIWKHVROXWLRQLQWUDSHULWRQHDOO\*UDIWVZHUHLQVHUWHGXQGHU
D:,/'VWHUHRRSHUDWLQJPLFURVFRSH0W\SH+HHUEUXJJ6ZLW]HUODQG
$WFHUWDLQSRLQWVGXULQJWKHRSHUDWLRQDQGLIQHFHVVDU\±PORIDQHVWKHWLF
VROXWLRQZDVJLYHQLQWUDSHULWRQHDOO\
4.2.3. GLYOXYLIC ACID-INDUCED FLUORESCENCE (I)
7KHJUDIWVDQGFRQWUROVSHFLPHQVZHUHWUHDWHGZLWKJO\R[\OLFDFLGJO\R[\OLFDFLG
PRQRK\GUDWHGLVVROYHGLQ0SKRVSKDWHEXIIHUS+DGMXVWHGWRZLWK1D2+
$IWHUDWOHDVWPLQXWHVRILPPHUVLRQWKHVSHFLPHQVZHUHEORWWHGGU\VWUHWFKHG
RQ PLFURVFRSH VOLGHV DQG GULHG ZLWK KRW DLU IURP D KDLU GU\HU 7KH VSHFLPHQV
ZHUHWKHQKHDWHGIRUVL[PLQXWHVDW R&DQGPRXQWHGLQ(QWHOODQ0HUFNDV
GHVFULEHGE\:DULVDQG3DUWDQHQ6DPSOHVIURPRQHDQLPDOZHUHWUHDWHGDV
DERYHEXWJO\R[\OLFDFLGZDVRPLWWHGIURPWKHSKRVSKDWHEXIIHU1RFDWHFKRODPLQH
ÀXRUHVFHQFHFRXOGEHGHPRQVWUDWHGLQWKHVDPSOHVIURPWKLVDQLPDO
0$7(5,$/6$1'0(7+2'6
4.2.4. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (II AND III)
$IWHU WKH VSHFLPHQV ZHUH FROOHFWHG WKH\ ZHUH GLVVHFWHG IUHH RI DQ\ UHPDLQLQJ
VXWXUH PDWHULDO RQ D 3HWUL GLVK UHVWLQJ RQ D EHG RI FUXVKHG LFH 7KH JUDIWV DQG
FRQWUROVSHFLPHQVZHUHWKHQGULHGTXLFNO\E\EORWWLQJRQWLVVXHSDSHUDQGVWRUHG
LQLQGLYLGXDOO\PDUNHGVPDOOSODVWLFEDJVLQDIUHH]HUDWR&7KH\ZHUHODWHU
WUDQVSRUWHGWRWKHODERUDWRU\IRUDQDO\VLVLQDVW\URIRDPFRQWDLQHUNHSWFROGZLWK
VROLGFDUERQGLR[LGH
)RUGHWHUPLQDWLRQRIQRUDGUHQDOLQHFRQWHQWWKHZHLJKHGVDPSOHRIDUWHU\ZDV
KRPRJHQL]HGLQ—OLFHFROG0+&O2VROXWLRQDQGWKHKRPRJHQDWHZDV
FHQWULIXJHGIRUPLQDW[J1RUDGUHQDOLQHIURP—ORIWKHVXSHUQDWDQWZDV
H[WUDFWHGLQWRDVROXWLRQRIPJ$O2LQPORIWULV+&OEXIIHUS+LQDPO
FRQLFDOWHVWWXEH'LK\GUR[\EHQ]\ODPLGHK\GUREURPLGH6LJPD6W/RXLV02
86$ZDVXVHGDVDQLQWHUQDOVWDQGDUGWRFRUUHFWIRUDEVROXWHUHFRYHU\YDULDWLRQV
RI QRUDGUHQDOLQH $IWHU ZDVKLQJ IRXU WLPHV ZLWK PO +2 QRUDGUHQDOLQH ZDV
HOXWHGLQWR—O0+&O2VROXWLRQ1RUDGUHQDOLQHLQWKHHOXDWHZDVPHDVXUHG
E\KLJKSUHVVXUHOLTXLGFKURPDWRJUDSK\ZLWKDQHOHFWURFKHPLFDOGHWHFWRU(6$
&RXORFKHP0XOWL(OHFWURGHPRGHO$(6$,QF&KHOPVIRUG0$86$$Q
(6$FDWHFKRODPLQH+5FROXPQîPP—PDQGPHWKDQROSKRVSKDWH
EXIIHUS+(6$FDW$3KDVHUHDJHQWDVPRELOHSKDVHZHUHXVHG7KHÀRZ
UDWH ZDV POPLQ )RU FDOLEUDWLRQ SXUSRVHV NQRZQ QRUDGUHQDOLQH VWDQGDUGV
ZHUHWUHDWHGLQWKHVDPHZD\DVVDPSOHVDQGWKHSHDNKHLJKWUDWLRUHODWLYHWRWKH
SHDNKHLJKWRIWKHLQWHUQDOVWDQGDUGRIWKHXQNQRZQQRUDGUHQDOLQHFRQFHQWUDWLRQ
LQWKHVDPSOHZDVFRPSDUHGZLWKWKDWRIWKHNQRZQV\QWKHWLFVWDQGDUG'
&KURPDWR,QWHJUDWRU+LWDFKL7RN\R-DSDQ
4.2.5. MICROSCOPIC EVALUATION (I AND IV)
)RU JO\R[\OLF DFLG ÀXRUHVFHQFH PLFURVFRSLF REVHUYDWLRQV ZHUH PDGH XVLQJ D
/HLW] $ULVWRSODQ GDUN ¿HOG ÀXRUHVFHQFH PLFURVFRSH 7\SH ZLWK
DQ DXWRPDWLF /HLW] PLFURVFRSH FDPHUD 7KH PLFURVFRSH ZDV HTXLSSHG ZLWK DQ
2VUDPXOWUDYLROHWHPLWWLQJEXOEW\SH+%27KH¿OWHUFRPELQDWLRQHPSOR\HG
IRUÀXRUHVFHQFHPLFURVFRS\FRQVLVWHGRI,QPEOXHDQGQP
JUHHQÀXRUHVFHLQ¿OWHUV
)RULPPXQRKLVWRFKHPLVWU\WKHVSHFLPHQVZHUHWKHQH[DPLQHGDQGSKRWRJUDSKHG
XVLQJD/HLW]9DULR2UWKRPDW(UQVW/HLW]*PE+:HW]ODU*HUPDQ\ÀXRUHVFHQFH
PLFURVFRSH)OXRUHVFHQFHZDVGHWHFWHGXVLQJUKRGDPLQHEORFN1H[FLWDWLRQ
±QPHPLVVLRQQPDQGRUÀXRUHVFHLQ,±QP±
QP¿OWHUV)RUPLFURSKRWRJUDSK\,OIRUG+3DQG.RGDN70$;$6$¿OPV
ZHUHXVHG
4.2.6. IMMUNOHISTOCHEMISTRY (IV)
*UDIWVZHUHFROOHFWHGXQGHUDQHVWKHVLDDWSUHVHWWLPHSRLQWVDQGWKH\ZHUHLPPHGLDWHO\
¿[HGE\ZD\RILPPHUVLRQIRUWRKRXUVLQLFHFROGSDUDIRUPDOGHK\GHLQ
SKRVSKDWHEXIIHUHGVDOLQH3%6S+)RUFU\RSURWHFWLRQWKHWLVVXHVZHUHWKHQ
LPPHUVHGLQVXFURVHIUR]HQDQGFXWLQWR—PWKLFNORQJLWXGLQDOFU\RVWDW
VHFWLRQV6HYHUDOVHFWLRQVZHUHVWDLQHGDQGDQDO\]HGIRUHDFKPDUNHUDQGWLPHSRLQW
7KHVHFWLRQVZHUHWKHQLQFXEDWHGZLWKQRUPDOVZLQHVHUXPLQSKRVSKDWH
EXIIHUHGVDOLQH3%6S+WKHQRYHUQLJKWZLWKWKHSULPDU\DQWLERGLHV7DEOH
7KHVDPSOHVZHUHULQVHGWZLFHLQ3%6IROORZHGE\LQFXEDWLRQIRURQHKRXU
ZLWKVHFRQGDU\DQWLERGLHV6HFRQGDU\DQWLERGLHVZHUHUKRGDPLQHRUÀXRUHVFHLQ
FRQMXJDWHG JRDW DQWLUDEELW ,J* -DFNVRQ ,PPXQR5HV /DE ,QF :HVW
*URYH3$86$IRUWKHSRO\FORQDODQWLERGLHVDQGÀXRUHVFHLQFRQMXJDWHGJRDW
DQWLPRXVH,J*-DFNVRQ,PPXQR5HV/DE,QF:HVW*URYH3$86$
IRUWKHPRQRFORQDODQWLERGLHV(DFKVOLGHZDVULQVHGWZLFHLQ3%6DQGDFRYHUVOLS
DSSOLHGXVLQJJO\FHURO1DYHURQDOPRXQWLQJPHGLXP
Table 4. Primary antibodies used in Study IV
ANTIBODY
SPECIES
DILUTION
SOURCE
REFERENCE
Tyrosine hydroxylase
rabbit, pc
1:1000
In collaboration
with Tong Joh
Vanhatalo 1995
neuronal Nitric Oxide
Synthase
rabbit, pc
1:1000–4000
EuroDiagnostica,
Malmö, Sweden
Endothelial Nitric Oxide
Synthase
mouse, mc
1:50–100
Transduction
Laboratories,
USA
commercial
product
Inducible Nitric Oxide
Synthase
mouse, mc
1:30–100
Transduction
Laboratories,
USA
commercial
product
GAP-43
mouse, mc
1:500–1000
BoeringerMannheim,
Germany
Schreyer and
Skene 1991
NEUROFILAMENT
mouse, mc
1:10–30
Gift from Prof.
I. Virtanen
Bäck et al. 1995
Alm 1993
mc = monoclonal antibodies
pc = polyclonal antibodies
1HXURQDO¿EHUSDWWHUQVDQGJHQHUDOFKDQJHVLQWKHPZHUHDQDO\]HGE\VWXG\LQJ
QHXUR¿ODPHQWV LPPXQRKLVWRFKHPLFDOO\ ,PPXQRUHDFWLYLW\ DJDLQVW QHXURQDO
JURZWKDVVRFLDWHGSURWHLQ*$3UHYHDOVDFWLYHD[RQDOJURZWK%HQRZLW]DQG
5RXWWHQEHUJ&DURQL,PPXQRUHDFWLYLW\WRZDUGVW\URVLQHK\GUR[\ODVH
7+ UHYHDOV V\PSDWKHWLF QHUYHV 9DVRDFWLYH LQWHVWLQDO SRO\SHSWLGH 9,3 DQG
QHXURQDOQLWULFR[LGHV\QWKDVHQ126DUHERWKSUHVHQWLQSDUDV\PSDWKHWLFQHUYHV
DQGZHLGHQWL¿HGWKHVHQHUYHVLPPXQRKLVWRFKHPLFDOO\<XQHWDO0RUULVHWDO
<XHWDO:HDOVRVWXGLHGWZRRWKHULVRIRUPVRIQLWULFR[LGHV\QWKDVH
0$7(5,$/6$1'0(7+2'6
HQGRWKHOLDOQLWULFR[LGHV\QWKDVHH126ZKLFKLVDQLPSRUWDQWUHJXODWRURIYDVFXODU
WRQH,DGHFROD*RRGVRQHWDO3OXWDHWDODQGLQGXFLEOH126
L126ZKLFKLVRIWHQDFWLYDWHGGXULQJWLVVXHWUDXPD1DWKDQ)RUVWHUPDQQ
7KH VSHFL¿FLWLHV RI WKH DQWLERGLHV XVHG LQ WKH SUHVHQW VWXG\ DUH GHVFULEHG
HOVHZKHUH DV LQGLFDWHG LQ 7DEOH 6SHFL¿FLW\ LQIRUPDWLRQ RQ WKH FRPPHUFLDO
L126DQGH126DQWLERGLHVLVQRWDYDLODEOH$OWKRXJKH[WHQVLYHVHTXHQFHKRPRORJ\
EHWZHHQ 126 LVRIRUPV PD\ UHVXOW LQ FURVVUHDFWLYLW\ RI WKHVH DQWLERGLHV WKH
GLVWULEXWLRQRIVWDLQLQJREVHUYHGLQWKHSUHVHQWVWXG\VXSSRUWVWKHDVVXPSWLRQWKDW
WKHL126DQGH126DQWLERGLHVVWDLQPDFURSKDJHVDQGHQGRWKHOLXPUHVSHFWLYHO\
,9
4.2.7. STATISTICAL ANALYSIS (II AND III)
7KHUHVXOWVDUHSUHVHQWHGDVPHDQVDQGVWDQGDUGGHYLDWLRQV6'V7KHUHVXOWVZHUH
DQDO\]HGDIWHUDWHVWRIQRUPDOLW\XVLQJRQHZD\DQDO\VLVRIYDULDQFH$QRYD
'LIIHUHQFHVEHWZHHQJUDIWDQGFRQWUROVDPSOHVZHUHDQDO\]HGE\SDLUHG6WXGHQW¶V
WWHVWV7KHOHYHORIVLJQL¿FDQFHZDVVHWDWS7XNH\¶VSRVWKRFWHVWZDVFDUULHG
RXWWRFRPSDUHWKHJUDIWJURXSVZLWKHDFKRWKHU6WDWLVWLFDODQDO\VLVZDVSHUIRUPHG
E\SURI3+XWWXQHQLQFRQMXQFWLRQZLWKWKH+3/&DQDO\VLV
5. RESULTS
5.1 GENERAL
$OO DQLPDOV VXUYLYHG WKH RSHUDWLRQ ,Q WKH KRXU JURXS WKHUH ZDV QR WLPH IRU
FRPSOLFDWLRQVWRRFFXUVLQFHWKHDQLPDOVZHUHVDFUL¿FHGLPPHGLDWHO\,QWKHKRXU
JURXSWKHDQLPDOVVWLOODSSHDUHGWREHXQGHUWKHHIIHFWVRIDQHVWKHVLD$WKRXUV
WKHDQLPDOVKDGIXOO\UHFRYHUHGIURPWKHVHHIIHFWV,QWKHKRXUJURXSWZRJUDIWV
VKRZHGHYLGHQFHRIWKURPERVLVWKHVHZHUHUHMHFWHGDQGQHZDQLPDOVZHUHRSHUDWHG
XSRQIRUWKLVJURXS
5.2. CONTROLS
,QWKH¿UVWVHWRIH[SHULPHQWVFRQWUDODWHUDOLQJXLQDODQGLQIHULRUHSLJDVWULFYHVVHOV
DVZHOODVGRUVDOVHJPHQWDOVXEFXWDQHRXVYHVVHOVZHUHXVHGDVFRQWUROV$W\SLFDO
ÀXRUHVFHQWQHWZRUNRIDGUHQHUJLFQHUYHVZDVVHHQLQWKHIHPRUDODUWHULHVDQGYHLQV
,QWKHDUWHULHVWKHQHUYHVIRUPHGDYHU\GHQVHQHWZRUNLQWKHDGYHQWLWLD,QWKH
YHLQVWKHQHXUDOQHWZRUNZDVOHVVGHQVHDQGWKH¿EHUVZHUHVSDFHGDWPRUHUHJXODU
LQWHUYDOVWKDQLQWKHDUWHULHVWKH\ZHUHDOVROHVVWRUWXRXVWKDQLQWKHDUWHULHV,Q
WKHLQIHULRUHSLJDVWULFDUWHULHVDQGYHLQVDVZHOODVLQWKHVHJPHQWDOGRUVDODUWHULHV
DQGYHLQVWKHSDWWHUQVZHUHVLPLODULQERWKW\SHVRIEORRGYHVVHO)LJ
,Q6WXGLHV,,DQG,,,ZHXVHGFRQWUDODWHUDOIHPRUDOYHVVHOVDVFRQWUROVDQGWKH
UHVXOWVDUHVKRZQLQ)LJXUHVDDQGEDDQGE7KHUHZDVQRVLJQL¿FDQWGLIIHUHQFH
EHWZHHQWKHKKKDQGKFRQWUROJURXSVSYDOXHV!,QWKHKRXU
FRQWUROJURXSWKHUHZDVDVWDWLVWLFDOO\VLJQL¿FDQWSIDOOLQWKHQRUDGUHQDOLQH
FRQWHQWFRPSDUHGZLWKWKHRWKHUFRQWUROJURXSV
,QLPPXQRKLVWRFKHPLVWU\VDPSOHVRIFDURWLGKRVWDQGIHPRUDODUWHULHVJUDIW
ZHUHDQDO\]HG,PPXQRUHDFWLYLW\DJDLQVWDQ\RIWKHPHQWLRQHGPDUNHUVZDVYHU\
VSDUVHLQWKHFDURWLGDUWHU\LQGLFDWLQJDVSDUVHLQQHUYDWLRQSDWWHUQLQJHQHUDO,Q
FRQWUDVWWKHUHZDVDPDUNHGO\GHQVHUSHULYDVFXODUQHUYHSOH[XVLQWKHIHPRUDO
DUWHU\FRPSDUHGZLWKWKHFDURWLGDUWHU\DQGLWH[KLELWHGLPPXQRUHDFWLYLW\DJDLQVW
DOOWKHPDUNHUVXVHGLQWKHSUHVHQWVWXG\7KHYLVXDODSSHDUDQFHRI¿EHUGHQVLWLHV
LQWKHIHPRUDODUWHULDOJUDIWVZDVFRPSDUDEOHWRWKDWVHHQLQLQWDFWIHPRUDODUWHULHV
5(68/76
Fig. 5. Control specimen of an non-operated rat femoral artery wall showing an intact neuronal
fluorescent network. Black bar, 100 —m.
5.3. GRAFTS
Graft weights
*UDIWVGLIIHUHGIURPFRQWUROVLQZHLJKW,QDOOJURXSVWKHJUDIWVZHUHKHDYLHUDQG
WKHGLIIHUHQFHZDVVWDWLVWLFDOO\VLJQL¿FDQWLQDOOJURXSVDSDUWIURPWKHKJURXS
ZSZSZSDQGZS 7KHJUDIWVZHUHDWWKHLU
KHDYLHVWRQHZHHNDIWHUWKHRSHUDWLRQFRUUHVSRQGLQJWRWKHFOLQLFDO¿QGLQJWKDW
WKH\ZHUHHGHPDWRXVDQGWKLFNZDOOHG7DEOH
Table 5. Graft and control weights in milligrams
WEEKS
CONTR. WEIGHT
N
MEAN
ST DEV
0
6
0.9683
0.1477
1
5
1.1060
0.2281
4
6
1.0967
0.2443
12
6
1.2583
0.1578
20
TOTAL
GRAFT WEIGHT
0
7
1,1243
0.1875
30
1.1243
0.2039
6
0.9567
0.3028
1
5
2.5620
0.7493
4
6
1.9017
0.4280
12
6
1.7550
0.2843
20
7
1.8829
0.3758
30
1.7890
0.6476
TOTAL
Table 6. Noradrenaline concentrations in the grafts and controls in each experimental animal group
shown as ng/mg of tissue, and standard deviations
Noradrenaline content of grafts and controls in this material
Mean grafts
Group
Mean controls
NA ng/mg
SD
NA ng/mg
SD
0h
0,7596
0,1963
1,1105
0,3695
6h
1,5575
1,1115
1,1972
0,518
12h
0,5645
0,1828
1,0113
0,1864
24h
0,1963
0,1556
0,9004
0,3594
48h
0,0293
0,05
0,4982
0,0205
1w
0,0176
0,0152
0,1163
0,2978
4w
0,2797
0,2196
0,1411
0,383
12w
0,3653
0,2796
0,1347
0,3187
24w
0,6646
0,4769
0,148
0,4398
0 hours (I–IV)
,QWKHJUDIWVDULFKDGUHQHUJLFQHWZRUNZDVVHHQDWKRXUV)LJE\XVLQJJO\R[\OLF
DFLGÀXRUHVFHQFH,7KLVQHWZRUNKRZHYHUZDVQRWH[DFWO\OLNHWKDWRIWKHFRQWURO
IHPRUDODUWHULHV)LJEXWVHHPHGWREHFRDUVHUDQGVRPHRIWKH¿ODPHQWVVHHPHG
WRKDYHVOLJKWO\ORVWWKHLUÀXRUHVFHQFH
5(68/76
,QWKHKJURXSWKHJUDIWVZHUHFROOHFWHGLPPHGLDWHO\DIWHURSHUDWLRQ,QWKLV
JURXSWKHQRUDGUHQDOLQHFRQWHQWLQWKHJUDIWVPHDVXUHGE\+3/&ZDVRI
WKHFRQWUROYDOXHEXWWKHGLIIHUHQFHZDVQRWVLJQL¿FDQW,,,,,6LQFHWKHUHZDVQR
GHOD\DIWHUWKHRSHUDWLRQLWVHHPVWKDWWKDWRSHUDWLRQLWVHOIFDXVHGWKLVGLIIHUHQFH
,Q LPPXQRKLVWRFKHPLVWU\ ERWK *$3 DQG 1) ZHUH SUHVHQW LQ WKH WXQLFD
DGYHQWLWLDLQDGHQVHQHWZRUNRI¿EHUVDVZHOODVWKHSDUDV\PSDWKHWLFPDUNHUV
Q126DQG9,3,9
Fig. 6. A graft harvested immediately after operation showing an intact neuronal network
6 hours (I and II)
$WKRXUVIHZHUÀXRUHVFHQW¿EHUVFRXOGEHYLVXDOL]HGE\PHDQVRIJO\R[\OLFDFLG
)LJ7KH\VWLOOIRUPHGDQHWZRUNDOWKRXJKLWDSSHDUHGWREHPRUHGLIIXVHDQG
WKH¿EHUVLQWHUUXSWHG7KHUHZDVDORVVRIÀXRUHVFHQFHFRPSDUHGZLWKWKHKRXU
JURXS,7KHUHZDVDVLJQL¿FDQWULVHLQQRUDGUHQDOLQHFRQFHQWUDWLRQZKLOHXVLQJ
+3/&ZKHQFRPSDUHGZLWKWKHKRXUJURXSS ,,)LJVDDQGE
Fig. 7. A graft six hours after operation showing a diminished amount of glyoxylic acid-induced
fluorescence. Black bar, 100 —m.
12 hours (II)
$WKRXUV+3/&VKRZHGWKDWWKHUHZDVDGHFUHDVHLQQRUDGUHQDOLQFRQFHQWUDWLRQ
ZKHQFRPSDUHGZLWKWKHKDQGKJURXSV)LJVDDQGE
24 hours (I, II and IV)
$WKRXUVDOPRVWDOO¿EHUVKDGGLVDSSHDUHGDQGRQO\GRWVRIJO\R[\OLFDLGLQGXFHG
ÀXRUHVFHQFHZHUHSUHVHQW7KHVHGRWVZHUHVFDWWHUHGLUUHJXODUO\RYHUWKHJUDIWDQG
ZHUHYHU\GLP$QDGUHQHUJLFQHXURQDOQHWZRUNFRXOGQRORQJHUEHLGHQWL¿HG,
)LJ
7KHUHZDVDVLJQL¿FDQWGHFOLQHLQQRUDGUHQDOLQHFRQFHQWUDWLRQLQ+3/&ZKHQ
FRPSDUHGZLWKWKHKRXUJURXS
,QLPPXQRKLVWRFKHPLVWU\7+SRVLWLYH¿EHUVZHUHHTXDOO\DEXQGDQWLQRSHUDWHG
IHPRUDO DUWHULHV DQG LQ FRQWURO VSHFLPHQV 1) DQG *$3 LPPXQRUHDFWLYLW\
VKRZHG WKH EHJLQQLQJ RI GHJHQHUDWLRQ DW WKLV SRLQW DQG WKH VWDLQLQJ ¿EHUV KDG
VZROOHQ,9
5(68/76
Fig. 8. A graft harvested 24 hours after operation showing scant fluorescence with no identifiable
neuronal structures. Black bar, 100 —m.
48 hours (II)
,QWKHKJURXSRIJUDIWVWKHUHZHUHWKUHHVSHFLPHQVLQZKLFKWKHFRQFHQWUDWLRQRI
QRUDGUHQDOLQHZDVEHORZWKHGHWHFWLRQOLPLWRIKLJKSUHVVXUHOLTXLGFKURPDWRJUDSK\
DQG WKH\ ZHUH PDUNHG SJPJ WKH ORZHU GHWHFWLRQ OLPLW RI RXU PHWKRG 7KH
QRUDGUHQDOLQHFRQFHQWUDWLRQKDGGURSSHGWRDQDOPRVWQRQH[LVWHQWOHYHO
72 hours (IV)
$WKRXUVERWK1)DQG*$3VWDLQLQJSDWWHUQVZHUHVSDUVHUEXWWKH\KDGQRW
GLVDSSHDUHGFRPSOHWHO\7+LPPXQRUHDFWLYLW\KDGGLVDSSHDUHGDOPRVWFRPSOHWHO\
DQGVRKDGWKDWRIWKHSDUDV\PSDWKHWLFPDUNHU9,3ZKLFKWHQGHGWRGLVDSSHDU
PRUHUDSLGO\WKDQWKHRWKHUSDUDV\PSDWKHWLFPDUNHUQ1266RPH¿EHUVSRVLWLYH
IRUQ126LPPXQRUHDFWLYLW\ZHUHVWLOOVHHQWKUHHGD\VDIWHUWKHRSHUDWLRQ
Fig. 9. Tyrosine hydroxylase immunoreactivity 1 (left) and 3 days (right) after the operation,
showing a clear decrease in the amount of fluorescence (IV). Arrows indicate the white lines of
tyrosine hydroxylase fluorescence
7 days (III and IV)
,Q+3/&WKHQRUDGUHQDOLQHFRQFHQWUDWLRQZDVDERXWRIWKDWRIWKHFRQWUROV
DWRQHZHHN,,,DYDOXHVLJQL¿FDQWO\ORZHUS
,Q LPPXQRKLVWRFKHPLVWU\ VRPH QHXUR¿ODPHQWV ZHUH VWLOO SUHVHQW DW WKH WD
WP ERUGHU 2QO\ VRPH YHU\ WKLQ ¿EHUV KDG LPPXQRUHDFWLYLW\ IRU *$3 7+
LPPXQRUHDFWLYLW\ZDVDOPRVWDEVHQWDWWKLVWLPH7KHSDUDV\PSDWKHWLFPDUNHUV
9,3 DQG Q12V KDG DOO EXW GLVDSSHDUHG ,9 2QH ZHHN DIWHU WKH RSHUDWLRQ WKH
GHQHUYDWLRQSURFHVVKDGEHHQFRPSOHWHG
4 weeks (III and IV)
$WWKLVSRLQWWKHPHDQQRUDGUHQDOLQHOHYHOLQWKHJUDIWVZDVRIWKDWLQWKH
FRQWUROVXVLQJ+3/&,,,
7+,5ZDVVHHQPRVWO\LQWKHWDEXWWKHUHZHUHRQO\DIHZ¿EHUV2QO\DIHZ
*$3SRVLWLYH¿EHUVZHUHHQFRXQWHUHGLQLPPXQRKLVWRFKHPLVWU\7KHUHZDVDQ
DEXQGDQFHRI1)VWDLQLQJ¿EHUVPRVWO\LQWKHWDDQGVRPHWLPHVLQWKHWP2QO\D
IHZQ126DQG9,3VWDLQLQJ¿EHUVZHUHVHHQDQGWKHVHZHUHPRVWO\LQWKHWD,9
5(68/76
12 weeks (III and IV)
$WZHHNVWKHJUDIW1$FRQFHQWUDWLRQPHDVXUHGE\+3/&ZDVVLJQL¿FDQWO\ORZHU
WKDQLQWKHFRQWUROVSDQGLQWKHPRQWKJUDIWJURXSEXWLWGLGQRW
VLJQL¿FDQWO\GLIIHUIURPWKDWLQZHHNDQGPRQWKJUDIWV,,,
7+,5 KDG LQFUHDVHG ZKHQ FRPSDUHG ZLWK WKH PRQWK JURXS DQG WKHUH
ZDV HYHQ VRPH LPPXQRUHDFWLYLW\ RQ WKH VXUIDFH RI WKH WXQLFD PHGLD *$3
LPPXQRUHDFWLYLW\ KDG SURIRXQGO\ LQFUHDVHG LQ WKH WXQLFD DGYHQWLWLD DQG 1)
LPPXQRUHDFWLYLW\ZDVDEXQGDQWERWKLQWKHWXQLFDDGYHQWLWLDDQGLQWKHWXQLFD
PHGLD 1) LPPXQRUHDFWLYLW\ ZDV IRXQG LQ UHODWLYHO\ WKLFN EXQGOHV 9HU\ GHQVH
Q126SRVLWLYHLPPXQRUHDFWLYLW\ZDVIRXQGLQRXWHUSDUWVRIWKHWXQLFDDGYHQWLWLD
DQGLQRFFDVLRQDO¿EHUVRQWKHVXUIDFHRIWKHWXQLFDPHGLD9,3VKRZHGDGLIIHUHQW
SDWWHUQRIGLVWULEXWLRQ±LWZDVIRXQGPRVWO\LQRXWHUSDUWVRIWKHWXQLFDPHGLD
ZLWKRQO\VRPH¿EHUVLQWKHWXQLFDDGYHQWLWLD
20 weeks (III and IV)
$IWHUPRQWKVQRUDGUHQDOLQHOHYHOVZHUHRIWKRVHLQWKHFRQWUROVDYDOXH
VLJQL¿FDQWO\ORZHUWKDQLQWKHFRQWUROVDQGVLJQL¿FDQWO\KLJKHUWKDQLQRQHZHHN
JUDIWVS EXWQRWVLJQL¿FDQWO\GLIIHUHQWIURPYDOXHVLQWKHZHHNRU
ZHHNJURXSV,,,
7+,5ZDVSUHVHQWERWKLQQHUYH¿EHUVDQGEXQGOHV*$3LPPXQRUHDFWLYLW\
KDGGHFUHDVHGWRDOHYHOFRUUHVSRQGLQJWRWKDWDWRQHPRQWKZKHUHDV1),5ZDV
REVHUYHGWREHLQFUHDVHGEXWRQO\LQWKLFNEXQGOHVQRWLQVLQJOH¿EHUV7KHUHZDV
DQLQFUHDVHLQWKHQXPEHURIQ126SRVLWLYH¿EHUVZKHQFRPSDUHGZLWKWKHWKUHH
PRQWKJURXS,9
eNOS and iNOS findings
,PPXQRKLVWRFKHPLVWU\ IRU L126 UHYHDOHG QR FHOOXODU SUR¿OHV LQ WKH FRQWURO
VSHFLPHQV $ VPDOO QXPEHU RI L126SRVLWLYH FHOOV ZDV REVHUYHG LQ WKH WXQLFD
DGYHQWLWLDGD\DIWHUJUDIWLQJZKHUHDVDWDQGGD\VDIWHUVXUJHU\WKHQXPEHU
RIWKHVHSRVLWLYHO\VWDLQHGFHOOVZDVVRPHZKDWLQFUHDVHG7KHL126SRVLWLYHFHOOV
ZHUHHTXDOO\GLVWULEXWHGDORQJWKHJUDIWSHULSKHU\DQGWKHJUDIWKRVWMXQFWLRQDQG
WKH\ZHUHURXQGLQPRUSKRORJ\UHVHPEOLQJDFWLYDWHGPDVWFHOOV+LJKLQWHQVLW\
VWDLQLQJIRUH126ZDVVHHQLQDOOWLVVXHVZLWKLQWDFWHQGRWKHOLXP7KHUHZDVQR
GHFUHDVHLQWKHVWDLQLQJIRUH126GXULQJWKHGD\VRIIROORZXSKRZHYHUWKH
HQGRWKHOLXP ZDV SDUWLDOO\ GHWDFKHG LQ VRPH RI WKH JUDIWV SUREDEO\ EHFDXVH RI
PLQRULQDGYHUWHQWWUDXPDWRWKHYHVVHOZDOOVGXULQJWKHJUDIWLQJSURFHGXUH,9
I
Fig. 10a. Noradrenaline concentrations (pg/—g tissue) for each group of rats. Bars show
mean values for the graft (Gr) and control (Cntr) groups, with SDs. The rise in noradrenaline
concentration in grafts at 6h was statistically insignificant (p>0.05). After 6h the concentration
in grafts fell significantly (p<0.05). In 48-h control specimens the decrease in noradrenaline
concentration was also statistically significant (p=0.04) compared with 0-h controls.
Fig. 10b shows the same results as in Fig. 10a, but as percentage of controls.
5(68/76
Fig. 11a. A chart showing noradrenaline concentrations (pg/mg tissue; mean ± SD) in graft
and control arteries during the postoperative period (weeks). The difference between controls
and grafts immediately after operation was statistically insignificant (p=0.053). One week
postoperatively the difference was statistically significant (p<0.001). In the grafts, noradrenaline
concentrations showed a rising trend from one to 20 weeks (statistically significant p=0.004).
Each group consisted of six rats except for the 20-week group, which consisted of seven rats.
The data was analyzed by means of Student’s t-test (paired).
Fig. 11b shows the same results as Fig. 11a, but as percentage of controls.
&RPELQHWUHVXOWVRIWKHRULJLQDODUWLFOHDUHVKRZQRQSDJHV7DEOH
5(68/76
Table 7. Results of the original articles
Method Ļ
Time ĺ
0h
6h
Glyoxylic acid- almost intact
induced
fluorecence
little
diminished
HPLC
significant
rise
compared
with 0-h
grafts
no significant
difference
between
grafts and
controls
12h
1d
none left
significant
fall in NA
concentration
significant
fall in NA
concentration
Histochemistry
Tyrosine
hydroxylase
initially rich
IR against TH
nNOS
initially rich
IR against
nNOS
fewer fibers
with IR
against nNOS
intense IR
against eNOS
eNOS
iNOS
few cells
present in
the ta
VIP
rich IR
against VIP
significantly
less IR
GAP-43
rich IR
throughout ta
less IR than
in 0-h group
Neurofilament
rich IR
throughout ta
less IR than
in 0-h group
2d
significant
fall in NA
concentration
3d
1w
1m
3m
5m
significant
difference
in NA
concentration
vs. 0-h group
significant
rise in NA
levels vs. 1-w
group
significant
rise in NA
levels vs. 1-w
group
No significant
difference
vs. 0-h
group in NA
concentration
IR becoming
sparse
IR against TH
disappeared
only
occasional
fibers with
TH IR
more fibers
with TH IR
still more IR
against TH
IR becoming
sparse
IR against
nNOS
disappeared
sparse IR
against nNOS
more IR
against nNOS
rich IR
against nNOS
intense IR
against eNos
intense IR
against eNOS
constitutive
expression
of IR against
eNOS
marked
increase in
the number
of cells with
IR
only a few
cells present
none present
completely
absent
sparse IR
against VIP
more than in
1-m group
rich IR
against VIP
less IR in ta
few fibers to
be seen
few nerve
fibers with IR
present
dense
network with
IR present
less IR than
in the 3-m
group
less IR in ta
fewer NFstaining
fibers to be
seen
',6&866,21
6. DISCUSSION
6.1. GENERAL METHODOLOGY
6HYHUDO PHWKRGV KDYH EHHQ HPSOR\HG WR DVVHVV DGUHQHUJLF QHUYH SOH[XVHV
TXDQWLWDWLYHO\7KHVHPHWKRGVLQFOXGHYLVXDODQGJULGFRXQWLQJWHFKQLTXHVRIQHUYH
GHQVLWLHV DQG HVWLPDWHV RI YLVXDO LQWHQVLW\ &RZHQ DQG %XUQVWRFN 2WKHU
PHWKRGV VXFK DV DXWRPDWLF TXDQWLWDWLYH LPDJH DQDO\VLV KDYH EHHQ LQWURGXFHG
IRU WKH VDPH SXUSRVH +LJKSHUIRUPDQFH OLTXLG FKURPDWRJUDSK\ +3/& ZLWK
HOHFWURFKHPLFDO GHWHFWLRQ LV D ZHOOHVWDEOLVKHG PHWKRG IRU PHDVXULQJ WLVVXH
FDWHFKRODPLQHV ,W LV D KLJKO\ VHQVLWLYH PHWKRG DQG FDSDEOH RI UHYHDOLQJ HYHQ
VPDOOFKDQJHVLQFDWHFKRODPLQHFRQWHQW7D\ORUHWDO&KHQJHWDO7KH
VHQVLWLYLW\RIWKHPHWKRGLVLQGLFDWHGE\WKHFKDQJHVLQWKHQRUDGUHQDOLQHFRQWHQW
RIWKHFRQWUROVDPSOHVGXULQJWKHSRVWRSHUDWLYHSHULRG6HYHUHGQHUYHD[RQVDUH
SUREDEO\ XQDEOH WR PDLQWDLQ ELRFKHPLFDO SURFHVVHV WKDW UHSOHQLVK WKH VWRUHV RI
QRUDGUHQDOLQHLQQHUYHHQGLQJV
7KHUHLVDQLQLWLDOEUHDNGRZQRID[RQDOHOHPHQWVLQWUDQVHFWHGQHUYHV:DOOHU
1RUDGUHQDOLQHLVH[SHFWHGWRGLVDSSHDUIURPYDVFXODUJUDIWVVKRUWO\DIWHUWKH
RSHUDWLRQGXHWR:DOOHULDQGHJHQHUDWLRQ7KHIDOOLQQRUDGUHQDOLQHFRQWHQWLQWKH
KJUDIWJURXSFDQEHDWWULEXWHGWRKDQGOLQJRIWKHJUDIWVGXULQJWKHRSHUDWLRQ
FRPSDUHGZLWKWKHFRQWUROV+RZHYHUWKHQRUDGUHQDOLQHFRQFHQWUDWLRQLQFRQWURO
VSHFLPHQVZDVZLWKLQWKHFRQ¿GHQFHLQWHUYDORIWKHFRQFHQWUDWLRQLQWKHJUDIWV
7KLVGHFUHDVHLQQRUDGUHQDOLQHFRQWHQWGXHWRSHULRSHUDWLYHKDQGOLQJLVFOLQLFDOO\
VLJQL¿FDQWDQGPD\EHWKHH[SODQDWLRQEHKLQGLQVWDQFHVRIYDVRVSDVPRFFXUULQJ
GXULQJDQGLPPHGLDWHO\DIWHURSHUDWLRQ
&DWHFKRODPLQHV KDYH D KDOIOLIH RI D IHZ PLQXWHV LQ WLVVXHV EXW WKH\ GR QRW
GLVDSSHDUIURPJUDIWVZLWKLQWKH¿UVWIHZKRXUVDVRQHPLJKWH[SHFW,QWKH
DQGKJURXSVWKHUHZDVDVWHDG\GHFUHDVHLQQRUDGUHQDOLQHFRQWHQW7KLVLV
ZHOOLQDFFRUGDQFHZLWKRWKHU¿QGLQJVUHSRUWHGLQWKHOLWHUDWXUH&RZHQHWDO
+XNNLHWDODQGDOVRDVUHJDUGVGLVWDOSDUWVRIGHQHUYDWHGDUWHULHV.DDUHOD
D.DDUHODHWDOF:DULV2XU¿QGLQJVFRQFHUQLQJJO\R[\OLFDFLG
LQGXFHGÀXRUHVFHQFHVXJJHVWWKDWQRUDGUHQDOLQHGLVDSSHDUVLQDOLQHDUIDVKLRQ
7KHUHODWLRQVKLSEHWZHHQÀXRUHVFHQFHDQGQRUDGUHQDOLQHFRQWHQWLVNQRZQQRW
WREHFRPSOHWHO\OLQHDU9DQ2UGHQHWDO,WLVDOVRYHU\GLI¿FXOWWRYLVXDOO\
HVWLPDWH WKH DPRXQW RI QRQQHXURQDO WLVVXH FDWHFKRODPLQHV :H SURSRVH WKDW
FRQWUDU\WRZKDWLVEHOLHYHGVHYHUHGD[RQVPLJKWUHWDLQVRPHDELOLW\WRWDNHXS
DQGFRQFHQWUDWHFDWHFKRODPLQHVIRUVRPHKRXUVDIWHUWUDQVSODQWDWLRQRIDQDUWHULDO
JUDIWLQWRDUHFLSLHQWYHVVHO7KLVFRXOGEHFRQQHFWHGZLWKWKHLQWHJULW\RID[RQDO
PHPEUDQHVDQGWKHSUHVHQFHRIHQHUJ\VWRUHVQHHGHGIRUELRFKHPLFDOSURFHVVHV
WKDWPDLQWDLQQRUDGUHQDOLQHVWRUHV,WPD\EHRIJUHDWFOLQLFDOVLJQL¿FDQFH
6.1.1. GLYOXYLIC ACID-INDUCED FLUORESCENCE
+LVWRFKHPLFDOGHPRQVWUDWLRQRIFDWHFKRODPLQHVE\ZD\RIIRUPDOGHK\GHLQGXFHG
ÀXRUHVFHQFHKDVORQJEHHQWKHPHWKRGRIFKRLFHIRUGHPRQVWUDWLRQRIWKHWUDQVPLWWHU
VXEVWDQFHLWVHOI*O\R[\OLFDFLGÀXRUHVFHQFHZDVRULJLQDOO\GHVFULEHGE\$[HOVVRQ
DQGRWKHUVDQGE\/LQGYDOODQG%M|NOXQG$[HOVVRQHWDO/LQGYDOOHWDO
DQGLWZDVDUH¿QHPHQWRIWKHIRUPDOGHK\GHLQGXFHGPHWKRGRULJLQDOO\GHVFULEHG
E\(UlQN|(UlQN|)DOFN7\URVLQHK\GUR[\ODVHDQHQ]\PHWKDW
LVUDWHOLPLWLQJLQWKHIRUPDWLRQRIFDWHFKRODPLQHVLQGLFDWHVRQO\LIFDWHFKRODPLQH
V\QWKHVLVLV SUHVHQW LQ WLVVXHV RU PRUHSUHFLVHO\LIVXFKFDSDELOLW\LVSUHVHQWLQ
WLVVXHV7KHUHIRUHDOWKRXJKJO\R[\OLFDFLGÀXRUHVFHQFHLVDQROGPHWKRGXVHGIRU
GHPRQVWUDWLRQRIFDWHFKRODPLQHVLWVWLOOKROGVLWVSODFHLQKLVWRFKHPLVWU\6WUHWFK
SUHSDUDWLRQVKDYHZLGHO\EHHQXVHGIRUGHPRQVWUDWLQJYDULRXVQHXURQDOVWUXFWXUHV
ERWKE\JO\R[\OLFDFLGLQGXFHGÀXRUHVFHQFHDQGE\LPPXQRKLVWRFKHPLVWU\:DULV
DQG3DUWDQHQ6HLJHUHWDO7KHUHVROXWLRQRIFDWHFKRODPLQHGHWHFWLRQ
LPSURYHGDIWHUJO\R[\OLFDFLGZDVLQWURGXFHGDVDFRQGHQVDWLRQDJHQW/LQGYDOODQG
%M|UNOXQG:DULVDQG3DUWDQHQDQGWKHUHKDYHEHHQPDQ\PRGL¿FDWLRQV
RIWKHJO\R[\OLFDFLGPHWKRG.RUNDODDQG:DULV:DULVHWDOD:DULVHWDO
F6HLJHUHWDO7KHJO\R[\OLFDFLGLQGXFHGÀXRUHVFHQFHPHWKRGKDVEHHQ
VKRZQWREHERWKVSHFL¿FDQGVHQVLWLYH:DULVD:DULVFDQGLWKDVEHHQ
IRXQGWREHXVHIXOLQKLVWRFKHPLFDOVWXGLHVRQWKHGHJHQHUDWLRQDQGUHJHQHUDWLRQ
RIDGUHQHUJLFQHUYHVIROORZLQJV\PSDWKHFWRP\.DDUHODHWDODEF
7KHXVHRIJO\R[\OLFDFLGLQGXFHGÀXRUHVFHQFHDOORZHGWKHWUDQVPLWWHUVXEVWDQFH
WREHGHPRQVWUDWHGQRWWKHHQ]\PHVRURWKHUSURWHLQFRPSRQHQWVRIWKHQHUYH
¿EHUVDVLQLPPXQRKLVWRFKHPLVWU\:DULV:DULVHWDODF,WH[SODLQV
WKHGLIIHUHQFHEHWZHHQWKHUHVXOWVLQ6WXG\,LQZKLFKFDWHFKRODPLQHVIURPWKH
DGUHQHUJLF QHUYHV RI DUWHULDO JUDIWV GLVDSSHDUHG ZLWKLQ KRXUV DQG WKRVH RI
6HLJHUHWDOZKRLPPXQRKLVWRFKHPLFDOO\GHPRQVWUDWHGWKHGLVDSSHDUDQFH
RIUHDFWLYHQHUYH¿EHUVLQWUDQVSODQWHGLULVHVDIWHURQHZHHN7KLV¿QGLQJRQWKH
RWKHUKDQGLVLQFRQFRUGDQFHZLWKWKH¿QGLQJVLQ6WXG\,9ZKHUH1),5SHUVLVWHG
IRUDZHHNDIWHUJUDIWVKDGEHHQLQVHUWHGLQWKHFDURWLGDUWHU\7KHGLVDSSHDUDQFH
RIFDWHFKRODPLQHÀXRUHVFHQFHIURPDUWHULDOJUDIWVZLWKLQRQHGD\FRUUHVSRQGVZHOO
ZLWKHDUOLHUREVHUYDWLRQV RQWKHGLVDSSHDUDQFHRIFDWHFKRODPLQHVIURPVXUJLFDO
ÀDSV:DULVHWDOEDQGIURPYHLQJUDIWV+XNNLHWDO2XUUHVXOWV
FRUUHVSRQGZHOOWRHDUOLHUREVHUYDWLRQVLQRWKHURUJDQVDGUHQHUJLFGHQHUYDWLRQ
RFFXUUHG±KRXUVDIWHUD[RWRP\LQFDWLULVHV0DOPIRUVDQG6DFKVDQG
DVLPLODUREVHUYDWLRQKDVEHHQREVHUYHGLQUDWLULVHV2OVRQDQG0DOPIRUV
',6&866,21
6.1.2. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
+LJKSHUIRUPDQFH OLTXLG FKURPDWRJUDSK\ LV D PHWKRG WKDW LV XVHG IRU DVVD\ RI
QXPHURXVFRPSRXQGVLQFOXGLQJELRJHQLFDPLQHV,WZDVGHYHORSHGLQWKHV
WR LPSURYH WKH H[LVWLQJ WHFKQLTXHV RI RSHQFROXPQ FKURPDWRJUDSK\ WKLQOD\HU
FKURPDWRJUDSK\DQGSDSHUFKURPDWRJUDSK\ZKLFKZHUHLQDGHTXDWHDVUHJDUGV
TXDQWL¿FDWLRQDQGGLIIHUHQWLDWLRQRIUHODWHGFRPSRXQGV,QSUHFLVHPHDVXUHPHQWV
RIWKHDPRXQWRIFDWHFKRODPLQHVLQWLVVXHVLWKDVSURYHQWREHDQLQYDOXDEOHWRRO
7D\ORUHWDO&KHQJHWDO7KHV\VWHPFRPSULVHVDVROYHQWUHVHUYRLUIURP
ZKLFKWKHVROYHQWLVGUDZQ7KHUHLVDQLQMHFWLRQSRUWWKURXJKZKLFKWKHVDPSOHLV
LQMHFWHGLQWRWKHV\VWHP7KHVDPSOHLVFDUULHGE\WKHVROYHQWWKURXJKWKHFROXPQ
ZKLFKLVDJODVVWXEHRI±PPLQWHUQDOGLDPHWHURUDPLFURFROXPQRI±—P
GLDPHWHU7KHJODVVFROXPQLV¿OOHGVXUIDFHFRDWHGZLWKDVXEVWDQFHWKDWVHSDUDWHV
WKHGLIIHUHQWFRPSRXQGVDVWKH\SDVVWKURXJKLW,GHQWL¿FDWLRQRIWKHFRPSRXQGLV
DFFRPSOLVKHGE\ZD\RIGHWHFWRUV7KHVHFDQEHRIGLIIHUHQWW\SHVQDPHO\UHIUDFWLYH
LQGH[XOWUDYLROHWÀXRUHVFHQWUDGLRFKHPLFDOHOHFWURFKHPLFDOQHDULQIUDUHGPDVV
VSHFWURVFRSLFQXFOHDUPDJQHWLFUHVRQDQFHDQGOLJKWVFDWWHULQJ(DFKRQHRIWKHVH
LQYROYHVXVHRIDGLIIHUHQWNLQGRISK\VLFDORUFKHPLFDOSKHQRPHQRQWRGHWHFWFKDQJHV
LQWKHFRQWHQWRIWKHVROYHQWSKDVHDQGDVLJQDOLVHPLWWHGZKHQVXFKDFKDQJHLV
GHWHFWHG(OHFWURFKHPLFDOGHWHFWRUVDVXVHGLQRXUVWXGLHVGHWHFWFRPSRXQGVWKDW
XQGHUJRR[LGL]DWLRQDQGUHGXFWLRQUHDFWLRQVRQWKHGHWHFWRUVXUIDFH7KHGHWHFWRULV
SODFHGMXVWEHKLQGWKHFROXPQLQWKHDUUD\4XDQWL¿FDWLRQLVDQLPSRUWDQWSDUWRI
WKHSURFHVV7KHVLJQDOIURPWKHXQNQRZQFRQFHQWUDWLRQLQWKHVDPSOHLVFRPSDUHG
ZLWKDVHULHVRISHDNVLQDFKURPDWRJUDPFUHDWHGE\DVHULHVRINQRZQFRQFHQWUDWLRQV
RIVDPSOHVROXWLRQV%\XVLQJDWULDQJXODUHTXDWLRQWKHDUHDVRIWKHVHSHDNVDUH
FDOFXODWHGWRFUHDWHDFDOLEUDWLRQFXUYHDQGWKHPHDVXUHPHQWREWDLQHGIURPWKH
VDPSOHLVWKHQFRPSDUHGZLWKWKLVFDOLEUDWLRQFXUYH
6.1.3. IMMUNOHISTOCHEMISTRY
,PPXQRKLVWRFKHPLVWU\ ZDV ¿UVW XVHG E\ 0DUUDFN WR GHPRQVWUDWH W\SKRLG DQG
FKROHUDRUJDQLVPV0DUUDFN&RRQVHWDOGHPRQVWUDWHGSQHXPRFRFFXV
W\SH,,,RUJDQLVPVXVLQJǃDQWKU\OLVRF\DQDWHFRQMXJDWHGDQWLERGLHVDJDLQVWWKHP
+HZDVDEOHWRGHPRQVWUDWHWKHGLIIHUHQFHEHWZHHQW\SHV,,DQG,,,SQHXPRFRFFL
7KLVZDVDGLUHFWUHDFWLRQDQGLWKDGSRRUVHQVLWLYLW\/DWHUDQLQGLUHFWPHWKRG
ZDVGHYHORSHGZLWKDQXQODEHOHGSULPDU\DQWLERG\DJDLQVWWKHWLVVXHDQWLJHQDQG
DODEHOHGVHFRQGDU\DQWLERG\DJDLQVWWKHSULPDU\RQH7KLVHQKDQFHGWKHFRORU
UHDFWLRQEHFDXVHVHYHUDOVHFRQGDU\DQWLERGLHVZHUHERXQGWRWKHSULPDU\DQWLERG\
7KHPHWKRGDOVRDOORZVVHYHUDOGLIIHUHQWNLQGVRIVHFRQGDU\DQWLERGLHVWREHXVHG
ZLWKWKHSULPDU\RQH)OXRUHVFHQWPDUNHUVDUHXQVWDEOHDQGODWHULQGHYHORSPHQWWKH
DQWLERGLHVZHUHOLQNHGZLWKHQ]\PHVVXFKDVKRUVHUDGLVKSHUR[LGDVH7KLVPDGHLW
SRVVLEOHWRGHPRQVWUDWHWKHDQWLERG\ZLWKDSHUPDQHQWVWDLQLQWKHVSHFLPHQEURXJKW
DERXWE\DGHSRVLWIURPDGLDPLQREHQ]LGLQHUHDFWLRQZLWKK\GURJHQSHUR[LGHDV
VXEVWUDWH/DWHUVWLOOWKHVHQVLWLYLW\RILPPXQRKLVWRFKHPLVWU\ZDVLQFUHDVHGZLWK
WKHLQWURGXFWLRQRIWKHSHUR[LGDVHDQWLSHUR[LGDVH3$3FRPSOH[DQGWKHDYLGLQ
ELRWLQFRPSOH[$%&1RUPDOVHUXPLVXVHGLQWKH¿UVWLQFXEDWLRQWREORFNRXW
QRQVSHFL¿FELQGLQJRISULPDU\DQWLERGLHV%XUQHWWHWDO
6.1.4. EXPERIMENTAL MODEL
%HFDXVHRIWKHDYDLODELOLW\RIUDWVLQRXUODERUDWRU\DOOUDWVZHUHLQWKHDJHUDQJHRI
±PRQWKVH[FHSWIRUWKRVHLQWKHKJURXSQ ZKLFKZHUHDJHGPRQWKV
6LQFHWKHFRQWUROYDOXHVLQWKLVJURXSZHUHQRGLIIHUHQWIURPWKRVHLQWKHRWKHU
FRQWUROJURXSVZLWKWKHH[FHSWLRQRIWKHKJURXSZHIRXQGWKDWDJHVKRXOG
KDYHQRHIIHFWRQWKHRXWFRPHRIWKHH[SHULPHQW$OVRVLQFHZHZHUHVWXG\LQJWKH
GLVDSSHDUDQFHRIQRUDGUHQDOLQHZKLFKUHVXOWVIURPWKHEUHDNGRZQRIWKHPROHFXOH
LQDQRQHQ]\PDWLFPDQQHURXUDVVXPSWLRQZDVWKDWWKHDJHRIWKHDQLPDOVZDV
QRWFULWLFDO,WZRXOGEHDFRPSOHWHO\GLIIHUHQWPDWWHULIZHKDGEHHQVWXG\LQJWKH
UHJHQHUDWLRQRIQHUYHVRUWKHV\QWKHVLVRIQRUDGUHQDOLQH+RZHYHUDJHKRPRJHQHLW\
VKRXOGEHFRQVLGHUHGLQIXWXUHVWXGLHV
:HXVHGVL[DQLPDOVSHUJURXS,QWKLVNLQGRIVWXG\¿YHDQLPDOVSHUJURXSLVWKH
PLQLPXPQHHGHG$ODUJHUJURXSRIDQLPDOVZRXOGUHYHDOVPDOOHUGLIIHUHQFHVEXW
WKDWZRXOGPHDQDJUHDWO\LQFUHDVHGODERULQSXWDQGVLQFHWKHUHLVDUHFRPPHQGDWLRQ
WKDWDVIHZODERUDWRU\DQLPDOVDVSRVVLEOHVKRXOGEHXVHGZHRSWHGIRUWKLVJURXS
VL]H
,QWKLVZRUNZHGHPRQVWUDWHGWKHUDWHRIGLVDSSHDUDQFHRIQRUDGUHQDOLQHLQ
DUWHULDOJUDIWV7KLVUDWHLVFRPSDUDEOHZLWKRWKHU¿QGLQJV&RZHQEXWLWLV
GH¿QLWHO\IDVWHUWKDQWKHGLVDSSHDUDQFHRIQHXURQDOVWUXFWXUHVIURPWUDQVSODQWHG
DUWHULHV
$WKWKHUHZDVDVLJQL¿FDQWULVHLQQRUDGUHQDOLQHFRQWHQWRIWKHJUDIWVFRPSDUHG
ZLWKWKHKJUDIWV7KHPHDQFRQWUROYDOXHIHOOZLWKLQWKHFRQ¿GHQFHLQWHUYDO
RIQRUDGUHQDOLQHFRQFHQWUDWLRQVLQWKHJUDIWVDWVL[KRXUV$OOFRQFHQWUDWLRQVLQRWKHU
JUDIWVZHUHEHORZFRQWUROYDOXHVHYHQLQWKHKJURXS7KHKJURXSGLIIHUHG
VLJQL¿FDQWO\IURPWKHDQGKJURXSVSEXWGLGQRWVLJQL¿FDQWO\
GLIIHUIURPLWVRZQFRQWUROJURXS1RFRUUHVSRQGLQJUHSRUWVLQWKHOLWHUDWXUHKDYH
EHHQIRXQG7KLVPD\EHGXHWRWKHIDFWWKDWWKHQHUYH¿EHUVGRQRWUHFHLYHVWLPXOL
WKDWZRXOGPDNHWKHPUHOHDVHQRUDGUHQDOLQHWRWKHV\QDSWLFYRLG$WWKHVDPHWLPH
WKHDFWLYHHQHUJ\GHSHQGHQWUHXSWDNHRIQRUDGUHQDOLQHRFFXUVLQWKHWHUPLQDO
HQGVRIWKH¿EHUV2QO\ZKHQWKHQHUYH¿EHUVORVHWKHLUHQHUJ\VWRUHVDQGVWDUWWR
GHJHQHUDWHGRWKH\OLEHUDWHWKHVHDFFXPXODWHGVWRUHVRIQRUDGUHQDOLQH
',6&866,21
7KHUHZDVQRVLJQL¿FDQWGLIIHUHQFHEHWZHHQWKHKKKDQGKFRQWURO
JURXSV ,Q WKH KRXU FRQWURO JURXS WKHUH ZDV D VWDWLVWLFDOO\ VLJQL¿FDQW IDOO LQ
QRUDGUHQDOLQHFRQWHQWFRPSDUHGZLWKWKHRWKHUFRQWUROJURXSV7KLVPD\EHEHFDXVH
WKHUHZDVDORQJHUSHULRGRIWLPHEHWZHHQWKHRSHUDWLRQDQGVDPSOHFROOHFWLRQDQG
KHQFHWKHUDWVZHUHQRORQJHUXQGHUJRLQJDPHWDEROLFUHVSRQVHWRVXUJHU\7KLV
FRXOGEHUHÀHFWHGLQWKHQRUDGUHQDOLQHOHYHOVVHHQLQWKHFRQWUROVSHFLPHQV$VVXFK
WKLVLVDFOLQLFDOO\LPSRUWDQW¿QGLQJVLQFHLWVKRZVWKDWGXULQJDQGLPPHGLDWHO\DIWHU
WKHRSHUDWLRQWKHUDWVZHUHXQGHUVWUHVVDQGH[SRVHGWRLQFUHDVHGDGUHQHUJLFVWLPXOL
7KH SUHVHQW UHVXOWV DUH FOLQLFDOO\ VLJQL¿FDQW LQ PLFURYDVFXODU DQG FRURQDU\
E\SDVVVXUJHU\LQZKLFKDUWHULDOJUDIWVDUHXVHG&DWHFKRODPLQHVOLEHUDWHGIURP
GHJHQHUDWLQJDGUHQHUJLFQHUYHVSURYLGHDVWURQJYDVRFRQVWULFWLYHVWLPXOXVWRDUWHULDO
ZDOOVPRRWKPXVFOHFHOOVDQGFDXVHYDVRFRQVWULFWLRQLQWKHJUDIW,WLVLPSRUWDQWWR
QRWHWKDWWKHUHLVDFULWLFDOSHULRGZKHQWKHUHLVDVLJQL¿FDQWULVHLQFDWHFKRODPLQH
FRQFHQWUDWLRQVLQDUWHULDOJUDIWVGXULQJWKH¿UVWSRVWRSHUDWLYHKRXUV7KLVPD\
YHU\ZHOOUDLVHWKHULVNRIYDVRVSDVPDQGPHDVXUHVWRPLQLPL]HWKLVULVNVKRXOG
SODQQHGZHOOLQDGYDQFH7KHRWKHULPSRUWDQWREVHUYDWLRQWKDWPD\GHVHUYHVSHFLDO
DWWHQWLRQLVWKHIDFWWKDWWKHGLVDSSHDUDQFHRIQRUDGUHQDOLQHIURPWKHJUDIWVZDV
VORZHUWKDQKDVEHHQSUHVXPHGGD\VLQVWHDGRIDIHZKRXUVZKLFKSURORQJV
WKH SRVWRSHUDWLYH SHULRG DVVRFLDWHG ZLWK D ULVN RI YDVRVSDVP :H SODQQHG RXU
H[SHULPHQWVWRFRYHUZHHNVRIIROORZXSVLQFHUHLQQHUYDWLRQUHDFKHVLWV¿QDO
OHYHODWZHHNVLQYHLQJUDIWV+XNNL
7KHHIIHFWVRISHULYDVFXODUV\PSDWKHFWRP\KDYHEHHQVWXGLHGLQDQLPDOPRGHOV
,W LV NQRZQ WKDW SHULYDVFXODU V\PSDWKHFWRP\ GRHV QRW UHPRYH GLVWDO YDVFXODU
DGUHQHUJLF LQQHUYDWLRQ .DDUHOD HW DO D .DDUHOD HW DO F :DULV HW DO
2QO\WKHVHJPHQWIURPZKLFKWKHDGYHQWLWLDLVUHPRYHGORVHVLWVDGUHQHUJLF
LQQHUYDWLRQ+RZHYHUDJUDIWVLJQL¿FDQWO\GLIIHUVIURPDV\PSDWKHFWRPL]HGYHVVHO
VLQFHLWKDVEHHQFRPSOHWHO\UHPRYHGIURPLWVVXUURXQGLQJVDQGDOOWKHDIIHUHQW
D[RQVKDYHEHHQVHYHUHG
1HXURQDOUHJHQHUDWLRQLVDVXEMHFWRIZLGHGHEDWHLQWKHFXUUHQWPHGLFDOOLWHUDWXUH
HVSHFLDOO\LQWUDQVSODQWDWLRQVXUJHU\)HUUHWWL7KHUHDUHFRQWUDGLFWRU\UHSRUWV
DERXWLWVRFFXUUHQFHDQGFOLQLFDOVLJQL¿FDQFHLQGLIIHUHQW¿HOGVRIVXUJHU\/HYHUHQ]
,Q PLFURYDVFXODU VXUJHU\ UHLQQHUYDWLRQ DQG DUWHULDO VSDVP GXULQJ WKH
LPPHGLDWHSRVWRSHUDWLYHSHULRGDUHLPSRUWDQWDVSHFWVZKHUHDVIRUH[DPSOHWKH
PHWDEROLFQHHGVRIDIUHH75$0ÀDS7UDQVYHUVH5HFWXV$EGRPLQLV0XVFOHLQ
EUHDVWUHFRQVWUXFWLRQRUDIUHHODWLVVLPXVGRUVLPXVFOHÀDSLQWKHUHSDLURIRSHQ
WLELDOIUDFWXUHDUHSHUKDSVQRWVRJUHDW
,QFRQFOXVLRQQRUDGUHQDOLQHGLVDSSHDUHGDOPRVWFRPSOHWHO\IURPDUWHULDOJUDIWV
LQUDWVZLWKLQRQHZHHNSRVWRSHUDWLYHO\7KHUHDIWHUQRUDGUHQDOLQHOHYHOVLQFUHDVHG
RYHUWKHIROORZXSSHULRGRIZHHNVPRVWSUREDEO\DVDUHVXOWRIUHLQQHUYDWLRQ
RIWKHJUDIWV,QUDWIHPRUDODUWHULDOJUDIWVWUDQVSODQWHGLQWRFDURWLGDUWHULHV1$
FRQFHQWUDWLRQVURVHWROHYHOVWKDWGLGQRWVLJQL¿FDQWO\GLIIHUIURPWKRVHLQWKHFRQWUROV
RUWKHGD\JURXS7KLVFDQEHLQWHUSUHWHGDVDVLJQRIUHLQQHUYDWLRQRIWKHJUDIWV
7KHUHZDVDQLQFUHDVHLQWKHVWDQGDUGGHYLDWLRQVDORQJZLWKWKHLQFUHDVHLQPHDQ
YDOXHVDIWHULPPHGLDWHPHDVXUHPHQWLQWKHJUDIWVEXWQRWLQWKHFRQWUROV7KLVPLJKW
UHÀHFWGLIIHUHQWUHJHQHUDWLYHDQGJURZWKFDSDFLWLHVRILQGLYLGXDOUDWVDVUHJDUGVWKH
JUDIWVEXWQRWWKHQRQRSHUDWHGFRQWURODUWHULDOVHJPHQWVZKHUHVXFKSURFHVVHV
DUHQRWH[SHFWHGWRWDNHSODFH
6.2. DEGENERATION
,QWKHSUHVHQWVWXG\ZHHOXFLGDWHGWKHSRVWRSHUDWLYHVHTXHQFHRIGHJHQHUDWLRQDQG
UHJHQHUDWLRQRIQHXURQDOF\WRVNHOHWDOVWUXFWXUHV1)VDQG*$3DQGFKDQJHV
LQV\PSDWKHWLFDQGSDUDV\PSDWKHWLFLQQHUYDWLRQ:KLOHRXU¿QGLQJVDUHLQJRRG
DJUHHPHQWZLWKWKRVHRISUHYLRXVVWXGLHVRQD[RQDOGHJHQHUDWLRQ:DOOHU
6FKODHSIHUDQG+DVOHU*ODVVDQG*ULI¿Q0LNLHWDODQGRQQHUYH
¿EHU JURZWK LQWR KRVW JUDIWV .DZDJXFKL HW DO 8EHUIXKU HW DO D E
8OULFK/DLDQG(QJODQGZHKDYHH[WHQGHGWKHH[LVWLQJGDWDE\VWXG\LQJ
WKHVHSKHQRPHQDLQDUWHULDOJUDIWV
,PPXQRUHDFWLYLW\ DJDLQVW WKH PDLQ D[RQDO F\WRVNHOHWDO SURWHLQ 1) DOPRVW
FRPSOHWHO\GLVDSSHDUHGGXULQJWKH¿UVWZHHNDIWHUJUDIWLQJ7KLVLQGLFDWHVGHVWUXFWLRQ
RIWKHD[RQDOHOHPHQWVLQGHJHQHUDWLQJ¿EHUV6FKODHSIHUDQG+DVOHU*ODVV
DQG*ULII¿Q0LNLHWDO7KHIHZ1)SRVLWLYHD[RQSUR¿OHVVHHQDWVHYHQ
GD\VDIWHUWKHRSHUDWLRQSUREDEO\UHSUHVHQWWKHUHPDLQVRIGHJHQHUDWLQJD[RQDO
IUDJPHQWVDQGQRWQHFHVVDULO\IXQFWLRQDO¿EHUVVLQFHDOOPDUNHUVRIWKHGLIIHUHQW
WUDQVPLWWHUVKDGGLVDSSHDUHGDWWKLVWLPHSRLQW
,QWKHSUHVHQWVWXG\WKHUHZDVVWLOOVRPH7+,5OHIWLQWKHHQODUJHGGHJHQHUDWLQJ
D[RQDOHQGLQJVRQHGD\DIWHUWKHRSHUDWLRQZKLOHDOO7+,5KDGGLVDSSHDUHGDW
ODWHUWLPHSRLQWV7KXVHQ]\PDWLFFDSDFLW\IRUWKHV\QWKHVLVRIFDWHFKRODPLQHVPD\
UHPDLQIRUDORQJHUWLPHWKDQWKHFDWHFKRODPLQHVWKHPVHOYHV/DFNRIFDWHFKRODPLQHV
LQWKHWHUPLQDOVRQHGD\SRVWRSHUDWLYHO\PD\EHDUHVXOWRIUDSLGUHOHDVHRUODFNRI
RWKHUPHFKDQLVPVUHTXLUHGIRUV\QWKHVLVHJODFNRISUHFXUVRUXSWDNHRUHQHUJ\
VWRUHV
6LPLODUO\ WR WKH PDUNHUV RI V\PSDWKHWLF LQQHUYDWLRQ ERWK PDUNHUV RI
SDUDV\PSDWKHWLF LQQHUYDWLRQ 9,3,5 DQG Q126,5 ZHUH DOVR ORVW GXULQJ WKH
¿UVWSRVWRSHUDWLYHZHHN,PPXQRUHDFWLYLW\WR9,3ZDVVHHQRQO\RQGD\RQHDIWHU
RSHUDWLRQZKLOHVRPHQ126,5ZDVVWLOOVHHQDWWKHWKLUGSRVWRSHUDWLYHGD\9,3LVD
SHSWLGHWUDQVPLWWHUVXEVWDQFHSHUVHDQGLWVGLVDSSHDUDQFHLVSUREDEO\H[SODLQHGE\
FHVVDWLRQRID[RQDOWUDQVSRUWRIQHZO\V\QWKHVL]HGSHSWLGHVIURPQHXURQDOVRPDWD
1HXURQDO126LVDQHQ]\PHRIQLWULFR[LGHV\QWKHVLVDQGWKHWLPHFRXUVHRILWV
GLVDSSHDUDQFHEHWWHUSDUDOOHOVWKDWRI7+LQWKHV\PSDWKHWLF¿EHUV7KHUHVXOWVRI
',6&866,21
QXPHURXVVWXGLHVKDYHVXJJHVWHGWKDWGHQRYRSURGXFWLRQRIQLWULFR[LGHLVRIWHQ
LQGXFHGLQLQMXUHGQHXURQV/XPPHHWDO*RQ]DOH]+HUQDQGH]DQG5XVWLRQL
DE6LQFHZHVWXGLHGGHJHQHUDWLQJD[RQDOIUDJPHQWVZLWKQRFRQQHFWLRQWR
WKHLUVRPDWDZHFDQVDIHO\SUHFOXGHDQ\GHQRYRH[SUHVVLRQRIQ126LQRXUJUDIWV
5DWKHUZHVXJJHVWWKDWQ126,5UHSUHVHQWVRQO\WKHUHPDLQVRIWKHHQ]\PHLQWKH
GHJHQHUDWLQJD[RQDOIUDJPHQWV
6.3. REGENERATION
:HREVHUYHGDUREXVWLQFUHDVHLQ¿QHQHUYH¿EHUVVKRZLQJ*$3LPPXQRUHDFWLYLW\
DWRQHPRQWKDIWHUWKHRSHUDWLRQ,QDUHFHQWVWXG\+DUGLQJDQGFRZRUNHUV
GHPRQVWUDWHG WKDW WKH H[SUHVVLRQ RI *$3 LV FRUUHODWHG ZLWK UDSLG D[RQDO
UHJHQHUDWLRQZKLOHVORZD[RQDOHORQJDWLRQPD\DOVRRFFXULQGHSHQGHQWO\RI*$3
SURWHLQ2XUREVHUYDWLRQVVXSSRUWVXFKDFRQFHSWZHVDZWKHKLJKHVWQXPEHU
RI*$3SRVLWLYH¿EHUVDWRQHPRQWKDIWHUWKHRSHUDWLRQZKLOHWKHQXPEHURI
QHXUDOHOHPHQWVDVUHYHDOHGE\1),5LQFUHDVHGWKURXJKRXWWKHREVHUYDWLRQSHULRG
,QWHUHVWLQJO\ZHIRXQGWKDWWKHJURZWKRIQHUYH¿EHUVSURFHHGHGRYHUWKHKRVWJUDIW
MXQFWLRQZKLFKLQGLFDWHVWKDWWKHRULJLQRIUHLQQHUYDWLRQRIWKHJUDIWHGIHPRUDO
DUWHU\LVWKHVDPHDVWKDWRIWKHKRVWFDURWLGDUWHU\+RZHYHUYLVXDODVVHVVPHQW
RILQQHUYDWLRQGHQVLWLHVVXJJHVWHGWKDWWKHQHUYHSOH[XVLQWKHIHPRUDODUWHU\JUDIW
ZDVGHQVHUWKDQLQWKHKRVWDUWHU\7KLVVXJJHVWVWKDWWKHUHPLJKWEHVRPHDV\HW
XQNQRZQLQWULQVLFSURSHUWLHVLQWKHIHPRUDODQGFDURWLGDUWHULHVWKDWGLIIHUHQWLDOO\
GHWHUPLQHWKHLULQQHUYDWLRQSDWWHUQV
,PPXQRUHDFWLYLW\DJDLQVW9,3DQGQ126DSSHDUHGVOLJKWO\HDUOLHUWKDQWKDW
DJDLQVW 7+ ZKLFK PD\ UHÀHFW GLIIHUHQW UDWHV RI D[RQDO UHJURZWK LQ GLIIHUHQW
QHXURQDOV\VWHPV&HUYLFDOV\PSDWKHWLFQHXURQVPD\VKRZGHQRYRH[SUHVVLRQRI
Q1269DQKDWDORHWDOEF/XPPHHWDODQGQHXURSHSWLGHVHJ9,3
6FKRW]LQJHUHWDOLQUHVSRQVHWRD[RQDOLQMXULHVDQGLWLVSRVVLEOHWKDWVRPH
RIWKH¿UVWQ126SRVLWLYHQHUYH¿EHUVUHSUHVHQWDVXESRSXODWLRQRIUHJHQHUDWLQJ
V\PSDWKHWLF QHXURQV :KLOH VXFK WUDQVLHQW FKDQJHV LQ WUDQVPLWWHU SKHQRW\SHV
FDQQRW EH H[FOXGHG RXU ¿QGLQJV VKRZ WKDW UHLQQHUYDWLRQ RI DQ DUWHULDO JUDIW LV
DVVRFLDWHGZLWKPDUNHGFKDQJHVLQLWVQHXURFKHPLFDOFRPSRVLWLRQ,WVHHPVORJLFDO
WRDVVXPHWKDWWKLVLVDOVRUHÀHFWHGLQDFKDQJLQJUROHRIQHUYHVLQFRQWURORIWKH
YDVFXODUWRQHLQWKHJUDIWZKLFKGHVHUYHVIXUWKHUVWXG\
,QQHUYDWLRQSDWWHUQVDUHNQRZQWRGLIIHUEHWZHHQGLIIHUHQWDUWHULHVDVVHHQLQ
WKHFXUUHQWVWXG\DVZHOODVLQRWKHUVWXGLHV'KDOOHWDO0RUULVHWDO
:HREVHUYHGWKDWLQQHUYDWLRQRIWKHJUDIWVUHVHPEOHGWKDWRIDQRUPDOIHPRUDO
DUWHU\PRUHWKDQWKDWRIWKHKRVWFDURWLGDUWHU\5HFHQWVWXGLHVLQGHYHORSPHQWDO
QHXURELRORJ\5DRDQG/DQGLV6FKRW]LQJHUHWDO.HQWURWLDQG9HUQDGDNLV
DQGRQYDULRXVPRGHOVRIWLVVXHWUDQVSODQWDWLRQ.L\RFKLHWDO7DQDQG
+DUYH\$VPXVHWDOKDYHUHSHDWHGO\GHPRQVWUDWHGWKDWWKHWDUJHWWLVVXH
LVRIFUXFLDOLPSRUWDQFHIRUGHWHUPLQDWLRQRIERWKWKHLQWHQVLW\DQGQHXURFKHPLFDO
LGHQWLW\RILQQHUYDWLRQ2XUREVHUYDWLRQVVXJJHVWWKDWYDVFXODUJUDIWVDUHDOVRDEOHWR
LQÀXHQFHWKHLURZQUHLQQHUYDWLRQ7KLVPD\KDYHIDUUHDFKLQJLPSOLFDWLRQVLQWKH
VHOHFWLRQRIDSSURSULDWHYDVFXODUJUDIWV'RQRUVLWHVIRUYDVFXODUJUDIWVDUHXVXDOO\
VHOHFWHGRQWKHEDVLVRIWKHLUDYDLODELOLW\ZKLOHWKHSUHVHQWUHVXOWVVXJJHVWWKDWRQH
PD\QHHGWRDWWHPSWWR¿QGYDVFXODUJUDIWVWKDWKDYHLQQHUYDWLRQSDWWHUQVFDXVLQJ
DVIHZFRPSOLFDWLRQVDVSRVVLEOHGXULQJERWKWKHLPPHGLDWHSRVWRSHUDWLYHSHULRG
DQGORQJHUIROORZXSSHULRGV
,PPXQRVWDLQLQJ IRU H126 GHPRQVWUDWHG WKDW WKLV HQ]\PH FRQWLQXHV LWV
FRQVWLWXWLYHH[SUHVVLRQLQJUDIWHGDUWHULHV5HFHQWVWXGLHVKDYHSURYLGHGHYLGHQFH
WKDW LQWUDYDVFXODU RSHUDWLRQV PD\ LQFUHDVH RU LQGXFH WKH H[SUHVVLRQ RI H126
%RVPDQV 3RSSD ZKLFK PD\ EH LPSRUWDQW IRU SURSHU UHOD[DWLRQ
DQGKHQFHIRUSUHYHQWLRQRIYDVRVSDVPLQUHFHQWO\RSHUDWHGDUWHULHV,QVRPHRI
RXUVDPSOHVZHREVHUYHGSDUWLDOGHWDFKPHQWRIWKHHQGRWKHOLXPZKLFKSUREDEO\
LPSDLUVQLWULFR[LGHGHSHQGHQWYDVRUHOD[DWLRQLQWKHJUDIWSUHGLVSRVLQJWKHJUDIW
WRYDVRVSDVP$WKLUGVRXUFHRIQLWULFR[LGHLQYDVFXODUVWUXFWXUHVLVYLDL1262XU
VWXG\,9VKRZVWKDWLWLVRQO\VHHQLQVRPHFHOOVLQWKHWXQLFDPHGLDSUREDEO\
PDFURSKDJHVDQGKHQFHRXUUHVXOWVGRQRWVXSSRUWDUROHIRUL126DVDQLPSRUWDQW
IDFWRULQFRQWUROOLQJYDVFXODUWRQH
6.4. CLINICAL CONSIDERATIONS
:LWKPRUHDQGPRUHSDWLHQWVXQGHUJRLQJUHRSHUDWLRQVEHFDXVHRIFRURQDU\DUWHU\
GLVHDVHDQGVLPXOWDQHRXVO\VXIIHULQJIURPLQWHUPLWWHQWFODXGLFDWLRQDQGFULWLFDO
LVFKHPLDRIWKHORZHUH[WUHPLWLHVFRURQDU\VXUJHRQVVRPHWLPHV¿QGWKHPVHOYHV
LQDVLWXDWLRQZKHUHWKH\KDYHQRYHLQVWRKDUYHVWIRUJUDIWV,QVXFKVLWXDWLRQV
FHUWDLQDUWHULHVPRVWO\UDGLDODUWHULHVIURPWKHXSSHUH[WUHPLWLHVDUHXVHG&KHQHW
DO7KHLQWHUQDOPDPPDU\DUWHU\IURPHLWKHUVLGHLVXVHGURXWLQHO\DQGWKH
ULJKWJDVWURHSLSORLFDUWHU\VRPHWLPHV*UDQGMHDQHWDOLQFRURQDU\E\SDVV
RSHUDWLRQV5DGLDODQGULJKWJDVWURHSLSORLFDUWHULHVUHSRUWHGO\EHFRPHVSDVWLFDQG
VXVFHSWLEOHWRK\SHUFRQWUDFWLELOLW\WKRXJKWWREHFDXVHGE\FDWHFKRODPLQHV&DWHHW
DODQGIRUWKLVUHDVRQWRSLFDOXVHRIVSDVPRO\WLFDJHQWVLVUHFRPPHQGHG+H
DQG<DQJGXULQJSUHSDUDWLRQRIWKHDUWHU\2XU¿QGLQJWKDWFDWHFKRODPLQHV
GLVDSSHDUVRRQDIWHUJUDIWLQJJLYHVDFOXHWRWKHXQGHUO\LQJPHFKDQLVP5HPHV
DQG FRZRUNHUV 5HPHV HW DO 5HPHV HW DO KDYH GHPRQVWUDWHG WKDW
LQVWUXPHQWDWLRQ FDXVHV FUXVK LQMXU\ WR WKH YHVVHO ZDOO ZLWK ORVV RI PHWDEROLF
DFWLYLW\DQGLQÀDPPDWRU\FHOOLQ¿OWUDWLRQ,QRXUZRUNDFUXVK]RQHZDVVHHQLQ
VHJPHQWDOGRUVDODUWHULHVDIWHUKDQGOLQJZLWKIRUFHSV,QWKHFHQWUDOSRUWLRQRIWKLV
',6&866,21
]RQHÀXRUHVFHQFHKDGDOPRVWFRPSOHWHO\GLVDSSHDUHGVXJJHVWLQJLQMXU\WRWKH
DGUHQHUJLFQHUYHVDQGUDSLGGLVDSSHDUDQFHRIWUDQVPLWWLQJVXEVWDQFHV
&OLQLFDODQGKLVWRFKHPLFDOVWXGLHVKDYHLQGLFDWHGWKDWZKHQRSHQKHDUWVXUJHU\
LVSHUIRUPHGFDWHFKRODPLQHVIUHHGIURPKHDUWWLVVXHFDQEHDULVNIDFWRU,IWKH
KHDUWWLVVXHFRQWDLQVODUJHDGUHQHUJLFQHUYHVZLWK³GURSOHW¿EHUV³RU³DGUHQDOLQH
ERPEV³FRQWDLQLQJKXJHORFDOLQWUDD[RQDOFRQFHQWUDWLRQVRIFDWHFKRODPLQHVPRVWO\
QRUDGUHQDOLQHWKHUHLVDQLQFUHDVHGULVNRIDUUK\WKPLDDQGHYHQGHDWK.\|VRODHW
DO,WKDVHYHQEHHQSURSRVHGWKDWǃEORFNLQJDJHQWVRUFDOFLXPDQWDJRQLVWV
VKRXOGEHXVHGIRUWKRVHSDWLHQWVZKRDUHDWDJUHDWHUWKDQQRUPDOULVN.\|VRODHW
DO.\|VRODHWDO+HDQG<DQJ,QPLFURVXUJHU\PHGLFDOWUHDWPHQW
KDVEHHQXVHGWRSUHYHQWDJJUHJDWLRQRIEORRGSODWHOHWV*DWHOH\HWDO5HQDXG
HWDODQGSDSDYHULQHDQGJXDQLGLQHDUHLQFOLQLFDOXVHWRSUHYHQWYDVRVSDVP
,QDGGLWLRQWKHORFDODQHVWKHWLFDJHQWOLGRFDLQHFDQEHXVHGWRSUHYHQWYDVRSDVP
DVFDQZDUPVDOLQHFRPSUHVVHV
1RUDGUHQDOLQHFRQFHQWUDWLRQVLQUDWVDUWHULDOJUDIWVDUHHOHYDWHGVLJQL¿FDQWO\
VL[KRXUVSRVWRSHUDWLYHO\UDLVLQJWKHULVNRIYDVRVSDVP7KH\DOVRWDNHORQJHUWKDQ
H[SHFWHGWREHFRPHUHGXFHGGURSWRE\KSRVWRSHUDWLYHO\3URSHUDWWHQWLRQ
LQWKLVHDUO\SRVWRSHUDWLYHSHULRGDIWHUDUWHULDOJUDIWLQJDQGVKRXOGEHFRQVLGHUHGWR
LPSURYHWKHRXWFRPHRIYDVFXODUJUDIWLQJ7KLVLVDQRYHO¿QGLQJZLWKQRSUHFHGLQJ
VLPLODUUHSRUWV7KHUHXSWDNHRIQRUDGUHQDOLQHLVDVXEVWUDWHGHSHQGHQWSURFHVV
WKDWGHULYHVLWVGULYLQJIRUFHIURPWKH1D.$73DVHJHQHUDWHG1DFRQFHQWUDWLRQ
JUDGLHQW%|QLFKDQG%UVV,QD]XHWDO+HQFHDVORQJDVWKHUHDUH
HQHUJ\VRXUFHVWRUXQWKLVSURFHVVWKHD[RQVZLOODFFXPXODWHQRUDGUHQDOLQH$W
WKHVDPHWLPHWKHUHDUHQRD[RQDOH[FLWDWLRQVLJQDOVDUULYLQJWRFDXVHWKHUHOHDVH
RIDGUHQDOLQHDQGWKLVGRHVQRWKDSSHQXQWLOD[RQDOGHJHQHUDWLRQZLWKO\VLVRIWKH
VWRUDJH YHVLFOHV WDNHV SODFH 1RUDGUHQDOLQH LV WKHQ UHOHDVHG WR WKH VXUURXQGLQJ
WLVVXHIURPWKHJUDIWZLWKLWVDGYHUVHHIIHFWRIYDVRVSDVP$IWHUWKLVSHULRGZLWKWKH
D[RQVGHDGDQGQRDGUHQHUJLFLQQHUYDWLRQWKHJUDIWLVVXEMHFWHGRQO\WRFLUFXODWLQJ
FDWHFKRODPLQHVXQWLOUHLQQHUYDWLRQWDNHVSODFH
1HXURQDOUHJHQHUDWLRQLVDVXEMHFWRIZLGHGHEDWHLQWKHFXUUHQWPHGLFDOOLWHUDWXUH
HVSHFLDOO\LQWUDQVSODQWDWLRQVXUJHU\2GDNDHWDO)HUUHWWLHWDO7KHUH
DUHFRQWUDGLFWRU\UHSRUWVDERXWLWVRFFXUUHQFHDQGFOLQLFDOVLJQL¿FDQFHLQGLIIHUHQW
¿HOGV RI VXUJHU\ /HYHUHQ] HW DO 0RQGHOOL HW DO ,Q PLFURYDVFXODU
VXUJHU\ UHLQQHUYDWLRQ DQG DUWHULDO VSDVP GXULQJ WKH LPPHGLDWH SRVWRSHUDWLYH
SHULRGDUHLPSRUWDQWDVSHFWVZKHUHDVIRUH[DPSOHWKHPHWDEROLFQHHGVRIDIUHH
75$0ÀDS7UDQVYHUVH5HFWXV$EGRPLQLV0XVFOHLQEUHDVWUHFRQVWUXFWLRQRUD
IUHHODWLVVLPXVGRUVLPXVFOHÀDSLQWKHUHSDLURIRSHQWLELDOIUDFWXUHDUHSHUKDSV
QRWVRJUHDW7KLVPD\EHEHFDXVHWKHÀRZUDWHVLQPLFURYDVFXODUÀDSVDUHVPDOO
WRPOPLQJRIWLVVXH/RUHQ]HWWLHWDO
5HLQQHUYDWLRQ SDWWHUQV KDYH EHHQ VKRZQ WR EH JUDIWGRPLQDWHG QRW KRVW
GRPLQDWHG ,9 6FKRW]LQJHU HW DO .L\RFKL HW DO &RZHQ HW DO $VPXVHWDODQGGLIIHUHQWLDWLRQRIWKHJURZLQJQHUYHVKDVEHHQVKRZQWR
EHDIIHFWHGE\WKHLUVXUURXQGLQJV&KRQJHWDO&RZHQHWDO
)URPRXUHDUOLHUVWXGLHVZHNQRZWKDWJO\R[\OLFDFLGLQGXFHGÀXRUHVFHQFHDQG
QRUDGUHQDOLQHLWVHOIGLVDSSHDUIURPDUWHULDOJUDIWVZLWKLQWRKRXUV,$IWHU
SHULYDVFXODU V\PSDWKHFWRP\ DGUHQHUJLF QHUYHV GR QRW GLVDSSHDU WRWDOO\ IURP
DUWHULHV)HUUHWWLHWDOEXWWKHVLWXDWLRQLVGLIIHUHQWZLWKDUWHULDOJUDIWV$
JUDIW LV E\ GH¿QLWLRQ FRPSOHWHO\ FXW RII IURP DQ\ VRXUFH RI LQWDFW QHUYH ¿EHUV
ERWKIURPD[LDOLQQHUYDWLRQDORQJWKHDUWHU\DQGIURPWKRVH¿EHUVHQWHULQJIURP
VXUURXQGLQJDUHDV
,QWKHSUHVHQWZRUNQRUDGUHQDOLQHOHYHOVZHUHVRKLJKDWWKHHQGRIWKHIROORZ
XSSHULRGWKDWLQRXUYLHZLWLVFOLQLFDOO\VLJQL¿FDQW,WLVLPSRUWDQWWRXQGHUVWDQG
WKDW D IUHH ÀDS LV QRW D VHSDUDWH SLHFH RI WLVVXH EXW ZLOO EH LQFRUSRUDWHG WR LWV
VXUURXQGLQJVFORVHO\DQGZLOOUHVSRQGWRQHUYRXVVWLPXOLDIWHUWKHUHLQQHUYDWLRQ
SURFHVVKDVEHHQFRPSOHWHG$QWLVSDVPRGLFPHGLFDWLRQVKRXOGEHDGPLQLVWHUHG
LPPHGLDWHO\DIWHUWKHRSHUDWLRQIRUDVORQJDVLWFDQEHVXSSRVHGWKDWQRUDGUHQDOLQH
LVOLEHUDWHGIURPWKHQHUYHV
,QFRQFOXVLRQQRUDGUHQDOLQHGLVDSSHDUHGDOPRVWFRPSOHWHO\IURPDUWHULDOJUDIWV
LQUDWVZLWKLQRQHZHHNSRVWRSHUDWLYHO\7KHUHDIWHUQRUDGUHQDOLQHOHYHOVLQFUHDVHG
RYHUWKHIROORZXSSHULRGRIZHHNVPRVWSUREDEO\DVDUHVXOWRIUHLQQHUYDWLRQ
RIWKHJUDIWV,QUDWIHPRUDODUWHULDOJUDIWVWUDQVSODQWHGLQWRFDURWLGDUWHULHV1$
FRQFHQWUDWLRQVURVHWROHYHOVWKDWGLGQRWVLJQL¿FDQWO\GLIIHUIURPWKRVHLQWKHFRQWUROV
RUWKHGD\JURXS7KLVFDQEHLQWHUSUHWHGDVDVLJQRIUHLQQHUYDWLRQRIWKHJUDIWV
7KHUHZDVDQLQFUHDVHLQWKHVWDQGDUGGHYLDWLRQVDORQJZLWKWKHLQFUHDVHLQPHDQ
YDOXHVDIWHULPPHGLDWHPHDVXUHPHQWLQWKHJUDIWVEXWQRWLQWKHFRQWUROV7KLVPLJKW
UHÀHFWGLIIHUHQWUHJHQHUDWLYHDQGJURZWKFDSDFLWLHVRILQGLYLGXDOUDWVDVUHJDUGVWKH
JUDIWVEXWQRWWKHQRQRSHUDWHGFRQWURODUWHULDOVHJPHQWVZKHUHVXFKSURFHVVHV
DUHQRWH[SHFWHGWRWDNHSODFH
2XU ¿QGLQJV VKRZ WKDW GHJHQHUDWLRQ RI LQQHUYDWLRQ RI DUWHULDO JUDIWV LQ UDWV
RFFXUVZLWKLQWKH¿UVWVHYHQGD\V5HJHQHUDWLRQLVDOUHDG\HYLGHQWRQHPRQWKDIWHU
RSHUDWLRQDQGLWFRQWLQXHVWRSURFHHGIRUDWOHDVWIRXUPRUHPRQWKV,WVHHPVWKDW
SDUDV\PSDWKHWLFQHUYHVPD\JURZVRPHZKDWIDVWHUWKDQV\PSDWKHWLFQHUYHV7KLV
PLJKWFDXVHWUDQVLHQWO\DOWHUHGQHXURQDOFRQWURORIYDVFXODUWRQHGXULQJWKH¿UVWIHZ
SRVWRSHUDWLYHPRQWKV)LQDOO\RXU¿QGLQJVVXJJHVWWKDWJUDIWWLVVXHGHWHUPLQHVWKH
SDWWHUQRILQQHUYDWLRQQRWWKHVXUURXQGLQJWLVVXHIURPZKLFKWKHQHUYHVVSURXW
',6&866,21
7. CONCLUSIONS
21(
,WZDVHVWDEOLVKHGWKDWFDWHFKRODPLQHLQGXFHGÀXRUHVFHQFHKDGGLVDSSHDUHGIURP
WKHJUDIWVZLWKLQKRXUVDIWHUWKHRSHUDWLRQ
7:2
8VLQJ+3/&ZHHVWDEOLVKHGWKDWQRUDGUHQDOLQHOHYHOVDUHVLJQL¿FDQWO\HOHYDWHGLQ
UDWDUWHULDOJUDIWVDWKRXUVDQGWDNHKRXUVWRFRPHGRZQ7KLVLVORQJHUWKDQ
RQHZRXOGH[SHFWIURPWKHÀXRUHVFHQFHVWXG\,7KLVZDVDQXQH[SHFWHG¿QGLQJ
7+5((
8VLQJ+3/&ZHHVWDEOLVKHGWKDWFDWHFKRODPLQHOHYHOVLQFUHDVHGRYHUWKHIROORZXS
SHULRGRIZHHNVZKLFKFDQEHLQWHUSUHWHGWREHDVLJQRIUHLQQHUYDWLRQRIUDW
IHPRUDODUWHULDOJUDIWVWUDQVSODQWHGLQWRFDURWLGDUWHULHV&RQFHQWUDWLRQVRI1$URVH
WROHYHOVWKDWGLGQRWVLJQL¿FDQWO\GLIIHUIURPWKRVHLQWKHFRQWUROVRUWKHGD\JURXS
)285 2XUKLVWRORJLFDO¿QGLQJVVKRZHGWKDWGHJHQHUDWLRQRILQQHUYDWLRQRIDUWHULDOJUDIWV
LQUDWVRFFXUVZLWKLQKRXUV7KHGLVVROXWLRQRIQHUYH¿EHUVLVFRPSOHWHZLWKLQ
RQH ZHHN DQG WKHUH LV JURZWK RI QHUYH ¿EHUV LQWR WKH JUDIW DIWHU RQH PRQWK ,W
VHHPVWKDWSDUDV\PSDWKHWLFQHUYHVUHJHQHUDWHVRRQHUWKDQV\PSDWKHWLFQHUYHV
7KHUHLQQHUYDWLRQSDWWHUQRIJUDIWHGIHPRUDODUWHULHVUHVHPEOHVWKDWRIDIHPRUDO
DUWHU\PRUHWKDQWKDWRIWKHKRVWFDURWLGDUWHU\
ACKNOWLEDGEMENTS
7KHSUHVHQWZRUNZDVFDUULHGRXWDW+HOVLQNL&LW\+RVSLWDO7DPSHUH8QLYHUVLW\
+RVSLWDO2XOX8QLYHUVLW\+RVSLWDODQGWKH'HSDUWPHQWVRI0D[LOORIDFLDO6XUJHU\
DQG3ODVWLF6XUJHU\DW+HOVLQNL8QLYHUVLW\+RVSLWDOEHWZHHQDQG
,ZRXOGOLNHWRWKDQN'RFHQW2XWL.DDUHOD0'3K'DQG3URIHVVRU6HSSR
6RLQLOD 0' 3K' IRU WKHLU FRQVWUXFWLYH DQG LQYDOXDEOH FULWLFLVP GXULQJ WKH
SUHSDUDWLRQRIWKLVPDQXVFULSW:LWKRXWLWWKLVWKHVLVZRXOGKDYHEHHQRIPXFK
OHVVYDOXH
,WZRXOGOLNHWRWKDQN'RFHQW7LPR:DULV0'3K'IRUPHUFKLHIRIWKH3ODVWLF
6XUJHU\8QLW'HSDUWPHQWRI6XUJHU\2XOX8QLYHUVLW\+RVSLWDOWKHVXSHUYLVRURI
WKLVZRUNIRUKLVHQGOHVVSDWLHQFHDQGLQQRYDWLYHLGHDVZKLOHVXSHUYLVLQJWKLVVWXG\
+HZDVDOVRDFRZRUNHUDQGDQLQVSLUDWLRQDO¿JXUHLQDOOWKHRULJLQDOSXEOLFDWLRQV
9HU\ LPSRUWDQW SHRSOH LQ WKLV VWXG\ KDYH EHHQ WKH FRZRUNHUV LQYROYHG LQ
WKH RULJLQDO SXEOLFDWLRQV 7KHVH LQFOXGH 3URIHVVRU 3LUNNR +XWWXQHQ IURP WKH
'HSDUWPHQW RI )RUHQVLF 6FLHQFH DW WKH 8QLYHUVLW\ RI 2XOX ZKR FDUULHG RXW WKH
+3/&DQDO\VHVIRUWKLVZRUN3URIHVVRU.DUOYRQ6PLWWHQIURPWKH%UHDVW6XUJHU\
8QLWZDVRIJUHDWLPSRUWDQFHERWKDWWKHHDUO\VWDJHVRIWKLVZRUNDQGDOVRZKHQ
WKLVWKHVLVZDV¿QDOL]HG3URIHVVRU1XUHGGLQ$VKDPPDNKLSURYLGHGKLVJUHDWKHOS
DQG H[SHULHQFH ZKLOH KHOSLQJ LQ SUHSDULQJ WKH PDQXVFULSWV IRU SXEOLFDWLRQ DQG
HGLWLQJWKLVPDQXVFULSW'RFHQW6DPSVD9DQKDWDORSOD\HGDQLPSRUWDQWUROHLQWKH
IRXUWKDQGODUJHVWRULJLQDOSXEOLFDWLRQDVZHOODVZKLOHSUHSDULQJWKHPDQXVFULSWRI
WKLVWKHVLV+HDOVRSURYLGHGYDOXDEOHFRQWDFWVWKDWZHUHFUXFLDOIRUWKHVXFFHVVIXO
RXWFRPHRIWKLVZRUN
, ZRXOG OLNH WR WKDQN P\ FROOHDJXH -HKDG $O6XNKXQ '' 3K' IRU KLV
IULHQGVKLSDQGKHOSZKLOHSUHSDULQJWKHPDQXVFULSWRIWKLVWKHVLV
,ZRXOGOLNHWRWKDQN3URIHVVRU(UNNL7XNLDLQHQIRUDFFHSWLQJWKLVWKHVLVIRU
SXEOLFDWLRQ XQGHU KLV DXWKRULW\ DW WKH 'HSDUWPHQW RI 3ODVWLF 6XUJHU\ +HOVLQNL
8QLYHUVLW\+RVSLWDO
,WDNHWKHRSSRUWXQLW\WRWKDQN'RFHQW1LFKRODV%ROWRQIRUOLQJXLVWLFUHYLVLRQ
RIWKLVPDQXVFULSW,ZLVKWRH[SUHVVVSHFLDOWKDQNVWR+DQQD+DNDODIRUKHUZRUN
RQWKHLOOXVWUDWLRQRQV\PSDWKHWLFQHXURQV
.DUL6DYHOLXVODERUDWRU\KHDGRIWKHH[SHULPHQWDODQLPDOXQLWDWWKH6XUJLFDO
+RVSLWDOODWHUWKHFKLHIRIH[SHULPHQWDOVXUJHU\XQLWDW0HLODKWL+RVSLWDOLVWKDQNHG
IRUKLVKHOSDQGVXSSRUWDWWKHHDUO\VWDJHVRIWKLVVWXG\+HDOVRSURYLGHGFRPSDQ\
DQGFRQYHUVDWLRQGXULQJPDQ\ORQJKRXUVRIODERUDWRU\ZRUN,ZRXOGOLNHWRWKDQN
WKHSHUVRQQHORIWKHODERUDWRU\DWWKH6XUJLFDO+RVSLWDOIRUWKHLUKHOSDQGVXSSRUW
LQDFFRPSOLVKLQJWKHQHFHVVDU\ODERUDWRU\ZRUNIRUWKH¿UVWRULJLQDOSXEOLFDWLRQ
5()(5(1&(6
REFERENCES:
$OP3/DUVVRQ((NEODG(6XQGOHU)DQG$QGHUVVRQ.(,PPXQRKLVWRFKHPLFDO
ORFDOL]DWLRQRISHULSKHUDOQLWULFR[LGHV\QWKDVHFRQWDLQLQJQHUYHVXVLQJ
DQWLERGLHVUDLVHGDJDLQVWV\QWKHVL]HG&DQG1WHUPLQDOIUDJPHQWVRIDFORQHG
HQ]\PHIURPUDWEUDLQ$FWD3K\VLRO6FDQG
$NDU.8\GHV'RJDQ%7XIDQ+$VODPDFL6.RNVR\&.DQ]LN,7KHFRPSDULVRQRI
WKHUHVSRQVLYHQHVVRIKXPDQLVRODWHGLQWHUQDOPDPPDU\DQGJDVWURHSLSORLF
DUWHULHVWROHYFURPDNDOLP$QDOWHUQDWLYHDSSURDFKWRWKHPDQDJHPHQWRI
JUDIWVSDVP%U-&OLQ3KDUPDFRO
$SDWK\61DFKZHOFKHU5LFKWXQJKLQVROOGLH1HUYHQOHKUHUHIRUPLHUWZHUGHQ%LRO
=HQWUDODO
$VPXV63DUVRQV6DQG/DQGLV&'HYHORSPHQWDOFKDQJHVLQWKHWUDQVPLWWHU
SURSHUWLHVRIV\PSDWKHWLFQHXURQVWKDWLQQHUYDWHWKHSHULRVWHXP-1HXURVFL
$WWZHOO'%DUERXU%DQG6]DWNRZVNL01RQYHVLFXODUUHOHDVHRIQHXURWUDQVPLWWHUV
1HXURQ
$[HOVVRQ 6 %M|UNOXQG $ /LQGYDOO 2 )OXRUHVFHQFH KLVWRFKHPLVWU\ RI ELRJHQLF
PRQRDPLQHVDVWXG\RIWKHFDSDFLW\RIYDULRXVFDUERQ\OFRPSRXQGVWRIRUP
ÀXRURSKRUHVZLWKELRJHQLFPRQRDPLQHVLQJDVSKDVHUHDFWLRQV-+LVWRFKHP
&\WRFKHP
%DDV 3 DQG %XVWHU ' 6ORZ $[RQDO 7UDQVSRUW DQG WKH *HQHVLV RI 1HXURQDO
0RUSKRORJ\-1HXURELRO
%HQPLOORXG 0 Y (XOHU 86 (IIHFWV RI EUHW\OLXP UHVHUSLQH JXDQHWKLGLQH DQG
V\PSDWKHWLFGHQHUYDWLRQRQWKHQRUDGUHQDOLQFRQWHQWRIWKHUDWVXEPD[LOODU\
JODQG$FWD3K\VLRO6FDQG
%HQRZLW]/DQG5RXWWHQEHUJ$*$3DQLQWULQVLFGHWHUPLQDQWRIQHXURQDO
GHYHORSPHQWDQGSODVWLFLW\7UHQGV1HXURVFL
%HUQDUG&,QÀXHQFHGXJUDQGV\PSDWKLTXHVXUODVHQVLELOLWpHWVXUODFDORUL¿FDWLRQ
&RPSWHVUHQGXVGHO¶$FDGGH6FLHQFHVVpDQFHGX0DUVW
%RVPDQV-%XOW+9ULQWV&.RFN[0DQG+HUPDQ$%DOORRQDQJLRSODVW\DQG
LQGXFWLRQRIQRQHQGRWKHOLDOQLWULFR[LGHV\QWKDVHLQUDEELWFDURWLGDUWHULHV
(XU-3KDUPDFRO
%UDGOH\ ( /DZ $ %HOO ' -RKQVRQ &' (IIHFWV RI YDU\LQJ LPSXOVH QXPEHU RQ
FRWUDQVPLWWHUFRQWULEXWLRQVWRV\PSDWKHWLFYDVRFRQVWULFWLRQLQUDWWDLODUWHU\
$P-3K\VLRO+HDUW&LUF3K\VLRO
%URGVNL&6FKDXEPDU$'HFKDQW*2SSRVLQJIXQFWLRQVRI*'1)DQG1*)LQ
WKHGHYHORSPHQWRIFKROLQHUJLFDQGQRUDGUHQHUJLFV\PSDWKHWLFQHXURQV0RO
&HOO1HXURVFL
%URZQ6HTXDUG & 3KLODGHOSKLD 0HGLFDO ([DPLQHU $XJ S 5HSULQWHG
LQ³([S5HVHDUFKHVDSSOLHGWR3K\VLRODQG3DWKRO´S1HZ<RUN
%XUQHWW5*XLFKDUG<DQG%DUDOH(,PPXQRKLVWRFKHPLVWU\IRUOLJKWPLFURVFRS\
LQ VDIHW\ HYDOXDWLRQ RI WKHUDSHXWLF DJHQWV DQ RYHUYLHZ 7R[LFRORJ\ %XUQVWRFN*'RVRPHQHUYHFHOOVUHOHDVHPRUHWKDQRQHWUDQVPLWWHU1HXURVFLHQFH
%XUQVWRFN * 1RUDGUHQDOLQH DQG $73 DV FRWUDQVPLWWHUV LQ V\PSDWKHWLF QHUYHV
1HXURFKHP,QW
%XUQVWRFN*&RWUDQVPLVVLRQ&XUUHQW2SLQLRQLQ3KDUPDFRORJ\±
%lFN 0 7\\QHOl 00 3RUWLHU , 9LUWDQHQ DQG 6 6RLQLOD 'LVWULEXWLRQ RI
QHXUR¿ODPHQWSURWHLQVDQGSHULSKHULQLQWKHUDWSLWXLWDU\JODQG1HXURVFL
5HV
%|PLVFK+DQG%UVV07KHQRUDGUHQDOLQWUDQVSRUWHURIWKHQHXURQDOSODVPD
PHPEUDQH$QQ1<$FDG6FL
&DMDO 6 (PEU\RJHQHVLV RI QHXUR¿EULOV 7UDE /DE ,QYHVWLJDFLRQHV %LRO 8QLY
0DGULG
&DURQL 3 ,QWULQVLF QHXURQDO GHWHUPLQDQWV WKDW SURPRWH D[RQDO VSURXWLQJ DQG
HORQJDWLRQ%LRHVVD\V
&DWH&0*LWWHU5-HWW.6SDVPRIWKHJDVWURHSLSORLFDUWHU\XVHGIRUFRURQDU\
DUWHU\E\SDVVJUDIWLQJ$P-&DUGLRO
&KDUFRXGLDQ16NLQEORRGÀRZLQDGXOWKXPDQWKHUPRUHJXODWLRQ+RZLWZRUNV
ZKHQLWGRHVQRWDQGZK\0D\R&OLQ3URF
&KHQ$+1DNDWR7%URGPDQ5)(DUO\3RVWRSHUDWLYHDQJLRJUDSKLFDVVHVVPHQWRI
UDGLDOJUDIWVXVHGIRUFRURQDU\DUWHU\E\SDVVJUDIWLQJ-7KRUDF&DUGLRYDVF
6XUJ
&KHQJ)X&KRX.XR-RQ6RQ+LJKSHUIRUPDQFHOLTXLG&KURPDWRJUDSKLFDQDO\VLV
ZLWKHOHFWURFKHPLFDOGHWHFWLRQRIELRJHQLFDPLQHVXVLQJPLFURERUHFROXPQV
-&KURPDWRJU%
&KRQJ 0 :RROI & 7XUPDLQH 0 (PVRQ 3 $QGHUVRQ 3 ,QWULQVLF YHUVXV
H[WULQVLFIDFWRUVLQGHWHUPLQLQJWKHUHJHQHUDWLRQRIWKHFHQWUDOSURFHVVHVRI
UDWGRUVDOURRWJDQJOLRQQHXURQVWKHLQÀXHQFHRIDSHULSKHUDOQHUYHJUDIW
-&RPS1HXURO
&RRQV $+ &UHHFK +- -RQHV 51 ,PPXQRORJLFDO SURSHUWLHV RI DQ DQWLERG\
FRQWDLQLQJDÀXRUHVFHQWJURXS3URF6RF([S%LRO0HG
&RRSHU-%ORRP)DQG5RWK7KHELRFKHPLFDOEDVLVRIQHXURSKDUPDFRORJ\,VW
HG1HZ<RUN2[IRUG8QLYHUVLW\3UHVV
5()(5(1&(6
&RKHQ5$3ODWHOHWLQGXFHGQHXURJHQLFFRURQDU\FRQWUDFWLRQVGXHWRDFFXPXODWLRQ
RIIDOVHQHXURWUDQVPLWWHUK\GUR[\WU\SWDPLQHQ-&OLQ,QYHVW
&RZHQ7DQG%XUQVWRFN*4XDQWLWDWLYHDQDO\VLVRIWKHGHQVLW\DQGSDWWHUQRI
DGUHQHUJLFLQQHUYDWLRQRIEORRGYHVVHOV+LVWRFKHPLVWU\
&RZHQ 7 0F&RUPLF ' 7RII : %XUQVWRFN * /XPOH\ - 7KH HIIHFW RI VXUJLFDO
SURFHGXUHRQEORRGYHVVHOLQQHUYDWLRQ$ÀXRUHVFHQFHKLVWRFKHPLFDOVWXG\RI
GHJHQHUDWLRQDQGUHJURZWKRISHULYDVFXODUDGUHQHUJLFQHUYHV%ORRG9HVVHOV
&RZHQ77KUDVLYRXORX&6KDZ6$EGHQ5DKPDQ77UDQVSODQWHGVZHDWJODQGV
IURPPDWXUHDQGDJHGGRQRUVGHWHUPLQHFKROLQHUJLFSKHQRW\SHDQGDOWHUHG
GHQVLW\RIKRVWV\PSDWKHWLFQHUYHV-$XWRQ1HUY6\VW
'KDOO 8 &RZHQ 7 +DYHQ $ DQG %XUQVWRFN * 3HULYDVFXODU QRUDGUHQHUJLF
DQGSHSWLGHFRQWDLQLQJQHUYHVVKRZGLIIHUHQWSDWWHUQVRIFKDQJHVGXULQJ
GHYHORSPHQWDQGDJHLQJLQWKHJXLQHDSLJ-$XWRQ1HUY6\VW
'RHULQJ/'UDFXS.0RVHU'&]HU/DQG3HWHU&+HPRG\QDPLFDGDSWDWLRQ
WRRUWKRVWDWLFVWUHVVDIWHURUWKRWRSLFKHDUWWUDQVSODQWDWLRQ+HDUW/XQJ
(KULQJHU%)DOFN%6SRUURQJ%$GUHQHUJLF¿EHUVWRWKHKHDUWDQGSHULSKHUDO
YHVVHOV%LEO$QDW
(UlQN|2'LVWULEXWLRQRIÀXRUHVFLQJLVOHWVDGUHQDOLQHDQGQRUDGUHQDOLQLQWKH
DGUHQDOPHGXOODRIWKHKDPVWHU$FWD(QGRFULQRO
(UlQN| 2 7KH SUDFWLFDO KLVWRFKHPLFDO GHPRQVWUDWLRQ RI FDWHFKRODPLQHV E\
IRUPDOGHK\GHLQGXFHGÀXRUHVFHQFH-50LFURVF6RF
(VOHU0''RXJODV0-3ODQQLQJIRUKHPRUUKDJH6WHSVDQHVWKHVLRORJLVWFDQWDNH
WROLPLWDQGWUHDWKHPRUUKDJHLQWKHREVWHWULFSDWLHQW$QHVWKHVLRO&OLQ1RUWK
$PHULFD
)DKUHQNUXJ-7UDQVPLWWHUUROHRIYDVRDFWLYHLQWHVWLQDOSHSWLGH3KDUPDFRO7R[LFRO
)DKUHQNUXJ-DQG+DQQLEDO-1HXURWUDQVPLWWHUVFRH[LVWLQJZLWK9,3DQG3$&$3
3HSWLGHV
)DOFN%2EVHUYDWLRQVRQWKHSRVVLELOLWLHVRIFHOOXODUORFDOL]DWLRQRIPRQRDPLQHV
E\DÀXRUHVFHQFHPHWKRG$FWD3K\VLRO6FDQG6XSSO
)DQVD+3ORJPHLHU.)HLVVWQHU+DQG6FKHLGHU:3ODVWLFLW\DQGIXQFWLRQ±WKH
IDWHRIDIUHHQHXURYDVFXODUPXVFOHJUDIWWHQ\HDUVSRVWUHFRQVWUXFWLRQ-
5HFRQVWU0LFURVXUJ
)HUUHWWL*0DUFRQL&$FKLOOL*HWDO7KHKHDUWUDWHUHVSRQVHWRH[HUFLVHDQG
FLUFXODWLQJFDWHFKRODPLQHVLQKHDUWWUDQVSODQWUHFLSLHQWV3ÀXJHUV$UFK
)RUEHV&'DQG/RZH*'2%ORRGYLVFRVLW\PHDVXUHPHQWLQ3K\VLFVLQ0HGLFLQH
DQG%LRORJ\(QF\FORSHGLD(GLW7)0F$LQVK3HUJDPRQ3UHVVSS
)RUVWHUPDQQ8&ORVV(3ROORFN-1DNDQH06FKZDU]3*DWK,DQG.OHLQHUW+
1LWULFR[LGHLVRHQ]\PHV&KDUDFWHUL]DWLRQSXUL¿FDWLRQPROHFXODUFORQLQJ
DQGIXQFWLRQV+\SHUWHQVLRQ
)XUQHVV - 0DUVKDOO - &RUUHODWLRQ RI WKH GLUHFWO\ REVHUYHG UHVSRQVHV RI WKH
PHVHQWHULF YHVVHOV RI WKH UDW WR QHUYH VWLPXODWLRQ DQG QRUDGUHQDOLQ ZLWK
WKHGLVWULEXWLRQRIDGUHQHUJLFQHUYHV-3K\VLRO
)X[H.$JQDWL/)9ROXPHWUDQVPLVVLRQLQWKHEUDLQQRYHOPHFKDQLVPVIRU
QHXUDOWUDQVPLVVLRQ1HZ<RUN5DYHQ3UHVV
*DVNHOO:+2QWKHRULJLQRIWKHFHQWUDOQHUYRXVV\VWHPRIYHUWHEUDWHV%UDLQ
/RQG;,,
*DVNHOO:+2QWKHUHODWLRQEHWZHHQWKHVWUXFWXUHIXQFWLRQGLVWULEXWLRQDQG
RULJLQRIWKHFUDQLDOQHUYHV7RJHWKHUZLWKDWKHRU\RIWKHRULJLQRIWKHQHUYRXV
V\VWHPRIWKHYHUWHEUDWD-3K\VLRO&DPEULGJH;
*DWHOH\ '5 0F$QXOW\ *5 0DUWLQ '/ ,QWUDYHQRXV LQIXVLRQ RI SURVWDF\FOLQ WR
SUHYHQW SODWHOHW WKURPEXV GXULQJ PLFURYDVFXODU DQDVWRPRVHV %U - 3ODVW
6XUJ
*LUHUG ; &KDPLRW&OHUF 3 &RSLH ; 5HQDXG - /DXUHQW 6 6DIDU 0 (IIHFWV RI
QRUHSLQHSKULQHRQWKHPHFKDQLFDOSURSHUWLHVRIWKHKXPDQUDGLDODUWHU\LQ
YLWUR$P+HDW-3W
*ODVV-DQG*ULI¿Q-1HXUR¿ODPHQWUHGLVWULEXWLRQLQWUDQVHFWHGQHUYHVHYLGHQFH
RIELGLUHFWLRQDOWUDQVSRUWRIQHXUR¿ODPHQWV-1HXURVFL
*RGGHQ'/LWWOH5:HVWRQ$*HHQVWHLQ$:RRGZDUGV5&DWHFKRODPLQHVHQVLWLYLW\
LQ WKH UDW IHPRUDO DUWHU\ DIWHU PLFURYDVFXODU DQDVWRPRVLV 0LFURVXUJHU\
D*RQ]DOH]+HUQDQGH]7DQG5XVWLRQL$([SUHVVLRQRIWKUHHIRUPVRIQLWULFR[LGH
V\QWKDVHLQSHULSKHUDOQHUYHUHJHQHUDWLRQ-1HXURVFL5HV
E *RQ]DOH]+HUQDQGH] 7 DQG 5XVWLRQL $ 1LWULF R[LGH V\QWKDVH DQG JURZWK
DVVRFLDWHG SURWHLQ DUH FRH[SUHVVHG LQ SULPDU\ VHQVRU\ QHXURQV DIWHU
SHULSKHUDOLQMXU\-&RPSDUDW1HXURO
*RRGVRQ $ /HLEROG - DQG *XWWHUPDQ ' ,QKLELWLRQ RI QLWULF R[LGH V\QWKHVLV
DXJPHQWVFHQWUDOO\LQGXFHGV\PSDWKHWLFFRURQDU\YDVRFRQVWULFWLRQLQFDWV
$P-3K\VLRO+
*R]HV , DQG )XUPDQ 6 3RWHQWLDO FOLQLFDO DSSOLFDWLRQV RI YDVRDFWLYH LQWHVWLQDO
SHSWLGH D VHOHFWHG XSGDWH %HVW 3UDFW 5HV &OLQ (QGRFULQRO 0HWDE *UDQGMHDQ-*0DULDQL0$(EHOV7&RURQDU\UHRSHUDWLRQYLDVPDOOODSDURWRP\
XVLQJULJKWJDVWURHSLSORLFDUWHU\ZLWKRXW&3%$QQ7KRU6XUJ
5()(5(1&(6
*UDQW03/DQGLV6&8QH[SHFWHGSODVWLFLW\DWDXWRQRPLFMXQFWLRQV(QYLURQPHQWDO
UHJXODWLRQRIQHXURWUDQVPLWWHUSKHQRW\SHDQGUHFHSWRUH[SUHVVLRQ%LRFKHP
3KDUPDFRO
*XVWDIVVRQ)+ROVWHQ5DWKORX1&RQGXFWHGYDVRPRWRUUHVSRQVHVLQDWHULROHV
&KDUDFWHULVWLFV PHFKDQLVPV DQG SK\VLRORJLFDO VLJQL¿FDQFH $FWD 3K\VLRO
6FDQG
*X\WRQDQG+DOOHG7H[WERRNRIPHGLFDOSK\VLRORJ\WKHGLWLRQ3KLODGHOSKLD
3D86$6DXGHUV
+DUGLQJ'*UHHQVPLWK/0DVRQ0$QGHUVRQ3DQG9UERYi*2YHUH[SUHVVLRQ
RI*$3LQGXFHVSURORQJHGVSURXWLQJDQGFDXVHVFHOOGHDWKRIDGXOWPRWRU
QHXURQV(XU-1HXURVFL
+H*<DQJ&&KDUDFWHULVWLFVRIDGUHQRUHFHSWRUVLQWKHKXPDQUDGLDODUWHU\&OLQLFDO
LPSOLFDWLRQV-7KRUDF&DUGLRYDVF6XUJ
+LJD7DQLJXFKL.6LOYD)6LOYD+0LFKHOLQL/DQG6WHUQ-([HUFLVHWUDLQLQJ
LQGXFHGUHPRGHOLQJRISDUDYHQWULFXODUQXFOHXVQRUDGUHQHUJLFLQQHUYDWLRQ
LQQRUPRWHQVLYHDQGK\SHUWHQVLYHUDWV$UWLFOHVLQ3UHV6$P-3K\VLRO5HJXO
,QWHJU&RPS3K\VLRO-DQXDU\
+LOODUS 1c 7KH FRQVWLWXWLRQ DQG IXQFWLRQDO RUJDQL]DWLRQ RI WKH DXWRQRPLF
LQQHUYDWLRQDSSDUDWXV$FWDSK\VLRO6FDQG6XSSO
+XNNL-$WWLOD0+DLNDOD+HWDO6HTXHQWLDOFKDQJHVRIQRUDGUHQDOLQFRQWHQW
RIYHLQJUDIWVLQUDWV4XDQWLWDWLYHHVWLPDWLRQE\KLJKSHUIRUPDQFHOLTXLG
FKURPDWRJUDSK\XVLQJHOHFWURFKHPLFDOGHWHFWLRQ0LFURVXUJ
+XV]WL='HOL0DQG-Ry)&DUULHUPHGLDWHGXSWDNHDQGUHOHDVHRIKLVWDPLQH
E\FXOWXUHGUDWFHUHEUDOHQGRWKHOLDOFHOOV1HXURVFL/HWW
,DGHFROD&5HJXODWLRQRIWKHFHUHEUDOPLFURFLUFXODWLRQGXULQJQHXUDODFWLYLW\LV
QLWULFR[LGHWKHPLVVLQJOLQN"7UHQGV1HXURVFL
,QD]X07DNHGD+DQG0DWVXPLD7)XQFWLRQDOH[SUHVVLRQRIWKHQRUHSLQHSKULQH
WUDQVSRUWHULQFXOWXUHGUDWDVWURF\WHV-1HXURFKHP
D.DDUHOD23HULYDVFXODUV\PSDWKHFWRP\RIWKHPHWDFDUSDODUWHU\RIWKHUDEELW
SDZ IDLO WR UHPRYH GLVWDO DGUHQHUJLF LQQHUYDWLRQ 6FDQG - 3ODVW 5HFRQVWU
+DQG6XUJ
E.DDUHOD25DDWLNDLQHQ7&DUOVRQ6+XRSDQLHPL7DQG:DULV7(IIHFWRI
SHULYDVFXODUV\PSDWHFWRP\RQGLVWDODGUHQHUJLFLQQHUYDWLRQLQWKHKDQGVRI
PRQNH\V-+DQG6XUJ%UYRO%
F.DDUHOD2+XWWXQHQ3-XQLOD-+DXNLSXUR.DQK:DULV7(IIHFWRISHULYDVFXODU
V\PSDWHFWRP\RQWKHQRUDGUHQDOLQFRQWHQWRIWKHGLVWDOYDVFXODWXUHRIWKH
UDEELWHDU(XU-3ODVW6XUJ
.DDUHOD 2 7RUQLDLQHQ 3 -XQLOD - DQG :DULV 7 (IIHFWV RI SHULYDVFXODU
V\PSDWKHFWRP\RQGLVWDOEORRGÀRZLQWKHUDEELWIRUHSDZ$QQ&KLU*\Q
.DQDJ\1/Į$GUHQHUJLFUHFHSWRUVLJQDOLQJLQK\SHUWHQVLRQ&OLQLFDO6FLHQFH
±
.DZDJXFKL $ 6KLUDL 0 <DPDQR 0 ,VKLEDVKL8HGD + <DPDWRGDQL $ DQG
.DZDVKLPD<$IIHUHQWUHLQQHUYDWLRQDIWHUOXQJWUDQVSODQWDWLRQLQWKHUDW
-+HDUW/XQJ7UDQVSODQW
.HQQH\0:HLVVDQG0+D\ZRRG-7KHSDUDYHQWULFXODUQXFOHXVDQLPSRUWDQW
FRPSRQHQW RI WKH FHQWUDO QHXURFLUFXLWU\ UHJXODWLQJ V\PSDWKHWLF QHUYH
RXWÀRZ$FWD3K\VLRO6FDQG±
.HQWURWL6DQG9HUQDGDNLV$(DUO\QHXUREODVWVDUHSOXULSRWHQWLDOFRORFDOL]DWLRQ
RIQHXURWUDQVPLWWHUVDQGQHXURSHSWLGHV-1HXURVFL5HV
.L\RFKL + 2QR $ <DPDPRWR 1 2QLVKL . 6KLPDKDUD < DQG .RED\DVKL 1
([WULQVLFV\PSDWKHWLFUHLQQHUYDWLRQDIWHULQWHVWLQDOWUDQVSODQWDWLRQLQUDWV
7UDQVSODQWDWLRQ
.MDHU0DQG6HFKHU1+([HUFLVHDQGRUJDQWUDQVSODQWDWLRQ6FDQG-0HG6FL
6SRUWV
.RUNDOD 2 DQG :DULV 7 7KH $FHW\OFKROLHVWHUDVH UHDFWLRQ DQG FDWHFKRODPLQH
ÀXRUHVFHQFHLQWKHJORPXVFHOOVRIUDWFDURWLGERG\([SHULHQWLD
.UROO666FKXVWHUPDQ0$5HHFH*3HWDO&KRLFHRIÀDSDQGLQFLGHQFHRIIUHHÀDS
VXFFHVV3ODVW5HFRQVWU6XUJ
.XSIHUPDQQ,)XQFWLRQDOVWXGLHVRQFRWUDQVPLVVLRQ3K\VLRO5HY
.\|VROD . :DULV 7 3HQWWLOl 2HW DO 0XOWLVFRUH HVWLPDWLRQ RI FDWHFKRODPLQH
ÀXRUHVFHQFHIRUFOLQLFDOSXUSRVHV$FWD+LVWRFKHP
.\|VROD.0DWWLOD7+DUMXOD$.\|VROD+:DULV7/LIHWKUHDWHQLQJFRPSOLFDWLRQV
RIFDUGLDFRSHUDWLRQVDQGRFFXUUHQFHRIP\RFDUGLDOFDWHFKRODPLQHERPEV
-7KRUDF&DUGLRYDVF6XUJ
/DQGLV6&4XLFNFKDQJHDUWLVWIURPH[FLWDWRU\WRLQKLELWRU\V\QDSVHLQPLQXWHV
1DW1HXURVFL
D/DQJOH\-2QWKHSK\VLRORJ\RIWKHVDOLYDU\VHFUHWLRQ-3K\VLRO&DPEULGJH
;
E/DQJOH\-2QWKHKLVWRORJ\RIWKHPXFRXVVDOLYDU\JODQGVDQGRQWKHEHKDYLRU
RIWKHLUPXFRXVFRQWHQWV-3K\VLRO&DPEULGJH;
/DVVPDQQ * DQG 3L]D + 7KH QHUYH UHVSRQVH DIWHU DXWRWUDQVSODQWDWLRQ RI WKH
UDEELWHDU-+DQG6XUJ$
5()(5(1&(6
/HH7./RLV-7URXSH-:LOVRQ7DQG<DWHV%7UDQVQHXURQDO7UDFLQJRI
1HXUDO3DWKZD\VWKDW5HJXODWH+LQGOLPE0XVFOH%ORRG)ORZ$UWLFOHVLQ3UHV
$P-3K\VLRO5HJXO,QWHJU&RPS3K\VLRO'HFHPEHUGRL
DMSUHJX
/HVWHU+0DJHU64XLFN0DQG&RUH\-3HUPHDWLRQSURSHUWLHVRIQHXURWUDQVPLWWHU
WUDQVSRUWHUV$QQ5HY3KDUPDFRO7R[LFRO
/HYHUHQ]-%0LOOHU0$'RELH'-3HVNLQG(55DVNLQG0$,QFUHDVHG
DOSKDDGUHQHUJLFUHFHSWRUELQGLQJLQORFXVFHUXOHXVSURMHFWLRQDUHDVLQ
GHPHQWLDZLWK/HZ\ERGLHV1HXURELRO$JLQJ
/LQGYDOO2%M|UNOXQG$7KHJO\R[\OLFDFLGÀXRUHVFHQFHPHWKRG$GHWDLOHGDFFRXQW
RIWKHPHWKRGRORJ\IRUWKHYLVXDOL]DWLRQRIFHQWUDOFDWHFKRODPLQHQHXURQV
+LVWRFKHPLVWU\
/LX4;LH)6LHGODN61XQRPXUD$+RQGD30RUHLUD3]KXD;6PLWK0
DQG3HUU\*1HXUR¿ODPHQWSURWHLQVLQQHXURGHJHQHUDWLYHGLVHDVHV&HOO
0RO/LIH6FL
/RUHQ]HWWL)6XRPLQHQ67XNLDLQHQ(.XRNNDQHQ+6XRPLQHQ(9XROD-DQG
$VNR6HOMDYDDUD6(YDOXDWLRQRIEORRGÀRZLQIUHHPLFURYDVFXODUÀDSV-
5HF0LFURVXUJ
/XPPH$9DQKDWDOR6DQG6RLQLOD6$[RQDOWUDQVSRUWRIQLWULFR[LGHV\QWKDVH
LQDXWRQRPLFQHUYHV-$XWRQ1HUY6\VW
/XQGEHUJ-bQJJnUG$)DKUHQNUXJ-+|NIHOW7DQG0XWW99DVRDFWLYHLQWHVWLQDO
SRO\SHSWLGH 9,3 LQ FKROLHQUJLF QHXURQV RI HQGRFULQH JODQGV IXQFWLRQDO
VLJQL¿FDQFHRIFRH[LVWLQJWUDQVPLWWHUVIRUYDVRGLODWLRQDQGVHFUHWLRQ3URF
1DWO$FDG6FL86$
0DOPIRUV76DFKV&'LUHFWVWXGLHVRQWKHGLVDSSHDUDQFHRIWKHWUDQVPLWWHUDQG
FKDQJHV LQ WKH XSWDNHVWRUDJH PHFKDQLVPV RI GHJHQHUDWLQJ DGUHQHUJLF
QHUYHV$FWD3K\VLRO6FDQG
0DUUDFN -5 'HULYHG DQWLJHQV DV D PHDQV RI VWXG\LQJ WKH UHODWLRQ RI VSHFL¿F
FRPELQDWLRQRIFKHPLFDOVWUXFWXUH3URF56RF0HGE0DUFK
0LNL $ )XMLPRWR ( 0L]RJXFKL $ HW DO 7UDXPDWLF GHJHQHUDWLRQ RI WUDQVHFWHG
P\HOLQDWHG ¿EHUV RI WKH PRXVH VFLDWLF QHUYH $UFK +LVWRO &\WRO ±
0RQGHOOL09HFFKLDUHOOL%5HDOH)0DUVLOL7*LDQQLQL)6\PSDWKHWLFVNLQ
UHVSRQVHEHIRUHDQGDIWHUVXUJLFDOUHOHDVHRIFDUSDOWXQQHOV\QGURPH0XVFOH
1HUYH
0RQFDGD 6 +LJJV $ 0HFKDQLVPV RI 'LVHDVH 7KH /$UJLQLQH1LWULF 2[LGH
3DWKZD\1(QJO-0HG
0RRUH ./ $JXU $0 (VVHQWLDO &OLQLFDO $QDWRP\ 7KLUG (GLWLRQ
%DOWLPRUH/LSSLQFRWW:LOOLDPV:LONLQV
0RUJDQ55HLVPDQ1DQG:LOJLV($QDWRPLFORFDOL]DWLRQRIV\PSDWKHWLFQHUYHV
LQWKHKDQG-+DQG6XUJ
0RUULV-*UDVE\'$QGHUVRQ5DQG*LEELQV,1HXURFKHPLFDOGLVWLQFWLRQEHWZHHQ
VNHOHWDOPXVFOHYDVRGLODWRUDQGSHOYLFYDVRGLODWRUQHXURQVLQJXLQHDSLJV-
$XWRQ1HUY6\VW
1DJD]X7/DYLWW0DQG8GHQIULHQG67\URVLQH+\GUR[\ODVH7KH,QLWLDO6WHSLQ
1RUHSLQHSKULQH%LRV\QWKHVLV-%LRO&KHP
1DWKDQ&1LWULFR[LGHDVDVHFUHWRU\SURGXFWRIPDPPDOLDQFHOOV)$6(%-
2%ULHQ%00RUULVRQ:$*XPOH\*-3ULQFLSOHVDQGWHFKQLTXHVRIPLFURYDVFXODU
VXUJHU\,Q0F&DUWK\-*HG3ODVWLFVXUJHU\3KLODGHOSKLD:%6DXQGHUV
&RPSDQ\
2GDND.YRQ6FKHLGW:=LHJOHU6,HWDO5HDSSHDUDQFHRIFDUGLDFSUHV\QDSWLF
V\PSDWKHWLFQHUYHWHUPLQDOVLQWKHWUDQVSODQWHGKHDUWFRUUHODWLRQEHWZHHQ
3(7XVLQJ&K\GUR[\HSKHGULQHDQGLQYDVLYHO\PHDVXUHGQRUHSLQHSKULQH
UHOHDVH-1XFO0HG
2OVRQ/0DOPIRUV7*URZWKFKDUDFWHULVWLFVRIDGUHQHUJLFQHUYHVLQDGXOWUDW$FWD
3K\VLRO6FDQG6XSSO
9DQ 2UGHQ / %HQFK . /DQJHU 6 7UHQGHOHQEXUJ 8 +LVWRFKHPLFDO DQG QH
VWUXFWXUDODVSHFWVRIWKHRQVHWRIGHQHUYDWLRQVXSHUVHQVLWLYLW\LQWKHQLFWLWDWLQJ
PHPEUDQHRIWKHVSLQDOFDW-$P3KDUPDFRO([S7KHU
3HUURQH&DSDQR&3HUQDV$ORQVR5DQGGL3RU]LR81HXUR¿ODPHQWKRPHRVWDVLV
DQGPRWRQHXURQHGHJHQHUDWLRQ%LRHVVD\V
3HW]ROG$O1HXUR¿ODPHQWSKRVSKRIRUPV6XUURJDWHPDUNHUVIRUD[RQDOLQMXU\
GHJHQHUDWLRQDQGORVV-RXUQDORIWKH1HXURORJLFDO6FLHQFHV
±
3OXWD57KRPSVRQ%'DZVRQ76Q\GHU6%RRFN5DQG2OG¿HOG(/RVVRI
QLWULFR[LGHV\QWKDVHLPPXQRUHDFWLYLW\LQFHUHEUDOYDVRVSDVP-1HXURVXUJ
3RSSD 9 0LDV\DVKLUR - &RUVRQ 0 DQG %HUN % (QGRWKHOLDO 12 V\QWKDVH LV
LQFUHDVHGLQUHJHQHUDWLQJHQGRWKHOLXPDIWHUGHQXGLQJLQMXU\RIWKHUDWDRUWD
$UWHULRVFOHU7KURPE9DVF%LRO
5DR 0 DQG /DQGLV 6 &HOO LQWHUDFWLRQV WKDW GHWHUPLQH V\PSDWKHWLF QHXURQ
WUDQVPLWWHU SKHQRW\SH DQG QHXURNLQHV WKDW PHGLDWH WKHP - 1HXURELRO
5HPHV9Y6PLWWHQ.:DULV75DHNDOOLR-+LVWRFKHPLFDOH[DPLQDWLRQRIHQHUJ\
PHWDEROLVPLQDRUWLFYHLQJUDIWV$FWD+LVWRFKHPLFD
5HPHV9Y6PLWWHQ.:DULV75DHNDOOLR-+LVWRFKHPLFDOH[DPLQDWLRQRIHQHUJ\
PHWDEROLVPLQDRUWLFYHLQJUDIWV&DUGLRYDVFXODU5HV2FW
5HPHV9Y6PLWWHQ.:DULV75DHNDOOLR-$VVHVVPHQWRIPLFURYDVFXODUFODPSLQJ
LQMXU\ RI UDW DRUWD E\ KLVWRFKHPLFDO H[DPLQDWLRQ RI R[LGRUHGXFWDVHV
0LFURVXUJHU\
5()(5(1&(6
5HQDXG)6XFFR($OHVV0&/HJUH5-XKDQ9DJXH,,ORSURVWDQGVDOYDJHRID
IUHHÀDS%U-3ODVW6XUJ
6DLG6DQG0XWW9$SHSWLGHIUDFWLRQIURPOXQJWLVVXHZLWKSURORQJHGSHULSKHUDO
YDVRGLODWRUDFWLYLW\6FDQG-&OLQ/DE,QYHVW6XSS
6DLG 6 DTQG 0XWW 9 ,VRODWLRQ IURP SRUFLQHLQWHVWLQDO ZDOO RI D YDVRDFWLYH
RFWDFRVDSHSWLGHUHODWHGWRVHFUHWLQDQGWRJOXFDFRQ(XU-%LRFKHP
6FKODHSIHU:+DVOHU07KHSHUVLVWHQFHDQGSRVVLEOHH[WHUQDOL]DWLRQRID[RQDO
GHEULV GXULQJ :DOOHULDQ GHJHQHUDWLRQ - 1HXURSDWKRO ([S 1HXURO ±
6FKRW]LQJHU5<LQ;DQG/DQGLV67DUJHWGHWHUPLQDWLRQRIQHXURWUDQVPLWWHU
SKHQRW\SHLQV\PSDWKHWLFQHXURQV-1HXURELRO
6FKUH\HU ' DQG 6NHQH - )DWH RI *$3 LQ DVFHQGLQJ VSLQDO D[RQV RI '5*
QHXURQVDIWHUSHULSKHUDOQHUYHLQMXU\GHOD\HGDFFXPXODWLRQRIFRUUHODWLRQ
ZLWKUHJHQHUDWLYHSRWHQWLDO-1HXURVFL
6FKURHGHU+HPRG\QDPLFSHUIRUPDQFHRIWKHKXPDQWUDQVSODQWHGKHDUW,Q5
&RUWHVLQLDQG)5DSSDSRUW(GV7UDQVSODQWDWLRQWRGD\3URFHHGLQJVRI
WKH6HYHQWK,QWHUQDWLRQDO&RQJUHVVRIWKH7UDQVSODQWDWLRQ6RFLHW\9RO9
*UXQHDQG6WUDWWRQ1HZ<RUNSS
6HJDO6,QWHJUDWLRQRIEORRGÀRZFRQWUROWRVNHOHWDOPXVFOH.H\UROHRIIHHGDUWHULHV
$FWD3K\VLRO6FDQG
6HLJHU c 'DKO ' $\HU/H/LHYUH & %M|UNOXQG + $SSHDUDQFH DQG GLVWULEXWLRQ
RI QHXUR¿ODPHQW LPPXQRUHDFWLYLW\ LQ ,ULV QHUYHV - &RPSDU 1HURO 6H\EROG960F&DUVRQ.(0HUPHOVWHLQ3**URWK5'$EUDKDPV/*&DOFLWRQLQ
JHQHUHODWHGSHSWLGHUHJXODWHVH[SUHVVLRQRIQHXURNLQLQUHFHSWRUVE\UDW
VSLQDOQHXURQV-1HXURVFL
6XWHUD636NDODN5³7KHKLVWRU\RI3RLVHXLOOH¶VODZ´$QQXDO5HYLHZRI)OXLG
0HFKDQLFV±
6X]XNL1+DUGHER-(2ZPDQ&+2ULJLQVDQGSDWKZD\VRIFHUHEURYDVFXODU
YDVRDFWLYHLQWHVWLQDOSRO\SHSWLGHSRVLWLYHQHUYHVLQWKHUDW-&HUHE%ORRG
)ORZ0HWDE±
6X]XNL1DQG+DUGHER-(.DKUVWURP-DQG2ZPDQ&+1HXURSHSWLGH<FRH[LVWV
ZLWKYDVRDFWLYHLQWHVWLQDOSRO\SHSWLGHDQGDFHW\OFKROLQHLQSDUDV\PSDWKHWLF
FHUHEURYDVFXODUQHUYHVRULJLQDWLQJLQWKHVSKHQRSDODWLQHRWLFDQGLQWHUQDO
FDURWLGJDQJOLDRIWKHUDW1HXURVFLHQFH±
6\NRYi(([WUDV\QDSWLFYROXPHWUDQVPLVVLRQSDUDPHWHUVRIWKHH[WUDFHOOXODUVSDFH
1HXURVFLHQFH±
7DNDFKL00DHGD76KLUDNXVDDQG<+D\DVKLGD6\PSDWKHWLFUHLQQHUYDWLRQ
RIXQLODWHUDOO\GHQHUYDWHGUDWOXQJ$FWD3K\VLRO6FDQG
7DQ0DQG+DUYH\$5HWLQDOD[RQUHJHQHUDWLRQLQSHULSKHUDOQHUYHWHFWDODQG
PXVFOHJUDIWVLQDGXOWUDWV-&RPS1HXURO
7DXROLV--LDQJ*&0LIIDW-'&RFNV706WRUDJHRIUDGLDODUWHU\JUDIWVLQEORRG
LQFUHDVHVYHVVHOUHDFWLYLW\WRYDVRFRQVWULFWLRQ$QQ7KRUDF6XUJ
7D\ORU 5% 5HLG 5 .HQGOH .( *HGGHV & &XUOH 3) $VVD\ SURFHGXUHV IRU
GHWHUPLQDWLRQRIELRJHQLFDPLQHVDQGWKHLUPHWDEROLWHVLQUDWK\SRWKDODPXV
XVLQJLRQSDLULQJUHYHUVHGSKDVHKLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\
-&URPDWRJU
7KHQJFKDLVUL1DQG5LYHUV55HPRWHDUWHULRODUGLODWLRQVFDXVHGE\PHWKDFKROLQH
DUROHIRU&*53VHQVRU\QHUYHV"$UWLFOHVLQ3UHV6$P-3K\VLRO+HDUW&LUF
3K\VLRO0DUFKGRLDMSKHDUW
7RLYROD'07DR*=+DEWH]LRQ$/LDR-DQG%LVKU2PDU\0&HOOXODULQWHJULW\
SOXV RUJDQHOOHUHODWHG DQG SURWHLQWDUJHWLQJ IXQFWLRQV RI LQWHUPHGLDWH
¿ODPHQWV7UHQGLQ&HOO%LRORJ\9RO1R1RYHPEHU
D 8EHUIXKU 3 )UH\ $ =LHJOHU 6 5HLFKDUW % DQG 6FKZDLJHU 0 6\PSDWKHWLF
UHLQQHUYDWLRQRIVLQXVQRGHDQGOHIWYHQWULFOHDIWHUKHDUWWUDQVSODQWDWLRQLQ
KXPDQV5HJLRQDOGLIIHUHQFHVDVVHVVHGE\KHDUWUDWHYDULDELOLW\DQGSRVLWURQ
HPLVVLRQWRPRJUDSK\-+HDUW/XQJ7UDQVSODQW
E 8EHUIXKU 3 =LHJOHU 6 6FKZDLEOPDLU 0 5HLFKDUW % DQG 6FKZDLJHU 0
,QFRPSOHWH V\PSDWKHWLF UHLQQHUYDWLRQ LQ WKH RUWKRWRSLFDOO\ WUDQVSODQWHG
KXPDQKHDUW2EVHUYDWLRQXSWR\HDUVDIWHUKHDUWWUDQVSODQWDWLRQ(XU
-&DUGLRWKRUDF6XUJ
8OULFK/DL<DQG(QJHODQG:5DWDGUHQDOWUDQVSODQWVDUHUHLQQHUYDWHGDQLQYDOLG
PRGHORIGHQHUYDWHGDGUHQDOWLVVXH-1HXURHQGRFULQRO
D69DQKDWDORDQG66RLQLOD'LUHFWQLWULFR[LGHFRQWDLQLQJLQQHUYDWLRQIURPWKH
UDWVSLQDOFRUGWRWKHSHQLV1HXURVFL/HWW
E9DQKDWDOR66RLQLOD6.DDUWLQHQ.DQG%lFN1&RORFDOL]DWLRQRIGRSDPLQHDQG
VHURWRQLQLQWKHUDWSLWXLWDU\JODQGDQGLQWKHQXFOHLLQQHUYDWLQJLW%UDLQ
5HV
F9DQKDWDOR66RLQLOD6'RSDPLQHUJLFSHULYHQWULFXORK\SRSK\VHDOQHUYHVKRZ
WU\SWRSKDQK\GUR[\ODVH LPPXQRUHDFWLYLW\ EXW ODFN VHURWRQLQ V\QWKHVLV
%UDLQ5HV
:DOOHU$([SHULPHQWVRQWKHVHFWLRQRIWKHJORVVRSKDU\QJHDODQGK\SRJORVVDO
QHUYHVRIWKHIURJDQGREVHUYDWLRQVRIWKHDOWHUDWLRQSURGXFHGWKHUHE\LQ
WKHVWUXFWXUHRIWKHLUSULPLWLYH¿EHUV3KLORV7UDQV56RF/RQG
:DULV73DUWDQHQ6'HPRQVWUDWLRQRIFDWHFKRODPLQHVLQSHULSKHULDODGUHQHUJLF
QHUYHVLQVWUHWFKSUHSDUDWLRQZLWKÀXRUHVFHQFHLQGXFHGE\DTXHRXVVROXWLRQ
RIJO\R[\OLFDFLG+LVWRFHPLVWU\
5()(5(1&(6
D:DULV75HFKDUGW/3DUWDQHQ66LPXOWDQHRXVDQGFRQVHFXWLYHKLVWRFKHPLFDO
GHPRQVWUDWLRQRIJO\R[\OLFDFLGLQGXFHGÀXRUHVFHQFHRIFDWHFKRODPLQHVDQG
FKROLQHVWHUDVHUHDFWLRQV&HOO0RO%LRO±
E :DULV 7 3DQXOD 3 5HFKDUG / 6KRUW FRPPXQLFDWLRQ 'LVWULEXWLRQ RI
FDWHFKRODPLQHV DQG DFHW\OFKROLQHVWHUDVH DFWLYLW\ LQ WKH VDPH FU\RWRPH
VHFWLRQRIUDWVQHRVWULDWXP&HOO0RO%LRO
F:DULV7'HJHQHUDWLRQDQGUHJHQHUDWLRQRIQHUYHVLQDGRUVDOVNLQÀDSLQWKHUDW
6FDQG-3ODVW5HFRQVWU6XUJ±
:DULV7.\|VROD.DQG3DUWDQHQ67KHDGUHQHUJLFLQQHUYDWLRQRIDUWHULRYHQRXV
DQDVWRPRVHVLQWKHVXEFXWDQHXVIDVFLDRIWKHUDWVNLQ6FDQG-3ODVW5HFRQVWU
6XUJ
:DULV7+XNNL-/lKWHHQPlNLW/|IVWHGW7.\|VROD.DQGYRQ6PLWWHQ.
$GUHQHUJLFLQQHUYDWLRQRIDRUWLFSDWFKJUDIWVLQUDWV6FDQG-3ODVW5HFRQVWU
6XUJ
:DULV7.DDUHOD2/DVDQHQ/-XQLOD-5XXVNDQHQ0.\|VROD.3HULYDVFXODU
V\PSDWKHFWRP\GRHVQRWUHPRYHDGUHQHUJLFQHUYHVIURPGLVWDQWYHVVHOV-
6XUJ5HV
9HQWXUD 6 'HZDODJDPD 5. /DX /& $GHQRVLQH WULSKRVSKDWH $73 LV DQ
H[FLWDWRU\ FRWUDQVPLWWHU ZLWK QRUDGUHQDOLQH WR VPRRWK PXVFOH RI WKH UDW
SURVWDWHJODQG%U-3KDUPDFRO
:UD\ ' 'RQDWR $ 8EHURL $ 0HUORQH - DQG 5LFKDUGVRQ 5 2QVHW H[HUFLVH
K\SHUDHPLDLQKXPDQVSDUWLWLRQLQJWKHFRQWULEXWRUV-3K\VLRO
SS±
;LH4:DQG1DWKDQ&0DFURSKDJHGHULYHGQLWULFR[LGHJHQHUDWLRQDQG
FRQVHTXHQFHV ,Q &HOOXODU DQG 0ROHFXODU 0HFKDQLVPV RI ,QÀDPPDWLRQ
&RFKUDQH & DQG *LPEURQH 0 HGV $FDGHPLF 3UHVV 1HZ <RUN 1HZ
<RUNSS
<DQJ%6ORQLPVN\-'%LUUHQ6-$UDSLGVZLWFKLQV\PSDWKHWLFQHXURWUDQVPLWWHU
UHOHDVHSURSHUWLHVPHGLDWHGE\WKHSUHFHSWRU1DW1HXURVFL
<RNRKDPD77RVD<.DGRPDWVX.6DWR.DQG+RVDND<$QRYHODSSURDFKIRU
SUHYHQWLQJ WKH GHYHORSPHQW RI SHUVLVWHQW YDVRVSDVPV DIWHU PLFURVXUJHU\
IRU WKH H[WUHPLWLHV LQWHUPLWWHQW WRSLFDO OLGRFDLQH DSSOLFDWLRQ - 5HFRQVWU
0LFURVXUJ)HE(SXE'HF
<X-.LPXUD7&KDQJ;DQG/HH76HJUHJDWLRQRI9,3HUJLFQLWULFR[LGHUJLFDQG
FKROLQHUJLFQLWULFR[LGHUJLFLQQHUYDWLRQLQSRUFLQHPLGGOHFHUHEUDODUWHULHV
%UDLQ5HVHDUFK
<X-73DWHO$-DQG0DODWD&07KHXVHRIWRSLFDOYDVRGLODWRUVLQPLFURYDVFXODU
VXUJHU\-3ODVW$HVWKHW6XUJ)HE(SXE0D\
<XQ+<'DZVRQ9DQG'DZVRQ71LWULFR[LGHLQKHDOWKDQGGLVHDVHRIWKH
QHUYRXVV\VWHP0RO3V\FK±
=DXJJ06FKDXE0*HQHWLF0RGXODWLRQRI$GUHQHUJLF$FWLYLW\LQWKH+HDUWDQG
9DVFXODWXUH,PSOLFDWLRQVIRU3HULRSHUDWLYH0HGLFLQH$QHVWKHVLRORJ\
±‹