TRAUMATIC BRAIN INJURY AND ACIDOSIS

8/13/2019 TRAUMATIC BRAIN INJURY AND ACIDOSIS
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TRAUMATIC BRAIN INJURY AND ACIDOSIS
by
LISA MARIE FORCE
A THESIS
Presented to the Deprt!ent o" B#o$o%y
nd the Honors Co$$e%e o" the Un#&ers#ty o" Ore%on
#n prt#$ "'$"#$$!ent o" the re('#re!ents"or the de%ree o" B)he$or o" S)#en)e*
No&e!ber +,,-

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Copyr#%ht +,,- L#s For)e
+

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An Abstr)t o" the Thes#s o" L#s For)e "or the de%ree o" B)he$or o" S)#en)e #n theDeprt!ent o" B#o$o%y to be t.en No&e!ber +,,-*
T#t$e/ Tr'!t#) Br#n In0'ry nd A)#dos#s
Appro&ed/11111111111111111111111111111111111111Nthn T'b$#t23 PhD*
Tr'!t#) Br#n In0'ry 4TBI5 #s s#%n#"#)nt )'se o" deth #n the Un#ted Sttes3 nd #s
the $ed#n% )'se o" d#sb#$#ty #n )h#$dren nd yo'n% d'$ts* Acidosis#s one o" the !ost
"re('ent se)ondry br#n #n0'r#es nd #s sso)#ted 6#th $on%er Intens#&e Cre nd
hosp#t$ $en%th o" sty* Th#s thes#s #n&est#%ted the #nter)t#on bet6een pr#!ry
!e)hn#)$ #n0'ry nd se)ondry acidosis#n0'ry 's#n% n #nno&t#&e e7per#!ent$ TBI
!ode$ )ons#st#n% o" organotypic)'$t'red rt hippocampus* Propidium Iodide#!%#n%
6s 'sed to ssess per)ent celldeth3 6#th res'$ts tentt#&e$y s'%%est#n% n dd#t#&e
rther thn sens#t#2#n% #nter)t#on bet6een TBInd acidosis* Ho6e&er3 n$ys#s o" the
dentte %yr's #nd#)ted tht there !y be both re%#on$ spe)#"#)#ty o" &'$nerb#$#ty nd
re%#on$ #nter)t#on bet6een TBInd acidosis* Amiloridendpsalmotoxin6ere
#n&est#%ted s poss#b$e ne'roprote)t#&e %ents3 6#thpsalmotoxinb't not amiloride
prod')#n% pro!#s#n% res'$ts "or red')#n% celldeth by acidosis* Immunocytochemistry
nd Western blotte)hn#('es 6ere 't#$#2ed to )on"#r! presen)e o" ASIC)hnne$s #n the
organotypicrt hippocampus)'$t'res3 6#th res'$ts de!onstrt#n% presen)e o" ASIC8
nd ASIC+ #n )ontro$ )'$t'res3 nd de)rese #n ASIC8 )on)entrt#on "o$$o6#n% TBI*
Dendr#t#) spine imaging6s 'sed to e7p$ore synpt#) str')t'r$ )hn%es res'$t#n% "ro!
TBInd acidosis* The dt obt#ned "ro! these #!%#n% st'd#es s'%%ests tht "o$$o6#n%
n #n0'ry dendr#t#) spinedens#ty de)reses 6h#$e varicositydens#ty #n)reses3 #!p$y#n%
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tht the !e)hn#s!s o" celldeth "or TBInd acidosisre $#.e$y not spine!ed#ted*
T.en to%ether3 these res'$ts "'rther o'r 'nderstnd#n% o" the #nter)t#ons bet6een TBI
nd acidosisnd re #!portnt "or )ons#der#n% the )$#n#)$ tret!ent o" TBI* F't're
reser)h sho'$d #n&est#%te "'rther the re%#on$ spe)#"#)#ty o" #n0'ry3psalmotoxins
poss#b$e ne'roprote)t#&e %ent3 nd the ro$e o" dendr#t#) spinere!ode$#n% "o$$o6#n% TBI
nd:or acidosis*
;

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ACEMENTS
The 'thor 6o'$d $#.e to %#&e Theres L'srd#3 #n"#n#te thn.s "or her )onstnt
en)o'r%e!ent3 s'pport3 nd s'%%est#ons* Thn. yo' $so to J#$$#n Hnsen "or herhe$p 6#th e7per#!ents nd so$'t#ons3 to ?h#%n% @#on% "or h#s #n"#n#te .no6$ed%e
bo't the s'b0e)t3 nd to S#!on Tho!pson "or the dendr#t#) sp#ne #!%#n% n$ys#s
too$s* In dd#t#on3 M#n%H' L#3 @#n%P#n% Ch'3

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TABLE OF CONTENTS
Chpter P%e
I* INTRODUCTION***************
II* MATERIALS AND METHODS****+9
III* RESULTS9
I* DISCUSSION**8
* FOOTNOTES**-,
I* >LOSSARY****-+
II* REFERENCES****-
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LIST OF FI>URES
F#%'re P%e
F#%'re 8 / pH s)$e 8,
F#%'re + / Opt#!$ )e$$ "'n)t#on bsed on pH 88F#%'re 9 / Lo)t#on o" the h#ppo)!p's #n the h'!n br#n 8;
F#%'re ; / Crossse)t#on o" the rt br#n 8
F#%'re / Pos#t#on o" the h#ppo)!p's #n the rt br#n 8
F#%'re - / Lo)t#on o" the s#7 !0or re%#ons o" the rt h#ppo)!p's 8-
F#%'re G / I!%e o" the rt h#ppo)!p's 8GF#%'re / D#%r! o" the !#n #ntr#ns#) )onne)t#ons o" the rt h#ppo)!p's 8G
F#%'re / Str')t're o" n #on )hnne$ 8F#%'re 8, / I$$'strt#on o" )on"or!t#ons o" $#%nd%ted #on )hnne$ 8
F#%'re 88 / PI #!%es +;
F#%'re 8+ / I$$'strt#on o" the !ode$ ne'ron +-F#%'re 89 / E7!p$e #!%e o" dendr#t#) sp#nes +G
F#%'re 8; / D#%r! o" sher str#n 98
F#%'re 8 / I!%es o" s#76e$$ p$te nd !#$$#)e$$ 98
F#%'re 8- / Tb$e o" e('#&$ent d#$ $e&e$s nd #!p)t &e$o)#t#es 9+F#%'re 8G / Crtoon dep#)t#on o" the TBI #n0'ry de)e 99
F#%'re 8 / Photo o" the TBI #n0'ry de)e 99
F#%'re 8 / T#!e )o'rse o" e7per#!ents 9;F#%'re +, / Do'b$e H#t )e$$ deth res'$ts "ro! PI dt 9
F#%'re +8 / Dentte >yr's )e$$ deth res'$ts "ro! PI dt ;,
F#%'re ++ / A!#$or#de )e$$ deth res'$ts "ro! PI dt ;+F#%'re +9 / eno! )e$$ deth res'$ts "ro! PI dt ;;
F#%'re +; / I!!'no)yto)he!#stry res'$ts ;
F#%'re + / ASIC8 =estern b$ot res'$ts ;-F#%'re +- / ASIC+ =estern b$ot res'$ts ;
F#%'re +G / Se$e)ted #!%es o" dendr#t#) sp#ne res'$ts ;
F#%'re + / Tb$e o" dendr#t#) sp#ne dens#ty nd &r#)os#ty dens#ty res'$ts ;
F#%'re + / Dendr#t#) sp#ne dens#ty res'$ts ,F#%'re 9, / r#)os#ty dens#ty res'$ts ,
G

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Introduction
One o" the !ost re!r.b$e3 )o!p$e73 nd !yster#o's str')t'res #n the 6or$d #s
the h'!n br#n* The br#n $$o6s "or per)ept#on3 behor3 tho'%ht3 $n%'%e3 $ern#n%3
nd !e!ory3 pro)esses tht de"#ne nd !.e so e7('#s#te o'r &ery e7#sten)e* The h'!n
br#n )ons#sts o" ppro7#!te$y 8,8+#nd#d'$ neurons3 the d#s)rete3 "'n)t#on$ 'n#ts o"
the br#n 4Tho!pson3 +,,,5* Th#s n'!ber o" neurons#s %reter thn $$ the strs #n the
ent#re M#$.y =y %$7y A$$ neuronsshre the s!e bs#) r)h#te)t're nd propert#es3
b't re b$e to prod')e &ery d#""erent3 )o!p$e7 res'$ts d'e to the 6y tht they re
)onne)ted 6#th e)h other 4

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Co!!on d#sb#$#t#es $so #n)$'de prob$e!s 6#th cognition4th#n.#n%3 !e!ory3 nd
reson#n%53 sensory processing4s#%ht3 her#n%3 to')h3 tste3 nd s!e$$53 communication
4e7press#on nd 'nderstnd#n%53 nd behavior or mental health4depress#on3 n7#ety3
person$#ty )hn%es3 %%ress#on3 )t#n% o't3 nd so)#$ #nppropr#teness5 4NIH 6ebs#te
85* =#th s')h h#%h !orb#d#ty3 !ort$#ty3 nd d#sb#$#ty3 the tret!ent nd pre&ent#on o"
TBIre top#)s o" s'bstnt#$ )$#n#)$ $#tert're nd re estb$#shed p'b$#) he$th )on)erns*
Ho6e&er3 the !e)hn#s!s 'nder$y#n% the d#""erent "or!s o" br#n #n0'ry nd e""e)t#&e
tret!ent re not 6e$$ 'nderstood*
TBI)'ses neurondeth s d#re)t res'$t "ro! the phys#)$3 nto!#)$ #n0'ry t
the t#!e o" #!p)t* Th#s type o" ne'ron deth #s d'e to the primary injury* For
#nstn)e3 #" b'$$et enters the br#n3 #t phys#)$$y d#sr'pts the br#ns str')t're3 .#$$#n% the
neurons$on% #ts pth* Ho6e&er3 TBIrre$y o))'rs #n #so$t#on the primary injury#s
typ#)$$y "o$$o6ed by secondaryinjury3 6h#)h #s 's'$$y de$yed nd !ore 6#despred
4Jere!#ts.y et $*3 +,,95* Typ#)$ secondary injuries#n)$'de metabolic imbalances
4prob$e!s 6#th the )he!#)$ pro)esses ne)essry "or the !#ntenn)e o" $#"e53 anoxia
4red')ed s'pp$y or bsen)e o" o7y%en53 hypoxia4#ns'""#)#ent $e&e$s o" o7y%en #n the
t#ss'e53 edema4s6e$$#n% o" the br#n53 excitotoxicity)o!p$#)t#ons 46hen the #n0'ry
)'ses the re$ese o" h#%h $e&e$s o" n excitatoryne'rotrns!#tter3 )'s#n% celldeth by
e7)ess#&e )t#&t#on o" #ts re)eptors53 ischemia4 de)rese #n the b$ood s'pp$y to prt
or $$ o" the t#ss'e53 nd:or acidosis4n bnor!$ #n)rese #n the )#d#ty o" the "$'#ds
s'rro'nd#n% the t#ss'e5 4Chesn't et $*3 +,,; >ennre$$#3 89 Jere!#ts.y et $*3 +,,9
Unterber% et $*3 +,,;5*

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The )#d#ty o" the b$ood nd interstitial luids4"$'#d o'ts#de the )rd#o&s)'$r
syste!5 #s !es'red $on% the KpH spe)tr'!3 6#th !ore )#d#) pHs be#n% $o6er &$'es
nd !ore bs#) pHs be#n% h#%her &$'es* The pH spe)tr'! r'ns "ro! pH o" , to pH
o" 8;3 6#th )#d#) #te!s $#.e $e!on 0'#)e hn% pH o" + nd bs#) #te!s $#.e b$e)h nd
sop hn% pH o" 89* P're 6ter hs pH o" G3 d#re)t$y #n the !#dd$e o" the spe)tr'!3
nd th's 6o'$d not be )$ss#"#ed s e#ther )#d#) or bs#)* Nor!$ physiologicalpH #s G*;
4see F#%'re 8 "or )$r#"#)t#on53 nd !ost cells"'n)t#on opt#!$$y t th#s prt#)'$r pH*
=hen the pH sh#"ts too "r "ro! G*; #n e#ther d#re)t#on3 the prod')t#ty o" cells
de)reses3 nd t "rther d#stn)es "ro! pH o" G*;3 the cellse&ent'$$y d#e* F#%'re +
#$$'strtes th#s )on)ept*
Figure 1* pH s)$e*Fro! 666*ep*%o&:***:s#te1st'dents:#!%es:phs)$e*%#"*
8,
http://www.epa.gov/.../site_students/images/phscale.gifhttp://www.epa.gov/.../site_students/images/phscale.gif

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Figure 2* The y7#s represents ho6 6e$$ the )e$$ #s "'n)t#on#n%*
Note ho63 the "'rther the d#stn)e "ro! the opt#!$ pH 4's'$$ythe phys#o$o%#)$ pH o" G*;53 the !ore tht the )e$$s proper
"'n)t#on#n% #s #!p#red* Fro!
http/::666*b#o$o%y)orner*)o!:reso'r)es:en2y!e1ph1%rph*%#"*
Fo$$o6#n% TBI3 the typ#)$ nor!$ "'n)t#ons o" neurons!y be $tered*
!euronsre )ont#n'$$y prod')#n% )#d#) !o$e)'$es3 b't the !e)hn#s!s "or re!on%
these !o$e)'$es "ro! the neuronsnd the s'rro'nd#n% extracellular luid!y be
)o!pro!#sed s res'$t o" the TBI* Th's3 "ter TBI3 the )#d#) !o$e)'$es )n
))'!'$te nd )'se "'rther hr! to the neuronsd'e to s')h h#%h )on)entrt#ons 4

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"'n)t#on 4@#on% et $*3 +,,;5* "lycolysis#s the 6y the body prod')es ener%y3 nd
anaerobic glycolysis#s the !ethod "or prod')#n% ener%y tht the body resorts to 6hen
there #s $#tt$e or no o7y%en present 4&ers's aerobic glycolysis3 6h#)h o))'rs #n the
presen)e o" o7y%en5 4S#$&erthorn3 +,,;5* Anaerobic glycolysisprod')es $)t#) )#d s
byproduct3 )'s#n% the br#ns pH to "$$ to $e&e$s ro'nd -*, 4

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shearstrain4!e)hn#)$ de"or!t#on5 o" the t#ss'e d'r#n% TBI4Morr#son et $*3 85*
To o&er)o!e these prob$e!s3 n in vitro#n0'ry !ode$ 6s de&e$oped by !e!bers o"
both the Do6 Ne'rob#o$o%y Lb nd the B#o!e)hn#)s Lb9* Th#s e7per#!ent$ !ode$
'ses threed#!ens#on$ organotypicbr#n )'$t'res rther thn !ono$yer o" cells* Th#s
threed#!ens#on$ t#ss'e preser&es the in vivoheterogeneouscellpop'$t#on nd
cytoarchitecture 4)e$$'$r rrn%e!ent53 prod#n% cells n)hored on $$ s#des to
s'rro'nd#n% cells* The organotypict#ss'e th's !ore ))'rte$y represents the cells
sens#n% o" nd response to !e)hn#)$ str#n 4>h6#$er et $*3 8G Stopp#n# et $*3
885* These organotypicbr#n s$#)es s'r&e #n )ontro$$ed )'$t're enron!ent "or
pro$on%ed per#ods o" t#!e;3 $$o6#n% "or )ontro$$ed sett#n% )hr)ter#st#) o" in vitro
e7per#!ents 4Bhr et $*3 85*
In the e7per#!ents des)r#bed #n th#s thes#s3 the organotypict#ss'e 'sed 6s
)'$t'red "ro! the rt br#n* Rts 6ere se$e)ted s the !ode$ or%n#s! s h'!n br#n
t#ss'e )$er$y )o'$d not be obt#ned3 nd !o'se 6ho$ebr#n )'$t'res $ose the#r %ross
integrity4str')t're5 t re$t#&e$y $o6 #n0'ry $e&e$s* Rt t#ss'e3 by )ontrst3 ret#ns #ts
%ross integritye&en t #n0'ry $e&e$s o&er t6#)e the ))e$ert#on $e&e$ 6here !o'se t#ss'e
deter#ortes 4person$ )o!!'n#)t#on 6#th T* L'srd#3 +,,-5* Pr#!ry rt )'$t'res re
one o" the !ost )on&en#ent nd )ost e""e)t#&e !ethods nd re 6#de$y 't#$#2ed to st'dy
&r#o's ne'ron$ deth !e)hn#s!s 4Cho# et $*3 8G >o"orth et $*3 85*
F'rther!ore3 rts re !ore )pb$e t per"or!#n% behor st'd#es thn re !#)e3 so 's#n%
rt t#ss'e &o#ds $#!#t#n% "'t're reser)h poss#b#$#t#es 6#th th#s !ode$* F#n$$y3 rts re
the $est phy$o%enet#)$$y d&n)ed n#!$ .no6n to shre phys#o$o%y nd ptho$o%#)$
response to TBI6#th h'!ns* In 's#n% rt t#ss'e3 the ss'!pt#on 6s !de tht h'!n
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TBI)n be rep$#)ted #n the rt nd tht the spe)#es d#""eren)es 4>ennre$$#3 8;53 6h#$e
)$er$y present3 6ere not s#%n#"#)nt eno'%h to pre&ent poss#b$e )on)$'s#ons be#n%
e7trpo$ted to the !e)hn#s!s nd tret!ent o" h'!ns*
The rt nd h'!n br#n re not homogenous* The br#n hs d#st#n)t "'n)t#on$
re%#ons3 e)h o" 6h#)h #s nto!#)$$y #dent#"#b$e* These re%#ons #n)$'de3 b't re not
$#!#ted to3 the hypothalamus4$o)ted #n the diencephalonnd respons#b$e "or 'to!t#)
"ee$#n%s s')h s h'n%er53 the basal ganglia4$o)ted #n the cerebral hemispheres#n the
orebrainnd re%'$tor o" !otor per"or!n)e53 nd the hippocampus4$so $o)ted #n
the )erebr$ hemispheres nd #n&o$&ed 6#th !e!ory stor%e5 4

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Figure 4* Crossse)t#on o" the rt br#n*
Fro! http/::666*b#o*ddson*ed':)o'rses:%eno!#)s:!ethod:br#n9*0p%*
Figure 5* Pos#t#on o" h#ppo)!p's #n the rt br#n*
Fro! http/::666*d'.e*ed':!6h#te:Ado$es)en)e:h#ppo)!p$s$#)e*0p%*
The hippocampus#s .no6n to "'n)t#on #n stor#n% Kep#sod#) !e!or#es3 or !e!or#es o"
e&ents nd ep#sodes 4

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o" !e!ory "or!t#on #s n #de$ port#on o" the br#n to st'dy #n e7p$or#n% TBIs#n)e
!e!ory dys"'n)t#on #s so pre&$ent "o$$o6#n% hed #n0'r#es-* F'rther!ore3 the
hippocampus#s re$t#&e$y homogenousnd 6e$$)hr)ter#2ed str')t're #n the rt
br#n nd ppers to be se$e)t#&e$y &'$nerb$e to #n0'ry 4>eddes et $*3 +,,;5*
The hippocampus#tse$" #s )o!posed o" se&er$ d#s)rete se)t#ons* The
hippocampus proper)ons#sts o" three "#e$ds 4CA83 CA+3 nd CA953 6h#)h3 to%ether
6#th "#&e other se)t#ons 4the dentte %yr's3 s'b#)'$'!3 pres'b#)'$'!3 prs'b#)'$'!3 nd
entorh#n$ )orte75 !.e 'p 6ht #s )o!!on$y )$$ed the hippocampus4A!r$ nd
=#tter3 85* F#%'res - nd G respe)t#&e$y dep#)t s)he!t#) d#%r! nd re$ #!%e o"
the hippocampus3 $be$ed to #d #n #dent#"y#n% these s#7 "ore!ent#oned re%#ons* The
!ost re$e&nt re%#ons "or th#s thes#s re the CA83 CA+3 CA93 6h#)h "or! KCshpe3
nd the dentte %yr's3 6h#)h "or!s s!$$er KCshpe )p ro'nd the CA9 end o" the
hippocampus proper*
Figure 6* Th#s "#%'re #$$'strtes the re$t#&e $o)t#on o" the s#7 !0or re%#ons o" the rt
h#ppo)!p's on hor#2ont$ s$#)e thro'%h the h#ppo)!p's* A* I!!'no)yto)he!#stry
#!%e* A* D#%r!/ ECEntorh#n$ Corte73 PSPrs'b#)'$'!3 PrSPres'b#)'$'!3SS'b#)'$'!3 CA8:+:9 Corn' A!!on#s3 D>Dentte >yr's* Fro! A!r$ nd
=#tter3 8*
8-

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Figure 7* An #!%e o" the rt h#ppo)!p's*
D>Dentte >yr's3 CA8:+:9 Corn' A!!on#s* Fro!
http/::)ontent*ns6ers*)o!:!#n:)ontent:6p:en::b:
H#ppo)!p$Re%#ons*0p%*
The hippocampus#s or%n#2ed $#.e n o&$ tr).3 6#th e)h o" the s#7 re%#ons
)onne)ted3 one to the ne7t3 by !ost$y 'n#d#re)t#on$ pro0e)t#ons* In"or!t#on "$o6s "ro!
the entorh#n$ )orte7 to the dentte %yr's the per"ornt pth6y3 the dentte %yr's
ne'rons 4.no6n s dentte %rn'$e )e$$s5 pro0e)t the#r !ossy "#bers to the CA9 "#e$d3 the
CA9 "#e$d trns!#ts #n"or!t#on to the CA8 s)h""er )o$$ter$sG3 nd the CA8 sends
#n"or!t#on to the s'b#)'$'! nd cortical4)orte73 the o'ter $yers o" the br#n 6h#)h
"'n)t#on #n cognition5 re%#ons o" the br#n* F#%'re #$$'strtes th#s 'n#('epttern o"
#n"or!t#on "$o6*
Figure 8* Th#s d#%r! dep#)ts the !#n #ntr#ns#) )onne)t#ons
o" the rt h#ppo)!p's* Note ho6 the !0or#ty o" pth6ys
re 'n#d#re)t#on$* ECEntorh#n$ Corte73 PSPrs'b#)'$'!3
PrSPres'b#)'$'!3 SS'b#)'$'!3 CA8:+:9Corn' A!!on#s3 D>Dentte >yr's*
Fro! A!r$ nd =#ter3 8*
8G
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On$y organotypicrt h#ppo)!p$ s$#)es 6ere 't#$#2ed #n the e7per#!ents #n th#s
thes#s* The s$#)es 6ere #n0'red 's#n% de)e tht 6#$$ be des)r#bed #n the Mter#$s nd
Methods se)t#on o" th#s thes#s* The #nno&t#&e TBI!ode$3 des#%ned spe)#"#)$$y "or
these e7per#!ents3 de$#&ers reprod')#b$e3 s)$b$e3 nd )$#n#)$$y re$e&nt shearstrain
res'$t nd #s 6e$$ s'#ted "or #dent#"y#n% )r#t#)$ pr!eters %o&ern#n% !e)hn#)$$y
#nd')ed sens#t#2t#on to s'bse('ent )#d#) #n0'ry*
The %o$ o" th#s thes#s 6s to )hr)ter#2e the #nter)t#on bet6een pr#!ry
!e)hn#)$ #ns'$t nd se)ondry acidosis#ns'$t 's#n% the "ore!ent#oned TBI!ode$*
It was my hypothesis that a sublethal mechanical injury would sensitie the hippocampus
to secondary acidosis! causing pre"entable neuronal death# It 6s hoped tht the #nter&$
bet6een these t6o #n0'r#es 6o'$d be re$#st#) tr%et "or therpe't#) #nter&ent#on3 nd
th's th#s reser)h 6o'$d not on$y d&n)e o'r 'nderstnd#n% o" the #nter)t#on bet6een
TBInd acidosis3 b't $so potent#$$y prode the too$s ne)essry "or d#s)o&er#n%
poss#b$e tret!ents*
To )o!prehend the e7per#!ent$ tret!ents e7!#ned #n th#s thes#s3 #t #s
ne)essry to .no6 $#tt$e !ore bo't neurons* A neuron#s seprted "ro! the
extracellular luid4#C$5 by the neurons cell membrane3 th#n3 "$'#d3 semipermeable
4se$e)t#&e5 brr#er tht )ontro$s the pss%e o" s'bstn)es #nto nd o't o" the neuron*
Interspersed thro'%ho't the cell membranere prote#ns tht $oo. $#.e do'%hn'ts3
prod#n% door6y "or entry o" !ter#$ #nto or o't o" the neuron* These prote#ns re
.no6n s ion channels3 nd there re n'!ero's types o" ion channels3 e)h spe)#$$y
des#%ned to $$o6 spe)#"#) type o" ion4e$e)tron#)$$y )hr%ed !o$e)'$e5 to pss
thro'%h* F#%'re dep#)ts the bs#) str')t're o" n ion channel*
8

19/74
Figure $* Th#s dr6#n% sho6s the "'nd!ent$ str')t're
o" n #on )hnne$* In the e7!p$e dep#)ted3 the #on
)hnne$ #s des#%ned "or the pss%e o" potss#'! #ons 4

20/74
One prt#)'$r type o" ligand%gated ion channel#s the )#dsens#n% #on )hnne$
4ASIC5* The ligand6h#)h activates4opens5 ASICs #s !o$e)'$e o" )#d* Th's3 #n
!ore )#d#) enron!ents 4de)res#n% pH53 ASICs re !ore $#.e$y to be #n the#r open
state* ASICs re expressed4present5 #n !ny types o" !!!$#n neurons 4A$&re2 de
$ Ros3 +,,+ Ch' et $*3 +,,; =$d!nn et $*3 8G =e!!#e et $*3 +,,+ @#on% et
$*3 +,,;53 nd )o!e #n se&er$ &r#et#es* S#7 ASICsubunits4prts tht !y be p't
to%ether #n d#""erent )o!b#nt#ons to )rete d#""erent &r#et#es o" ASICs5 h&e been
d#s)o&ered/ ASIC83 ASIC8Q3 ASIC+3 ASIC+Q3 ASIC93 nd ASIC; 4Ch' et $*3 +,,;5*
O" these subunits3 homomeric4)ons#st#n% o" on$y one type o" subunit5 ASIC8 nd
ASIC+ )hnne$s re prt#)'$r$y pre&$ent3 o))'rr#n% #n both sensory nd )entr$
neurons 4Ch' et $*3 +,,;53 nd th's these 6ere the )hnne$s se$e)ted "or "'rther n$ys#s
#n th#s thes#s*
ASIC)hnne$s re not the on$y type o" ion channels "o'nd #n neurons
!e!brne there re h'ndreds o" d#""erent types o" )hnne$s* Th's3 #n order to #so$te the
ASIC)hnne$ "'n)t#on nd deter!#ne the celldeth res'$t#n% "ro! acidosis$one3 #t 6s
ne)essry to bloc&the e""e)ts o" these other )hnne$s* Th#s 6s ))o!p$#shed by dd#n%
!#7t're o" se&er$ dr'%s3 .no6n s 'C!4M

21/74
neurons nt'r$ b$n)e so !')h tht the neurons !y e&en d#e3 thereby %%r&t#n%
the #n#t#$ TBI* Th's3 b$o).#n% the ASICs 4spe)#"#)$$y ASIC8 nd ASIC+5 "ro!
"'n)t#on#n% sho'$d de)rese the o&er$$ )#d#nd')ed celldeth obser&ed*
Se&er$ e7per#!ents h&e de!onstrted th#s de)rese #n )e$$ deth 6#th spe)#"#)
dr'%s tht b$o). ASICs* One s')h dr'% #s .no6n s amiloride3 6h#)h hs !ny e""e)ts
rn%#n% "ro! #d#n% #n the tret!ent o" hypertens#on to b$o).#n% ASICs* Amiloridehs
been de!onstrted to be nonspeciicb$o).er o" ASIC )hnne$ )t#&t#on #n se&er$
electrophysiologicalst'd#es 4Pr#)e et $*3 +,,, =$d!nn et $*3 8G @#on% et $*3
+,,;5* The ter! nonspeciichere re"ers to ho6 amiloride)ts/ the dr'% does not
d#s)r#!#nte bet6een 6h#)h type o" ASICtht #t b$o).s3 #t s#!p$y b$o).s ny ASIC
)hnne$3 re%rd$ess o" subunittype* Psalmotoxin 4or PcT)5 #s nother dr'% nd #s
obt#ned "ro! the &eno! o" the trnt'$&salmopoeus cambridgei* PcT)hs been
sho6n to be spe)#"#) ASIC8 b$o).er 4#t on$y b$o).s ASIC8 "'n)t#on5 4Es)o'bs et $*3
+,,, @#on% et $*3 +,,;5*
To e""e)t#&e$y 'se these dr'%s to bloc&ASIC)hnne$ "'n)t#on3 #t #s ne)essry to
h&e ASIC)hnne$s present #n the cell membrane* Mny te)hn#('es !y be 'sed to
&er#"y the e7#sten)e nd:or $o)$#2t#on o" ASICs#n t#ss'e3 #n)$'d#n%
immunocytochemistrynd Western blot analysis3 t6o pro)ed'res 6h#)h 6#$$ be
e7p$#ned #n the Mter#$s nd Methods se)t#on "o$$o6#n%* Preo's reser)h stron%$y
s'pports both ASIC8 nd ASIC+ e7#sten)e #n !!!$#n h#ppo)!p$ t#ss'e3 $tho'%h
d#""erent 6ys o" prepr#n% nd #so$t#n% t#ss'e )'$t'res !y ""e)t the pre&$en)e o" the
ASICs4=$d!nn et $*3 8G =e!!#e et $*3 +,,+5*
+8

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The e7p$ort#on o" ASIC)hnne$ presen)e3 $o)$#2t#on3 nd b$o).%e #n th#s
thes#s 6s #n dd#t#on to the pr#!ry thes#s ('est#on* It 6s n tte!pt to be%#n the
enor!o's ts. o" deter!#n#n% poss#b$e tret!ents nd thereby de)rese the )e$$ deth
#nd')ed by acidosis3 prod#n% poss#b$e )$#n#)$$y re$e&nt tret!ent opt#ons "or h'!ns
#n the $on%ter!* Ho6e&er3 the pr#!ry reser)h hypothes#s re!#ned tht a primary
TBIsensities the hippocampusto secondary acidosis*
++

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'aterials and 'ethods
'urgery and (issue &rotocol
H#ppo)!p$ s$#)e )'$t'res 6ere prepred ))ord#n% to !od#"#ed &ers#on o" the
#nter")e !ethod 'sed by Stopp#n# et $* 4885* The Le%)y An#!$ Cre nd Use
Co!!#ttee o" Le%)y C$#n#)$ Reser)h nd Te)hno$o%y Center ppro&ed the 'se o" the
rt br#ns "or )'$t're* =#ster rts 6ere nesthet#2ed 6#th h$othne dys postnt$
4"ter the !#%rtory phse o" ner&e )e$$s hs "#n#shed5 be"ore be#n% de)p#tted* The
br#n 6s then re!o&ed nd p$)ed #n #)e)o$d d#sse)t#n% !ed#'! 4Hn.s BSS3 !%:!$
%$')ose3 8M HEPES5 "or !#n'tes pr#or to h#ppo)!p$ d#sse)t#on nd re!o&$* The
h#ppo)!p$ t#ss'e 6s s$#)ed ;,,! th#). 's#n% t#ss'e s$#)er 4M)I$6#n3 Stoe$t#n%
Co*3 =ood D$e3 IL53 "$oted #n #)e)o$d d#sse)t#n% !ed#'!3 nd tr#!!ed to the
hippocampusnd d0)ent entorh#n$ )orte7 t#ss'e* Three t#ss'e s$#)es 6ere trns"erred
to 'illicell)! )'$t're p$te #nsert 6#th !#)roporo's !e!brne 4PICM,R>,3
M#$$#pore3 Bed"ord3 MA5 #n )'$t're !ed#'!* The s$#)es 6ere then p$)ed #n #n)'btors3
6#th n #de$ enron!ent o" 9-C3 CO+enr#)hed3 h'!#d#"#ed t!osphere !#nt#ned*
The )'$t're !ed#'! 6s rep$)ed e&ery 9; dys )'$t'res 6ere 'sed 898 dys
"o$$o6#n% p$t#n%* Essent#$$y $$ the ner&e nd glial)e$$ types s'r&e #n organotypic
br#n )'$t'res3 b't 6#th de)resed dens#ty o" synpt#) str')t'res #n the h#ppo)!p$
s$#)es "o$$o6#n% the s$#)#n% pro)ess*
&ropidium Iodide )&I* +pta,e
On)e s$#)e 'nder%oes des#%nted experimental condition4e*%* TBI3 n )#d
#n0'ry3 both TBInd )#d #n0'ry3 no #n0'ry3 et)*53 #t #s ne)essry to deter!#ne tht s$#)es
$e&e$ o" )e$$ deth* Th#s #s done by $$o6#n% the s$#)e to re)o&er "or ppro7#!te$y +;
+9

24/74
ho'rs3 then e7pos#n% #t to s'bstn)e tht labels4!r.s5 on$y the ded )e$$s* Th#s
!r.er #s .no6n s PI4Prop#d#'! Iod#de53 nd #t te)hn#)$$y on$y $be$s )e$$s 6#th
)o!pro!#sed cellular membranes3 "et're typ#)$ o" ded )e$$s* =hen the s$#)e #s
#!%ed on conocal!#)ros)ope3 the s$#)e ppers 6h#te #n res o" )e$$ deth nd b$).
#n res o" $#n% )e$$s* F#%'re 88 portrys n e7!p$e o" s$#)e $be$ed 6#th PI"ter
TBI45 nd "ter e7pos're to .no6n to7#) s'bstn)e 4b5* Th's3 's#n% PI#!%#n%
$$o6s "or $o)$#2t#on 46here the #n0'ry )'sed the !ost hr!5 s 6e$$ s ('nt#"#)t#on
o" celldeth*
Figure 11* The PI #!%e to the $e"t 45 sho6s )e$$ deth 4the 6h#te re%#ons5 #n the CA8 nd thedentte %yr's port#ons o" the h#ppo)!p$ s$#)e "o$$o6#n% TBI* The PI #!%e to the r#%ht 4b5 sho6s
)e$$ deth #n $$ o" the re%#ons p#)t'red 4CA83 CA+3 CA93 nd the dentte %yr's5 "o$$o6#n% +;ho'rs
o" e7pos're to 8!M NMDA nd 8,'M %$y)#ne !ed#* Fro! thes#s e7per#!ents* S)$e brs/ +,,'!
"or both #!%es*
Pr#or to #!%#n%3 )'$t'res 6ere e7posed "or t $est one ho'r to PI4Mo$e)'$r
Probes3 +'%:!$ o" 8 !%:!$ sto).5* I!%es 6ere )pt'red 6#th Ret#% 89,, PMT
's#n% )pt're so"t6re 4#!%#n%5 on Le#) DMR 'pr#%ht !#)ros)ope 4=et2$r3
>er!ny5 #$$'!#nted by !er)'ry r) $!p nd "#$tered 's#n% ;nM:,nM
4e7)#tt#on:e!#ss#on5 "#$ter set* An e7pos're t#!e o" e#ther 8,,!s or +,!s 6s 'sed
+;
a b

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dt presented #n the Res'$ts se)t#on re $$ "ro! +,!s e7pos're t#!e* Fo$$o6#n% the
"#rst set o" PI#!%#n% +; ho'rs "ter the TBI3 the )'$t'res 6ere then e)h e7posed to
8!M NMDA nd 8,'M %$y)#ne #n ser'!"ree )'$t're !ed#'! "or +; ho'rs3 nd "#n$$y
#!%ed ; ho'rs "o$$o6#n% the primary injury* C'$t'res treted 6#th the
NMDA:%$y)#ne )o!b#nt#on ser&ed s K)ontro$ #n0'r#es3 represent#n% the !7#!$
ne'ron$ celldeth tht the )'$t'res )o'$d e7per#en)e* r#t#on bet6een #nd#d'$
)'$t'res bsed on d#""erent#$ s$#)e he$th 6s th's )o!pensted "or s#n)e e)h s$#)es cell
deth 6s nor!$#2ed to #ts o6n !7#!$ celldeth*
-endritic 'pine Imaging
=h#$e PI#!%#n% #s n e""e)t#&e !es're o" the o&er$$ he$th o" rt
h#ppo)!p$ s$#)e3 )o!p$#!entry !ethod o" n$y2#n% s$#)e he$th #s .no6n s spine
imaging* =#th spine imaging#t #s poss#b$e to 2oo! #n to the $e&e$ o" the neuron#tse$"3
prod#n% !ore sens#t#&e !es're o" s$#)e he$th* The !0or#ty o" neurons)ons#st o"
"o'r "'n)t#on$ se)t#ons/ n #np't se)t#on3 n #nte%rt#&e or tr#%%er se)t#on3 )ond')t#$e
se)t#on3 nd n o'tp't se)t#on 4

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Figure 12* The !ode$ ne'ron3 )ons#st#n% o" "o'rse)t#ons* In"or!t#on "$o6s "ro! the top o" the
d#%r! to the botto! nd on to the ne7t
ne'ron4s5 #n the pth6y* Fro!

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trns"er5 t tht s#te3 s'%%est#n% de)rese #n #n"or!t#on )o!!'n#)t#on to tht neuron*
S#n)e the '$t#!te p'rpose o" neuron#s to trns"er #n"or!t#on3 #" dendr#t#) spines
de)rese3 #t #nd#)tes tht the neuron#s not per"or!#n% #ts "'n)t#on nd #s 'nhe$thy
re$t#&e to #ts preo's stte* F#%'re 89 d#sp$ys he$thy s$#)e 6#th p$ethor o"
dendr#t#) spines* The rro6s po#nt to se&er$ d#""erent shpes o" spinespresent $on% th#s
prt#)'$r dendrite*
Figure 13* Dendr#t#) sp#nes present #n the !otor )orte7 o" pyr!#d$ ne'ron#n n d'$t rt* Note the sp#ne shpe &r#ety3 po#nted o't by the 6h#te rro6s*
S)$e br/ '!* Fro! Hr!s et $*3 +,,-*
Mny reser)hers s'%%est tht dendr#t#) spinesp$y s#%n#"#)nt ro$e #n br#n
plasticity8+3 or the b#$#ty o" the br#n to )hn%e3 s#n)e $tert#ons #n the spines
the!se$&es !y be )orre$ted 6#th $tert#ons #n the pth6ys o" #n"or!t#on "$o6
4Hr!s et $*3 +,,- Her#n% nd Shen%3 +,,8 L#pp!n et $*3 +,,5* Th's3 by n$y2#n%
the )hn%es #n spinedens#ty #n rt h#ppo)!p$ s$#)es "or these e7per#!ents3 n
e7tre!e$y sens#t#&e %'%e o" s$#)e he$th #s poss#b$e*
C'$t'res 6ere $be$ed 6#th D#O 4939Vd#$#no$ey$o7)rbo)yn#ne per)h$orte3
Intro%en53 "$'ores)ent3 lipophilic!r.er t.en 'p by neurons nd !.#n% the#r
dendr#tes s#b$e3 ))ord#n% to te)hn#('e o't$#ned #n D#$ey nd S!#th3 8-* A
)p#$$ry p'$$er 4Nr#sh#%# !ode$ PP9,5 6s 'sed to p'$$ %$ss )p#$$ry t'be3
prod')#n% &ery "#ne t#p* Th#s t#p 6s then )oted 6#th so$#d D#O )ryst$s 4no d#$'t#on5
nd #nserted #nto !#)ro!n#p'$tor* The t#p 6s )re"'$$y !ne'&ered to pre)#se
+G

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$o)t#ons on the organotypich#ppo)!p$ s$#)e 's#n% +@ ob0e)t#&e on !#)ros)ope*
The t#p 6#th the dye 6s bre$y3 %ent$y to')hed to the s'r")e o" the t#ss'e3 depos#t#n%
D#O )ryst$s* A$$ prts o" th#s pro)ed're 6ere per"or!ed #n the dr. s !')h s poss#b$e3
s#n)e D#O #s sens#t#&e to $#%ht*
Dendr#t#) spine#!%#n% 6s )rr#ed o't ppro7#!te$y - ho'rs "o$$o6#n% $be$#n%
o" the s$#)e* The #!%#n% 6s )o!p$eted 's#n% n 'pr#%ht )o!po'nd !#)ros)ope 4Le#)
DMR3 =et2$r3 >er!ny5 nd Le#) Con"o)$ so"t6re* L#&e 898 dy o$d )'$t'red
s$#)es 4one t t#!e5 6ere !o'nted #n per"'s#on )h!ber nd p$)ed on the st%e o" the
conocal!#)ros)ope* The s$#)e 6s per"'sed 6#th n #C$so$'t#on t pH o" G*;* D#O
$be$ed prote#ns 6ere #!%ed t ;n! e7)#tt#on 4Ar $ser53 nd e!#ss#on "#$tered t ,,
9n!* A -,@ 6ter#!!ers#on $ens 6s 'sed to e6 the spines* I!%es 6ere
)('#red t s)nn#n% speed o" ;,,H2 nd 6ere typ#)$$y 8,+;@8,+; reso$'t#on 6#th
$#ne &er%#n% set to ; s)ns per $#ne* ?oo! 6s %ener$$y !#nt#ned t $ess thn or
e('$ to ;@3 s %reter 2oo! t 8,+;@8,+; reso$'t#on 6o'$d h&e e7)eeded the
opt#)$ reso$'t#on o" the ob0e)t#&e* S#n)e dendr#t#) spinese7tend #n three d#!ens#ons
6#th#n the s$#)e3 st).s o" #!%es 6ere )o$$e)ted o&er se)t#ons spnn#n% G+G'! #n the ?
d#!ens#on 6#th ,*8*'! step s#2e #n order to ens're tht str')t'res s s!$$ s spines
6ere "'$$y )pt'red*
Immunocytochemistry
Immunocytochemistry#s te)hn#('e tht 't#$#2es luorescent4%$o6#n%5
antibodies4!r.ers5 to pre)#se$y dete)t nd $o)$#2e 6here )e$$'$r )o!ponents $#.e
ASIC)hnne$s re* The stndrd immunocytochemistryproto)o$ 6s 'sed "or the
e7per#!ents #n th#s thes#s3 $tho'%h so!e d0'st!ents to the t#!#n% s)he!e 6ere !de to
+

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))o!!odte "or the #n)resed th#).ness o" organotypict#ss'e s$#)es* A 8/8,, d#$'t#on
6s 't#$#2ed "or the Ne'rotr)e 4Mo$e)'$r Probes53 nd 8/8,, d#$'t#on "or the ASIC8
4S#%!3 S G;;5 nd ASIC+ 4A$ph D#%nost#)3 MDE>88A5*
.estern /lot 0nalysis
Another !ethod #s .no6n s the Western blotte)hn#('e3 nd #t $$o6s "or the
#dent#"#)t#on nd !es're!ent o" the re$t#&e !o'nt o" prt#)'$r prote#n 4e*%* the
homomericASIC8 )hnne$5* Western blots 'se gel electrophoresis4e$e)tr#)#ty
thro'%h %e$t#no's !ter#$5 to seprte prote#ns bsed on the#r respe)t#&e molecular
*eights 4s#2e5* Fo$$o6#n% trns"er o" the dt "ro! the %e$ to pper 4 !ore per!nent
"or!53 spe)#"#) prote#n 4e*%* ASIC85 #s dete)ted by 's#n% probe894 !r.er5*
H#ppo)!p$ s$#)es 6ere ho!o%en#2ed #n ,'L o" $ys#s b'""er 4,*8M NC$3
,*,+M Tr#sHC$3 8!M EDTA3 8 NP;,3 8*!%:!L Aprot#n#n3 8,!%:!L Le'pept#n3
8!%:!L MeOH Pepstt#n3 8,!%:!L PMSF5 * Three h#ppo)!p$ s$#)es 6ere )o$$e)ted
"or e)h )ond#t#on 4e*%* three )ontro$ s$#)es3 three TBIt 8,*9!ph s$#)es5* I" the
)ond#t#on 6s TBI3 the s$#)es 6ere )o$$e)ted t + ho'rs "o$$o6#n% the pr#!ry TBI3 to
obt#n the s$#)es t st%e tht 6o'$d !#!#) the enron!ent present 6hen secondary
4)#d5injury6o'$d be pp$#ed* Prote#n )on)entrt#ons 6ere !es'red 's#n% 8!L
Brd"ord re%ent 4S#%!5 6#th 9*9'L o" e)h s!p$e* E('$ prote#n !o'nts 6ere
reso$&ed #n SDS8, po$y)ry$!#de %e$s t 8+,!A nd trns"erred to n#tro)e$$'$ose
!e!brnes 4B#oRd5 o&ern#%ht* The !e!brnes 6ere b$o).ed 6#th dry !#$.
T6een so$'t#on3 #n)'bted 6#th 8/8,,, pr#!ry antibodyd#$'t#on t ;C o&ern#%ht*
A"ter se&er$ r#nses #n PBST3 horserd#sh pero7#dse 4HRP5 $#n.ed nt#!o'se
se)ondry antibody4Che!#)on5 6s 'sed to dete)t pr#!ry antibodiest d#$'t#on o"
+

30/74
8/+,,, "or , !#n'tes t roo! te!pert're* The ASIC8 nd ASIC+ antibodies6ere
obt#ned "ro! S#%! 4S G;;5 nd A$ph D#%nost#) 4MDE>88A5 respe)t#&e$y* The
pos#t#&e )ontro$s "or both ASIC8 nd ASIC+ 6ere "ro! HE< 4H'!n E!bryon#)
ennre$$# et $*3 8+5* For
in vitroTBI!ode$#n%3 the b#o$o%#)$ #n0'ry )s)de sho'$d be #n#t#ted by !e)hn#)$
st#!'$'s !ost ppropr#te to in vivoTBI)'st#on* Hen)e3 te! o" reser)hers "ro!
the Do6 Ne'rob#o$o%y Lb nd the B#o!e)hn#)s Lb de&e$oped n organotypic
!euronal Shear Injury (!SI+!ode$ 6h#)h e!p$oys #nert#dr#&en shear strain8;to
%enerte s)$b$e !e)hnotr'!* Th#s !ode$ )retes )hn%e #n ))e$ert#on3 6h#)h
)'ses #nert#3 6h#)h #n t'rn )'ses the shear strain4Bott$n% et $*3 +,,-5* Th's3 the
"ster the ))e$ert#on )hn%e3 the !ore se&ere the shear strain*
Shear straino))'rs by !e)hn#s! s#!#$r to 6ht hppens 6hen person
p'shes st). o" pr#nter pper t n n%$e $$ o" the #nd#d'$ p%es re!#n 'nh'rt3 b't
the st). hs st#$$ been $tered* Th's3 the hor#2ont$ ne'ron$ cellso" the t#ss'e re!#n
9,

31/74
re$t#&e$y 'nto')hed nd 'n#n0'red3 6h#$e the &ert#)$ ne'ron$ cells'nder%o s#%n#"#)nt
de"or!t#on 4see F#%'re 8;5* The #n0'ry de)e #s b$e to prod')e #nert#$ ))e$ert#ons
tht rese!b$e the !%n#t'des o" shear strainobser&ed in vivo3 th's !#!#).#n% the
!e)hn#s! by 6h#)h h'!n hed tr'! )'ses ne'ron$ #n0'ry 4rp#d ))e$ert#on
#nd')ed shear strain#n0'ry53 s #s obser&ed #n )o!!on tr""#) ))#dents*
Figure 14* H#ppo)!p$ s$#)e de"or!t#on* Note ho6 the hor#2ont$ ne'ron re!#ns
#nt)t3 6h#$e the &ert#)$ ne'ron #s s#%n#"#)nt$y stret)hed d'r#n% the #n0'ry*
A )rtoon dep#)t#on 4F#%'re 8G5 nd )t'$ photo o" the #n0'ry de)e 4F#%'re 85
!y he$p s'$$y e7p$#n ho6 th#s !)h#ne 6or.s* The millicells3 )ont#n#n% the
)'$t'red t#ss'e 4see F#%'re 853 re p$)ed on the ))e$ert#on !od'$e3 6h#)h )n ho$d 'p
to - M#$$#pore millicellst t#!e #n stndrd s#76e$$ rrn%e!ent* A trns$')ent
P$e7#%$s )o&er #s s$#d on top o" these millicellsnd $o).ed #nto p$)e3 prote)t#n% the
)'$t'res %#nst )ont!#nt#on nd #r"$o6 d'r#n% e7per#!ents*
Figure 15* A* A stndrd s#76e$$ p$te #s d#sp$yed* E)h 6e$$ )ont#ns one M#$$#pore !#$$#)e$$ !e!brne
#n 8!L o" )'$t're !ed#'!* E)h M#$$#pore hs three or%notyp#) rt h#ppo)!p$ s$#)es p$ted on #t* B* A
)$oser e6 o" the $o6er r#%ht 6e$$* Note the )'$t're !ed#3 6h#)h #s the $#%ht orn%e $#('#d3 nd the three
d#st#n)t h#ppo)!p$ s$#)es p$ted on the M#$$#pore*
dDeformation
Recovery
98
A B

32/74
The ))e$ert#on !od'$e #s then #nserted #nto the $#ner s$#de3 rest#n% %#nst the p#ston o"
$#ner )t'tor* The )t'tor ))e$erted the !od'$e o&er ,!! stro.e $en%th nd #s
dr#&en by "$y6hee$ !e)hn#s! en%%ed by so$eno#dpo6ered )$'t)h*
r#o's ))e$ert#ons )n be prod')ed by the de)e bsed on the $e&e$ o"
press're d#$ed #nto the p#ston* The $e&e$s o" #n0'ry 'sed "or th#s e7per#!ent 6ere D,3
D,3 D893 nd D+ 4s 6e$$ s ,!ph "or so$e$y )#d #n0'r#es nd shamexperimental
conditions53 6#th the KD nott#on re"err#n% to the D#$ on the #n0'ry de)e* These d#$
$e&e$s )orrespond to the #!p)t &e$o)#t#es dep#)ted #n F#%'re 8-3 6h#)h 6ere deter!#ned
's#n% h#%hspeed )!er syste!* The )!er syste! 4Fst)! U$t#! AP@ RS3
Photron3 Itron73 CA5 )pt'red 8+@8+ p#7e$s:"r!e t 9-3,,, "r!es:s o" the trns#ent
de"or!t#on o" the )'$t're s'r")e d'r#n% #!p)t* Note tht the #!p)t &e$o)#ty &$'es3
6h#)h 6#$$ be 'sed "ro! th#s po#nt on to re"er to the $e&e$ o" TBI3 re not d#re)t$y
)o!prb$e to typ#)$ h'!n tr""#) ))#dents* H'!n br#ns h&e !')h %reter mass3
re en)sed #n bony s.'$$3 nd re prote)ted by cerebrospinal luid (CS$+* In dd#t#on3
#n !ost 'to!ob#$e )rshes3 $ot o" the #nert#$ ener%y o" the #!p)t #s t.en 'p #n the )r
s prt o" the s"ety "et'res 4e*%* the b'!pers3 "ront nd rer )r'!p$e 2ones3 set be$t3
nd #r b%53 6h#)h re des#%ned to red')e the o))'pnts ))e$ert#on )hn%e* Th's3 the
#n0'r#es to the rt h#ppo)!p$ t#ss'e des)r#bed s res'$t#n% "ro! #!p)t &e$o)#t#es #n !ph
do not d#re)t$y #nd#)te the #n0'r#es 6h#)h 6o'$d o))'r "or h'!ns*
Dial Level Impact Velocity (m/s) Impact Velocity (mph)
0 2 4.5
8 4.6 10.
1 6.6 14.8
25 10.4 2.
Figure 16* The #!p)t &e$o)#t#es #n both !:s nd !ph 6h#)h
)orrespond to the d#$ $e&e$s on the #n0'ry de)e 'sed*
9+

33/74
Figure 17* Crtoon dep#)t#on o" the TBI In0'ry De)e*
Figure18* Photo o" the TBI In0'ry De)e*
aterials
#xtracellular luid4#C$5 6s prepred "o$$o6#n% #nstr')t#ons by @#on% et $*
4+,,;5 nd 6s 'sed s the !ed#'! "or pp$y#n% the secondary injury* #C$6s !de
t both pH o" -*, 4the )#d#) &r#b$e5 nd pH o" G*; 4!#!#).#n% phys#o$o%#)$ pH nd
p$)ed on shamsecondary injury)'$t'res5* The #C$6s $e"t on )'$t'res tht
'nder6ent secondary injury"or e#ther 8 or 9 ho'rs3 depend#n% on 6h#)h d'rt#on those
)'$t'res 6ere ss#%ned "or the e7per#!ent*
C'$t're !ed# 6s prepred s , MEM3 + horse ser'!3 + HBSS3 8M
HEPES3 8!M %$'t!#ne 4>$'t!73 Intro%en3 Cr$sbd3 CA53 nd +!M %$')ose*
Ser'!"ree )'$t're !ed#'! 6s $so prepred 's#n% th#s proto)o$3 e7)ept 6#tho't the
horse ser'!3 nd 6#th so!e d0'st!ents "or os!o$r#ty*
99
A))e$ert#on Mod'$e
B'!perP#ston

34/74
'C!#s n )rony! 'sed to des#%nte )'$t're !ed# s'pp$e!ented 6#th three
)he!#)$s/ 8,'M M

35/74
Th#s 9, !#n'te e7pos're to 'C!6s sp$#t #nto t6o 8 !#n'tes 6shes* At the end o"
the 9, !#n'tes3 #" the )'$t're #s 'nder%o#n% secondary injury3 #t 6s p$)ed #nto pH-*,
#C$"or d'rt#on o" e#ther 8 or 9 ho'rs* I" the )'$t're #s des#%nted s sham
se)ondry #n0'ry3 #t 6#$$ be #n)'bted #n pHG*; #C$"or e#ther 8 or 9 ho'rs* Note tht3 "or
)'$t're 'nder%o#n% ,h de$y e7per#!ent$ )ond#t#on3 the tr'e de$y #s 9, !#n'tes d'e
to the 'C!pretret!ent* S#!#$r$y3 "or )'$t're 'nder%o#n% +h de$y experimental
condition3 the tr'e de$y #s +* ho'rs on ))o'nt o" the 9, !#n 'C!pretret!ent*
On)e the spe)#"#ed d'rt#on o" secondary injury#s )o!p$ete3 the )'$t'res re
p$)ed b). #nto ser'!"ree )'$t're !ed#'! nd $$o6ed to #n)'bte o&ern#%ht* T6enty
three ho'rs "ter the #n#t#$ TBI#n0'ry3 +'$:!$ o" PI"ro! 8!%:!$ sto). so$'t#on #s
#n0e)ted #nto the )'$t're !ed#'! "or 'pt.e by the t#ss'e3 nd 8 ho'r $ter 4+; ho'rs
"o$$o6#n% #n#t#$ TBI53 the )'$t'res re #!%ed* A"ter th#s "#rst ro'nd o" PI#!%#n%3 e)h
)'$t're #s e7posed to 8!M NMDA nd 8,'M %$y)#ne "or +; ho'rs3 nd #!%ed se)ond
t#!e to obt#n the !7#!$ celldeth #!%es 4; ho'rs "o$$o6#n% #n#t#$ TBI5* F#%'re 8
d#sp$ys th#s t#!e )o'rse proto)o$*
Note tht d'e to t#!e restr#)t#ons nd the ne)ess#ty o" #n)res#n% !4s!p$e s#2e5
&$'es3 de$y o" + ho'rs 4)t'$$y +* ho'rs )ons#der#n% the 'C!pretret!ent53 TBI
$e&e$ o" 8,*9!ph3 nd n )#d #n0'ry o" -*,#C$"or 9 ho'rs 6s )hosen to "o)'s on*
-ata ollection
On)e the PI#!%es re )pt'red nd s&ed on the )o!p'ter3 they !'st be
n$y2ed* I!%eJ 8*9-b so"t6re 4NIH5 #s 'sed to "#nd the !en br#%htness o" the #!%e
4the br#%hter the #!%e3 the !ore cells tht re e""e)t#&e$y ded5* These !en &$'es re
then d#ded by the !en &$'e obt#ned "ro! the PI#!%e o" the !7#!$ celldeth "or
9

36/74
tht s!e respe)t#&e h#ppo)!p$ s$#)e 4by NMDA see F#%'re 88 "or )$r#"#)t#on5*
=hen th#s "r)t#on #s !'$t#p$#ed by 8,,3 #t %#&es per)ent%e o" the tot$ )'$t're tht #s
ded s res'$t o" the spe)#"#) #n0'ry pres)r#bed to tht %#&en )'$t're* A$$ reported &$'es
re "or the +; ho'r t#!epo#nt "ter #!p)t 4TBI5 d#ded by the !7#!'! #ntens#ty 4"ter
e7pos're to NMDA5 "or the s!e s$#)e* The )$)'$t#ons "or these per)ent%es 6ere
per"or!ed #n M#)roso"t E7)e$ +,,9*
The n$ys#s o" the per)ent celldeth "or spe)#"#) re%#on o" the hippocampus3
e*%* the dentte %yr's3 6s )o!p$eted by "o$$o6#n% the s!e proto)o$ "ore!ent#oned "or
the o&er$$ #!%e* The on$y d#""eren)e #s tht the Freehnd Se$e)t#ons too$ 6s 'sed to
!n'$$y dr6 n o't$#ne ro'nd the re%#on #n ('est#on rther thn 's#n% the o&er$$
#!%e to obt#n !en #ntens#ty* The s!e o't$#ne 6s 'sed "or both the +; ho'r cell
deth nd the !7#!$ celldeth #!%es3 nd then per)ent%e celldeth 6s )$)'$ted
by the s!e pro)ess $redy e7p$#ned*
I!%e J 8*9-b 6s $so 'sed to n$y2e the dendr#t#) spine images* A"ter sett#n%
the s)$e to %$ob$ &$'e o" ;*9,8 p#7e$s:'!3 st). o" dendr#t#) spine images6s then
2pro0e)ted 's#n% the !7#!'! #ntens#ty o" e)h #!%e* >reen or %rys)$e $oo.'p
tb$es 6ere pr#!r#$y 'sed d'r#n% the n$ys#s pro)ess* The Ce$$ Co'nter too$ #n I!%e J
6s then 'sed to tr). the n'!ber o" dendr#t#) spinesnd:or varicosities$on% $en%th
o" dendrite* The $en%th o" the dendrite6s deter!#ned by 's#n% the se%!ented $#ne
se$e)t#on too$ #n I!%e J* O&er$$ dens#t#es o" spinesnd varicositiesper !#)ron 6ere
)$)'$ted #n M#)roso"t E7)e$ +,,9*
'tatistical 0nalysis
9-

37/74
M#)roso"t E7)e$ +,,9 spredsheets 6ere 'p$oded #nto Stt#e6 "or stt#st#)$
n$ys#s* Pre$#!#nry %rph#n% 6s per"or!ed #n Stt#e63 6h#$e "#n$ %rph#n% 6s
per"or!ed #n E7)e$* S#%n#"#)n)e 6s deter!#ned by 's#n% the S)he""e ANOA Post
ho) test t stndrd )on"#den)e $e&e$ o" * Th#s prt#)'$r type o" ANOA 6s
se$e)ted be)'se the s!p$e s#2es 4!5 "or the &r#o's e7per#!ent$ )ond#t#ons 6ere not
e('$* The S)he""e postho) test $$o6ed "or the 'ne('$ &r#n)es nd 'ne('$ n'!ber
o" spe)#!ens to be ))o'nted "or* A stndrd ANOA test 6s $so 'sed "or
deter!#nt#on o" #nter)t#on bet6een t6o &r#b$es3 6h#)h #n th#s thes#s 6ere the t6o
#n0'r#es* In $$ res'$ts nd %rphs !en &$'es re represented W one stndrd error rther
thn stndrd det#on*
,esults
9G

38/74
&I esults
The "'nd!ent$ e7per#!ent per"or!ed 6s the double hite7per#!ent to
deter!#ne the #nter)t#on bet6een e)h #nd#d'$ Kh#t3 or #n0'ry 4TBInd acidosis5*
The per)ent celldeth res'$t#n% "ro! )ontro$ 4No TBI5 experimental conditions 6#th
nd 6#tho't )#d3 sham4Sham TBI5 )ond#t#ons 6#th nd 6#tho't )#d3 nd TBIs 4t
8,*9!ph5 6#th nd 6#tho't )#d 6ere deter!#ned t )#d $e&e$s o" ph-*, "or 9 ho'rs*
Contro$ experimental conditions sho6ed *G W ,*- celldeth 6#tho't )#d 4No
TBInd No A)#d5 nd 8*; W ;*8 celldeth 6#th )#d 4No TBInd -*,49h55*
Sham experimental conditions sho6ed -*-; W ,*, celldeth 6#tho't )#d 4Sham
TBInd No A)#d5 nd 8;*- W 8*9 celldeth 6#th )#d 4Sham TBInd -*,49h55*
TBI experimental conditions sho6ed 88*; W +*+8 celldeth 6#tho't )#d 48,*9!ph
nd No A)#d5 nd +*G8 W +*98 celldeth 6#th )#d 48,*9!ph nd -*,49h55*
The res'$ts #n re%rd to the pr#!ry hypothes#s re d#sp$yed #n F#%'re +,* Note
tht both the TBI$one nd the )#d 4-*, "or 9 ho'rs5 #n0'ry $one re s#%n#"#)nt$y
d#""erent thn the )ontro$ experimental condition#n the 6ho$e h#ppo)!p$ s$#)e
4pX,*,,,8 nd p,*,,+ respe)t#&e$y5* In dd#t#on3 the double hit#n0'ry 4TBIt $e&e$
o" 8,*9!ph3 + ho'r de$y3 nd s'bse('ent -*, #n0'ry "or 9 ho'rs5 #s s#%n#"#)nt$y
d#""erent "ro! the TBIt 8,*9!ph $one 4p,*,,,95* F#n$$y3 stndrd ANOA test o"
th#s double hitdt set prod')es s#%n#"#)n)e "or both TBI$one 4p,*,,,+5 nd )#d
$one 4pX,*,,,853 b't does not prod')e s#%n#"#)n)e "or n #nter)t#on bet6een the t6o
4p,*8,;5*
9
Double Hit Results
0
5
10
15
20
25
0
o&'I ham&'I 10.mph
TBI Condition
%C
ellDeath
o -cid
6.0(h)

39/74
Figure 2%* The res'$ts obt#ned "ro! the PI dt o" the do'b$e h#t res'$ts3 tht #s3 the TBI nd )#d #n0'r#es
$one nd to%ether3 6#th +h de$y* Error brs represent stndrd error* The s#n%$e str 45 #nd#)tes tht
the )#d #n0'ry $one 4-*, "or 9 ho'rs3 se)ond )o$'!n #n "ro! the $e"t5 d#""ers s#%n#"#)nt$y "ro! the )ontro$
4No TBI nd No A)#d:"r $e"t5 4pX,*,,,85* The do'b$e str 45 #nd#)tes tht the TBI #n0'ry $one 4on$y
8,*9!ph #n)$'ded3 6#tho't )#d #n0'ry5 d#""ers s#%n#"#)nt$y "ro! the )ontro$ 4p,*,,+5* The tr#p$e str
45 #nd#)tes tht the do'b$e h#t #n0'ry 4"r r#%ht )o$'!n3 both TBI o" 8,*9!ph nd )#d #n0'ry o" -*, "or 9
ho'rs5 d#""ers s#%n#"#)nt$y "ro! the pr#!ry TBI $one 4p,*,,,95* N &$'es "ro! $e"t to r#%ht/ ;+3 8;3 +8383 +83 8*
'$#tt#&e obser&t#on o" the PI#!%es )o!pr#s#n% the double hitdt
s'%%ested tht n )#d #n0'ry3 e#ther $one or #n double hitexperimental condition3
se$e)t#&e$y res'$ted #n celldeth #n the dentte %yr's re%#on o" the hippocampus*
'nt#tt#&e s'pport "or th#s obser&t#on 6s p'rs'ed by se$e)t#&e$y n$y2#n% the !en
p#7e$ #ntens#ty "or the dentte %yr's re%#on $one* Contro$ experimental conditions
sho6ed 8*, W 9*-; celldeth 6#tho't )#d 4No TBInd No A)#d5 nd 8*9G W
9*+, celldeth 6#th )#d 4No TBInd -*,49h55* TBI experimental conditions sho6ed
+9*G W *+ celldeth 6#tho't )#d 48,*9!ph nd No A)#d5 nd -G*+ W ;*;- cell
deth 6#th )#d 48,*9!ph nd -*,49h55*
9
-
---
--

40/74
F#%'re +8 d#sp$ys the res'$ts "ro! th#s ('nt#tt#&e n$ys#s* The double hit
#n0'ry #s s#%n#"#)nt$y d#""erent "ro! the )#d #n0'ry $one 4p,*,8G53 s 6e$$ s &ery
h#%h$y s#%n#"#)nt$y d#""erent "ro! the TBI$one 4pX,*,,,85* The TBI$one 6s not
s#%n#"#)nt$y d#""erent "ro! the )ontro$ experimental condition3 6h#$e the )#d #n0'ry
$one 6s &ery h#%h$y s#%n#"#)nt$y d#""erent "ro! the )ontro$ )ond#t#on 4pX,*,,,85*
F'rther!ore3 stndrd ANOA test o" the dentte %yr's re%#on $one prod')ed
s#%n#"#)n)e "or the )#d #n0'ry $one 4pX,*,,,853 s 6e$$ s "or the #nter)t#on bet6een
the t6o #n0'r#es 4TBInd acidosis5 4p,*,+-5*
Dentate Gyrus Cell Death Results
0
10
20
0
40
50
60
30
80
o&'I 10.mph
TBI Condition
%C
ellDeath
o -cid
6.0 (h)
Figure 21* The res'$ts obt#ned "ro! the PI dt o" the do'b$e h#t res'$ts 6#th +h de$y "or the dentte
%yr's re%#on o" the h#ppo)!p's* Error brs represent stndrd error* The s#n%$e str 45 #nd#)tes tht the
)#d #n0'ry $one 4-*, "or 9 ho'rs3 se)ond )o$'!n #n "ro! the $e"t5 d#""ers &ery h#%h$y s#%n#"#)nt$y "ro! the)ontro$ 4No TBI nd No A)#d:"r $e"t5 4pX,*,,,85* The do'b$e str 45 #nd#)tes tht the do'b$e h#t #n0'ry
4TBI t 8,*9!ph3 )#d #n0'ry t -*,ECF "or 9 ho'rs5 d#""ers s#%n#"#)nt$y "ro! the )#d #n0'ry $one
4p,*,8G5 nd "ro! the TBI $one 4pX,*,,,85* Note tht the TBI $one )ond#t#on d#d not s#%n#"#)nt$y
d#""er "ro! the )ontro$ )ond#t#on* N &$'es "ro! $e"t to r#%ht/ ;+3 8;3 +83 8*
There 6s hope tht amiloride)o'$d be potent#$ dr'% to b$o). the e""e)ts o"
se)ondry )#dos#s "o$$o6#n% the pr#!ry TBI3 s#n)e amiloridehs been de!onstrted to
;,
-
---

41/74
nonspe)#"#)$$y b$o). ASIC)hnne$ )t#&t#on #n electrophysiologicalst'd#es 4Pr#)e et
$*3 +,,, =$d!nn et $*3 8G @#on% et $*3 +,,;5* The per)ent celldeth res'$t#n%
"ro! )ontro$ 4No Dr'%5 experimental conditions 6#th nd 6#tho't )#d3 )on)entrt#on
one 48,,'M amiloride5 )ond#t#ons 6#th nd 6#tho't )#d3 nd )on)entrt#on t6o 4t
8!M amiloride5 )ond#t#ons 6#th nd 6#tho't )#d 6ere deter!#ned t )#d $e&e$s o"
ph-*, "or 9 ho'rs* Contro$ experimental conditions sho6ed *G W ,*- celldeth
6#tho't )#d 4No Dr'% nd No A)#d5 nd 8*88 W +*; celldeth 6#th )#d 4No Dr'%
nd -*,49h55* Con)entrt#on oneexperimental conditions sho6ed -*8- W ,*G, cell
deth 6#tho't )#d 48,,'M amiloridend No A)#d5 nd 8*8; W 8*8 celldeth 6#th
)#d 48!M amiloridend -*,49h55* Con)entrt#on t6oexperimental conditions sho6ed
+;*,; W ,*G9 celldeth 6#tho't )#d 48,,'M amiloridend No A)#d5 nd 9,*8 W
8*+; celldeth 6#th )#d 48!M amiloridend -*,49h55*
As F#%'re ++ d#sp$ys3 t h#%h )on)entrt#ons3 amiloridee&en ppered to be
to7#)3 )'s#n% &ery h#%h$y s#%n#"#)nt #n)rese #n celldeth 4pX,*,,,85* At $o6er
)on)entrt#ons3 the amiloride6s #ne""e)t#&e #n e#ther #n)res#n% or de)res#n% cell
deth* The celldeth res'$t#n% "ro! n )#d #n0'ry $one nd n )#d #n0'ry 6#th
amiloridere not s#%n#"#)nt$y d#""erent 4see F#%'re ++5* Th's3 6hen amiloride6s
'sed #n n tte!pt to pre&ent )#d #n0'ry $one3 #t ppered 'nb$e to do so*
;8

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Amiloride Neuroprotection
0
5
10
15
20
25
0
5
oD"% 100% 1m
[Amiloride]
%CellDeath
o -cid
6.0(h)
Figure 22* PI res'$ts "ro! e7per#!ents 6#th !#$or#de ne'roprote)t#on* The s#n%$e str 45 #nd#)tes tht
the 8!M !#$or#de 6#tho't )#d 4se)ond )o$'!n #n "ro! the r#%ht5 d#""ers s#%n#"#)nt$y "ro! the )ontro$
)ond#t#on 4No Dr'%3 No A)#d53 6h#)h #s d#sp$yed #n the "r $e"t )o$'!n 4pX,*,,,85* Note tht 6h#$e the8,,'M !#$or#de 6#tho't )#d d#d not d#""er s#%n#"#)nt$y "ro! the )ontro$ )ond#t#on3 the 8,,'M !#$or#de
6#th )#d 4-*,5 "or 9 ho'rs $so d#d not d#""er s#%n#"#)nt$y "ro! the )#d $one )ond#t#on 4-*,49h55* Error
brs represent stndrd error* N &$'es "ro! $e"t to r#%ht/ ;+3 +83 -3 -3 93 9*
As amilorided#d not pper to be e""e)t#&e3psalmotoin4PcT):&eno!5 6s
p'rs'ed s potent#$ b$o).er to sens#t#2t#on "o$$o6#n% pr#!ry TBI* &salmotoin#s
spe)#"#) b$o).er o" ASIC8 )hnne$s3 nd th#s spe)#"#)#ty !y red')e the to7#)#ty
sso)#ted 6#th amiloride* The per)ent celldeth res'$t#n% "ro! )ontro$ 4No Dr'%5
experimental conditions 6#th nd 6#tho't )#d3 nd se&er$ PcT))on)entrt#on 48,,3
9,,3 ;,,3 ,,3 -,,3 G,3 ,,3 8,,,3 8,,3 nd 9,,,n%:!L &eno!5 )ond#t#ons 6#th nd
6#tho't )#d 6ere deter!#ned t )#d $e&e$s o" ph-*, "or 9 ho'rs* Contro$ experimental
conditions sho6ed G*;, W ,*G, celldeth 6#tho't )#d 4No Dr'% nd No A)#d5 nd
+,*-+ W 8*-8 celldeth 6#th )#d 4No Dr'% nd -*,49h55* 8,,n%:!L &eno! sho6ed
;+
-

43/74
98*9 W +*9 celldeth 6#th )#d 48,,n%:!L &eno! nd -*,49h55 nd n e7per#!ent$
)ond#t#on 6#tho't )#d 6s not per"or!ed* 9,,n%:!L &eno! sho6ed *;- W +*9- cell
deth 6#tho't )#d 49,,n%:!L &eno! nd No A)#d5 nd +8*8+ W 8*;, celldeth 6#th
)#d 49,,n%:!L &eno! nd -*,49h55* ;,,n%:!L &eno! sho6ed *-8 W 8*;- cell
deth 6#tho't )#d 4;,,n%:!L &eno! nd No A)#d5 nd 8-*, W +*88 celldeth 6#th
)#d 4;,,n%:!L &eno! nd -*,49h55* ,,n%:!L &eno! sho6ed G*;, W ,*-9 cell
deth 6#tho't )#d 4,,n%:!L &eno! nd No A)#d5 nd 8-*- W 8*;, celldeth 6#th
)#d 4,,n%:!L &eno! nd -*,49h55* -,,n%:!L &eno! sho6ed -*+- W 8*; cell
deth 6#tho't )#d 4-,,n%:!L &eno! nd No A)#d5 nd 8*8, W 8*9 celldeth 6#th
)#d 4-,,n%:!L &eno! nd -*,49h55* G,n%:!L &eno! sho6ed -*,8 W 8*,8 cell
deth 6#tho't )#d 4G,n%:!L &eno! nd No A)#d5 nd 8G*,9 W 8*G celldeth 6#th
)#d 4G,n%:!L &eno! nd -*,49h55* ,,n%:!L &eno! sho6ed *, W ,*-8 cell
deth 6#tho't )#d 4,,n%:!L &eno! nd No A)#d5 nd 89*+; W +*8 celldeth 6#th
)#d 4,,n%:!L &eno! nd -*,49h55* 8,,,n%:!L &eno! sho6ed 8;*-, W ;*- cell
deth 6#tho't )#d 48,,,n%:!L &eno! nd No A)#d5 nd 8*9 W 8*- celldeth 6#th
)#d 48,,,n%:!L &eno! nd -*,49h55* 8,,n%:!L &eno! sho6ed 9*-+ W ,*-- cell
deth 6#tho't )#d 48,,n%:!L &eno! nd No A)#d5 nd +9*,9 W G*89 celldeth
6#th )#d 48,,n%:!L &eno! nd -*,49h55* 9,,,n%:!L &eno! sho6ed G*+ W 9*8G
celldeth 6#th )#d 49,,,n%:!L &eno! nd -*,49h55 nd 6#tho't )#d e7per#!ent$
)ond#t#on 6s not per"or!ed*
As F#%'re +9 de!onstrtes3 pre$#!#nry dt e7!#n#n% the dose response o"
&eno! sho6 thtpsalmotoin#s not to7#) to the )'$t'res e7)ept t e7tre!e$y h#%h $e&e$s*
In dd#t#on3 th#s dt de!onstrtes tht t #nter!ed#te )on)entrt#ons3psalmotoin!y
;9

44/74
be e""e)t#&e t red')#n% celldeth* =h#$e these res'$ts 6ere not s#%n#"#)nt3 the dt
portryed #n F#%'re +9 re "ro! on$y one 6ee.s 6orth o" e7per#!ents* Hd there been
!ore t#!e3 n #n)resed !!y h&e s'pported s#%n#"#)n)e #n the #nter!ed#te &eno!
)on)entrt#ons*
enom Neuroprotection
0
10
20
0
40
50
60
30
oD"% 100 00 400 500 600 350 00 1000 1500 000
[enom] n!"m#
%C
ellDeath
o -cid
6.0(h)
Figure 23* PI res'$ts "ro! e7per#!ents 6#th &eno! ne'roprote)t#on* Note tht none o" these res'$ts re
s#%n#"#)nt by the S)he""e ANOA postho) test3 $tho'%h the ,,3 -,,3 nd ,,n%:!L o" &eno! 6#th )#d
hd $o6er p&$'es re$t#&e to the rest o" the res'$ts* N &$'es "ro! $e"t to r#%ht/ ;3 83 93 93 8+3 93 93 +3 3
+3 93 -3 8+3 93 93 -3 83 93 93 9*
;;

45/74
Immunocytochemistry esults
Figure 24* I!!'no)yto)he!#stry res'$ts* The $e"t #!%e sho6s s$#)e $be$ed 6#th ne'rotr)e3 nd the
r#%ht #!%e sho6s s$#)e $be$ed 6#th n ASIC+ nt#body* Note tht the ASIC+ hs s$#%ht$y "o$ded
o&er re%#on #n the 'pper $e"thnd )orner o" the #!%e3 b't tht th#s "o$d does not pre&ent )on"#dent
#nterprett#on o" the res'$ts* S)$e brs/ ,,'! "or both #!%es*
Ne'rotr)e $be$s the N#ss$ s'bstn)e 4endop$s!#) ret#)'$'!5 o" neurons3 nd
the $e"t #!%e #n F#%'re +; )$er$y d#sp$ys the $o)$#2t#on o" the ne'rotr)e thro'%ho't
the organotypicrt h#ppo)!p$ s$#)e* The ne'rotr)e #!%e sho6s tht neurons re
present #n $$ re%#ons o" the hippocampus4CA83 CA93 nd dentte %yr's5* The r#%ht
#!%e #n F#%'re +; d#sp$ys the res'$ts o" immunocytochemistry's#n% n ASIC+
antibody* The $o)$#2t#on o" ASIC+ "o$$o6s pttern 6h#)h )$ose$y !#rrors tht o" the
ne'rotr)e3 pper#n% to be present #n $$ res o" the organotypicrt h#ppo)!p$ s$#)e*
Immunocytochemistry6#th n ASIC8 antibody6s tte!pted3 b't d#d not prod')e
res'$ts 6h#)h )o'$d )on"#dent$y be s'%%ested s ASIC8 4the 'to"$'ores)en)e 6s
prt#)'$r$y h#%h both t#!es ASIC8 immunocytochemistry6s tte!pted5*
;
!eurotrace ASIC.a

46/74
.estern /lot esults
Figure 25# =estern B$ot de&e$oped on :8:,-* ASIC8 se)ondry nt#body 'sed* E7pos're t#!e o" +,
!#n'tes* Note $so the dr. bnd pper#n% t ;.D #n the 6ho$e br#n nd )orte7 t#ss'e*
The Western Blotser&ed to 85 prode "'rther s'pport tht ASIC)hnne$s re
present #n the organotypicrt h#ppo)!p$ s$#)es )'$t'red #n the $b nd to +5
de!onstrte the re$t#&e !o'nt o" ASICprote#n present be"ore nd "ter TBI* Both
ASIC8 nd ASIC+ prote#ns h&e theoret#)$ molecular *eighto" ppro7#!te$y
.D3 b't #n pr)t#)e )t'$$y pper t bo't G.D molecular *eight4A$&re2 de $
Ros et $*3 +,,+5*
The ASIC8 Western blot#s sho6n bo&e #n F#%'re +* =h#$e there re se&er$
$ess thn opt#!$ prts to th#s prt#)'$r Western blot4the ho$e #n the !#dd$e nd the
&ert#)$ sh#"t #n the %e$53 #t ne&erthe$ess st#$$ prodes s#%n#"#)nt !o'nt o" #!portnt
#n"or!t#on* The h#%h$#%hted ye$$o6 bo7es "et're the !ost essent#$ dt* The b$ot hs
the r#nbo6 $dder 4sho6s 6here .no6n molecular *eightsre $o)ted on the %e$5 s the
&ery r#%hthnd )o$'!n* The )ontro$ t#ss'e #s the se)ond $ne #n "ro! the r#%ht3 nd the
;-
G.D
--.D
;.D
Ne%t#&eContro$
Pos#t#&e
Contro$
Contro$
Ldder
TBI/8,*9!ph
H#ppo)
!p's
Corte7
=ho$eB
r#n
Cerebe$$'!
Br#nSt
e!

47/74
th#rd $ne #n #s organotypicrt h#ppo)!p$ t#ss'e 4; s$#)es5 )o$$e)ted + ho'rs "o$$o6#n%
the pr#!ry TBI* Th's3 th#s $ne sho6s ho6 !')h ASIC8 #s present #n the )'$t'res
6hen the secondary4)#d5 injury#s pp$#ed*
The $nes #n the !#dd$e on the Western blotre $$ &r#o's prts o" the rt br#n*
Th's3 they re $$ )ontro$s3 s#n)e ASICs sho'$d pper #n $$ re%#ons o" the rt br#n* It
ppers tht th#s #s #ndeed the )se3 s there #s )$er$y !ore #ntense bnd t bo't G.D
"or these $nes3 $tho'%h the presen)e o" the ho$e #n the b$ot does pre&ent ny de"#n#t#&e
ssert#on* The "r $e"t $nes 4the se)ond ye$$o6 h#%h$#%hted bo75 sho6 the pos#t#&e nd
ne%t#&e )ontro$s "or ASIC8 presen)e 4"ro! r#%ht to $e"t respe)t#&e$y5* The pos#t#&e
)ontro$ #s s'$$y dr.er thn the ne%t#&e )ontro$3 #nd#)t#n% spe)#"#)#ty o" the ASIC8
antibody* Th's3 "or $$ the "$6s #n the ASIC8 Western blot3 #t s'%%ests the presen)e
o" ASIC8 prote#n #n )ontro$ organotypicrt h#ppo)!p$ s$#)es3 s 6e$$ s re$t#&e
drop #n the )on)entrt#on o" these )hnne$ prote#ns "o$$o6#n% TBInd s'bse('ent +
ho'r de$y*
;G

48/74
Figure 26* =estern b$ot de&e$oped on :8:,- 6#th +, !#n'te e7pos're* ASIC+ nt#body 'sed*
The ASIC+ Western blotprod')ed &ery 6e. res'$ts3 s sho6n bo&e #n F#%'re
+-* Th#s #s not s'rpr#s#n%3 s ASICantibodiesre notor#o's$y 'ns'))ess"'$* The
ASIC+ b$ot does 6e.$y de!onstrte presen)e o" ASIC+ )hnne$ prote#ns t
ppro7#!te$y the )orre)t molecular *eight3 tho'%h3 s'pport#n% the dt obt#ned "ro!
immunocytochemistry6h#)h sho6s ASIC+ present thro'%ho't the hippocampus*
-endritic 'pine esults
Figure 27* The $e"t #!%e 45 #s o" )ontro$ s$#)e* The r#%ht #!%e 4b5 #s o" do'b$eh#t 4TBI nd )#d
#n0'r#es5 s$#)e 6#th +h de$y* Note the h#%h dendr#t#) sp#ne dens#ty #n the )ontro$ s$#)e nd h#%h &r#)os#ty
dens#ty #n the do'b$eh#t s$#)e* S)$e br/ '! "or both #!%es*
;
--.D
a b
Ne%t#&
eContro$
Pos#t#&e
Contro$
Ldder
Contro$
TBI/8,
*9!ph
H#ppo)!p's
Corte7
Cerebe$$'!
=ho$eBr#n
Br#nS
te!
G.D

49/74
F#%'re +G d#sp$ys ('$#tt#&e representt#ons o" the dendr#t#) spine imaging* The
)ontro$ s$#)e hs re$t#&e$y h#%h spinedens#ty )o!pred to the double hit#n0'ry3 6h#$e
the double hit#n0'ry hs re$t#&e$y h#%h varicositydens#ty )o!pred to the )ontro$*
F#%'re +3 tb$e3 "et'res the ('nt#tt#&e res'$ts "or both spines:8,'! nd
varicosities:8,'! "or the bs#) experimental conditions 4double hit:not #n)$'d#n% ny
dr'% tret!ents5*
pe"ime#talo#ditio# pi#es/10%m
pi#esta#da"d ""o" Va"icosities/10%m
Va"icositiesta#da"d ""o"
o#t"ol 2.53 0.4 0.24 0.08
&'I 0.mph 0 0 0.61 0.0
-cid 6.0(h) 0.06 0.05 0.55 0.15
Do%7le hit (2h delay) 0 0 0.80 0.03Figure 28* Tb$e d#sp$y#n% the dens#ty o" dendr#t#) sp#nes nd &r#)os#t#es per !#)ron o" dendr#te $en%th*
These &$'es re $so portryed #n F#%'res + nd 9,3 #n %rph#)$ "or!* As F#%'re +
de!onstrtes3 the )ontro$ spinedens#ty #s &ery h#%h$y s#%n#"#)nt$y d#""erent "ro! e&ery
other experimental conditions spinedens#ty 44pX,*,,,85* In dd#t#on3 s F#%'re 9,
sho6s3 the double hitvaricositydens#ty #s s#%n#"#)nt$y d#""erent "ro! the )ontro$
varicositydens#ty 4p,*,8853 6h#$e no other experimental conditions d#""er
s#%n#"#)nt$y "ro! the )ontro$ varicositydens#ty*
;

50/74
Dendritic $pine Density
0
0.5
1
1.5
2
2.5
o#t"ol 10.mph 6.0(h) 10.mph2h delay6.0(h)
%&perimental condition
$pines"'(u
Figure 2$# Dendr#t#) sp#ne dens#ty "or e)h bs#) e7per#!ent$ )ond#t#on* Note tht the do'b$e h#t #n0'ry
4the "r r#%ht )o$'!n5 #s 6#th + ho'r de$y* Note $so tht the sp#ne dens#ty #s portryed #n sp#nes:8,'!
rther thn per 8'!3 #n order to $$o6 )o!pr#son to the &r#)os#ty dens#ty* Error brs sho6n re stndrd
errors* The s#n%$e str 45 #nd#)tes tht the )ontro$ )ond#t#on #s &ery h#%h$y s#%n#"#)nt$y d#""erent "ro!
e&ery other )ond#t#on 4pX,*,,,8 "or e&ery other e7per#!ent$ )ond#t#on5* N &$'es "ro! $e"t to r#%ht/ 8;3
8+3 3 -*
aricosity Density
0
0.1
0.2
0.
0.4
0.5
0.6
0.3
0.8
0.
o#t"ol 10.mph 6.0(h) 10.mph2h delay6.0(h)
%&perimental condition
aricosities"'(
Figure 3%* r#)os#ty dens#ty "or e)h bs#) e7per#!ent$ )ond#t#on* Note tht the do'b$e h#t #n0'ry 4the"r r#%ht )o$'!n5 #s 6#th + ho'r de$y* Note $so tht th#s %rph #s on d#""erent y&$'e s)$e #n order to
$$o6 )o!pr#son o" the d#""erent #nd#d'$ e7per#!ent$ )ond#t#on he#%hts3 nd tht &r#)os#ty dens#ty #s
portryed per 8,'!* Error brs sho6n re stndrd errors* The s#n%$e str 45 #nd#)tes tht on$y the
do'b$e h#t #n0'ry 6s s#%n#"#)nt$y d#""erent "ro! the )ontro$ #n ter!s o" &r#)os#ty dens#ty 4p,*,885* N
&$'es "ro! $e"t to r#%ht/ G3 8+3 3 -*
,
-
-

51/74
/iscussion
It 6s !y hypothes#s tht s'b$eth$ !e)hn#)$ #n0'ry 6o'$d sens#t#2e the
hippocampusto se)ondry acidosis3 )'s#n% pre&entb$e ne'ron$ deth* The
obser&t#ons nd dt presented #n th#s thes#s s'%%est tht )o!b#ned TBIo" 8,*9!ph
nd acidosist pH o" -*, res'$t #n n #n0'ry %reter thn e#ther #nd#d'$ #n0'ry 4see
F#%'re +,5* =h#$e the PIdt "or the double hit#n0'ry #s s#%n#"#)nt$y d#""erent "ro! the
TBIt 8,*9!ph $one 4p,*,,,953 #t #s not s#%n#"#)nt$y d#""erent "ro! the )#d #n0'ry
$one3 #nd#)t#n% tht sens#t#2t#on by TBIto s'bse('ent acidosis#s not o))'rr#n%*
F'rther!ore3 stndrd ANOA does not prod')e s#%n#"#)n)e "or the #nter)t#on
bet6een TBInd acidosis3 $tho'%h e)h #n0'ry $one #s s#%n#"#)nt* Th's3 these results
do not seem to support the hypothesis that a primary TBIsensities the hippocampusto
secondary acidosis3 $tho'%h n #n)resed !6o'$d $$o6 "or !ore so$#d s'pport o" th#s
)on)$'s#on*
The )on)$'s#on tht pr#!ry TBIdoes not see! to sens#t#2e to se)ondry
acidosis#n the hippocampusdoes not #!p$y tht the res'$ts re #ns#%n#"#)nt* As
e$borted #n the Introd')t#on3 acidosis)o!!on$y o))'rs s secondary injury
"o$$o6#n% TBI* In ")t3 o" $$ se)ondry br#n #n0'ry ")tors3 acidosis3 hypo)pn#3 nd
hypotens#on o))'r !ost "re('ent$y3 6h#$e acidosisnd hypoxiare re$ted to $on%er
Intens#&e Cre nd hosp#t$ $en%th o" sty 4Jer#!#ts.y et $*3 +,,95* F'rther!ore3 acidosis
sso)#ted 6#th TBItends to be e7tre!e 6#th pH $e&e$s "$$#n% be$o6 -*3 nd th's
acidosistends to %%r&te br#n #n0'ry 4

52/74
hippocampus3 the other opt#on #s tht TBInd acidosisd#sp$y n dd#t#&e re$t#onsh#p*
In other 6ords3 TBInd acidosissee! to be t6o #ndependent #n0'r#es o))'rr#n% by
seprte pth6ys rther thn s#n%$e3 )o!!on pth6y s sens#t#2t#on 6o'$d #!p$y*
M#$d TBInd )#d #n0'r#es 6ere de$#berte$y se$e)ted to 'n!#st.b$y deter!#ne
sens#t#2t#on #nter)t#on3 s the doublehit experimental condition6o'$d be e7pe)ted to
res'$t #n !')h h#%her3 s#%n#"#)nt celldeth re$t#&e to e)h #nd#d'$ experimental
condition4TBInd acidosis5* A$tho'%h the res'$ts d#d not s'pport the hypothes#s3 they
see! to #nd#)te n dd#t#&e e""e)t* It #s #!portnt3 ho6e&er3 to note tht ny
)on)$'s#ons tentt#&e$y !de pp$y on$y to the spe)#"#) TBInd )#d #n0'r#es 'sed #n th#s
thes#s* I" !ore or $ess se&ere #n0'r#es 6ere #n&est#%ted3 #t #s poss#b$e tht the#r
#nter)t#on 6o'$d not be dd#t#&e*
L)tte Dehydro%ense 4LDH53 n en2y!e re$esed by d!%ed cells nd th's
nother te)hn#('e tht #nd#)tes %ross celldeth 6s )ons#dered s !ethod o"
('nt#"y#n% celldeth "or th#s thes#s* Ho6e&er3 #t 6s re0e)ted be)'se 85 o" poss#b$e
#nter)t#ons o" the LDH 6#th the )'$t're !ed#3 6h#)h 6o'$d prod')e 'nre$#b$e res'$ts3
nd +5 the d#sd&nt%e o" not $$o6#n% "or )$ose n$ys#s o" the s$#)e3 on$y o&er$$
h#ppo)!p$ celldeth* The de)#s#on to 't#$#2e PIs the pr#!ry !ethod o" ('nt#"y#n%
celldeth ppers to h&e been d&nt%eo's3 s#n)e PI#!%es $$o6 "or n$ys#s o" the
%%re%te h#ppo)!p$ celldeth3 s 6e$$ s re%#on$ n$ys#s* D'r#n% n$ys#s o" the
%%re%te 4$$ h#ppo)!p$ re%#ons5 PIdt3 #t 6s ('$#tt#&e$y obser&ed tht the dentte
%yr's ppered to h&e h#%her celldeth thn the other h#ppo)!p$ re%#ons "o$$o6#n%
n )#d #n0'ry 4$one or double hit5* The d&nt%e o" PI#s tht #t $$o6s "or se)ond
ro'nd o" n$ys#s o" celldeth3 spe)#"#) to e)h h#ppo)!p$ re%#on*
+

53/74
=h#$e the PIdt o&er$$ s'%%est seprte TBInd acidosis!e)hn#s!s "or
)'s#n% celldeth3 n$ys#s o" the dentte %yr's dt "or the s!e #!%es s #n)$'ded #n
the double hitdt set )o!p$#)te th#s ssert#on* A$tho'%h the TBI$one 6s not
s#%n#"#)nt$y d#""erent "ro! the )ontro$ experimental condition3 th#s 6s hypothes#2ed3
s the TBI6s e7pe)ted to be s'b$eth$ s#n)e !#$d #ntens#ty 6s se$e)ted* The )#d
$one 6s &ery h#%h$y s#%n#"#)nt$y d#""erent "ro! the )ontro$ )ond#t#on 4pX,*,,,853 s
6e$$ s "ro! the double hit#n0'ry 4p,*,8G5* F#n$$y3 the double hit#n0'ry 6s $so
&ery h#%h$y s#%n#"#)nt$y d#""erent "ro! the TBI$one 4pX,*,,,85* T.en to%ether3 the
dentte %yr's res'$ts #nd#)te tht th#s prt#)'$r re%#on o" the hippocampussee!s to be
se$e)t#&e$y &'$nerb$e to )#d #n0'ry3 $tho'%h t th#s po#nt there #s no )ert#n dt to
deter!#ne 6hether tht #n)resed celldeth "o$$o6#n% acidosis#s d'e to n dd#t#&e e""e)t
or to sens#t#2t#on* The res'$ts o" stndrd ANOA do #nd#)te so!e #nter)t#on
bet6een the t6o #n0'r#es 4p,*,+-53 b't the pre)#se #nter)t#on #s 'n.no6n* The
#!portnt )on)$'s#on "ro! n$ys#s o" the dentte %yr's re%#on #s tht the !e)hn#s!s o"
celldeth !y d#""er bet6een h#ppo)!p$ re%#ons nd tht e)h re%#on !y prod')e
res'$ts spe)#"#) to tht re%#on* Th's3 n$ys#s o" %%re%te celldeth "or s$#)e !y "#$
to spot )r')#$ res'$ts* An$ys#s o" the "'n)t#on$$y d#st#n)t re%#ons o" the hippocampus
4CA83 CA93 nd dentte %yr's5 #s )$er$y 6rrnted3 both "or "'t're st'd#es nd "or the
dt )o!pr#s#n% th#s thes#s*
Fo$$o6#n% &ery tentt#&e s'%%est#on o" celldeth o))'rr#n% by seprte
pth6ys "or TBInd acidosis3 e7p$or#n% ne'roprote)t#&e strte%#es "or the )'te t#!e
"r!e "o$$o6#n% TBIsee!ed nt'r$ s pth to p'rs'e3 s#n)e b$o).#n% the e""e)t o" the
acidosis)o!ponent $one sho'$d theoret#)$$y de)rese the o&er$$ double hitcelldeth
9

54/74
to $e&e$s ro'nd tht o" TBI$one* Be"ore red')t#on o" double hitdeth !y be
n$y2ed 6#th prt#)'$r dr'%3 the dr'% !'st be pro&en to red')e celldeth s res'$t
o" n )#d #n0'ry $one* By pron% th#s3 n opt#!$ )on)entrt#on "or the dr'% #s
deter!#ned3 s 6e$$ s n ""#r!t#on tht the spe)#"#) dr'% #s )t'$$y e""e)t#&e #n
!e$#ort#n% deth )'sed by acidosis"or the )'$t're type be#n% 'sed*
F#%'re ++ d#sp$ys the amilorideres'$ts3 nd the $). o" s#%n#"#)n)e bet6een
)#d #n0'ry $one nd 8,,'M amiloride#!p$#es tht amiloride#s not e""e)t#&e t
b$o).#n% celldeth )'sed by acidosis* Note tht th#s does not ne)essr#$y #!p$y tht
amiloride#s 'b#('#to's$y #ne""e)t#&e #t !y be #ne""e)t#&e here d'e to the type o"
)'$t'red s$#)es3 the %e o" the s$#)es3 or !ny other poss#b#$#t#es* Preo's st'd#es 6h#)h
s'%%ested tht amiloride#s nonspeciicb$o).er o" ASICs re pr#!r#$y
electrophysiological4Pr#)e et $*3 +,,, =$d!nn et $*3 8G @#on% et $*3 +,,;53
te)hn#('e 6h#)h n$y2es neurons d'r#n% &ery short t#!e per#od* In these )ses3
amilorideneed on$y be pp$#ed o&er short t#!e 4!#n'tes53 6h#)h #s )ons#derb$y
d#""erent "ro! the $on%er d'rt#on tht amiloride6s pp$#ed #n the e7per#!ents
d#sp$yed #n F#%'re ++ 4ppro7#!te$y 9* ho'rs or the tot$ d'rt#on o" )#d nd 'C!
tret!ent5* Amiloride#s .no6n to h&e n'!ero's e""e)ts3 #n)$'d#n% b$o).#n% the
sod#'!:proton nt#port3 pro!ot#n% sod#'! e7)ret#on nd potss#'! retent#on3 s 6e$$ s
b$o).#n% ASICs* It #s $#.e$y tht the #n)resed celldeth obser&ed s res'$t o"
amiloride#n F#%'re ++ #s d'e to one o" amiloride0snonASIC#n&o$&ed e""e)ts3 6h#)h
o&er n e7tended per#od o" t#!e )o'$d )ert#n$y ne%t#&e$y #!p)t cell"'n)t#on#n%3 e&en
'$t#!te$y res'$t#n% #n celldeth*
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As amilorided#d not pre&ent celldeth by acidosis3 the sp#der &eno! PcT)
6s p'rs'ed s poss#b#$#ty to de)rese celldeth res'$t#n% "ro! acidosis* PcT)hs
been sho6n to be spe)#"#) ASIC8 b$o).er 4Es)o'bs et $*3 +,,, @#on% et $*3 +,,;53
$so thro'%h 'se #n electrophysiologyst'd#es* Ho6e&er3 be)'se PcT)#s &ery
spe)#"#)3 #t 6s hoped tht e7tended e7pos're o" PcT)to the )'$t'red s$#)es 6o'$d be
b$e to de)rese celldeth )'sed by acidosisnd not #n)rese celldeth by d#st'rb#n%
other )e$$'$r !e)hn#s!s s amilorideprobb$y d#d* F#%'re +9 d#sp$ys the &eno!
res'$ts3 6h#)h re $so not s#%n#"#)nt* Ho6e&er3 th#s does not #nd#)te tht the res'$ts re
#ns#%n#"#)nt* '$#tt#&e$y nd e&en ('nt#tt#&e$y there #s so!e s'%%est#on tht
#nter!ed#te )on)entrt#ons o" &eno! !y de)rese celldeth res'$t#n% "ro! )#d #n0'ry
$one3 &ery pro!#s#n% s'%%est#on "or on$y one 6ee.s 6orth o" dt* Th's3 PcT)
sho6s pro!#se s poss#b$e ne'roprote)t#&e %ent %#nst se)ondry acidosis"o$$o6#n%
TBI3 nd the res'$ts portryed #n F#%'re +9 6rrnt "'rther #n&est#%t#on*
Ho6e&er3 s both amiloridend &eno! res'$ts d#d not prod')e s#%n#"#)n)e3 the
('est#on r#ses s to #" ASIC)hnne$s re e&en present #n )'$t'red3 898 dy o$d
organotypicrt h#ppo)!p$ s$#)es* ASIC)hnne$s re .no6n to be present #n !ny
types o" !!!$#n neurons 4A$&re2 de $ Ros3 +,,+ Ch' et $*3 +,,; =$d!nn et
$*3 8G =e!!#e et $*3 +,,+ @#on% et $*3 +,,;53 b't the te)hn#('es nd proto)o$ "or
t#ss'e preprt#on #n th#s thes#s )o'$d poss#b$y )'se ASIC)hnne$ e7press#on to
de)rese or be )o!p$ete$y bsent* Th's3 immunocytochemistry6s p'rs'ed #n order to
)on"#r! tht ASIC)hnne$s 6ere present #n the )'$t'res 'sed #n th#s thes#s* ASIC+
)$er$y ppers to be present #n $$ !0or re%#ons o" the hippocampus4CA83 CA93 nd
dentte %yr's53 s d#sp$yed #n F#%'re +;*

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Un"ort'nte$y3 ASIC8 d#d not prod')e s )$er immunocytochemistryres'$ts
d'e to 'to"$'ores)en)e* By )onse('en)e3 Western blotting6s p'rs'ed #n n tte!pt
85 to s'pport the presen)e o" ASIC)hnne$s3 prt#)'$r$y ASIC83 #n the organotypic
h#ppo)!p$ s$#)es3 nd +5 to sho6 ho6 TBI""e)ts the re$t#&e )on)entrt#on o" the
ASIC)hnne$s #n the s$#)es* As F#%'re + portrys3 ASIC8 #s )$er$y present #n the
)ontro$ )'$t'red s$#)es3 b't "o$$o6#n% TBInd s'bse('ent + ho'r de$y3 the ASIC8
)on)entrt#on #s drst#)$$y red')ed* Th#s red')t#on #n ASIC8 "o$$o6#n% TBI!y
$#!#t the e""#))y o" the &eno!3 s &eno! #s spe)#"#) ASIC8 b$o).er3 nd 6#th $ess
ASIC8 present there re "e6er ASIC8 )hnne$s present "or the &eno! to )t on*
Ho6e&er3 the Western blots'%%ests tht there #s st#$$ t $est so!e ASIC8 present #n the
)'$t'res "o$$o6#n% TBIs#n)e the bnd #s st#$$ )$er$y present b't not#)eb$y "#nter thn
tht o" the )ontro$* Hen)e3 &eno! sho'$d st#$$ be e7p$ored s poss#b$e ne'roprote)t#&e
%ent "o$$o6#n% TBI*
The immunocytochemistrynd Western blotres'$ts $so $$o6 "or "'rther
poss#b$e #!p$#)t#ons 6hen )ons#dered 6#th the PIdt preo's$y d#s)'ssed* The
double hitdt !y pper to be n dd#t#&e res'$t o" the #nd#d'$ TBInd )#d
#n0'r#es be)'se o" "e6 poss#b#$#t#es* F#rst3 TBInd acidosis!y be s#!p$y dd#t#&e3
)t#n% seprte pth6ys to )'se %reter double hit celldeth* Se)ond3 e)h
h#ppo)!p$ re%#on !y prod')e res'$ts spe)#"#) to tht prt#)'$r re%#on3 6h#)h 6hen
n$y2ed to%ether #n the o&er$$ !en #ntens#ty )'se $oss o" s#%n#"#)n)e* Th#rd3 the
ASIC8 !y de)rese s )o!penstory !e)hn#s! "o$$o6#n% TBI* In th#s 6y3 the
cell6o'$d be prote)t#n% #tse$" %#nst "'rther hr! "ro! )#d by pre&ent#n% one ro'te "or
)#d to )t on the cell* Th#s drop #n ASIC8 obser&ed "ro! the Western blot res'$ts
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58/74
"or tret!ent* F'rther!ore3 the 6#ndo6 o" &'$nerb#$#ty "o$$o6#n% TBI!'st be
deter!#ned #n order to s)ert#n #" the !e)hn#s!4s5 o" TBInd )#d #n0'ry re!#n
)ons#stent the ent#re postTBIper#od3 or #" #nter)t#on o))'rs d'r#n% one t#!e #nter&$3
nd dd#t#on d'r#n% nother*
One 6y o" #n&est#%t#n% "'rther the !e)hn#s!4s5 #n&o$&ed #s to e7!#ne 6ht
hppens t the )e$$'$r $e&e$ to )ontr#b'te to the celldeth obser&ed 6#th PI* Dendr#t#)
spine#!%#n% $$o6s "or ('#te sens#t#&e n$ys#s o" s$#)e he$th nd n #nd#re)t !es're
o" s$#)e "'n)t#on3 nd )n be 'sed not on$y to s'pport the res'$ts obt#ned 6#th PI3 b't
$so to )o!pre the str')t'r$ )hn%es "o$$o6#n% TBIor acidosis* C$#n#)$$y3 TBI#s
sso)#ted 6#th both trns#ent nd per!nent !e!ory $oss 4>eddes et $*3 +,,9 No&).
et $*3 +,,, =n% et $*3 8G53 #nd#re)t$y #nd#)t#n% h#ppo)!p$ d!%e* In dd#t#on3
preo's st'd#es h&e #!p$#)ted dendr#t#) spines #n ne'ron$ plasticitysso)#ted 6#th
$ern#n% nd !e!ory "or!t#on 4Hr!s et $*3 +,,- Her#n% nd Shen%3 +,,8 L#pp!n
et $*3 +,,5* These st'd#es s'%%est tht the str')t'res o" the spines the!se$&es !y be
)orre$ted 6#th $tert#ons #n the pth6ys o" #n"or!t#on "$o6* It "o$$o6s tht the
neurons !y 'se these $tert#ons #n the spines to #n)rese or de)rese the#r synpt#)
trns!#ss#on "ter TBI#n order to prote)t "ro! "'rther celldeth or to )o!penste "or
the celldeth tht o))'rred*
F#%'res +G+ sho6 tht #n0'r#n% the s$#)e 4TBI$one3 )#d $one3 or double hit5
#n)reses the dens#ty o" varicositiesnd de)reses the dens#ty o" spines* Note tht 6h#$e
the double hit)ond#t#on #s s#%n#"#)nt )o!pred to the )ontro$ "or the varicositydens#ty3
th#s )o'$d be res'$t o" n dd#t#&e re$t#onsh#p bet6een the t6o #nd#d'$ #n0'r#es #t
does not ne)essr#$y #nd#)te sens#t#2t#on* Moreo&er3 s the spines3 the str')t'res

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$$o6#n% "or communicationt synpse3 pper to dr!t#)$$y de)rese "o$$o6#n% n
#n0'ry3 #t see!s !ost $#.e$y tht the celldeth obser&ed #n the PIdt #s the res'$t o"
!e)hn#s! other thn spine!od'$t#on* In essen)e3 the obser&ed de)rese #n spine
dens#ty 6o'$d see! to #!p$y short ter! prote)t#on by red')t#on o" synpt#) stren%th
4trns#ent prote)t#on %#nst excitotoxicity5 nd de)rese3 rther thn the obser&ed
#n)rese3 #n o&er$$ celldeth*
As preo's$y d#s)'ssed3 spines re )ons#dered to be plasticstr')t'res* Th's3
$tho'%h spinedens#ty drst#)$$y de)resed nd varicositydens#ty drst#)$$y #n)resed
"o$$o6#n% #n0'ry3 #t #s p$'s#b$e tht3 %#&en s'bstnt#$ d'rt#on o" t#!e3 the spine
dens#ty )o'$d #n)rese ner$y b). to #ts or#%#n$ &$'e nd the varicositydens#ty )o'$d
de)rese b). to #ts or#%#n$ &$'e* I" th#s re!ode$#n% does o))'r3 #t s'%%ests tht
re)o&ery !y be poss#b$e nd tht the celldeth o))'rr#n% "o$$o6#n% TBIor acidosis
#n0'ry )o'$d poss#b$y be )o!pensted "or by ne6 synpt#) pth6ys* F't're reser)h
sho'$d )ert#n$y e7p$ore 6hether the dendr#t#) spines e&er repper3 nd #" they do3 ny
potent#$ ne'roprote)t#&e dr'%s sho'$d be )on"#r!ed to not h#nder th#s #n)rese #n spine
dens#ty d'r#n% re)o&ery*
Th's3 t.en to%ether these res'$ts on the s'r")e tentt#&e$y s'%%est tht TBInd
acidosis)t #n n dd#t#&e !nner* Ho6e&er3 pre$#!#nry dt ppers to s'%%est !ore
)o!p$e7 re$t#onsh#p3 6#th re%#on$ &r#t#on* These res'$ts3 6h#$e pre$#!#nry3 do
"'rther 'nderstnd#n% o" the TBIacidosis#nter)t#on nd d&n)e .no6$ed%e "or
poss#b$e tret!ents o" TBIs #n 6h#)h acidosis#s #n&o$&ed*

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$ootnotes
8D#sb#$#t#es res'$t#n% "ro! TBIdepend on the se&er#ty nd $o)t#on o" the #n0'ry3 s
6e$$ s the %e nd he$th o" the pt#ent*+A red')t#on #n o7#dt#&e !etbo$#s! nd $tered %$')ose !etbo$#s! )n $so res'$t
"ro! TBI6#tho't br#n ischemia4esp et $*3 +,,5*9B#o!e)hn#)s Lbortory3 Le%)y Reser)h Center3 8++ NE +ndA&en'e3 Port$nd3 OR
G+9+*;The !0or#ty o" $$ ner&e nd glial)e$$ types s'r&e #n the )'$t're enron!ent3$tho'%h #n the hippocampusthe dens#ty o" synpt#) str')t'res de)reses 4F#nsen et $3
8+5*=h#$e rts shre !ny physiologicalnd ptho$o%#)$ responses to TBI6#th h'!ns3 #t#s notb$e tht the rt t#ss'e 'sed #n these e7per#!ents 6s re!o&ed "ro! the s.'$$*
Hen)e3 the #n0'ry $e&e$s nd res'$t#n% ne'ron$ deth sho'$d not be t.en s d#re)t$y
)o!prb$e to h'!n hed #n0'ry o" the s!e &e$o)#ty* H'!ns h&e br#n t#ss'e tht
#s en)sed 6#th#n hrd3 bony s.'$$ nd 6h#)h #s s'rro'nded by cerebrospinal luid
4CS$53 both o" 6h#)h )t to prote)t the br#n nd bsorb the "or)e o" the #!p)t*F'rther!ore3 pre&ent#on sho'$d $6ys be the "#rst $#ne o" de"ense3 6h#)h #s the reson
beh#nd e7tens#&e !r.et#n% to pro!ote 6er#n% o" he$!ets 6hen r#d#n% b#)y)$es or6er#n% o" setbe$ts 6hen #n !otor &eh#)$e*-=ho$e br#n s$#)es 6ere re0e)ted "or 'se be)'se o" s$#)etos$#)e heterogeneity
4&r#b#$#ty53 6h#)h 6o'$d #!pede ))'rte )o!pr#sons o" e7per#!ents3 nd $so d'e tode!onstrted $oss o" %ross integrityt h#%h TBI))e$ert#ons 4Morr#son et $3 +,,;5*GThe CA+3 "#e$d bet6een the CA9 nd CA8 "#e$ds #n the hippocampusproper3 #s
re)o%n#2ed #n so!e te7ts nd not #n others* In essen)e3 the re re!#ns3 b't so!e$#tert're d#des the re%#on des#%nted s CA+ #nto the CA9 nd CA8 "or &r#o's
resons* Th's3 #n the e7p$nt#on "or h#ppo)!p$ #n"or!t#on "$o63 s 6e$$ s #n F#%'re
G3 the CA+ hs been e""e)t#&e$y Kbsorbed #nto the CA9 nd CA83 b't #s st#$$ present*Th#s pttern o" #n"or!t#on "$o6 #s 'n#('e be)'se Kone6y pro0e)t#ons re e7tre!e$ytyp#)$ "or )ort#)o)ort#)$ )onne)t#ons 4A!r$ nd =#tter3 85* Most cortical
re%#ons )ont#n re)#pro)$ )onne)t#ons*A K!o$e)'$e o" )#d #s )t'$$y proton 4H5* Th's3 !ore )#d#) 4h#%her )on)entrt#ono" protons5 enron!ents 6#$$ h&e h#%her probb#$#ty o" one o" these protons b#nd#n% to
n ASICnd thereby )t#&t#n% #t* The #!portnt po#nt #s s#!p$y tht !ore )#d#)
enron!ent #s !ore $#.e$y to open ASICs*8,M

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#s )$er$y not $6ys poss#b$e3 s ser#o's sy!pto!s do r#se "ro! TBI3 b't does o))'r3
nd prodes hope "or rehb#$#tt#on nd therpy e""orts "o$$o6#n% TBI*89Probes'sed #n Western blotsre typ#)$$y antibodies*8;Un#"or! br#n t#ss'e shear strainres'$ts3 ss'!#n% tht the t#ss'e #n #n)o!press#b$e*
-8

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"lossary
Acidosis/ The )ond#t#on o" #n)resed )#d#ty3 )'sed by the ))'!'$t#on o" )#ds #n the
"$'#ds s'rro'nd#n% the t#ss'e* An bnor!$ )ond#t#on "or the h'!n body tht #s !r.ed
by s6eet breth3 hed)he3 n'se3 &o!#t#n%3 nd s'$ d#st'rbn)es*
Activate/ To $$o6 "'n)t#on o" !o$e)'$e* Here3 )t#&te re"ers to open#n% n ASIC)hnne$ so tht #ons )n pss thro'%h*
Amiloride/ A dr'% tht #s nonspe)#"#) b$o).er o" ASIC )hnne$s*
Anaerobic glycolysis/ A )e$$'$r pro)ess o" !.#n% ener%y tht $).s o7y%en* It )'sesthe b'#$d'p o" )#d#) !o$e)'$es #n the )e$$3 6h#)h )n be dn%ero's to the )e$$s "'n)t#on
nd he$th3 e&en res'$t#n% #n )e$$ deth*
Anoxia/ A red')t#on or tot$ bsen)e #n the s'pp$y o" o7y%en*
Antibody/ A !o$e)'$e 6h#)h "'n)t#ons s the e""e)tor o" n #!!'ne response3 b't 6h#)h
#n th#s thes#s #s 'sed3 #n &r#o's te)hn#('es3 s !r.er tht #nd#)tes the presen)e or
bsen)e o" prote#n by b#nd#n% to #t* The prote#ns #n ('est#on "or th#s thes#s re ASIC
)hnne$s o" d#""erent types3 e)h o" 6h#)h hs #ts o6n spe)#"#) nt#body*
ASIC4A)#dSens#n% Ion Chnne$5/ A $#%nd%ted #on )hnne$ tht opens 6hen n )#d#)!o$e)'$e 4 proton3 the $#%nd5 #s bo'nd to #t* It $$o6s )#d to h&e n e""e)t on the )e$$
by be#n% present #n the )e$$s !e!brne*
Axon/ A )$ss#)$$y $on% e7tens#on or pro)ess "ro! the ne'rons )e$$ body* It )rr#es
ner&e s#%n$s 6y "ro! the )e$$ body nd to6rd other tr%et )e$$s*
Basal ganglia/ A port#on o" the br#n tht #s $o)ted #n the )erebr$ he!#spheres #n the"orebr#n* It #s .no6n to "'n)t#on s re%'$tor o" !otor per"or!n)e*
Behavior/ Obser&b$e )t#ty3 the %%re%te o" responses to both #ntern$ nd e7tern$
st#!'$#*
Bind/ A trns#ent stte 6here t6o !o$e)'$es tt)h to%ether*
Bloc&/ The )t or b#$#ty to stop prt#)'$r )t#on* Here3 b$o). re"ers to p$'%%#n% the
pss%e6y #n ASIC )hnne$s so tht #ons )nnot pss thro'%h*Byproduct/ A se)ondry prod')t or s#dee""e)t !o$e)'$e3 's'$$y 'n#ntent#on$*
Cell/ A !#)ros)op#) str')t're )ont#n#n% n')$e's 4DNA5 nd )ytop$s!#) !ter#$3 nd
en)$osed by se!#per!eb$e !e!brne* There re p$ethor o" )e$$ types3 nd ne'rons
re one type*
Cell body/ The se)t#on o" )e$$ tht )ont#ns the n')$e's3 6h#)h #n t'rn )ont#ns the
%enet#) !ter#$ o" the )e$$* Typ#)$$y3 the dendr#tes br#n% #n"or!t#on to the )e$$ body
nd the 7ons trns!#t the #n"or!t#on 6y "ro! the )e$$ body*
Cell membrane/ A th#n3 "$'#d3 se!#per!eb$e brr#er tht )ontro$s the pss%e o"
s'bstn)es #nto nd o't o" the ne'ron*
Cerebrospinal luid/ See CSF*
Cognition/ The )t or pro)ess o" .no6#n%* Per)ept#on3 th#n.#n%3 !e!ory3 nd reson#n%re $$ prt o" )o%n#t#on*
Communication/ A)t by 6h#)h #n"or!t#on #s trns"erred* E7press#on nd
'nderstnd#n% re prt o" )o!!'n#)t#on*
Conocal/ A type o" !#)ros)ope tht $$o6s the obser&er to s'$#2e ob0e)ts #n s#n%$e
p$)e o" "o)'s3 )ret#n% shrper #!%e*
-+

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Cortical/ O" the )erebr$ )orte73 port#on o" the br#n 6h#)h #s on the s'r")e* It #s the
$r%est nd !ost )o!p$e7 prt o" the !!!$#n br#n nd )ont#ns sensory nd !otor
ner&e )e$$ bod#es* It #s .no6n to "'n)t#on #n )o%n#t#on*
CS$ (Cerebrospinal luid+/ A )$er "$'#d tht "#$$s the &entr#)$es o" the br#n nd the
)entr$ )n$ o" the sp#n$ )ord* It )ont#ns s!$$ ('nt#t#es o" %$')ose nd prote#ns*
Cytoarchitecture/ Ce$$'$r rrn%e!ent nd )onne)t#ons*/eclarative/ One type o" #n"or!t#on tht #s stored by the h#ppo)!p's* It #s e7p$#)#t
#n"or!t#on3 the K6ht3 e*%* ")ts nd e&ents*
/endrite/ One o" typ#)$$y !ny short3 h#%h$y brn)hed pro)esses o" ne'ron* It)on&eys ner&e s#%n$s to6rd the )e$$ body*
/endritic spine/ A !'shroo!shped protr's#on "ro! dendr#tes tht represents the
pr#n)#p$ s#te o" presynpt#) ter!#nt#on onto postsynpt#) ne'ron* Tho'%ht to be &ery sens#t#&e #nd#)tor o" )e$$ he$th*
/iencephalon/ The poster#or se)t#on o" the "orebr#n* It )onne)ts the !#dbr#n to the
)erebr$ he!#spheres*
/issociated/ C'$t'res o" $#n% ne'rons tht re bro.en prt "ro! one nother "o$$o6#n%
d#sse)t#on "ro! !ode$ or%n#s!*/ouble hit/ In th#s thes#s3 th#s ter! re"ers to both pr#!ry 4TBI5 #n0'ry nd se)ondry
4)#d5 #n0'ry ""e)t#n% the s!e s$#)e* It #s n e7per#!ent$ )ond#t#on #n 6h#)h one s$#)e'nder%oes both types o" #n0'r#es3 #n prt#)'$r se('en)e3 nd 6#th prt#)'$r de$y
bet6een the t6o #n0'r#es*
#C$ (#xtra%Cellular $luid+/ The #nterst#t#$ "$'#d nd the p$s! 4e*%* the "$'#d e7#st#n%o'ts#de o" the )e$$s #n n or%n#s!5*
#dema/ S6e$$#n% o" the br#n*
#lectrophysiological/ A te)hn#('e 6h#)h !es'res the stren%th o" ne'rons s#%n$
4te)hn#)$$y3 the ne'rons )'rrent53 n e7tre!e$y rp#d o))'rren)e*
#pileptic/ A trns#ent ne'ro$o%#)$ e&ent #n 6h#)h )ert#n prts o" the br#n re o&er
st#!'$ted3 )'s#n% bnor!$ !otor pheno!en3 $oss o" ttent#on nd )ons)#o'sness3 nd3
t t#!es3 se&ere )on&'$s#ons*
#xcitatory/ Ind')#n% e7)#tt#on or pth6y tht #n)reses ne'ron$ st#!'$t#on*
#xcitotoxicity/ A theory tht s'%%ests tht #n0'ry st#!'$tes the re$ese o" h#%h $e&e$s o"
e7)#ttory ne'rotrns!#tters3 )'s#n% )e$$ deth by the e7)ess#&e )t#&t#on o" re)eptors"or tht prt#)'$r ne'rotrns!#tter*
#xperimental condition/ Re"ers to the spe)#"#) e&ents tht prt#)'$r s$#)e 'nder%oes
d'r#n% %#&en e7per#!ent* For #nstn)e3 #n th#s thes#s3 there re n'!ero's e7per#!ent$)ond#t#ons/ )ontro$3 sh! TBI3 sh! )#d #n0'ry3 TBI3 )#d #n0'ry3 do'b$e h#t 6#th +h
de$y3 do'b$e h#t 6#th ,h de$y3 et)* None o" these e7!p$es #n)$'de ny o" the dr'%
e7per#!ent$ )ond#t#ons r'n*
#xpressed/ Present*
#xtra%Cellular $luid/ See ECF*
$luorescent/ E!#tt#n% $#%ht d'r#n% e7pos're to rd#t#on "ro! n e7tern$ so'r)e*
Bs#)$$y3 br#%ht$y )o$ored3 $!ost %$o6#n% !r.er*
$orebrain/ The nter#or o" the three pr#!ry d#s#ons o" the br#n #n the &ertebrte
e!bryo* It #s the prt o" the d'$t br#n tht #n)$'des the d#en)eph$ons nd the
te$en)eph$on*
-9

64/74
"el electrophoresis/ The seprt#on o" prote#ns on the bs#s o" the#r s#2e nd e$e)tr#)$
)hr%e by the rte o" prote#n !o&e!ent thro'%h n e$e)tr#)$ "#e$d #n %e$*
"lia/ A )$ss o" )e$$s #n the br#n nd sp#n$ )ord tht "or! s'pport#n% str')t're "or thene'rons nd prode )ert#n ne'rons 6#th #ns'$t#on3 #n)res#n% the#r speed o" s#%n$
trns!#ss#on*
"lycolysis (aerobic+/ The pro)ess o" sp$#tt#n% %$')ose3 pr#!ry ener%y so'r)e3 #ntopyr'&te* It #s the be%#nn#n% po#nt "or erob#) resp#rt#on nd the '$t#!te prod')t#on o"
ener%y* Th#s pro)esss needs o7y%en #n order to "'n)t#on*
1emisphere/ In re"eren)e to the br#n3 e#ther o" the $ter$ h$&es o" the )erebr'!*
1eterogeneity21eterogeneous/ Be#n% )o!posed "ro! d#ss#!#$r prts3 &r#b$e*
1ippocampus/ A prt o" the br#n $o)ted #n the )erebr$ he!#spheres nd 6h#)h p$ys
)entr$ ro$e #n !e!ory pro)esses3 spe)#"#)$$y !e!ory stor%e* It #s )o!posed o" s#7!#n se)t#ons .no6n s the h#ppo)!p's proper3 dentte %yr's3 s'b#)'$'!3
pres'b#)'$'!3 prs'b#)'$'!3 nd entorh#n$ )orte7*
1ippocampus proper/ The three se)t#ons o" the h#ppo)!p's .no6n s the CA83 CA+3
nd CA9*
1omogeneity21omogeneous/ A$$ o" the s!e or s#!#$r .#nd or nt're*1omomeric/ A type o" s'b'n#t 6here e)h s'b'n#t #s the s!e 4the prote#n or #on
)hnne$ )ons#sts o" on$y one type o" s'b'n#t5*
1ypothalamus/ The &entr$ prt o" the &ertebrte "orebr#n* It #s .no6n to "'n)t#on #n
!#nt#n#n% ho!eosts#s nd )oord#nt#n% the endo)r#n

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