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,
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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


22/74

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*

++


23/74

'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


25/74

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


26/74

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!


27/74

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


28/74

$o)t#ons on the organotypich#ppo)!p$ 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

+


29/74

))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


42/74

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*

;


55/74

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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57/74


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


59/74

$$o6#n% "or communicationt synpse3 pper to dr!t#)$$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


62/74

"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*

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"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

TRAUMATIC BRAIN INJURY AND ACIDOSIS

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