TRAUMATIC BRAIN INJURY AND ACIDOSIS

by LISA MARIE FORCE

A THESIS Presented to the Dep rt!ent o" B#o$o%y nd the Honors Co$$e%e o" the Un#&ers#ty o" Ore%on #n p rt# $ "'$"#$$!ent o" the re('#re!ents "or the de%ree o" B )he$or o" S)#en)e* No&e!ber +,,-

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

An Abstr )t o" the Thes#s o" L#s For)e "or the de%ree o" B )he$or o" S)#en)e #n the Dep rt!ent o" B#o$o%y to be t .en No&e!ber +,,-* T#t$e/ Tr '! t#) Br #n In0'ry nd A)#dos#s Appro&ed/11111111111111111111111111111111111111 N th n T'b$#t23 PhD* Tr '! t#) Br #n In0'ry 4TBI5 #s s#%n#"#) nt ) 'se o" de th #n the Un#ted St tes3 nd #s the $e d#n% ) 'se o" d#s b#$#ty #n )h#$dren nd yo'n% d'$ts* Acidosis #s one o" the !ost "re('ent se)ond ry br #n #n0'r#es nd #s sso)# ted 6#th $on%er Intens#&e C re nd hosp#t $ $en%th o" st y* Th#s thes#s #n&est#% ted the #nter )t#on bet6een pr#! ry !e)h n#) $ #n0'ry nd se)ond ry acidosis #n0'ry 's#n% n #nno& t#&e e7per#!ent $ TBI !ode$ )ons#st#n% o" organotypic )'$t'red r t hippocampus* Propidium Iodide #! %#n% 6 s 'sed to ssess per)ent cell de th3 6#th res'$ts tent t#&e$y s'%%est#n% n dd#t#&e r ther th n sens#t#2#n% #nter )t#on bet6een TBI nd acidosis* Ho6e&er3 n $ys#s o" the dent te %yr's #nd#) ted th t there ! y be both re%#on $ spe)#"#)#ty o" &'$ner b#$#ty nd re%#on $ #nter )t#on bet6een TBI nd acidosis* Amiloride nd psalmotoxin 6ere #n&est#% ted s poss#b$e ne'roprote)t#&e %ents3 6#th psalmotoxin b't not amiloride prod')#n% pro!#s#n% res'$ts "or red')#n% cell de th by acidosis* Immunocytochemistry nd Western blot te)hn#('es 6ere 't#$#2ed to )on"#r! presen)e o" ASIC )h nne$s #n the organotypic r t hippocampus )'$t'res3 6#th res'$ts de!onstr t#n% presen)e o" ASIC8 nd ASIC+ #n )ontro$ )'$t'res3 nd de)re se #n ASIC8 )on)entr t#on "o$$o6#n% TBI* Dendr#t#) spine imaging 6 s 'sed to e7p$ore syn pt#) str')t'r $ )h n%es res'$t#n% "ro! TBI nd acidosis* The d t obt #ned "ro! these #! %#n% st'd#es s'%%ests th t "o$$o6#n% n #n0'ry dendr#t#) spine dens#ty de)re ses 6h#$e varicosity dens#ty #n)re ses3 #!p$y#n%

; th t the !e)h n#s!s o" cell de th "or TBI nd acidosis re $#.e$y not spine !ed# ted* T .en to%ether3 these res'$ts "'rther o'r 'nderst nd#n% o" the #nter )t#ons bet6een TBI nd acidosis nd re #!port nt "or )ons#der#n% the )$#n#) $ tre t!ent o" TBI* F't're rese r)h sho'$d #n&est#% te "'rther the re%#on $ spe)#"#)#ty o" #n0'ry3 psalmotoxin s poss#b$e ne'roprote)t#&e %ent3 nd the ro$e o" dendr#t#) spine re!ode$#n% "o$$o6#n% TBI nd:or acidosis*

AC<NO=LED>EMENTS The 'thor 6o'$d $#.e to %#&e Theres L's rd#3 #n"#n#te th n.s "or her )onst nt en)o'r %e!ent3 s'pport3 nd s'%%est#ons* Th n. yo' $so to J#$$# n H nsen "or her he$p 6#th e7per#!ents nd so$'t#ons3 to ?h#% n% @#on% "or h#s #n"#n#te .no6$ed%e bo't the s'b0e)t3 nd to S#!on Tho!pson "or the dendr#t#) sp#ne #! %#n% n $ys#s too$s* In dd#t#on3 M#n%AH' L#3 @# n%AP#n% Ch'3 <o#)h# Ino'e3 Josh Seeds3 nd S'A Yo'ne Ch n% deser&e th n.s "or the#r s'pport #n ter!s o" s'pp$#es nd #nstr')t#on* Th n.s to N th n T'b$#t2 "or h#s o'ts#de &#e6 nd ed#ts to th#s thes#s* Th n. yo' to the Le% )y =hee$er Fo'nd t#on "or "#n n)# $ s'pport* The 'thorBs " !#$y deser&es %re t th n. yo' "or the#r $o&e nd %'#d n)e o&er the $ st ++ ye rs* F#n $$y3 th n. yo' to >od "or he$p#n% !e 6#th the " #th to be$#e&e #n !yse$"*

TABLE OF CONTENTS Ch pter I* II* III* IF* F* FI* FII* P %e INTRODUCTIONDDDDDDDDDDDDDDDDD***************E MATERIALS AND METHODSDDDDDDDDDDDDDD****+9 RESULTSDDDDDDDDDDDDDDDDDDDDDDDD9E DISCUSSIONDDDDDDDDDDDDDDDDDDDDD**DC8 FOOTNOTESDDDDDDDDDDDDDDDDDDDDD**D-, >LOSSARYDDDDDDDDDDDDDDDDDDDDD**D**-+ REFERENCESDDDDDDDDDDDDDDDDDDDD**D**-E

LIST OF FI>URES F#%'re F#%'re 8 / pH s) $e F#%'re + / Opt#! $ )e$$ "'n)t#on b sed on pH F#%'re 9 / Lo) t#on o" the h#ppo) !p's #n the h'! n br #n F#%'re ; / CrossAse)t#on o" the r t br #n F#%'re C / Pos#t#on o" the h#ppo) !p's #n the r t br #n F#%'re - / Lo) t#on o" the s#7 ! 0or re%#ons o" the r t h#ppo) !p's F#%'re G / I! %e o" the r t h#ppo) !p's F#%'re E / D# %r ! o" the ! #n #ntr#ns#) )onne)t#ons o" the r t h#ppo) !p's F#%'re H / Str')t're o" n #on )h nne$ F#%'re 8, / I$$'str t#on o" )on"or! t#ons o" $#% ndA% ted #on )h nne$ F#%'re 88 / PI #! %es F#%'re 8+ / I$$'str t#on o" the !ode$ ne'ron F#%'re 89 / E7 !p$e #! %e o" dendr#t#) sp#nes F#%'re 8; / D# %r ! o" she r str #n F#%'re 8C / I! %es o" s#7A6e$$ p$ te nd !#$$#)e$$ F#%'re 8- / T b$e o" e('#& $ent d# $ $e&e$s nd #!p )t &e$o)#t#es F#%'re 8G / C rtoon dep#)t#on o" the TBI #n0'ry de&#)e F#%'re 8E / Photo o" the TBI #n0'ry de&#)e F#%'re 8H / T#!e )o'rse o" e7per#!ents F#%'re +, / Do'b$e H#t )e$$ de th res'$ts "ro! PI d t F#%'re +8 / Dent te >yr's )e$$ de th res'$ts "ro! PI d t F#%'re ++ / A!#$or#de )e$$ de th res'$ts "ro! PI d t F#%'re +9 / Feno! )e$$ de th res'$ts "ro! PI d t F#%'re +; / I!!'no)yto)he!#stry res'$ts F#%'re +C / 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 +E / T b$e o" dendr#t#) sp#ne dens#ty nd & r#)os#ty dens#ty res'$ts F#%'re +H / Dendr#t#) sp#ne dens#ty res'$ts F#%'re 9, / F r#)os#ty dens#ty res'$ts P %e 8, 88 8; 8C 8C 88G 8G 8H 8H +; ++G 98 98 9+ 99 99 9; 9H ;, ;+ ;; ;C ;;E ;E ;H C, C,

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 $$o6s "or per)ept#on3 beh &#or3 tho'%ht3 $ n%' %e3 $e rn#n%3 nd !e!ory3 pro)esses th t de"#ne nd ! .e so e7('#s#te o'r &ery e7#sten)e* The h'! n br #n )ons#sts o" ppro7#! te$y 8,8+ #nd#&#d' $ neurons3 the d#s)rete3 "'n)t#on $ 'n#ts o" the br #n 4Tho!pson3 +,,,5* Th#s n'!ber o" neurons #s %re ter th n $$ the st rs #n the ent#re M#$.y = y % $ 7yI A$$ neurons sh re the s !e b s#) r)h#te)t're nd propert#es3 b't re b$e to prod')e &ery d#""erent3 )o!p$e7 res'$ts d'e to the 6 y th t they re )onne)ted 6#th e )h other 4< nde$ et $*3 +,,,5* These pre)#se n to!#) $ )#r)'#ts3 r ther th n spe)# $#2 t#on o" the #nd#&#d' $ neurons3 %#&e r#se to the )o!p$e7#ty o" $#"e* =hen neuron #s #n0'red or d#es3 #t typ#) $$y $oses the b#$#ty to de(' te$y "'n)t#on s p rt o" #ts )#r)'#t* The "$o6 o" #n"or! t#on $on% th#s p th #s there"ore #nterr'pted3 ) 's#n% the #n"or! t#on to be $ost3 nd $e d#n% to & r#ety o" poss#b$e $osses o" "'n)t#on depend#n% on the p rt#)'$ r )#r)'#t nd the e7tent o" d ! %e* Traumatic Brain Injury (TBI5 #s ) 'se o" s#%n#"#) nt de th nd d#s b#$#ty #n the Un#ted St tes* E )h ye r ppro7#! te$y 8*C to + !#$$#on peop$e e7per#en)e TBI3 8 !#$$#on peop$e 6#th n #n0'ry to the he d re tre ted #n hosp#t $ e!er%en)y roo!s3 nd +9,3,,, peop$e re hosp#t $#2ed s res'$t o" TBI nd s'r&#&e 4NIH 6ebs#te83 NIH 6ebs#te +5* TBI #s the $e d#n% ) 'se o" d#s b#$#ty #n )h#$dren nd yo'n% d'$ts3 nd e&ery ye r bo't E,3,,, peop$e s'""er TBI so ser#o's th t they re $e"t 6#th per! nent3 #n) p )#t t#n% $osses o" "'n)t#on3 s#%n#"#) nt$y $ter#n% the#r "'t're p$ ns3 $#"esty$e3 nd )re t#n% $ r%e !ed#) $ e7penses8 4NIH 6ebs#te +5* Chron#) phys#) $ h r! #s on$y the be%#nn#n%3 tho'%h*

H Co!!on d#s b#$#t#es $so #n)$'de prob$e!s 6#th cognition 4th#n.#n%3 !e!ory3 nd re son#n%53 sensory processing 4s#%ht3 he r#n%3 to')h3 t ste3 nd s!e$$53 communication 4e7press#on nd 'nderst nd#n%53 nd behavior or mental health 4depress#on3 n7#ety3 person $#ty )h n%es3 %%ress#on3 )t#n% o't3 nd so)# $ #n ppropr# teness5 4NIH 6ebs#te 85* =#th s')h h#%h !orb#d#ty3 !ort $#ty3 nd d#s b#$#ty3 the tre t!ent nd pre&ent#on o" TBI re top#)s o" s'bst nt# $ )$#n#) $ $#ter t're nd re est b$#shed p'b$#) he $th )on)erns* Ho6e&er3 the !e)h n#s!s 'nder$y#n% the d#""erent "or!s o" br #n #n0'ry nd e""e)t#&e tre t!ent re not 6e$$ 'nderstood* TBI ) 'ses neuron de th s d#re)t res'$t "ro! the phys#) $3 n to!#) $ #n0'ry t the t#!e o" #!p )t* Th#s type o" ne'ron de th #s d'e to the primary injury* For #nst n)e3 #" b'$$et enters the br #n3 #t phys#) $$y d#sr'pts the br #nBs str')t're3 .#$$#n% the neurons $on% #ts p th* Ho6e&er3 TBI r re$y o))'rs #n #so$ t#onJ the primary injury #s typ#) $$y "o$$o6ed by secondary injury3 6h#)h #s 's' $$y de$ yed nd !ore 6#despre d 4Jere!#ts.y et $*3 +,,95* Typ#) $ secondary injuries #n)$'de metabolic imbalances 4prob$e!s 6#th the )he!#) $ pro)esses ne)ess ry "or the ! #nten n)e o" $#"e53 anoxia 4red')ed s'pp$y or bsen)e o" o7y%en53 hypoxia 4#ns'""#)#ent $e&e$s o" o7y%en #n the t#ss'e53 edema 4s6e$$#n% o" the br #n53 excitotoxicity )o!p$#) t#ons 46hen the #n0'ry ) 'ses the re$e se o" h#%h $e&e$s o" n excitatory ne'rotr ns!#tter3 ) 's#n% cell de th by e7)ess#&e )t#& t#on o" #ts re)eptors53 ischemia 4 de)re se #n the b$ood s'pp$y to p rt or $$ o" the t#ss'e53 nd:or acidosis 4 n bnor! $ #n)re se #n the )#d#ty o" the "$'#ds s'rro'nd#n% the t#ss'e5 4Chesn't et $*3 +,,;J >enn re$$#3 8HH9J Jere!#ts.y et $*3 +,,9J Unterber% et $*3 +,,;5*

8, The )#d#ty o" the b$ood nd interstitial luids 4"$'#d o'ts#de the ) rd#o& s)'$ r syste!5 #s !e s'red $on% the KpH spe)tr'!3L 6#th !ore )#d#) pHs be#n% $o6er & $'es nd !ore b s#) 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 h &#n% pH o" + nd b s#) #te!s $#.e b$e )h nd so p h &#n% pH o" 89* P're 6 ter h s 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 b s#)* Nor! $ physiological pH #s G*; 4see F#%'re 8 "or )$ r#"#) t#on53 nd !ost cells "'n)t#on opt#! $$y t th#s p rt#)'$ r pH* =hen the pH sh#"ts too " r "ro! G*; #n e#ther d#re)t#on3 the prod')t#&#ty o" cells de)re ses3 nd t " rther d#st n)es "ro! pH o" G*;3 the cells e&ent' $$y d#e* F#%'re + #$$'str tes th#s )on)ept*

Figure 1* pH s) $e* Fro! 666*ep *%o&:***:s#te1st'dents:#! %es:phs) $e*%#"*

88

Figure 2* The yA 7#s represents ho6 6e$$ the )e$$ #s "'n)t#on#n%* Note ho63 the "'rther the d#st n)e "ro! the opt#! $ pH 4's' $$y the phys#o$o%#) $ pH o" G*;53 the !ore th t the )e$$Bs proper "'n)t#on#n% #s #!p #red* Fro! http/::666*b#o$o%y)orner*)o!:reso'r)es:en2y!e1ph1%r ph*%#"*

Fo$$o6#n% TBI3 the typ#) $ nor! $ "'n)t#ons o" neurons ! y be $tered* !eurons re )ont#n' $$y prod')#n% )#d#) !o$e)'$es3 b't the !e)h n#s!s "or re!o&#n% these !o$e)'$es "ro! the neurons nd 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 h r! to the neurons d'e to s')h h#%h )on)entr t#ons 4< .' et $*3 8HH95* In dd#t#on3 #t h s been s'%%ested th t e$e)tr#) $ st#!'$ t#on #nd')es e7tr )e$$'$ r )#d#"#) t#on 4@#on% nd Str#n%er3 +,,,53 #!p$y#n% th t b'#$dA'p o" )#d#) !o$e)'$es ! y o))'r s res'$t o" & r#o's e&ents th t pro!ote neuron e7)#t b#$#ty s')h s n epileptic e&ent or n #n0'ry $#.e TBI* These #n0'r#es re )h r )ter#2ed by the e7)ess#&e re$e se o" excitatory !o$e)'$es "ro! #n0'red )e$$s 4<o'r et $*3 8HHEJ Se $ et $*3 8HHH5* Pr#! ry TBIs )o!!on$y ) 'se so!e "or! o" o7y%en dep$et#on3 typ#) $$y by p rt# $$y )'tt#n% o"" b$ood )#r)'$ t#on3 the !ed#'! by 6h#)h o7y%en #s d#str#b'ted to the br #n+* Th#s o7y%en dep$et#on "or)es the br #n to 'se anaerobic glycolysis #n order to

8+ "'n)t#on 4@#on% et $*3 +,,;5* "lycolysis #s the 6 y the body prod')es ener%y3 nd anaerobic glycolysis #s the !ethod "or prod')#n% ener%y th t 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 glycolysis prod')es $ )t#) )#d s byproduct3 ) 's#n% the br #nBs pH to " $$ to $e&e$s ro'nd -*, 4< .' et $*3 8HH9J Neder% rd et $*3 8HH8J @#on% et $*3 +,,;J @' et $*3 8HHE5* Th's3 acidosis 4the b'#$d 'p o" )#d#) !o$e)'$es $#.e $ )t#) )#d #n the t#ss'e5 p$ ys )r')# $ ro$e #n br #n #n0'ry* Rese r)h s'%%ests th t s$#%ht$y )#d#) en&#ron!ent 4 ppro7#! te$y pH-*EApHG*;5 #s )t' $$y prote)t#&e to neurons3 6h#$e !ore e7tre!e acidosis 4p rt#)'$ r$y pH & $'es $o6er th n -*;5 %%r & tes ne'ron $ #n0'ry 4< .' et $*3 8HH9J @#on% et $*3 +,,;5* S#n)e pH typ#) $$y " $$s to $e&e$s bet6een -*, nd -*C3 nd e&en be$o6 -*, #n se&ere ) ses "o$$o6#n% TBI3 acidosis tends to %%r & te br #n #n0'ry 4< .' et $*3 8HH9J @#on% et $*3 +,,;5* To st'dy the ro$e o" acidosis "o$$o6#n% TBI3 #t #s ne)ess ry to h &e n ))'r te nd )$#n#) $$y re$e& nt br #n #n0'ry !ode$* It #s e7tre!e$y d#""#)'$t3 #" not #!poss#b$e3 to )h r )ter#2e )o!p$e7 !e)h n#) $ #ns'$t de$#&ered in vivo 46#th#n $#&#n% or% n#s!5* Th's3 n in vitro 4#n n rt#"#)# $ en&#ron!ent5 #n0'ry !ode$ 6 s 'sed #n th#s rese r)h pro0e)t* Us#n% in vitro )'$t'res $$o6s "or e7pos're to de"#ned st#!'$#3 )ontro$$ed !e)h n#) $ nd b#o)he!#) $ e7per#!ent $ )ond#t#ons3 pre)#se !e s're!ent o" )e$$'$ r response to #n0'ry3 nd the "$e7#b#$#ty to s#!'$ te re%'$ ted secondary injury* The ! 0or#ty o" pre&#o's in vitro #n0'ry !ode$s h &e 'sed !ono$ yer dissociated )e$$ )'$t'res3 6h#)h do not ))'r te$y rese!b$e the #nter)e$$'$ r ! tr#7 nd heterogeneous 4& r# b$e5 cell pop'$ t#on o" in vivo br #n t#ss'e3 nor )$ose$y represent the

89 shear strain 4!e)h n#) $ de"or! t#on5 o" the t#ss'e d'r#n% TBI 4Morr#son et $*3 8HHE5* To o&er)o!e these prob$e!s3 n in vitro #n0'ry !ode$ 6 s de&e$oped by !e!bers o" both the Do6 Ne'rob#o$o%y L b nd the B#o!e)h n#)s L b9* Th#s e7per#!ent $ !ode$ 'ses threeAd#!ens#on $ organotypic br #n )'$t'res r ther th n !ono$ yer o" cells* Th#s threeAd#!ens#on $ t#ss'e preser&es the in vivo heterogeneous cell pop'$ t#on nd cytoarchitecture 4)e$$'$ r rr n%e!ent53 pro&#d#n% cells n)hored on $$ s#des to s'rro'nd#n% cells* The organotypic t#ss'e th's !ore ))'r te$y represents the cellsB sens#n% o" nd response to !e)h n#) $ str #n 4>Mh6#$er et $*3 8HHGJ Stopp#n# et $*3 8HH85* These organotypic br #n s$#)es s'r&#&e #n )ontro$$ed )'$t're en&#ron!ent "or pro$on%ed per#ods o" t#!e;3 $$o6#n% "or )ontro$$ed sett#n% )h r )ter#st#) o" in vitro e7per#!ents 4B hr et $*3 8HHC5* In the e7per#!ents des)r#bed #n th#s thes#s3 the organotypic t#ss'e 'sed 6 s )'$t'red "ro! the r t br #n* R ts 6ere se$e)ted s the !ode$ or% n#s! s h'! n br #n t#ss'e )$e r$y )o'$d not be obt #ned3 nd !o'se 6ho$eAbr #n )'$t'res $ose the#r %ross integrity 4str')t're5 t re$ t#&e$y $o6 #n0'ry $e&e$s* R t t#ss'e3 by )ontr st3 ret #ns #ts %ross integrity e&en t #n0'ry $e&e$s o&er t6#)e the ))e$er t#on $e&e$ 6here !o'se t#ss'e deter#or tes 4person $ )o!!'n#) t#on 6#th T* L's rd#3 +,,-5* Pr#! ry r t )'$t'res re one o" the !ost )on&en#ent nd )ost e""e)t#&e !ethods nd re 6#de$y 't#$#2ed to st'dy & r#o's ne'ron $ de th !e)h n#s!s 4Cho# et $*3 8HEGJ >o"orth et $*3 8HHH5* F'rther!ore3 r ts re !ore ) p b$e t per"or!#n% beh &#or st'd#es th n re !#)e3 so 's#n% r t t#ss'e &o#ds $#!#t#n% "'t're rese r)h poss#b#$#t#es 6#th th#s !ode$* F#n $$y3 r ts re the $e st phy$o%enet#) $$y d& n)ed n#! $ .no6n to sh re phys#o$o%y nd p tho$o%#) $ response to TBI 6#th h'! nsC* In 's#n% r t t#ss'e3 the ss'!pt#on 6 s ! de th t h'! n

8; TBI ) n be rep$#) ted #n the r t nd th t the spe)#es d#""eren)es 4>enn re$$#3 8HH;53 6h#$e )$e r$y present3 6ere not s#%n#"#) nt eno'%h to pre&ent poss#b$e )on)$'s#ons be#n% e7tr po$ ted to the !e)h n#s!s nd tre t!ent o" h'! ns* The r t nd h'! n br #n re not homogenous* The br #n h s d#st#n)t "'n)t#on $ re%#ons3 e )h o" 6h#)h #s n to!#) $$y #dent#"# b$e* These re%#ons #n)$'de3 b't re not $#!#ted to3 the hypothalamus 4$o) ted #n the diencephalon nd respons#b$e "or 'to! t#) "ee$#n%s s')h s h'n%er53 the basal ganglia 4$o) ted #n the cerebral hemispheres #n the orebrain nd re%'$ tor o" !otor per"or! n)e53 nd the hippocampus 4 $so $o) ted #n the )erebr $ hemispheres nd #n&o$&ed 6#th !e!ory stor %e5 4< nde$ et $*3 +,,;53 the $ tter o" 6h#)h #s the "o)'s o" th#s st'dy* The h#ppo) !p's #s deepA$y#n% str')t're o" the orebrain3 nd "or!s #n both hemispheres 4the t6o s#des o" the br #n5 d'r#n% de&e$op!ent* F#%'res 9 thro'%h C dep#)t the pos#t#on o" the hippocampus 6#th#n the o&er $$ )onte7t o" the h'! n 4F#%'re 95 nd r t 4F#%'re ; nd F#%'re C5 br #n*

Figure 3* Lo) t#on o" the h#ppo) !p's #n the h'! n br #n* Fro! 666*!orphon#7*)o!*

8C

Figure 4* CrossAse)t#on o" the r t br #n* Fro! http/::666*b#o*d &#dson*ed':)o'rses:%eno!#)s:!ethod:br #n9*0p%*

Figure 5* Pos#t#on o" h#ppo) !p's #n the r t br #n* Fro! http/::666*d'.e*ed':N !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#es3L or !e!or#es o" e&ents nd ep#sodes 4< nde$ et $*3 +,,,53 nd th's #t #s #!port nt #n the !e!or#2 t#on o" both declarative 4e7p$#)#t3 the K6h t3L e*%* " )ts nd e&ents5 nd nondeclarative 4#!p$#)t3 the Kho63L e*%* pro)ed'r $ se('en)es nd e!ot#on $ responses5 #n"or! t#on 4Tho!pson3 +,,,5* Be#n% so h#%h$y #n&o$&ed #n $e rn#n% nd the "or! t#on o" ne6 !e!or#es3 #t "o$$o6s th t h#ppo) !p $ d ! %e h s been d#re)t$y )orre$ ted to $e rn#n% d#s b#$#t#es nd !e!ory $oss 4>eddes et $*3 +,,9J = n% et $*3 8HHG5* The NIH h s st ted th t the $oss o" spe)#"#) !e!or#es nd the #n b#$#ty to "or! ne6 ones #s the K!ost )o!!on cognitive #!p #r!ent !on% se&ere$y he dA#n0'red p t#ents #s !e!ory $ossL 4NIH 6ebs#te 85* C$#n#) $$y3 TBI #s sso)# ted 6#th both tr ns#ent nd per! nent !e!ory $oss 4No& ). et $*3 +,,,53 #nd#re)t$y #nd#) t#n% h#ppo) !p $ d ! %e* Th's3 the hippocampusA the s#te

8o" !e!ory "or! t#onA #s n #de $ port#on o" the br #n to st'dy #n e7p$or#n% TBI s#n)e !e!ory dys"'n)t#on #s so pre& $ent "o$$o6#n% he d #n0'r#es-* F'rther!ore3 the hippocampus #s re$ t#&e$y homogenous nd 6e$$A)h r )ter#2ed str')t're #n the r t br #n nd ppe rs to be se$e)t#&e$y &'$ner b$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$ds 4CA83 CA+3 nd CA953 6h#)h3 to%ether 6#th "#&e other se)t#ons 4the dent te %yr's3 s'b#)'$'!3 pres'b#)'$'!3 p r s'b#)'$'!3 nd entorh#n $ )orte75 ! .e 'p 6h t #s )o!!on$y ) $$ed the hippocampus 4A! r $ nd =#tter3 8HHC5* F#%'res - nd G respe)t#&e$y dep#)t s)he! t#) d# %r ! nd re $ #! %e o" the hippocampus3 $ be$ed to #d #n #dent#"y#n% these s#7 "ore!ent#oned re%#ons* The !ost re$e& nt re%#ons "or th#s thes#s re the CA83 CA+3 CA93 6h#)h "or! KCLAsh pe3 nd the dent te %yr's3 6h#)h "or!s s! $$er KCLAsh pe ) p ro'nd the CA9 end o" the hippocampus proper*

Figure 6* Th#s "#%'re #$$'str tes the re$ t#&e $o) t#on o" the s#7 ! 0or re%#ons o" the r t 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 !/ ECOEntorh#n $ Corte73 P SOP r s'b#)'$'!3 PrSOPres'b#)'$'!3 SOS'b#)'$'!3 CA8:+:9O Corn' A!!on#s3 D>ODent te >yr's* Fro! A! r $ nd =#tter3 8HHC*

8G

Figure 7* An #! %e o" the r t h#ppo) !p's* D>ODent te >yr's3 CA8:+:9O 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 dent te %yr's &# the per"or nt p th6 y3 the dent te %yr's ne'rons 4.no6n s dent te %r n'$e )e$$s5 pro0e)t the#r !ossy "#bers to the CA9 "#e$d3 the CA9 "#e$d tr ns!#ts #n"or! t#on to the CA8 &# s)h ""er )o$$ ter $sG3 nd the CA8 sends #n"or! t#on to the s'b#)'$'! nd cortical 4)orte73 the o'ter $ yers o" the br #n 6h#)h "'n)t#on #n cognition5 re%#ons o" the br #n* F#%'re E #$$'str tes th#s 'n#('eE p ttern o" #n"or! t#on "$o6*

Figure 8* Th#s d# %r ! dep#)ts the ! #n #ntr#ns#) )onne)t#ons o" the r t h#ppo) !p's* Note ho6 the ! 0or#ty o" p th6 ys re 'n#d#re)t#on $* ECOEntorh#n $ Corte73 P SOP r s'b#)'$'!3 PrSOPres'b#)'$'!3 SOS'b#)'$'!3 CA8:+:9OCorn' A!!on#s3 D>ODent te >yr's* Fro! A! r $ nd =#ter3 8HHC*

8E On$y organotypic r t 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 th t 6#$$ be des)r#bed #n the M ter# $s nd Methods se)t#on o" th#s thes#s* The #nno& t#&e TBI !ode$3 des#%ned spe)#"#) $$y "or these e7per#!ents3 de$#&ers reprod')#b$e3 s) $ b$e3 nd )$#n#) $$y re$e& nt shear strain res'$t nd #s 6e$$ s'#ted "or #dent#"y#n% )r#t#) $ p r !eters %o&ern#n% !e)h n#) $$y #nd')ed sens#t#2 t#on to s'bse('ent )#d#) #n0'ry* The %o $ o" th#s thes#s 6 s to )h r )ter#2e the #nter )t#on bet6een pr#! ry !e)h n#) $ #ns'$t nd se)ond ry acidosis #ns'$t 's#n% the "ore!ent#oned TBI !ode$* It was my hypothesis that a sublethal mechanical injury would sensiti e the hippocampus to secondary acidosis! causing pre"entable neuronal death# It 6 s hoped th t the #nter& $ bet6een these t6o #n0'r#es 6o'$d be re $#st#) t r%et "or ther pe't#) #nter&ent#on3 nd th's th#s rese r)h 6o'$d not on$y d& n)e o'r 'nderst nd#n% o" the #nter )t#on bet6een TBI nd acidosis3 b't $so potent# $$y pro&#de the too$s ne)ess ry "or d#s)o&er#n% poss#b$e tre t!ents* To )o!prehend the e7per#!ent $ tre t!ents e7 !#ned #n th#s thes#s3 #t #s ne)ess ry to .no6 $#tt$e !ore bo't neurons* A neuron #s sep r ted "ro! the extracellular luid 4#C$5 by the neuronBs cell membrane3 th#n3 "$'#d3 semipermeable 4se$e)t#&e5 b rr#er th t )ontro$s the p ss %e o" s'bst n)es #nto nd o't o" the neuron* Interspersed thro'%ho't the cell membrane re prote#ns th t $oo. $#.e do'%hn'ts3 pro&#d#n% door6 y "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" ion 4e$e)tron#) $$y )h r%ed !o$e)'$e5 to p ss thro'%h* F#%'re H dep#)ts the b s#) str')t're o" n ion channel*

8H

Figure $* Th#s dr 6#n% sho6s the "'nd !ent $ str')t're o" n #on )h nne$* In the e7 !p$e dep#)ted3 the #on )h nne$ #s des#%ned "or the p ss %e o" pot ss#'! #ons 4<P5 #nto the )e$$ !e!br ne* Fro! < nde$ et $*3 +,,,*

So!e ion channels re open 4 $$o6#n% the p ss %e o" spe)#"#) ion5 $$ the t#!e3 so!e open d'e to )h n%es #n the &o$t %e o" the )e$$ !e!br ne3 so!e re opened "o$$o6#n% stret)h#n% o" the neuron3 nd so!e )h nne$s depend on ligands 4s#%n $#n% !o$e)'$es5 to dopt the#r open state* Ch nne$s th t need to bind 4 tt )h to5 ligand #n order to be #n the#r open )on"or! t#on re .no6n s ligand%gated ion channels* The ligand3 $!ost $#.e .ey3 !'st be present #n the extracellular luid s 6e$$ s bo'nd to the ligandA% ted )h nne$ "or the )h nne$Bs door to open* =#tho't th#s .ey3 the ligandA % ted )h nne$ re! #ns sh't* F#%'re 8, portr ys s)he! t#) #$$'str t#on o" the )$osed nd open states o" ligand%gated ion channel*

Figure 1%* An #$$'str t#on o" the )$osed nd open )on"or! t#ons o" $#% ndA% ted #on )h nne$* Note th t #n order "or the )h nne$ to dopt the open )on"or! t#on3 the $#% nd !'st be bo'nd to the )h nne$* The open )on"or! t#on $$o6s "or the p ss %e o" s'bst n)e spe)#"#) to th t )h nne$ thro'%h the !e!br ne* Fro! < nde$ et $*3 +,,,*

+, One p rt#)'$ r type o" ligand%gated ion channel #s the )#dAsens#n% #on )h nne$ 4ASIC5* The ligand 6h#)h activates 4opens5 ASICs #s !o$e)'$e o" )#dH* Th's3 #n !ore )#d#) en&#ron!ents 4de)re s#n% pH53 ASICs re !ore $#.e$y to be #n the#r open state* ASICs re expressed 4present5 #n ! ny types o" ! !! $# n neurons 4A$& re2 de $ Ros 3 +,,+J Ch' et $*3 +,,;J = $d! nn et $*3 8HHG J =e!!#e et $*3 +,,+J @#on% et $*3 +,,;53 nd )o!e #n se&er $ & r#et#es* S#7 ASIC subunits 4p rts th t ! y be p't to%ether #n d#""erent )o!b#n t#ons to )re te d#""erent & r#et#es o" ASICs5 h &e been d#s)o&ered/ ASIC8 3 ASIC8Q3 ASIC+ 3 ASIC+Q3 ASIC93 nd ASIC; 4Ch' et $*3 +,,;5* O" these subunits3 homomeric 4)ons#st#n% o" on$y one type o" subunit5 ASIC8 ASIC+ )h nne$s re p rt#)'$ r$y pre& $ent3 o))'rr#n% #n both sensory nd )entr $ neurons 4Ch' et $*3 +,,;53 nd th's these 6ere the )h nne$s se$e)ted "or "'rther n $ys#s #n th#s thes#s* ASIC )h nne$s re not the on$y type o" ion channels "o'nd #n neuronBs !e!br neJ there re h'ndreds o" d#""erent types o" )h nne$s* Th's3 #n order to #so$ te the ASIC )h nne$ "'n)t#on nd deter!#ne the cell de th res'$t#n% "ro! acidosis $one3 #t 6 s ne)ess ry to bloc& the e""e)ts o" these other )h nne$s* Th#s 6 s ))o!p$#shed by dd#n% !#7t're o" se&er $ dr'%s3 .no6n s 'C! 4M<AE,83 CNR@3 nd N#!od#p#ne8,53 thereby b$o).#n% $$ re$e& nt )h nne$s e7)ept "or the ASICs* Any cell de th obser&ed 6 s there"ore the e""e)t o" )#d $one3 not res'$t o" the & r#o's other se)ond ry #n0'ry poss#b#$#t#es "ore!ent#oned* The "'rther n $ys#s p'rs'ed #n re% rd to ASICs 6 s #n tte!pt to bloc& the#r e""e)ts on the neuron* =hen )#d b#nds to n ASIC "o$$o6#n% n #n0'ry3 #t $$o6s "or the ASIC to $et #n & r#o's ions88 !ore th n 's' $* Th#s e7)ess o" ions thro6s o"" the nd

+8 neuronBs n t'r $ b $ n)e so !')h th t 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)re se the o&er $$ )#dA#nd')ed cell de th obser&ed* Se&er $ e7per#!ents h &e de!onstr ted th#s de)re se #n )e$$ de th 6#th spe)#"#) dr'%s th t b$o). ASICs* One s')h dr'% #s .no6n s amiloride3 6h#)h h s ! ny e""e)ts r n%#n% "ro! #d#n% #n the tre t!ent o" hypertens#on to b$o).#n% ASICs* Amiloride h s been de!onstr ted to be nonspeci ic b$o).er o" ASIC )h nne$ )t#& t#on #n se&er $ electrophysiological st'd#es 4Pr#)e et $*3 +,,,J = $d! nn et $*3 8HHG J @#on% et $*3 +,,;5* The ter! nonspeci ic here re"ers to ho6 amiloride )ts/ the dr'% does not d#s)r#!#n te bet6een 6h#)h type o" ASIC th t #t b$o).s3 #t s#!p$y b$o).s ny ASIC )h nne$3 re% rd$ess o" subunit type* Psalmotoxin ( 4or PcT)(5 #s nother dr'% nd #s obt #ned "ro! the &eno! o" the t r nt'$ &salmopoeus cambridgei* PcT)( h s been sho6n to be spe)#"#) ASIC8 b$o).er 4#t on$y b$o).s ASIC8 "'n)t#on5 4Es)o'b s et $*3 +,,,J @#on% et $*3 +,,;5* To e""e)t#&e$y 'se these dr'%s to bloc& ASIC )h nne$ "'n)t#on3 #t #s ne)ess ry to h &e ASIC )h nne$s present #n the cell membrane* M ny te)hn#('es ! y be 'sed to &er#"y the e7#sten)e nd:or $o) $#2 t#on o" ASICs #n t#ss'e3 #n)$'d#n% immunocytochemistry nd Western blot analysis3 t6o pro)ed'res 6h#)h 6#$$ be e7p$ #ned #n the M ter# $s nd Methods se)t#on "o$$o6#n%* Pre&#o's rese r)h stron%$y s'pports both ASIC8 nd ASIC+ e7#sten)e #n ! !! $# n h#ppo) !p $ t#ss'e3 $tho'%h

d#""erent 6 ys o" prep r#n% nd #so$ t#n% t#ss'e )'$t'res ! y ""e)t the pre& $en)e o" the ASICs 4= $d! nn et $*3 8HHG J =e!!#e et $*3 +,,+5*

++ The e7p$or t#on o" ASIC )h nne$ presen)e3 $o) $#2 t#on3 nd b$o). %e #n th#s thes#s 6 s #n dd#t#on to the pr#! ry thes#s ('est#on* It 6 s n tte!pt to be%#n the enor!o's t s. o" deter!#n#n% poss#b$e tre t!ents nd thereby de)re se the )e$$ de th #nd')ed by acidosis3 pro&#d#n% poss#b$e )$#n#) $$y re$e& nt tre t!ent opt#ons "or h'! ns #n the $on%Ater!* Ho6e&er3 the pr#! ry rese r)h hypothes#s re! #ned th t a primary TBI sensiti es the hippocampus to secondary acidosis*

+9

'aterials and 'ethods

'urgery and (issue &rotocol H#ppo) !p $ s$#)e )'$t'res 6ere prep red ))ord#n% to !od#"#ed &ers#on o" the #nter" )e !ethod 'sed by Stopp#n# et $* 48HH85* The Le% )y An#! $ C re nd Use Co!!#ttee o" Le% )y C$#n#) $ Rese r)h nd Te)hno$o%y Center ppro&ed the 'se o" the r t br #ns "or )'$t're* =#ster r ts 6ere n esthet#2ed 6#th h $oth ne CAE d ys postn t $ 4 "ter the !#%r tory ph se o" ner&e )e$$s h s "#n#shed5 be"ore be#n% de) p#t ted* The br #n 6 s then re!o&ed nd p$ )ed #n #)eA)o$d d#sse)t#n% !ed#'! 4H n.Bs BSS3 C!%:!$ %$')ose3 8M HEPES5 "or C !#n'tes pr#or to h#ppo) !p $ d#sse)t#on nd re!o& $* The h#ppo) !p $ t#ss'e 6 s s$#)ed ;,,! th#). 's#n% t#ss'e s$#)er 4M)I$6 #nS3 Stoe$t#n% Co*3 =ood D $e3 IL53 "$o ted #n #)eA)o$d d#sse)t#n% !ed#'!3 nd tr#!!ed to the hippocampus nd d0 )ent entorh#n $ )orte7 t#ss'e* Three t#ss'e s$#)es 6ere tr ns"erred to 'illicellA)! )'$t're p$ te #nsert 6#th !#)roporo's !e!br ne 4PICM,R>C,3 M#$$#pore3 Bed"ord3 MA5 #n )'$t're !ed#'!* The s$#)es 6ere then p$ )ed #n #n)'b tors3 6#th n #de $ en&#ron!ent o" 9-TC3 CU CO+ enr#)hed3 h'!#d#"#ed t!osphere ! #nt #ned* The )'$t're !ed#'! 6 s rep$ )ed e&ery 9A; d ysJ )'$t'res 6ere 'sed 89A8C d ys "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)re sed dens#ty o" syn pt#) str')t'res #n the h#ppo) !p $ s$#)es "o$$o6#n% the s$#)#n% pro)ess* &ropidium Iodide )&I* +pta,e On)e s$#)e 'nder%oes des#%n ted experimental condition 4e*%* TBI3 n )#d #n0'ry3 both TBI nd )#d #n0'ry3 no #n0'ry3 et)*53 #t #s ne)ess ry to deter!#ne th t s$#)eBs $e&e$ o" )e$$ de th* Th#s #s done by $$o6#n% the s$#)e to re)o&er "or ppro7#! te$y +;

+; ho'rs3 then e7pos#n% #t to s'bst n)e th t labels 4! r.s5 on$y the de d )e$$s* Th#s ! r.er #s .no6n s PI 4Prop#d#'! Iod#de53 nd #t te)hn#) $$y on$y $ be$s )e$$s 6#th )o!pro!#sed cellular membranes3 "e t're typ#) $ o" de d )e$$s* =hen the s$#)e #s #! %ed on con ocal !#)ros)ope3 the s$#)e ppe rs 6h#te #n re s o" )e$$ de th nd b$ ). #n re s o" $#&#n% )e$$s* F#%'re 88 portr ys n e7 !p$e o" s$#)e $ be$ed 6#th PI "ter TBI 4 5 nd "ter e7pos're to .no6n to7#) s'bst n)e 4b5* Th's3 's#n% PI #! %#n% $$o6s "or $o) $#2 t#on 46here the #n0'ry ) 'sed the !ost h r!5 s 6e$$ s (' nt#"#) t#on o" cell de th*

Figure 11* The PI #! %e to the $e"t 4 5 sho6s )e$$ de th 4the 6h#te re%#ons5 #n the CA8 nd the dent te %yr's port#ons o" the h#ppo) !p $ s$#)e "o$$o6#n% TBI* The PI #! %e to the r#%ht 4b5 sho6s )e$$ de th #n $$ o" the re%#ons p#)t'red 4CA83 CA+3 CA93 nd the dent te %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 b rs/ +,,'! "or both #! %es*

Pr#or to #! %#n%3 )'$t'res 6ere e7posed "or t $e st one ho'r to PI 4Mo$e)'$ r Probes3 +'%:!$ o" 8 !%:!$ sto).5* I! %es 6ere ) pt'red 6#th Ret#% 89,, PMT 's#n% R) pt're so"t6 re 4R#! %#n%5 on Le#) DMR 'pr#%ht !#)ros)ope 4=et2$ r3 >er! ny5 #$$'!#n ted by !er)'ry r) $ !p nd "#$tered 's#n% C;CnM:CH,nM 4e7)#t t#on:e!#ss#on5 "#$ter set* An e7pos're t#!e o" e#ther 8,,!s or +C,!s 6 s 'sedJ

+C d t presented #n the Res'$ts se)t#on re $$ "ro! +C,!s e7pos're t#!e* Fo$$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'!A"ree )'$t're !ed#'! "or +; ho'rs3 nd "#n $$y #! %ed ;E ho'rs "o$$o6#n% the primary injury* C'$t'res tre ted 6#th the NMDA:%$y)#ne )o!b#n t#on ser&ed s K)ontro$ #n0'r#es3L represent#n% the ! 7#! $ ne'ron $ cell de th th t the )'$t'res )o'$d e7per#en)e* F r# t#on bet6een #nd#&#d' $ )'$t'res b sed on d#""erent# $ s$#)e he $th 6 s th's )o!pens ted "or s#n)e e )h s$#)eBs cell de th 6 s nor! $#2ed to #ts o6n ! 7#! $ cell de th* -endritic 'pine Imaging =h#$e PI #! %#n% #s n e""e)t#&e !e s're o" the o&er $$ he $th o" r t h#ppo) !p $ s$#)e3 )o!p$#!ent ry !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$"3 pro&#d#n% !ore sens#t#&e !e s're o" s$#)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%r t#&e or tr#%%er se)t#on3 )ond')t#$e se)t#on3 nd n o'tp't se)t#on 4< nde$ et $*3 +,,,5* F#%'re 8+ dep#)ts these "o'r se)t#ons #n &ery b s#) "or! t*

+-

Figure 12* The !ode$ ne'ron3 )ons#st#n% o" "o'r se)t#ons* In"or! t#on "$o6s "ro! the top o" the d# %r ! to the botto! nd on to the ne7t ne'ron4s5 #n the p th6 y* Fro! < nde$ et $3 +,,,*

The top3 st rAsh ped port#on o" the neuron3 $ be$ed s the K#nte%r t#&eL se)t#on3 #s the cell body3 or the re%#on 6here the neuronBs %enet#) #n"or! t#on #s stored3 s 6e$$ s 6here s#%n $s "ro! & r#o's other neurons re #nte%r ted 4hen)e the t#t$e K#nte%r t#&eL5 to prod')e one o&er $$ s#%n $* Th#s one s#%n $3 #" #t #s $ r%e eno'%h3 then tr &e$s do6n the )ond')t#&e se)t#on3 $so .no6n s the axon3 to re )h the o'tp't se)t#on3 6here the s#%n $ #s tr ns!#tted to the ne7t neuron #n the p th6 y3 nd the "$o6 o" #n"or! t#on )ont#n'es* These & r#o's s#%n $s rr#&e #n the cell body &# $on% pro)esses e7tend#n% o't6 rd "ro! the cell body* These "#bro's e7tens#ons re .no6n s dendrites3 6h#)h "'n)t#on s re)e#&ers o" #n"or! t#on "ro! other ne'rons 4the K#np'tL se)t#on5* A$on% the $en%th o" he $thy3 "'n)t#on#n% dendr#tes re t#ny protr's#ons ter!ed dendritic spines* Spines )o!e #n 6#de & r#ety o" sh pes nd s#2es3 nd re re! r. b$y dyn !#) str')t'res 4H r!s et $*3 +,,-J Her#n% nd Shen%3 +,,85* =hen spine n'!ber nd !orpho$o%y de)re se3 there #s p r $$e$ de)re se #n synaptic transmission 4#n"or! t#on

+G tr ns"er5 t th t s#te3 s'%%est#n% de)re se #n #n"or! t#on )o!!'n#) t#on to th t neuron* S#n)e the '$t#! te p'rpose o" neuron #s to tr ns"er #n"or! t#on3 #" dendr#t#) spines de)re se3 #t #nd#) tes th t the neuron #s not per"or!#n% #ts "'n)t#on nd #s 'nhe $thy re$ t#&e to #ts pre&#o's st te* 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 sh pes o" spines present $on% th#s p rt#)'$ r dendrite*

Figure 13* Dendr#t#) sp#nes present #n the !otor )orte7 o" pyr !#d $ ne'ron #n n d'$t r t* Note the sp#ne sh pe & r#ety3 po#nted o't by the 6h#te rro6s* S) $e b r/ C'!* Fro! H r!s et $*3 +,,-*

M ny rese r)hers s'%%est th t dendr#t#) spines p$ y s#%n#"#) nt ro$e #n br #n plasticity8+3 or the b#$#ty o" the br #n to )h n%e3 s#n)e $ter t#ons #n the spines the!se$&es ! y be )orre$ ted 6#th $ter t#ons #n the p th6 ys o" #n"or! t#on "$o6 4H r!s et $*3 +,,-J Her#n% nd Shen%3 +,,8J L#pp! n et $*3 +,,C5* Th's3 by n $y2#n% the )h n%es #n spine dens#ty #n r t 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$ed 6#th D#O 4939VAd#$#no$ey$o7 ) rbo)y n#ne per)h$or te3 In&#tro%en53 "$'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 8HH-* A ) p#$$ ry p'$$er 4N r#sh#%# !ode$ PPAE9,5 6 s 'sed to p'$$ %$ ss ) p#$$ ry t'be3 prod')#n% &ery "#ne t#p* Th#s t#p 6 s then )o ted 6#th so$#d D#O )ryst $s 4no d#$'t#on5 nd #nserted #nto !#)ro! n#p'$ tor* The t#p 6 s ) re"'$$y ! ne'&ered to pre)#se

+E $o) t#ons on the organotypic h#ppo) !p $ s$#)e 's#n% +@ ob0e)t#&e on !#)ros)ope* The t#p 6#th the dye 6 s b re$y3 %ent$y to')hed to the s'r" )e o" the t#ss'e3 depos#t#n% D#O )ryst $s* A$$ p rts o" th#s pro)ed're 6ere per"or!ed #n the d r. s !')h s poss#b$e3 s#n)e D#O #s sens#t#&e to $#%ht* Dendr#t#) spine #! %#n% 6 s ) rr#ed o't ppro7#! te$y - ho'rs "o$$o6#n% $ be$#n% o" the s$#)e* The #! %#n% 6 s )o!p$eted 's#n% n 'pr#%ht )o!po'nd !#)ros)ope 4Le#) DMR3 =et2$ r3 >er! ny5 nd Le#) Con"o) $ so"t6 re* L#&e 89A8C d y 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 con ocal !#)ros)ope* The s$#)e 6 s per"'sed 6#th n #C$ so$'t#on t pH o" G*;* D#O $ be$ed prote#ns 6ere #! %ed t ;EEn! e7)#t t#on 4Ar $ ser53 nd e!#ss#on "#$tered t C,,A C9Cn!* A -,@ 6 terA#!!ers#on $ens 6 s 'sed to &#e6 the spines* I! %es 6ere )('#red t s) nn#n% speed o" ;,,H2 nd 6ere typ#) $$y 8,+;@8,+; reso$'t#on 6#th $#ne &er %#n% set to ; s) ns per $#ne* ?oo! 6 s %ener $$y ! #nt #ned t $ess th n or e(' $ to ;@3 s %re ter 2oo! t 8,+;@8,+; reso$'t#on 6o'$d h &e e7)eeded the opt#) $ reso$'t#on o" the ob0e)t#&e* S#n)e dendr#t#) spines e7tend #n three d#!ens#ons 6#th#n the s$#)e3 st ).s o" #! %es 6ere )o$$e)ted o&er se)t#ons sp nn#n% GA+G'! #n the ?A d#!ens#on 6#th ,*CA8*E'! step s#2e #n order to ens're th t str')t'res s s! $$ s spines 6ere "'$$y ) pt'red* Immunocytochemistry Immunocytochemistry #s te)hn#('e th t 't#$#2es luorescent 4%$o6#n%5 antibodies 4! r.ers5 to pre)#se$y dete)t nd $o) $#2e 6here )e$$'$ r )o!ponents $#.e ASIC )h nne$s re* The st nd rd immunocytochemistry proto)o$ 6 s '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

+H ))o!!od te "or the #n)re sed th#).ness o" organotypic t#ss'e s$#)es* A 8/8,, d#$'t#on 6 s 't#$#2ed "or the Ne'rotr )e 4Mo$e)'$ r Probes53 nd 8/8,, d#$'t#on "or the ASIC8 4S#%! 3 S GH;;5 nd ASIC+ 4A$ph D# %nost#)3 MDE>88AA5* .estern /lot 0nalysis Another !ethod #s .no6n s the Western blot te)hn#('e3 nd #t $$o6s "or the #dent#"#) t#on nd !e s're!ent o" the re$ t#&e !o'nt o" p rt#)'$ r prote#n 4e*%* the homomeric ASIC8 )h nne$5* Western blots 'se gel electrophoresis 4e$e)tr#)#ty thro'%h %e$ t#no's ! ter# $5 to sep r te prote#ns b sed on the#r respe)t#&e molecular *eights 4s#2e5* Fo$$o6#n% tr ns"er o" the d t "ro! the %e$ to p per 4 !ore per! nent "or!53 spe)#"#) prote#n 4e*%* ASIC8 5 #s dete)ted by 's#n% probe89 4 ! r.er5* H#ppo) !p $ s$#)es 6ere ho!o%en#2ed #n C,'L o" $ys#s b'""er 4,*8M N C$3 ,*,+M Tr#sAHC$3 8!M EDTA3 8U NPA;,3 8*C!%:!L Aprot#n#n3 8,!%:!L Le'pept#n3 8!%:!L MeOH Pepst t#n3 8,!%:!L PMSF5 * Three h#ppo) !p $ s$#)es 6ere )o$$e)ted "or e )h )ond#t#on 4e*%* three )ontro$ s$#)es3 three TBI t 8,*9!ph s$#)es5* I" the )ond#t#on 6 s TBI3 the s$#)es 6ere )o$$e)ted t + ho'rs "o$$o6#n% the pr#! ry TBI3 to obt #n the s$#)es t st %e th t 6o'$d !#!#) the en&#ron!ent present 6hen secondary 4 )#d5 injury 6o'$d be pp$#ed* Prote#n )on)entr t#ons 6ere !e s'red 's#n% 8!L Br d"ord re %ent 4S#%! 5 6#th 9*9'L o" e )h s !p$e* E(' $ prote#n !o'nts 6ere reso$&ed #n SDSA8,U po$y )ry$ !#de %e$s t 8+,!A nd tr ns"erred to n#tro)e$$'$ose !e!br nes 4B#oR d5 o&ern#%ht* The !e!br nes 6ere b$o).ed 6#th CU dry !#$.A T6een so$'t#on3 #n)'b ted 6#th 8/8,,, pr#! ry antibody d#$'t#on t ;TC o&ern#%ht* A"ter se&er $ r#nses #n PBSAT3 horseAr d#sh pero7#d se 4HRP5 $#n.ed nt#A!o'se se)ond ry antibody 4Che!#)on5 6 s 'sed to dete)t pr#! ry antibodies t d#$'t#on o"

9, 8/+,,, "or H, !#n'tes t roo! te!per t're* The ASIC8 nd ASIC+ antibodies 6ere

obt #ned "ro! S#%! 4S GH;;5 nd A$ph D# %nost#) 4MDE>88AA5 respe)t#&e$y* The pos#t#&e )ontro$s "or both ASIC8 nd ASIC+ 6ere "ro! HE< 4H'! n E!bryon#)

<#ndey5 )e$$s tr ns"e)ted 6#th th t p rt#)'$ r type o" ASIC )h nne$* The ne% t#&e )ontro$ "or ASIC 6ere 'n#nd')ed HE< )e$$s 4not tr ns"e)ted 6#th the ASIC )h nne$5* Fo$$o6#n% e7tens#&e 6 shes #n PBSAT3 HRP pos#t#&e b nds on the b$ots 6ere dete)ted by )he!#$'!#nes)en)e 4Per.#nE$!er5 "or 8 !#n'te* Che!#$'!#nes)en)e 6 s dete)ted on photo%r ph#) "#$! 4<od .5 "or +, !#n'tesJ the "#$! 6 s de&e$oped #n n 'to! t#) de&e$oper 4<od .5* Th's3 the te)hn#('es o" immunocytochemistry nd Western blotting $$o6 "or )on"#r! t#on o" ASIC )h nne$ presen)e #n )'$t'red t#ss'e* (/I 1odel R p#d ))e$er t#on or de)e$er t#on o" the he d d'r#n% )$osedAhe d TBI #!p )t #s be$#e&ed to $e d to t#ss'e shear strain by inertial loading 4>enn re$$# et $*3 8HE+5* For in vitro TBI !ode$#n%3 the b#o$o%#) $ #n0'ry ) s) de sho'$d be #n#t# ted by !e)h n#) $ st#!'$'s !ost ppropr# te to in vivo TBI ) 's t#on* Hen)e3 te ! o" rese r)hers "ro! the Do6 Ne'rob#o$o%y L b nd the B#o!e)h n#)s L b de&e$oped n organotypic !euronal Shear Injury (!SI+ !ode$ 6h#)h e!p$oys #nert# Adr#&en shear strain8; to %ener te s) $ b$e !e)h notr '! * Th#s !ode$ )re tes )h n%e #n ))e$er t#on3 6h#)h ) 'ses #nert# 3 6h#)h #n t'rn ) 'ses the shear strain 4Bott$ n% et $*3 +,,-5* Th's3 the " ster the ))e$er t#on )h n%e3 the !ore se&ere the shear strain* Shear strain o))'rs by !e)h n#s! s#!#$ r to 6h t h ppens 6hen person p'shes st ). o" pr#nter p per t n n%$eA $$ o" the #nd#&#d' $ p %es re! #n 'nh'rt3 b't the st ). h s st#$$ been $tered* Th's3 the hor#2ont $ ne'ron $ cells o" the t#ss'e re! #n

98 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$er t#ons th t rese!b$e the ! %n#t'des o" shear strain obser&ed in vivo3 th's !#!#).#n% the !e)h n#s! by 6h#)h h'! n he d tr '! ) 'ses ne'ron $ #n0'ry 4r p#d ))e$er t#onA #nd')ed shear strain #n0'ry53 s #s obser&ed #n )o!!on tr ""#) ))#dents*

Deformation

Recovery
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 8E5 ! y he$p &#s' $$y e7p$ #n ho6 th#s ! )h#ne 6or.s* The millicells3 )ont #n#n% the )'$t'red t#ss'e 4see F#%'re 8C53 re p$ )ed on the ))e$er t#on !od'$e3 6h#)h ) n ho$d 'p to - M#$$#pore millicells t t#!e #n st nd rd s#7A6e$$ rr n%e!ent* A tr ns$')ent P$e7#%$ s )o&er #s s$#d on top o" these millicells nd $o).ed #nto p$ )e3 prote)t#n% the )'$t'res % #nst )ont !#n t#on nd #r"$o6 d'r#n% e7per#!ents*

Figure 15* A* A st nd rd s#7A6e$$ p$ te #s d#sp$ yed* E )h 6e$$ )ont #ns one M#$$#pore !#$$#)e$$ !e!br ne #n 8!L o" )'$t're !ed#'!* E )h M#$$#pore h s three or% notyp#) r t h#ppo) !p $ s$#)es p$ ted on #t* B* A )$oser &#e6 o" the $o6er r#%ht 6e$$* Note the )'$t're !ed# 3 6h#)h #s the $#%ht or n%e $#('#d3 nd the three d#st#n)t h#ppo) !p $ s$#)es p$ ted on the M#$$#pore*

9+ The ))e$er t#on !od'$e #s then #nserted #nto the $#ne r s$#de3 rest#n% % #nst the p#ston o" $#ne r )t' tor* The )t' tor ))e$er ted the !od'$e o&er H,!! stro.e $en%th nd #s dr#&en by "$y6hee$ !e)h n#s! en% %ed by so$eno#dApo6ered )$'t)h* F r#o's ))e$er t#ons ) n be prod')ed by the de&#)e b sed 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,E3 D893 nd D+C 4 s 6e$$ s ,!ph "or so$e$y )#d #n0'r#es nd sham experimental conditions53 6#th the KDL not t#on re"err#n% to the D# $ on the #n0'ry de&#)e* These d# $ $e&e$s )orrespond to the #!p )t &e$o)#t#es dep#)ted #n F#%'re 8-3 6h#)h 6ere deter!#ned 's#n% h#%hAspeed ) !er syste!* The ) !er syste! 4F st) ! U$t#! AP@ RS3 Photron3 Itron73 CA5 ) pt'red C8+@C8+ p#7e$s:"r !e t 9-3,,, "r !es:s o" the tr ns#ent de"or! t#on o" the )'$t're s'r" )e d'r#n% #!p )t* Note th t the #!p )t &e$o)#ty & $'es3 6h#)h 6#$$ be 'sed "ro! th#s po#nt on to re"er to the $e&e$ o" TBI3 re not d#re)t$y )o!p r b$e to typ#) $ h'! n tr ""#) ))#dents* H'! n br #ns h &e !')h %re ter 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 )r shes3 $ot o" the #nert# $ ener%y o" the #!p )t #s t .en 'p #n the ) r s p rt o" the s "ety "e t'res 4e*%* the b'!pers3 "ront nd re r )r'!p$e 2ones3 se t be$t3 nd #r b %53 6h#)h re des#%ned to red')e the o))'p ntBs ))e$er t#on )h n%e* Th's3 the #n0'r#es to the r t 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*

99

A))e$er t#on Mod'$e

P#ston

B'!per

Figure 17* C rtoon dep#)t#on o" the TBI In0'ry De&#)e*

Figure 18* Photo o" the TBI In0'ry De&#)e*

1aterials #xtracellular luid 4#C$5 6 s prep red "o$$o6#n% #nstr')t#ons by @#on% et $* 4+,,;5 nd 6 s 'sed s the !ed#'! "or pp$y#n% the secondary injury* #C$ 6 s ! de t both pH o" -*, 4the )#d#) & r# b$e5 nd pH o" G*; 4!#!#).#n% phys#o$o%#) $ pH nd p$ )ed on sham secondary injury )'$t'res5* The #C$ 6 s $e"t on )'$t'res th t 'nder6ent secondary injury "or e#ther 8 or 9 ho'rs3 depend#n% on 6h#)h d'r t#on those )'$t'res 6ere ss#%ned "or the e7per#!ent* C'$t're !ed# 6 s prep red s C,U MEM3 +CU horse ser'!3 +CU HBSS3 8M HEPES3 8!M %$'t !#ne 4>$'t ! 73 In&#tro%en3 C r$sb d3 CA53 nd +C!M %$')ose* Ser'!A"ree )'$t're !ed#'! 6 s $so prep red '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*

9; 'C! #s n )rony! 'sed to des#%n te )'$t're !ed# s'pp$e!ented 6#th three )he!#) $s/ 8,'M M<AE,8 4 NMDA nt %on#st53 +,'M CNR@ 4 n AMPA nd . #n#te re)eptor nt %on#st53 nd C'M N#!od#p#ne* The p'rpose o" th#s 'C! !#7t're #s to #so$ te the e""e)t o" )#d#ty on the )'$t'res* In essen)e3 the 'C! b$o).s the e""e)t th t )#d 6o'$d h &e on hypoxia3 excitotoxicity3 nd the n'!ero's other poss#b#$#t#es3 nd $$o6s 6 y to pre&ent $$ cell de th e7)ept s d#re)t ) 'se o" the )#d#) )ond#t#ons* 'C! "'n)t#ons by #so$ t#n% the ASIC 4A)#dASens#n% Ion Ch nne$s5 "'n)t#on #n the t#ss'e3 s e7p$ #ned #n the Introd')t#on* Amiloride nd sp#der &eno! 4PcT)(5 6ere 'sed #n th#s thes#s s poss#b$e ne'roprote)t#&e %ents* Amiloride 6 s p'r)h sed "ro! S#%! 3 nd the PcT)( 6 s obt #ned #n po6der "or! "ro! Sp#derPh r!3 In)* D#$'t#ons 6ere ! de "ro! these b s#) )on)entr t#ons* (ime 2ourse A"ter des#%n t#n% 6h#)h millicells 6#$$ be$on% to 6h#)h experimental condition 4e*%* )ontro$3 sham "or )#dos#s3 sham "or TBI3 both )#dos#s nd TBI #n0'r#es3 )#d or TBI $one53 the )'$t'res th t 'nder%o TBI re #n0'red 's#n% the de&#)e t one o" se&er $ $e&e$s 4,!ph3 ;*C!ph3 8,*9!ph3 8;*E!ph3 or +9*9!ph5* A"ter the TBI3 $$ )'$t'res e7)ept the )ontro$ nd ! 7#!'! )'$t'res 6ere p$ )ed #nto ser'!A"ree )'$t're !ed#'! "or d'r t#on o" e#ther ,h 4no de$ y5 or +h* Fo$$o6#n% the de$ y3 the )'$t'res 6ere p$ )ed
!"ima"y I#$%"y Acceleration Delay in SFCM (Oh or 2h) 30min MCN pre-treatment Se)ond ry In0'ry

-uration
-cid I#$%"y (1 o" ho%"s) (,ildtyype

+ecove"y (24ho%"s) +eplace acid ,ith )/01 !I Ima.i#.

t1 (&'I( )ham* 4.5* 10. * 14.8* 2 . mph)

&,o 15mi# ,ashes

Figure 1$/ T#!e )o'rse o" )o!b#ned ))e$er t#on nd )#dos#s e7per#!ents* #nto 'C! "or 9, !#n'tes 4eno'%h t#!e to e""e)t#&e$y #so$ te ASIC )h nne$ "'n)t#on5*

9C Th#s 9, !#n'te e7pos're to 'C! 6 s sp$#t #nto t6o 8C !#n'tes 6 shes* At the end o" the 9, !#n'tes3 #" the )'$t're #s 'nder%o#n% secondary injury3 #t 6 s p$ )ed #nto pH-*, #C$ "or d'r t#on o" e#ther 8 or 9 ho'rs* I" the )'$t're #s des#%n ted s sham se)ond ry #n0'ry3 #t 6#$$ be #n)'b ted #n pHG*; #C$ "or e#ther 8 or 9 ho'rs* Note th t3 "or )'$t're 'nder%o#n% ,h de$ y e7per#!ent $ )ond#t#on3 the tr'e de$ y #s 9, !#n'tes d'e to the 'C! preAtre t!ent* S#!#$ r$y3 "or )'$t're 'nder%o#n% +h de$ y experimental condition3 the tr'e de$ y #s +*C ho'rs on ))o'nt o" the 9, !#n 'C! preAtre t!ent* On)e the spe)#"#ed d'r t#on o" secondary injury #s )o!p$ete3 the )'$t'res re p$ )ed b ). #nto ser'!A"ree )'$t're !ed#'! nd $$o6ed to #n)'b te o&ern#%ht* T6entyA 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$$o6#n% #n#t# $ TBI53 the )'$t'res re #! %ed* A"ter th#s "#rst ro'nd o" PI #! %#n%3 e )h )'$t're #s e7posed to 8!M NMDA nd 8,'M %$y)#ne "or +; ho'rs3 nd #! %ed se)ond t#!e to obt #n the ! 7#! $ cell de th #! %es 4;E ho'rs "o$$o6#n% #n#t# $ TBI5* F#%'re 8H d#sp$ ys th#s t#!e )o'rse proto)o$* Note th t d'e to t#!e restr#)t#ons nd the ne)ess#ty o" #n)re s#n% ! 4s !p$e s#2e5 & $'es3 de$ y o" + ho'rs 4 )t' $$y +*C ho'rs )ons#der#n% the 'C! preAtre t!ent53 TBI $e&e$ o" 8,*9!ph3 nd n )#d #n0'ry o" -*,#C$ "or 9 ho'rs 6 s )hosen to "o)'s on* -ata 2ollection On)e the PI #! %es re ) pt'red nd s &ed on the )o!p'ter3 they !'st be n $y2ed* I! %eJ 8*9-b so"t6 re 4NIH5 #s 'sed to "#nd the !e n br#%htness o" the #! %e 4the br#%hter the #! %e3 the !ore cells th t re e""e)t#&e$y de d5* These !e n & $'es re then d#&#ded by the !e n & $'e obt #ned "ro! the PI #! %e o" the ! 7#! $ cell de th "or

9th t s !e respe)t#&e h#ppo) !p $ s$#)e 4by NMDAJ see F#%'re 88 "or )$ r#"#) t#on5* =hen th#s "r )t#on #s !'$t#p$#ed by 8,,3 #t %#&es per)ent %e o" the tot $ )'$t're th t #s de d s res'$t o" the spe)#"#) #n0'ry pres)r#bed to th t %#&en )'$t're* A$$ reported & $'es re "or the +; ho'r t#!epo#nt "ter #!p )t 4TBI5 d#&#ded by the ! 7#!'! #ntens#ty 4 "ter e7pos're to NMDA5 "or the s !e s$#)e* The ) $)'$ t#ons "or these per)ent %es 6ere per"or!ed #n M#)roso"t E7)e$ +,,9* The n $ys#s o" the per)ent cell de th "or spe)#"#) re%#on o" the hippocampus3 e*%* the dent te %yr's3 6 s )o!p$eted by "o$$o6#n% the s !e proto)o$ "ore!ent#oned "or the o&er $$ #! %e* The on$y d#""eren)e #s th t the Freeh nd Se$e)t#ons too$ 6 s 'sed to ! n' $$y dr 6 n o't$#ne ro'nd the re%#on #n ('est#on r ther th n 's#n% the o&er $$ #! %e to obt #n !e n #ntens#ty* The s !e o't$#ne 6 s 'sed "or both the +; ho'r cell de th nd the ! 7#! $ cell de th #! %es3 nd then per)ent %e cell de th 6 s ) $)'$ ted by the s !e pro)ess $re dy e7p$ #ned* I! %e J 8*9-b 6 s $so 'sed to n $y2e the dendr#t#) spine images* A"ter sett#n% the s) $e to %$ob $ & $'e o" ;*9,8 p#7e$s:'!3 st ). o" dendr#t#) spine images 6 s then 2Apro0e)ted 's#n% the ! 7#!'! #ntens#ty o" e )h #! %e* >reen or %r ys) $e $oo.A'p t b$es 6ere pr#! r#$y 'sed d'r#n% the n $ys#s pro)ess* The Ce$$ Co'nter too$ #n I! %e J 6 s then 'sed to tr ). the n'!ber o" dendr#t#) spines nd:or varicosities $on% $en%th o" dendrite* The $en%th o" the dendrite 6 s deter!#ned by 's#n% the se%!ented $#ne se$e)t#on too$ #n I! %e J* O&er $$ dens#t#es o" spines nd varicosities per !#)ron 6ere ) $)'$ ted #n M#)roso"t E7)e$ +,,9* 'tatistical 0nalysis

9G M#)roso"t E7)e$ +,,9 spre dsheets 6ere 'p$o ded #nto St tF#e6 "or st t#st#) $ n $ys#s* Pre$#!#n ry %r ph#n% 6 s per"or!ed #n St tF#e63 6h#$e "#n $ %r ph#n% 6 s per"or!ed #n E7)e$* S#%n#"#) n)e 6 s deter!#ned by 's#n% the S)he""e ANOFA PostA ho) test t st nd rd )on"#den)e $e&e$ o" CU* Th#s p rt#)'$ r type o" ANOFA 6 s 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 postAho) test $$o6ed "or the 'ne(' $ & r# n)es nd 'ne(' $ n'!ber o" spe)#!ens to be ))o'nted "or* A st nd rd ANOFA test 6 s $so 'sed "or deter!#n t#on o" #nter )t#on bet6een t6o & r# b$es3 6h#)h #n th#s thes#s 6ere the t6o #n0'r#es* In $$ res'$ts nd %r phs !e n & $'es re represented W one st nd rd error r ther th n st nd rd de&# t#on*

,esults

9E &I 3esults The "'nd !ent $ e7per#!ent per"or!ed 6 s the double hit e7per#!ent to deter!#ne the #nter )t#on bet6een e )h #nd#&#d' $ Kh#t3L or #n0'ry 4TBI nd acidosis5* The per)ent cell de th res'$t#n% "ro! )ontro$ 4No TBI5 experimental conditions 6#th nd 6#tho't )#d3 sham 4Sham TBI5 )ond#t#ons 6#th nd 6#tho't )#d3 nd TBIs 4 t 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 C*GC W ,*C-U cell de th 6#tho't )#d 4No TBI nd No A)#d5 nd 8H*H; W ;*8EU cell de th 6#th )#d 4No TBI nd -*,49h55* Sham experimental conditions sho6ed -*-; W ,*E,U cell de th 6#tho't )#d 4Sham TBI nd No A)#d5 nd 8;*-H W 8*E9U cell de th 6#th )#d 4Sham TBI nd -*,49h55* TBI experimental conditions sho6ed 88*;C W +*+8U cell de th 6#tho't )#d 48,*9!ph nd No A)#d5 nd +C*G8 W +*98U cell de th 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 th t both the TBI $one nd the )#d 4-*, "or 9 ho'rs5 #n0'ry $one re s#%n#"#) nt$y d#""erent th n the )ontro$ experimental condition #n the 6ho$e h#ppo) !p $ s$#)e 4pX,*,,,8 nd pO,*,,+C respe)t#&e$y5* In dd#t#on3 the double hit #n0'ry 4TBI t $e&e$

Double Hit Results

o" 8,*9!ph3 + ho'r de$ y3 nd s'bse('ent -*, #n0'ry "or 9 ho'rs5 #s s#%n#"#) nt$y
0

d#""erent "ro! the TBI t 8,*9!ph $one 4pO,*,,,95* F#n $$y3 st nd rd ANOFA test o" 2o -cid
h) t set prod')es s#%n#"#) n)e "or both TBI $one 4pO,*,,,+5 nd )#d th#s double6.0( hit d
25

% Cell Death

20 4pX,*,,,853 b't does not prod')e s#%n#"#) n)e "or n #nter )t#on bet6een the t6o $one

4pO,*8E,;5* 15
10

2o&'I

)ham&'I

10. mph

TBI Condition

9H

----

Figure 2%* The res'$ts obt #ned "ro! the PI d t o" the do'b$e h#t res'$ts3 th t #s3 the TBI nd )#d #n0'r#es $one nd to%ether3 6#th +h de$ y* Error b rs represent st nd rd error* The s#n%$e st r 4Y5 #nd#) tes th t 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 st r 4YY5 #nd#) tes th t 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$ 4pO,*,,+C5* The tr#p$e st r 4YYY5 #nd#) tes th t the do'b$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 4pO,*,,,95* N & $'es "ro! $e"t to r#%ht/ ;+3 8;3 +83 8C3 +83 8E*

R' $#t t#&e obser& t#on o" the PI #! %es )o!pr#s#n% the double hit d t s'%%ested th t n )#d #n0'ry3 e#ther $one or #n double hit experimental condition3 se$e)t#&e$y res'$ted #n cell de th #n the dent te %yr's re%#on o" the hippocampus* R' nt#t t#&e s'pport "or th#s obser& t#on 6 s p'rs'ed by se$e)t#&e$y n $y2#n% the !e n p#7e$ #ntens#ty "or the dent te %yr's re%#on $one* Contro$ experimental conditions sho6ed 8H*H, W 9*-;U cell de th 6#tho't )#d 4No TBI nd No A)#d5 nd C8*9G W 9*+,U cell de th 6#th )#d 4No TBI nd -*,49h55* TBI experimental conditions sho6ed +9*EG W C*C+U cell de th 6#tho't )#d 48,*9!ph nd No A)#d5 nd -G*+C W ;*;-U cell de th 6#th )#d 48,*9!ph nd -*,49h55*

;, F#%'re +8 d#sp$ ys the res'$ts "ro! th#s (' nt#t t#&e n $ys#s* The double hit #n0'ry #s s#%n#"#) nt$y d#""erent "ro! the )#d #n0'ry $one 4pO,*,8HG53 s 6e$$ s &ery h#%h$y s#%n#"#) nt$y d#""erent "ro! the TBI $one 4pX,*,,,85* The TBI $one 6 s not s#%n#"#) nt$y d#""erent "ro! the )ontro$ experimental condition3 6h#$e the )#d #n0'ry $one 6 s &ery h#%h$y s#%n#"#) nt$y d#""erent "ro! the )ontro$ )ond#t#on 4pX,*,,,85* F'rther!ore3 st nd rd ANOFA test o" the dent te %yr's re%#on $one prod')ed s#%n#"#) n)e "or the )#d #n0'ry $one 4pX,*,,,853 s 6e$$ s "or the #nter )t#on bet6een the t6o #n0'r#es 4TBI nd acidosis5 4pO,*,+-H5*

Dentate Gyrus Cell Death Results

80

30

2o -cid 6.0 ( h)

---

60

% Cell Death

50

40

20

10

2o&'I

10. mph

TBI Condition

Figure 21* The res'$ts obt #ned "ro! the PI d t o" the do'b$e h#t res'$ts 6#th +h de$ y "or the dent te %yr's re%#on o" the h#ppo) !p's* Error b rs represent st nd rd error* The s#n%$e st r 4Y5 #nd#) tes th t 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 st r 4YY5 #nd#) tes th t 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 4pO,*,8HG5 nd "ro! the TBI $one 4pX,*,,,85* Note th t 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 8E*

There 6 s hope th t amiloride )o'$d be potent# $ dr'% to b$o). the e""e)ts o" se)ond ry )#dos#s "o$$o6#n% the pr#! ry TBI3 s#n)e amiloride h s been de!onstr ted to

;8 nonAspe)#"#) $$y b$o). ASIC )h nne$ )t#& t#on #n electrophysiological st'd#es 4Pr#)e et $*3 +,,,J = $d! nn et $*3 8HHG J @#on% et $*3 +,,;5* The per)ent cell de th res'$t#n% "ro! )ontro$ 4No Dr'%5 experimental conditions 6#th nd 6#tho't )#d3 )on)entr t#on one 48,,'M amiloride5 )ond#t#ons 6#th nd 6#tho't )#d3 nd )on)entr t#on t6o 4 t 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 C*GC W ,*C-U cell de th 6#tho't )#d 4No Dr'% nd No A)#d5 nd 8H*88 W +*H;U cell de th 6#th )#d 4No Dr'% nd -*,49h55* Con)entr t#on one experimental conditions sho6ed -*8- W ,*G,U cell de th 6#tho't )#d 48,,'M amiloride nd No A)#d5 nd 8C*8; W 8*E8U cell de th 6#th )#d 48!M amiloride nd -*,49h55* Con)entr t#on t6o experimental conditions sho6ed +;*,; W ,*G9U cell de th 6#tho't )#d 48,,'M amiloride nd No A)#d5 nd 9,*8H W 8*+;U cell de th 6#th )#d 48!M amiloride nd -*,49h55* As F#%'re ++ d#sp$ ys3 t h#%h )on)entr t#ons3 amiloride e&en ppe red to be to7#)3 ) 's#n% &ery h#%h$y s#%n#"#) nt #n)re se #n cell de th 4pX,*,,,85* At $o6er )on)entr t#ons3 the amiloride 6 s #ne""e)t#&e #n e#ther #n)re s#n% or de)re s#n% cell de th* The cell de th res'$t#n% "ro! n )#d #n0'ry $one nd n )#d #n0'ry 6#th amiloride re not s#%n#"#) nt$y d#""erent 4see F#%'re ++5* Th's3 6hen amiloride 6 s 'sed #n n tte!pt to pre&ent )#d #n0'ry $one3 #t ppe red 'n b$e to do so*

;+

Amiloride Neuroprotection
5

2o -cid
0

6.0( h)
25

% Cell Death

20

15

10

0 2oD"%. 100%1 1m1

[Amiloride]
Figure 22* PI res'$ts "ro! e7per#!ents 6#th !#$or#de ne'roprote)t#on* The s#n%$e st r 4Y5 #nd#) tes th t the 8!M !#$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 th t 6h#$e the 8,,'M !#$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 b rs represent st nd rd error* N & $'es "ro! $e"t to r#%ht/ ;+3 +83 -3 -3 93 9*

As amiloride d#d not ppe r to be e""e)t#&e3 psalmoto4in 4PcT)(:&eno!5 6 s p'rs'ed s potent# $ b$o).er to sens#t#2 t#on "o$$o6#n% pr#! ry TBI* &salmoto4in #s spe)#"#) b$o).er o" ASIC8 )h nne$s3 nd th#s spe)#"#)#ty ! y red')e the to7#)#ty sso)# ted 6#th amiloride* The per)ent cell de th res'$t#n% "ro! )ontro$ 4No Dr'%5 experimental conditions 6#th nd 6#tho't )#d3 nd se&er $ PcT)( )on)entr t#on 48,,3 9,,3 ;,,3 C,,3 -,,3 GC,3 H,,3 8,,,3 8C,,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,U cell de th 6#tho't )#d 4No Dr'% nd No A)#d5 nd +,*-+ W 8*-8U cell de th 6#th )#d 4No Dr'% nd -*,49h55* 8,,n%:!L &eno! sho6ed

;9 98*C9 W +*9EU cell de th 6#th )#d 48,,n%:!L &eno! nd -*,49h55 nd n e7per#!ent $ )ond#t#on 6#tho't )#d 6 s not per"or!ed* 9,,n%:!L &eno! sho6ed E*;- W +*9-U cell de th 6#tho't )#d 49,,n%:!L &eno! nd No A)#d5 nd +8*8+ W 8*;,U cell de th 6#th )#d 49,,n%:!L &eno! nd -*,49h55* ;,,n%:!L &eno! sho6ed E*-8 W 8*;-U cell de th 6#tho't )#d 4;,,n%:!L &eno! nd No A)#d5 nd 8-*E, W +*88U cell de th 6#th )#d 4;,,n%:!L &eno! nd -*,49h55* C,,n%:!L &eno! sho6ed G*;, W ,*-9U cell de th 6#tho't )#d 4C,,n%:!L &eno! nd No A)#d5 nd 8-*-C W 8*;,U cell de th 6#th )#d 4C,,n%:!L &eno! nd -*,49h55* -,,n%:!L &eno! sho6ed -*+- W 8*H;U cell de th 6#tho't )#d 4-,,n%:!L &eno! nd No A)#d5 nd 8C*8, W 8*9EU cell de th 6#th )#d 4-,,n%:!L &eno! nd -*,49h55* GC,n%:!L &eno! sho6ed -*,8 W 8*,8U cell de th 6#tho't )#d 4GC,n%:!L &eno! nd No A)#d5 nd 8G*,9 W 8*GCU cell de th 6#th )#d 4GC,n%:!L &eno! nd -*,49h55* H,,n%:!L &eno! sho6ed C*C, W ,*-8 U cell de th 6#tho't )#d 4H,,n%:!L &eno! nd No A)#d5 nd 89*+; W +*8C U cell de th 6#th )#d 4H,,n%:!L &eno! nd -*,49h55* 8,,,n%:!L &eno! sho6ed 8;*-, W ;*C- U cell de th 6#tho't )#d 48,,,n%:!L &eno! nd No A)#d5 nd 8H*H9 W 8*-EU cell de th 6#th )#d 48,,,n%:!L &eno! nd -*,49h55* 8C,,n%:!L &eno! sho6ed 9*-+ W ,*-- U cell de th 6#tho't )#d 48C,,n%:!L &eno! nd No A)#d5 nd +9*,9 W G*89 U cell de th 6#th )#d 48C,,n%:!L &eno! nd -*,49h55* 9,,,n%:!L &eno! sho6ed CG*+C W 9*8G U cell de th 6#th )#d 49,,,n%:!L &eno! nd -*,49h55 nd 6#tho't )#d e7per#!ent $ )ond#t#on 6 s not per"or!ed* As F#%'re +9 de!onstr tes3 pre$#!#n ry d t e7 !#n#n% the dose response o" &eno! sho6 th t psalmoto4in #s not to7#) to the )'$t'res e7)ept t e7tre!e$y h#%h $e&e$s* In dd#t#on3 th#s d t de!onstr tes th t t #nter!ed# te )on)entr t#ons3 psalmoto4in ! y

;; be e""e)t#&e t red')#n% cell de th* =h#$e these res'$ts 6ere not s#%n#"#) nt3 the d t portr yed #n F#%'re +9 re "ro! on$y one 6ee.Bs 6orth o" e7per#!ents* H d there been !ore t#!e3 n #n)re sed ! ! y h &e s'pported s#%n#"#) n)e #n the #nter!ed# te &eno! )on)entr t#ons*
enom Neuroprotection
30

60

2o -cid 6.0( h)

50

% Cell Death

40

20

10

0 2oD"%. 100 00 400 500 600 [ enom] n!"m# 350 400 1000 1500 000

Figure 23* PI res'$ts "ro! e7per#!ents 6#th &eno! ne'roprote)t#on* Note th t none o" these res'$ts re s#%n#"#) nt by the S)he""e ANOFA postAho) test3 $tho'%h the C,,3 -,,3 nd H,,n%:!L o" &eno! 6#th )#d h d $o6er pA& $'es re$ t#&e to the rest o" the res'$ts* N & $'es "ro! $e"t to r#%ht/ E;3 C83 93 93 8+3 93 93 +3 H3 +3 93 -3 8+3 93 93 -3 8E3 93 93 9*

;C Immunocytochemistry 3esults

!eurotrace

ASIC.a

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$ed 6#th n ASIC+ nt#body* Note th t the ASIC+ h s s$#%ht$y "o$ded o&er re%#on #n the 'pper $e"tAh nd )orner o" the #! %e3 b't th t th#s "o$d does not pre&ent )on"#dent #nterpret t#on o" the res'$ts* S) $e b rs/ C,,'! "or both #! %es*

Ne'rotr )e $ be$s the N#ss$ s'bst n)e 4endop$ s!#) ret#)'$'!5 o" neurons3 nd the $e"t #! %e #n F#%'re +; )$e r$y d#sp$ ys the $o) $#2 t#on o" the ne'rotr )e thro'%ho't the organotypic r t h#ppo) !p $ s$#)e* The ne'rotr )e #! %e sho6s th t neurons re present #n $$ re%#ons o" the hippocampus 4CA83 CA93 nd dent te %yr's5* The r#%ht #! %e #n F#%'re +; d#sp$ ys the res'$ts o" immunocytochemistry 's#n% n ASIC+ antibody* The $o) $#2 t#on o" ASIC+ "o$$o6s p ttern 6h#)h )$ose$y !#rrors th t o" the ne'rotr )e3 ppe r#n% to be present #n $$ re s o" the organotypic r t h#ppo) !p $ s$#)e* Immunocytochemistry 6#th n ASIC8 antibody 6 s tte!pted3 b't d#d not prod')e res'$ts 6h#)h )o'$d )on"#dent$y be s'%%ested s ASIC8 4the 'to"$'ores)en)e 6 s p rt#)'$ r$y h#%h both t#!es ASIC8 immunocytochemistry 6 s tte!pted5*

;.estern /lot 3esults HG.D

--.D

;C.D

Ne% t#&e Contro$

Pos#t#&e Contro$

TBI/ 8,*9!ph

H#ppo) !p's

=ho$e Br #n

Cerebe$$'!

Br #n Ste!

Contro$

Figure 25# =estern B$ot de&e$oped on H:8H:,-* ASIC8 se)ond ry nt#body 'sed* E7pos're t#!e o" +, !#n'tes* Note $so the d r. b nd ppe r#n% t ;C.D #n the 6ho$e br #n nd )orte7 t#ss'e*

The Western Blot ser&ed to 85 pro&#de "'rther s'pport th t ASIC )h nne$s re present #n the organotypic r t h#ppo) !p $ s$#)es )'$t'red #n the $ b nd to +5 de!onstr te the re$ t#&e !o'nt o" ASIC prote#n present be"ore nd "ter TBI* Both ASIC8 nd ASIC+ prote#ns h &e theoret#) $ molecular *eight o" ppro7#! te$y

CE.D 3 b't #n pr )t#)e )t' $$y ppe r t bo't GE.D molecular *eight 4A$& re2 de $ Ros et $*3 +,,+5* The ASIC8 Western blot #s sho6n bo&e #n F#%'re +C* =h#$e there re se&er $ $ess th n opt#! $ p rts to th#s p rt#)'$ r Western blot 4the ho$e #n the !#dd$e nd the &ert#) $ sh#"t #n the %e$53 #t ne&erthe$ess st#$$ pro&#des s#%n#"#) nt !o'nt o" #!port nt #n"or! t#on* The h#%h$#%hted ye$$o6 bo7es "e t're the !ost essent# $ d t * The b$ot h s the r #nbo6 $ dder 4sho6s 6here .no6n molecular *eights re $o) ted on the %e$5 s the &ery r#%htAh nd )o$'!n* The )ontro$ t#ss'e #s the se)ond $ ne #n "ro! the r#%ht3 nd the

L dder

Corte7

;G th#rd $ ne #n #s organotypic r t 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 )'$t'res 6hen the secondary 4 )#d5 injury #s pp$#ed* The $ nes #n the !#dd$e on the Western blot re $$ & r#o's p rts o" the r t br #n* Th's3 they re $$ )ontro$s3 s#n)e ASICs sho'$d ppe r #n $$ re%#ons o" the r t br #n* It ppe rs th t th#s #s #ndeed the ) se3 s there #s )$e r$y !ore #ntense b nd t bo't GE.D "or these $ nes3 $tho'%h the presen)e o" the ho$e #n the b$ot does pre&ent ny de"#n#t#&e ssert#on* The " r $e"t $ nes 4the se)ond ye$$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' $$y d r.er th n the ne% t#&e )ontro$3 #nd#) t#n% spe)#"#)#ty o" the ASIC8 antibody* Th's3 "or $$ the "$ 6s #n the ASIC8 Western blot3 #t s'%%ests the presen)e o" ASIC8 prote#n #n )ontro$ organotypic r t h#ppo) !p $ s$#)es3 s 6e$$ s re$ t#&e drop #n the )on)entr t#on o" these )h nne$ prote#ns "o$$o6#n% TBI nd s'bse('ent + ho'r de$ y*

;E

HG.D --.D

Ne% t#&e Contro$

Pos#t#&e Contro$

Figure 26* =estern b$ot de&e$oped on H:8H:,- 6#th +, !#n'te e7pos're* ASIC+

TBI/ 8,*9!ph Contro$ L dder

Corte7 H#ppo) !p's

=ho$e Br #n

Cerebe$$'!

Br #n Ste!

nt#body 'sed*

The ASIC+ Western blot prod')ed &ery 6e . res'$ts3 s sho6n bo&e #n F#%'re +-* Th#s #s not s'rpr#s#n%3 s ASIC antibodies re notor#o's$y 'ns'))ess"'$* The ASIC+ b$ot does 6e .$y de!onstr te presen)e o" ASIC+ )h nne$ prote#ns t ppro7#! te$y the )orre)t molecular *eight3 tho'%h3 s'pport#n% the d t obt #ned "ro! immunocytochemistry 6h#)h sho6s ASIC+ present thro'%ho't the hippocampus* -endritic 'pine 3esults

Figure 27* The $e"t #! %e 4 5 #s o" )ontro$ s$#)e* The r#%ht #! %e 4b5 #s o" do'b$eAh#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$#)e nd h#%h & r#)os#ty dens#ty #n the do'b$eAh#t s$#)e* S) $e b r/ C'! "or both #! %es*

;H F#%'re +G d#sp$ ys (' $#t t#&e represent t#ons o" the dendr#t#) spine imaging* The )ontro$ s$#)e h s re$ t#&e$y h#%h spine dens#ty )o!p red to the double hit #n0'ry3 6h#$e the double hit #n0'ry h s re$ t#&e$y h#%h varicosity dens#ty )o!p red to the )ontro$* F#%'re +E3 t b$e3 "e t'res the (' nt#t t#&e res'$ts "or both spines:8,'! nd varicosities:8,'! "or the b s#) experimental conditions 4double hit:not #n)$'d#n% ny dr'% tre t!ents5*
56pe"ime#tal )pi#es Va"icosities 0o#ditio# )pi#es/10%m )ta#da"d 5""o" Va"icosities/10%m )ta#da"d 5""o" 0o#t"ol 2.53 0.4 0.24 0.08 &'I( 0. mph 0 0 0.61 0.04 -cid( 6.0( h) 0.06 0.05 0.55 0.15 Do%7le hit (2h delay) 0 0 0.80 0.03 Figure 28* T b$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 portr yed #n F#%'res +H nd 9,3 #n %r ph#) $ "or!* As F#%'re +H de!onstr tes3 the )ontro$ spine dens#ty #s &ery h#%h$y s#%n#"#) nt$y d#""erent "ro! e&ery other experimental conditionBs spine dens#ty 44pX,*,,,85* In dd#t#on3 s F#%'re 9, sho6s3 the double hit varicosity dens#ty #s s#%n#"#) nt$y d#""erent "ro! the )ontro$ varicosity dens#ty 4pO,*,8C853 6h#$e no other experimental conditions d#""er s#%n#"#) nt$y "ro! the )ontro$ varicosity dens#ty*

C,

Dendritic $pine Density

2.5 2 $pines"'(um

1.5

0.5

0 0o#t"ol 10. mph %&perimental condition 6.0( h) 10. mph82h delay86.0( h)

Figure 2$# Dendr#t#) sp#ne dens#ty "or e )h b s#) e7per#!ent $ )ond#t#on* Note th t the do'b$e h#t #n0'ry 4the " r r#%ht )o$'!n5 #s 6#th + ho'r de$ y* Note $so th t the sp#ne dens#ty #s portr yed #n sp#nes:8,'! r ther th n per 8'!3 #n order to $$o6 )o!p r#son to the & r#)os#ty dens#ty* Error b rs sho6n re st nd rd errors* The s#n%$e st r 4Y5 #nd#) tes th t the )ontro$ )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 E3 -*
aricosity Density
0.4

0.8

0.3

aricosities"'(um

0.6

0.5

0.4

0.

0.2

0.1

0 0o#t"ol 10. mph %&perimental condition 6.0( h) 10. mph82h delay86.0( h)

Figure 3%* F r#)os#ty dens#ty "or e )h b s#) e7per#!ent $ )ond#t#on* Note th t the do'b$e h#t #n0'ry 4the " r r#%ht )o$'!n5 #s 6#th + ho'r de$ y* Note $so th t th#s %r ph #s on d#""erent yA& $'e s) $e #n order to $$o6 )o!p r#son o" the d#""erent #nd#&#d' $ e7per#!ent $ )ond#t#on he#%hts3 nd th t & r#)os#ty dens#ty #s portr yed per 8,'!* Error b rs sho6n re st nd rd errors* The s#n%$e st r 4Y5 #nd#) tes th t on$y the do'b$e h#t #n0'ry 6 s s#%n#"#) nt$y d#""erent "ro! the )ontro$ #n ter!s o" & r#)os#ty dens#ty 4pO,*,8C85* N & $'es "ro! $e"t to r#%ht/ G3 8+3 E3 -*

C8

/iscussion
It 6 s !y hypothes#s th t s'b$eth $ !e)h n#) $ #n0'ry 6o'$d sens#t#2e the hippocampus to se)ond ry acidosis3 ) 's#n% pre&ent b$e ne'ron $ de th* The obser& t#ons nd d t presented #n th#s thes#s s'%%est th t )o!b#ned TBI o" 8,*9!ph nd acidosis t pH o" -*, res'$t #n n #n0'ry %re ter th n e#ther #nd#&#d' $ #n0'ry 4see F#%'re +,5* =h#$e the PI d t "or the double hit #n0'ry #s s#%n#"#) nt$y d#""erent "ro! the TBI t 8,*9!ph $one 4pO,*,,,953 #t #s not s#%n#"#) nt$y d#""erent "ro! the )#d #n0'ry $one3 #nd#) t#n% th t sens#t#2 t#on by TBI to s'bse('ent acidosis #s not o))'rr#n%* F'rther!ore3 st nd rd ANOFA does not prod')e s#%n#"#) n)e "or the #nter )t#on bet6een TBI nd 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 TBI sensiti es the hippocampus to secondary acidosis3 $tho'%h n #n)re sed ! 6o'$d $$o6 "or !ore so$#d s'pport o" th#s )on)$'s#on* The )on)$'s#on th t pr#! ry TBI does not see! to sens#t#2e to se)ond ry acidosis #n the hippocampus does not #!p$y th t the res'$ts re #ns#%n#"#) nt* As e$ bor ted #n the Introd')t#on3 acidosis )o!!on$y o))'rs s secondary injury "o$$o6#n% TBI* In " )t3 o" $$ se)ond ry br #n #n0'ry " )tors3 acidosis3 hypo) pn# 3 nd hypotens#on o))'r !ost "re('ent$y3 6h#$e acidosis nd hypoxia re re$ ted to $on%er Intens#&e C re nd hosp#t $ $en%th o" st y 4Jer#!#ts.y et $*3 +,,95* F'rther!ore3 acidosis sso)# ted 6#th TBI tends to be e7tre!e 6#th pH $e&e$s " $$#n% be$o6 -*C3 nd th's acidosis tends to %%r & te br #n #n0'ry 4< .' et $*3 8HH9J @#on% et $*3 +,,;5* The re$ t#onsh#p bet6een TBI nd acidosis #s e7tre!e$y #!port nt to deter!#ne3 nd3 be) 'se the d t do not s'pport TBI sens#t#2#n% to se)ond ry acidosis #n the

C+ hippocampus3 the other opt#on #s th t TBI nd acidosis d#sp$ y n dd#t#&e re$ t#onsh#p* In other 6ords3 TBI nd acidosis see! to be t6o #ndependent #n0'r#es o))'rr#n% by sep r te p th6 ys r ther th n s#n%$e3 )o!!on p th6 y s sens#t#2 t#on 6o'$d #!p$y* M#$d TBI nd )#d #n0'r#es 6ere de$#ber te$y se$e)ted to 'n!#st . b$y deter!#ne sens#t#2 t#on #nter )t#on3 s the double hit experimental condition 6o'$d be e7pe)ted to res'$t #n !')h h#%her3 s#%n#"#) nt cell de th re$ t#&e to e )h #nd#&#d' $ experimental condition 4TBI nd 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 #!port nt3 ho6e&er3 to note th t ny

)on)$'s#ons tent t#&e$y ! de pp$y on$y to the spe)#"#) TBI nd )#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 th t the#r #nter )t#on 6o'$d not be dd#t#&e* L )t te Dehydro%en se 4LDH53 n en2y!e re$e sed by d ! %ed cells nd th's nother te)hn#('e th t #nd#) tes %ross cell de th 6 s )ons#dered s !ethod o" (' nt#"y#n% cell de th "or th#s thes#s* Ho6e&er3 #t 6 s 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#s d& nt %e o" not $$o6#n% "or )$ose n $ys#s o" the s$#)e3 on$y o&er $$ h#ppo) !p $ cell de th* The de)#s#on to 't#$#2e PI s the pr#! ry !ethod o" (' nt#"y#n% cell de th ppe rs to h &e been d& nt %eo's3 s#n)e PI #! %es $$o6 "or n $ys#s o" the %%re% te h#ppo) !p $ cell de th3 s 6e$$ s re%#on $ n $ys#s* D'r#n% n $ys#s o" the %%re% te 4 $$ h#ppo) !p $ re%#ons5 PI d t 3 #t 6 s (' $#t t#&e$y obser&ed th t the dent te %yr's ppe red to h &e h#%her cell de th th n the other h#ppo) !p $ re%#ons "o$$o6#n% n )#d #n0'ry 4 $one or double hit5* The d& nt %e o" PI #s th t #t $$o6s "or se)ond ro'nd o" n $ys#s o" cell de th3 spe)#"#) to e )h h#ppo) !p $ re%#on*

C9 =h#$e the PI d t o&er $$ s'%%est sep r te TBI nd acidosis !e)h n#s!s "or ) 's#n% cell de th3 n $ys#s o" the dent te %yr's d t "or the s !e #! %es s #n)$'ded #n the double hit d t set )o!p$#) te th#s ssert#on* A$tho'%h the TBI $one 6 s not s#%n#"#) nt$y d#""erent "ro! the )ontro$ experimental condition3 th#s 6 s hypothes#2ed3 s the TBI 6 s e7pe)ted to be s'bA$eth $ s#n)e !#$d #ntens#ty 6 s se$e)ted* The )#d $one 6 s &ery h#%h$y s#%n#"#) nt$y d#""erent "ro! the )ontro$ )ond#t#on 4pX,*,,,853 s 6e$$ s "ro! the double hit #n0'ry 4pO,*,8HG5* F#n $$y3 the double hit #n0'ry 6 s $so &ery h#%h$y s#%n#"#) nt$y d#""erent "ro! the TBI $one 4pX,*,,,85* T .en to%ether3 the dent te %yr's res'$ts #nd#) te th t th#s p rt#)'$ r re%#on o" the hippocampus see!s to be se$e)t#&e$y &'$ner b$e to )#d #n0'ry3 $tho'%h t th#s po#nt there #s no )ert #n d t to deter!#ne 6hether th t #n)re sed cell de th "o$$o6#n% acidosis #s d'e to n dd#t#&e e""e)t or to sens#t#2 t#on* The res'$ts o" st nd rd ANOFA do #nd#) te so!e #nter )t#on bet6een the t6o #n0'r#es 4pO,*,+-H53 b't the pre)#se #nter )t#on #s 'n.no6n* The #!port nt )on)$'s#on "ro! n $ys#s o" the dent te %yr's re%#on #s th t the !e)h n#s!s o" cell de th ! y d#""er bet6een h#ppo) !p $ re%#ons nd th t e )h re%#on ! y prod')e res'$ts spe)#"#) to th t re%#on* Th's3 n $ys#s o" %%re% te cell de th "or s$#)e ! y " #$ to spot )r')# $ res'$ts* An $ys#s o" the "'n)t#on $$y d#st#n)t re%#ons o" the hippocampus 4CA83 CA93 nd dent te %yr's5 #s )$e r$y 6 rr nted3 both "or "'t're st'd#es nd "or the d t )o!pr#s#n% th#s thes#s* Fo$$o6#n% &ery tent t#&e s'%%est#on o" cell de th o))'rr#n% by sep r te p th6 ys "or TBI nd acidosis3 e7p$or#n% ne'roprote)t#&e str te%#es "or the )'te t#!e "r !e "o$$o6#n% TBI see!ed n t'r $ s p th to p'rs'e3 s#n)e b$o).#n% the e""e)t o" the acidosis )o!ponent $one sho'$d theoret#) $$y de)re se the o&er $$ double hit cell de th

C; to $e&e$s ro'nd th t o" TBI $one* Be"ore red')t#on o" double hit de th ! y be n $y2ed 6#th p rt#)'$ r dr'%3 the dr'% !'st be pro&en to red')e cell de th s res'$t o" n )#d #n0'ry $one* By pro&#n% th#s3 n opt#! $ )on)entr t#on "or the dr'% #s deter!#ned3 s 6e$$ s n ""#r! t#on th t the spe)#"#) dr'% #s )t' $$y e""e)t#&e #n !e$#or t#n% de th ) 'sed by acidosis "or the )'$t're type be#n% 'sed* F#%'re ++ d#sp$ ys the amiloride res'$ts3 nd the $ ). o" s#%n#"#) n)e bet6een )#d #n0'ry $one nd 8,,'M amiloride #!p$#es th t amiloride #s not e""e)t#&e t b$o).#n% cell de th ) 'sed by acidosis* Note th t th#s does not ne)ess r#$y #!p$y th t amiloride #s 'b#('#to's$y #ne""e)t#&eJ #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* Pre&#o's st'd#es 6h#)h s'%%ested th t amiloride #s nonspeci ic b$o).er o" ASICs re pr#! r#$y electrophysiological 4Pr#)e et $*3 +,,,J = $d! nn et $*3 8HHG J @#on% et $*3 +,,;53 te)hn#('e 6h#)h n $y2es neurons d'r#n% &ery short t#!e per#od* In these ) ses3 amiloride need on$y be pp$#ed o&er short t#!e 4!#n'tes53 6h#)h #s )ons#der b$y d#""erent "ro! the $on%er d'r t#on th t amiloride 6 s pp$#ed #n the e7per#!ents d#sp$ yed #n F#%'re ++ 4 ppro7#! te$y 9*C ho'rs or the tot $ d'r t#on o" )#d nd 'C! tre t!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 pot ss#'! retent#on3 s 6e$$ s b$o).#n% ASICs* It #s $#.e$y th t the #n)re sed cell de th obser&ed s res'$t o" amiloride #n F#%'re ++ #s d'e to one o" amiloride0s nonAASIC #n&o$&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 cell de th*

CC As amiloride d#d not pre&ent cell de th by acidosis3 the sp#der &eno! PcT)( 6 s p'rs'ed s poss#b#$#ty to de)re se cell de th res'$t#n% "ro! acidosis* PcT)( h s been sho6n to be spe)#"#) ASIC8 b$o).er 4Es)o'b s et $*3 +,,,J @#on% et $*3 +,,;53 $so thro'%h 'se #n electrophysiology st'd#es* Ho6e&er3 be) 'se PcT)( #s &ery spe)#"#)3 #t 6 s hoped th t e7tended e7pos're o" PcT)( to the )'$t'red s$#)es 6o'$d be b$e to de)re se cell de th ) 'sed by acidosis nd not #n)re se cell de th by d#st'rb#n% other )e$$'$ r !e)h n#s!s s amiloride prob b$y d#d* F#%'re +9 d#sp$ ys the &eno! res'$ts3 6h#)h re $so not s#%n#"#) nt* Ho6e&er3 th#s does not #nd#) te th t the res'$ts re #ns#%n#"#) nt* R' $#t t#&e$y nd e&en (' nt#t t#&e$y there #s so!e s'%%est#on th t #nter!ed# te )on)entr t#ons o" &eno! ! y de)re se cell de th res'$t#n% "ro! )#d #n0'ry $one3 &ery pro!#s#n% s'%%est#on "or on$y one 6ee.Bs 6orth o" d t * Th's3 PcT)( sho6s pro!#se s poss#b$e ne'roprote)t#&e %ent % #nst se)ond ry acidosis "o$$o6#n% TBI3 nd the res'$ts portr yed #n F#%'re +9 6 rr nt "'rther #n&est#% t#on* Ho6e&er3 s both amiloride nd &eno! res'$ts d#d not prod')e s#%n#"#) n)e3 the ('est#on r#ses s to #" ASIC )h nne$s re e&en present #n )'$t'red3 89A8C d y o$d organotypic r t h#ppo) !p $ s$#)es* ASIC )h nne$s re .no6n to be present #n ! ny types o" ! !! $# n neurons 4A$& re2 de $ Ros 3 +,,+J Ch' et $*3 +,,;J = $d! nn et $*3 8HHG J =e!!#e et $*3 +,,+J @#on% et $*3 +,,;53 b't the te)hn#('es nd proto)o$ "or t#ss'e prep r t#on #n th#s thes#s )o'$d poss#b$y ) 'se ASIC )h nne$ e7press#on to de)re se or be )o!p$ete$y bsent* Th's3 immunocytochemistry 6 s p'rs'ed #n order to )on"#r! th t ASIC )h nne$s 6ere present #n the )'$t'res 'sed #n th#s thes#s* ASIC+ )$e r$y ppe rs to be present #n $$ ! 0or re%#ons o" the hippocampus 4CA83 CA93 nd dent te %yr's53 s d#sp$ yed #n F#%'re +;*

CUn"ort'n te$y3 ASIC8 d#d not prod')e s )$e r immunocytochemistry res'$ts d'e to 'to"$'ores)en)e* By )onse('en)e3 Western blotting 6 s p'rs'ed #n n tte!pt 85 to s'pport the presen)e o" ASIC )h nne$s3 p rt#)'$ r$y ASIC8 3 #n the organotypic h#ppo) !p $ s$#)es3 nd +5 to sho6 ho6 TBI ""e)ts the re$ t#&e )on)entr t#on o" the ASIC )h nne$s #n the s$#)es* As F#%'re +C portr ys3 ASIC8 #s )$e r$y present #n the )ontro$ )'$t'red s$#)es3 b't "o$$o6#n% TBI nd s'bse('ent + ho'r de$ y3 the ASIC8 )on)entr t#on #s dr st#) $$y red')ed* Th#s red')t#on #n ASIC8 "o$$o6#n% TBI ! y $#!#t the e""#) )y o" the &eno!3 s &eno! #s spe)#"#) ASIC8 b$o).er3 nd 6#th $ess ASIC8 present there re "e6er ASIC8 )h nne$s present "or the &eno! to )t on* Ho6e&er3 the Western blot s'%%ests th t there #s st#$$ t $e st so!e ASIC8 present #n the )'$t'res "o$$o6#n% TBI s#n)e the b nd #s st#$$ )$e r$y present b't not#)e b$y " #nter th n th t o" the )ontro$* Hen)e3 &eno! sho'$d st#$$ be e7p$ored s poss#b$e ne'roprote)t#&e %ent "o$$o6#n% TBI* The immunocytochemistry nd Western blot res'$ts $so $$o6 "or "'rther poss#b$e #!p$#) t#ons 6hen )ons#dered 6#th the PI d t pre&#o's$y d#s)'ssed* The double hit d t ! y ppe r to be n dd#t#&e res'$t o" the #nd#&#d' $ TBI nd )#d #n0'r#es be) 'se o" "e6 poss#b#$#t#es* F#rst3 TBI nd acidosis ! y be s#!p$y dd#t#&e3 )t#n% &# sep r te p th6 ys to ) 'se %re ter double hit cell de th* Se)ond3 e )h h#ppo) !p $ re%#on ! y prod')e res'$ts spe)#"#) to th t p rt#)'$ r re%#on3 6h#)h 6hen n $y2ed to%ether #n the o&er $$ !e n #ntens#ty ) 'se $oss o" s#%n#"#) n)e* Th#rd3 the ASIC8 ! y de)re se s )o!pens tory !e)h n#s! "o$$o6#n% TBI* In th#s 6 y3 the cell 6o'$d be prote)t#n% #tse$" % #nst "'rther h r! "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

CG )o'$d o))'r d'r#n% the s !e t#!e th t ASIC+ sens#t#2es3 prod')#n% o&er $$ res'$ts 6h#)h ppe r to be dd#t#&e 6hen the #nd#&#d' $ #n0'r#es re )t' $$y #nter )t#n%* ASIC+ #s )$e r$y present #n )ontro$ )'$t'res s F#%'re +; de!onstr tes3 b't 6h t h ppens to the )on)entr t#on o" ASIC+ "o$$o6#n% TBI #s )'rrent$y 'n.no6n* Th's3 these other poss#b#$#t#es !'st be e7p$ored be"ore n dd#t#&e e""e)t ! y be tent t#&e$y )on)$'ded* Th#s #n&est#% t#on sho'$d #n)$'de n n $ys#s o" ASIC+ )on)entr t#ons #n )ontro$ t#ss'e nd $so "o$$o6#n% TBI nd t6o ho'r de$ y3 s 6e$$ s tte!pt#n% to pre&ent the ASIC8 drop "o$$o6#n% TBI #n order to see #" the $e&e$ o" cell de th sh#"ts* Deter!#n t#on o" 6h#)h !e)h n#s! #s be#n% e!p$oyed by the h#ppo) !p $ neurons #s )r')# $ to #!pro&#n% tre t!ent o" TBI* =h#$e pre&ent#on 4he$!et3 se t be$t3 b'!per3 )r'!p$e 2one3 #r b %3 et)*5 o""ers s#%n#"#) nt red')t#on #n the se&er#ty o" TBI3 the %'#de$#nes "or tre t#n% TBI on)e #t o))'rs re #n de(' te* The d#""#)'$ty #n tre t#n% TBI r#ses be) 'se o" the 6#de r n%e o" secondary injuries th t o))'r 6#th TBI* For #nst n)e3 d!#n#ster#n% dr'% th t 6o'$d de)re se the )#d#ty o" the h#ppo) !p $ t#ss'e 6o'$d see! n ob&#o's tre t!ent #n order to pre&ent "'rther cell de th* Ho6e&er3 s#n)e s$#%ht$y )#d#) en&#ron!ent 4 ppro7#! te$y pH-*EApHG*;5 ) n )t' $$y be prote)t#&e to neurons 4< .' et $*3 8HH9J @#on% et $*3 +,,;53 dr'% th t 6o'$d de)re se acidosis 6o'$d on$y be ppropr# te t )ert #n t#!es 4e*%* "ter deter!#ned $e&e$ o" TBI5* In the ) se o" th#s thes#s3 sep r te TBI nd acidosis !e)h n#s!s )o'$d be tre ted sep r te$y3 b't #" these #nd#&#d' $ #n0'r#es do sh re !e)h n#s! "or ) 's#n% the#r respe)t#&e #n0'r#es3 then the#r tre t!ent ) n on$y be )ons#dered to%ether* Beyond )on"#dent$y deter!#n#n% the !e)h n#s! o" TBI nd acidosis #n0'r#es 4 dd#t#&e or #nter )t#n%53 #t #s #!port nt to est b$#sh )$#n#) $ t#!e"r !e

CE "or tre t!ent* F'rther!ore3 the 6#ndo6 o" &'$ner b#$#ty "o$$o6#n% TBI !'st be deter!#ned #n order to s)ert #n #" the !e)h n#s!4s5 o" TBI nd )#d #n0'ry re! #n )ons#stent the ent#re postATBI per#od3 or #" #nter )t#on o))'rs d'r#n% one t#!e #nter& $3 nd dd#t#on d'r#n% nother* One 6 y o" #n&est#% t#n% "'rther the !e)h n#s!4s5 #n&o$&ed #s to e7 !#ne 6h t h ppens t the )e$$'$ r $e&e$ to )ontr#b'te to the cell de th obser&ed 6#th PI* Dendr#t#) spine #! %#n% $$o6s "or ('#te sens#t#&e n $ys#s o" s$#)e he $th nd n #nd#re)t !e s're o" s$#)e "'n)t#on3 nd ) n be 'sed not on$y to s'pport the res'$ts obt #ned 6#th PI3 b't $so to )o!p re the str')t'r $ )h n%es "o$$o6#n% TBI or acidosis* C$#n#) $$y3 TBI #s sso)# ted 6#th both tr ns#ent nd per! nent !e!ory $oss 4>eddes et $*3 +,,9J No& ). et $*3 +,,,J = n% et $*3 8HHG53 #nd#re)t$y #nd#) t#n% h#ppo) !p $ d ! %e* In dd#t#on3 pre&#o's st'd#es h &e #!p$#) ted dendr#t#) spines #n ne'ron $ plasticity sso)# ted 6#th $e rn#n% nd !e!ory "or! t#on 4H r!s et $*3 +,,-J Her#n% nd Shen%3 +,,8J L#pp! n et $*3 +,,C5* These st'd#es s'%%est th t the str')t'res o" the spines the!se$&es ! y be )orre$ ted 6#th $ter t#ons #n the p th6 ys o" #n"or! t#on "$o6* It "o$$o6s th t the neurons ! y 'se these $ter t#ons #n the spines to #n)re se or de)re se the#r syn pt#) tr ns!#ss#on "ter TBI #n order to prote)t "ro! "'rther cell de th or to )o!pens te "or the cell de th th t o))'rred* F#%'res +GA+H sho6 th t #n0'r#n% the s$#)e 4TBI $one3 )#d $one3 or double hit5 #n)re ses the dens#ty o" varicosities nd de)re ses the dens#ty o" spines* Note th t 6h#$e the double hit )ond#t#on #s s#%n#"#) nt )o!p red to the )ontro$ "or the varicosity dens#ty3 th#s )o'$d be res'$t o" n dd#t#&e re$ t#onsh#p bet6een the t6o #nd#&#d' $ #n0'r#esJ #t does not ne)ess r#$y #nd#) te sens#t#2 t#on* Moreo&er3 s the spines3 the str')t'res

CH $$o6#n% "or communication t syn pse3 ppe r to dr ! t#) $$y de)re se "o$$o6#n% n #n0'ry3 #t see!s !ost $#.e$y th t the cell de th obser&ed #n the PI d t #s the res'$t o" !e)h n#s! other th n spine !od'$ t#on* In essen)e3 the obser&ed de)re se #n spine dens#ty 6o'$d see! to #!p$y short ter! prote)t#on by red')t#on o" syn pt#) stren%th 4tr ns#ent prote)t#on % #nst excitotoxicity5 nd de)re se3 r ther th n the obser&ed #n)re se3 #n o&er $$ cell de th* As pre&#o's$y d#s)'ssed3 spines re )ons#dered to be plastic str')t'res* Th's3 $tho'%h spine dens#ty dr st#) $$y de)re sed nd varicosity dens#ty dr st#) $$y #n)re sed "o$$o6#n% #n0'ry3 #t #s p$ 's#b$e th t3 %#&en s'bst nt# $ d'r t#on o" t#!e3 the spine dens#ty )o'$d #n)re se ne r$y b ). to #ts or#%#n $ & $'e nd the varicosity dens#ty )o'$d de)re se b ). to #ts or#%#n $ & $'e* I" th#s re!ode$#n% does o))'r3 #t s'%%ests th t re)o&ery ! y be poss#b$e nd th t the cell de th o))'rr#n% "o$$o6#n% TBI or acidosis #n0'ry )o'$d poss#b$y be )o!pens ted "or by ne6 syn pt#) p th6 ys* F't're rese r)h sho'$d )ert #n$y e7p$ore 6hether the dendr#t#) spines e&er re ppe r3 nd #" they do3 ny potent# $ ne'roprote)t#&e dr'%s sho'$d be )on"#r!ed to not h#nder th#s #n)re se #n spine dens#ty d'r#n% re)o&ery* Th's3 t .en to%ether these res'$ts on the s'r" )e tent t#&e$y s'%%est th t TBI nd acidosis )t #n n dd#t#&e ! nner* Ho6e&er3 pre$#!#n ry d t ppe rs to s'%%est !ore

)o!p$e7 re$ t#onsh#p3 6#th re%#on $ & r# t#on* These res'$ts3 6h#$e pre$#!#n ry3 do "'rther 'nderst nd#n% o" the TBIAacidosis #nter )t#on nd d& n)e .no6$ed%e "or poss#b$e tre t!ents o" TBIs #n 6h#)h acidosis #s #n&o$&ed*

-,

$ootnotes
8

D#s b#$#t#es res'$t#n% "ro! TBI depend on the se&er#ty nd $o) t#on o" the #n0'ry3 s 6e$$ s the %e nd he $th o" the p t#ent* + A red')t#on #n o7#d t#&e !et bo$#s! nd $tered %$')ose !et bo$#s! ) n $so res'$t "ro! TBI 6#tho't br #n ischemia 4Fesp et $*3 +,,C5* 9 B#o!e)h n#)s L bor tory3 Le% )y Rese r)h Center3 8++C NE +nd A&en'e3 Port$ nd3 OR HG+9+* ; The ! 0or#ty o" $$ ner&e nd glial )e$$ types s'r&#&e #n the )'$t're en&#ron!ent3 $tho'%h #n the hippocampus the dens#ty o" syn pt#) str')t'res de)re ses 4F#nsen et $3 8HH+5* C =h#$e r ts sh re ! ny physiological nd p tho$o%#) $ responses to TBI 6#th h'! ns3 #t #s not b$e th t the r t t#ss'e 'sed #n these e7per#!ents 6 s re!o&ed "ro! the s.'$$* Hen)e3 the #n0'ry $e&e$s nd res'$t#n% ne'ron $ de th sho'$d not be t .en s d#re)t$y )o!p r b$e to h'! n he d #n0'ry o" the s !e &e$o)#ty* H'! ns h &e br #n t#ss'e th t #s en) sed 6#th#n h rd3 bony s.'$$ nd 6h#)h #s s'rro'nded by cerebrospinal luid 4CS$53 both o" 6h#)h )t to prote)t the br #n nd bsorb the "or)e o" the #!p )t* F'rther!ore3 pre&ent#on sho'$d $6 ys be the "#rst $#ne o" de"ense3 6h#)h #s the re son beh#nd e7tens#&e ! r.et#n% to pro!ote 6e r#n% o" he$!ets 6hen r#d#n% b#)y)$es or 6e r#n% o" se tbe$ts 6hen #n !otor &eh#)$e* =ho$e br #n s$#)es 6ere re0e)ted "or 'se be) 'se o" s$#)eAtoAs$#)e heterogeneity 4& r# b#$#ty53 6h#)h 6o'$d #!pede ))'r te )o!p r#sons o" e7per#!ents3 nd $so d'e to de!onstr ted $oss o" %ross integrity t h#%h TBI ))e$er t#ons 4Morr#son et $3 +,,;5* G The CA+3 "#e$d bet6een the CA9 nd CA8 "#e$ds #n the hippocampus proper3 #s re)o%n#2ed #n so!e te7ts nd not #n others* In essen)e3 the re re! #ns3 b't so!e $#ter t're d#&#des the re%#on des#%n ted s CA+ #nto the CA9 nd CA8 "or & r#o's re sons* Th's3 #n the e7p$ n t#on "or h#ppo) !p $ #n"or! t#on "$o63 s 6e$$ s #n F#%'re G3 the CA+ h s been e""e)t#&e$y K bsorbedL #nto the CA9 nd CA83 b't #s st#$$ present* E Th#s p ttern o" #n"or! t#on "$o6 #s 'n#('e be) 'se KoneA6 y pro0e)t#ons re e7tre!e$y typ#) $ "or )ort#)o)ort#) $ )onne)t#onsL 4A! r $ nd =#tter3 8HHC5* Most cortical re%#ons )ont #n re)#pro) $ )onne)t#ons* H A K!o$e)'$e o" )#dL #s )t' $$y proton 4HP5* Th's3 !ore )#d#) 4h#%her )on)entr t#on o" protons5 en&#ron!ents 6#$$ h &e h#%her prob b#$#ty o" one o" these protons b#nd#n% to n ASIC nd thereby )t#& t#n% #t* The #!port nt po#nt #s s#!p$y th t !ore )#d#) en&#ron!ent #s !ore $#.e$y to open ASICs* 8, M<AE,8 #s n NMDA re)eptor nt %on#st3 CNR@ #s n AMPA nd . #n#te re)eptor nt %on#st3 nd N#!od#p#ne #s ) $)#'! )h nne$ b$o).er* 88 =h#)h #ons re $$o6ed !o&e!ent by the ASIC #s dependent on 6h#)h p rt#)'$ r ASIC )h nne$ #s be#n% re"eren)ed* For #nst n)e3 homomeric ASIC8 )h nne$s re permeable 4 $$o6 thro'%h the )e$$ !e!br ne5 to both sod#'! 4N P5 nd ) $)#'! 4C +P53 6h#$e ASIC8Q h s no ) $)#'! permeability 4Ch' et $*3 +,,;5* 8+ It sho'$d be noted th t3 6h#$e the e7per#!ents 6h#)h ! .e 'p th#s thes#s "o)'s on )e$$ de th nd #n0'ry3 the br #n #s re! r. b$e plastic* Th's3 K6hen one "'n)t#on $ re%#on or p th6 y #s d ! %ed3 others ! y be b$e to )o!pens te p rt# $$y "or the $ossL 4< nde$ et $3 H5 by reor% n#2#n% the#r $#n. %es* The re! #n#n%3 'n#n0'red port#ons o" the br #n essent# $$y ))o!!od te "or the bre .do6n #n #n"or! t#on tr ns"er #n nother re * Th#s

-8 #s )$e r$y not $6 ys poss#b$e3 s ser#o's sy!pto!s do r#se "ro! TBI3 b't does o))'r3 nd pro&#des hope "or reh b#$#t t#on nd ther py e""orts "o$$o6#n% TBI* 89 Probes 'sed #n Western blots re typ#) $$y antibodies* 8; Un#"or! br #n t#ss'e shear strain res'$ts3 ss'!#n% th t the t#ss'e #n #n)o!press#b$e*

-+

"lossary
Acidosis/ The )ond#t#on o" #n)re sed )#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 th t #s ! r.ed by s6eet bre th3 he d )he3 n 'se 3 &o!#t#n%3 nd &#s' $ d#st'rb n)es* Activate/ To $$o6 "'n)t#on o" !o$e)'$e* Here3 )t#& te re"ers to open#n% n ASIC )h nne$ so th t #ons ) n p ss thro'%h* Amiloride/ A dr'% th t #s nonspe)#"#) b$o).er o" ASIC )h nne$s* Anaerobic glycolysis/ A )e$$'$ r pro)ess o" ! .#n% ener%y th t $ ).s o7y%en* It ) 'ses the b'#$dA'p o" )#d#) !o$e)'$es #n the )e$$3 6h#)h ) n be d n%ero's to the )e$$Bs "'n)t#on nd he $th3 e&en res'$t#n% #n )e$$ de th* Anoxia/ A red')t#on or tot $ bsen)e #n the s'pp$y o" o7y%en* Antibody/ A !o$e)'$e 6h#)h "'n)t#ons s the e""e)tor o" n #!!'ne response3 b't 6h#)h #n th#s thes#s #s 'sed3 #n & r#o's te)hn#('es3 s ! r.er th t #nd#) tes the presen)e or bsen)e o" prote#n by b#nd#n% to #t* The prote#ns #n ('est#on "or th#s thes#s re ASIC )h nne$s o" d#""erent types3 e )h o" 6h#)h h s #ts o6n spe)#"#) nt#body* ASIC 4A)#dASens#n% Ion Ch nne$5/ A $#% ndA% ted #on )h nne$ th t opens 6hen n )#d#) !o$e)'$e 4 proton3 the $#% nd5 #s bo'nd to #t* It $$o6s )#d to h &e n e""e)t on the )e$$ by be#n% present #n the )e$$Bs !e!br ne* Axon/ A )$ ss#) $$y $on% e7tens#on or pro)ess "ro! the ne'ronBs )e$$ body* It ) rr#es ner&e s#%n $s 6 y "ro! the )e$$ body nd to6 rd other t r%et )e$$s* Basal ganglia/ A port#on o" the br #n th t #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#!'$#* Bind/ A tr ns#ent st te 6here t6o !o$e)'$es tt )h to%ether* Bloc&/ The )t or b#$#ty to stop p rt#)'$ r )t#on* Here3 b$o). re"ers to p$'%%#n% the p ss %e6 y #n ASIC )h nne$s so th t #ons ) nnot p ss thro'%h* Byproduct/ A se)ond ry prod')t or s#deAe""e)t !o$e)'$e3 's' $$y 'n#ntent#on $* Cell/ A !#)ros)op#) str')t're )ont #n#n% n')$e's 4DNA5 nd )ytop$ s!#) ! ter# $3 nd en)$osed by se!#per!e b$e !e!br ne* There re p$ethor o" )e$$ types3 nd ne'rons re one type* Cell body/ The se)t#on o" )e$$ th t )ont #ns the n')$e's3 6h#)h #n t'rn )ont #ns the %enet#) ! ter# $ o" the )e$$* Typ#) $$y3 the dendr#tes br#n% #n"or! t#on to the )e$$ body nd the 7ons tr ns!#t the #n"or! t#on 6 y "ro! the )e$$ body* Cell membrane/ A th#n3 "$'#d3 se!#per!e b$e b rr#er th t )ontro$s the p ss %e o" s'bst n)es #nto nd o't o" the ne'ron* Cerebrospinal luid/ See CSF* Cognition/ The )t or pro)ess o" .no6#n%* Per)ept#on3 th#n.#n%3 !e!ory3 nd re son#n% re $$ p rt o" )o%n#t#on* Communication/ A)t by 6h#)h #n"or! t#on #s tr ns"erred* E7press#on nd 'nderst nd#n% re p rt o" )o!!'n#) t#on* Con ocal/ A type o" !#)ros)ope th t $$o6s the obser&er to &#s' $#2e ob0e)ts #n s#n%$e p$ )e o" "o)'s3 )re t#n% sh rper #! %e*

-9 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 p rt o" the ! !! $# n br #n nd )ont #ns sensory nd !otor ner&e )e$$ bod#es* It #s .no6n to "'n)t#on #n )o%n#t#on* CS$ (Cerebrospinal luid+/ A )$e r "$'#d th t "#$$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 rr n%e!ent nd )onne)t#ons* /eclarative/ One type o" #n"or! t#on th t #s stored by the h#ppo) !p's* It #s e7p$#)#t #n"or! t#on3 the K6h t3L e*%* " )ts nd e&ents* /endrite/ One o" typ#) $$y ! ny short3 h#%h$y br n)hed pro)esses o" ne'ron* It )on&eys ner&e s#%n $s to6 rd the )e$$ body* /endritic spine/ A !'shroo!Ash ped protr's#on "ro! dendr#tes th t represents the pr#n)#p $ s#te o" preAsyn pt#) ter!#n t#on onto postAsyn pt#) ne'ron* Tho'%ht to be &ery sens#t#&e #nd#) tor o" )e$$ he $th* /iencephalon/ The poster#or se)t#on o" the "orebr #n* It )onne)ts the !#dbr #n to the )erebr $ he!#spheres* /issociated/ C'$t'res o" $#&#n% ne'rons th t re bro.en p rt "ro! one nother "o$$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)ond ry 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$#)e 'nder%oes both types o" #n0'r#es3 #n p rt#)'$ r se('en)e3 nd 6#th p rt#)'$ 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 !e s'res the stren%th o" ne'ronBs s#%n $ 4te)hn#) $$y3 the ne'ronBs )'rrent53 n e7tre!e$y r p#d o))'rren)e* #pileptic/ A tr ns#ent ne'ro$o%#) $ e&ent #n 6h#)h )ert #n p rts o" the br #n re o&erA st#!'$ ted3 ) 's#n% bnor! $ !otor pheno!en 3 $oss o" ttent#on nd )ons)#o'sness3 nd3 t t#!es3 se&ere )on&'$s#ons* #xcitatory/ Ind')#n% e7)#t t#on or p th6 y th t #n)re ses ne'ron $ st#!'$ t#on* #xcitotoxicity/ A theory th t s'%%ests th t #n0'ry st#!'$ tes the re$e se o" h#%h $e&e$s o" e7)#t tory ne'rotr ns!#tters3 ) 's#n% )e$$ de th by the e7)ess#&e )t#& t#on o" re)eptors "or th t p rt#)'$ r ne'rotr ns!#tter* #xperimental condition/ Re"ers to the spe)#"#) e&ents th t p rt#)'$ r s$#)e 'nder%oes d'r#n% %#&en e7per#!ent* For #nst n)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 r d# t#on "ro! n e7tern $ so'r)e* B s#) $$y3 br#%ht$y )o$ored3 $!ost %$o6#n% ! r.er* $orebrain/ The nter#or o" the three pr#! ry d#&#s#ons o" the br #n #n the &ertebr te e!bryo* It #s the p rt o" the d'$t br #n th t #n)$'des the d#en)eph $ons nd the te$en)eph $on*

-; "el electrophoresis/ The sep r t#on o" prote#ns on the b s#s o" the#r s#2e nd e$e)tr#) $ )h r%e by the r te o" prote#n !o&e!ent thro'%h n e$e)tr#) $ "#e$d #n %e$* "lia/ A )$ ss o" )e$$s #n the br #n nd sp#n $ )ord th t "or! s'pport#n% str')t're "or the ne'rons nd pro&#de )ert #n ne'rons 6#th #ns'$ t#on3 #n)re s#n% the#r speed o" s#%n $ tr ns!#ss#on* "lycolysis (aerobic+/ The pro)ess o" sp$#tt#n% %$')ose3 pr#! ry ener%y so'r)e3 #nto pyr'& te* It #s the be%#nn#n% po#nt "or erob#) resp#r t#on nd the '$t#! te prod')t#on o" ener%y* Th#s pro)esss needs o7y%en #n order to "'n)t#on* 1emisphere/ In re"eren)e to the br #n3 e#ther o" the $ ter $ h $&es o" the )erebr'!* 1eterogeneity21eterogeneous/ Be#n% )o!posed "ro! d#ss#!#$ r p rts3 & r# b$e* 1ippocampus/ A p rt o" the br #n $o) ted #n the )erebr $ he!#spheres nd 6h#)h p$ ys )entr $ ro$e #n !e!ory pro)esses3 spe)#"#) $$y !e!ory stor %e* It #s )o!posed o" s#7 ! #n se)t#ons .no6n s the h#ppo) !p's proper3 dent te %yr's3 s'b#)'$'!3 pres'b#)'$'!3 p r s'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$$ o" the s !e or s#!#$ r .#nd or n t're* 1omomeric/ A type o" s'b'n#t 6here e )h s'b'n#t #s the s !e 4the prote#n or #on )h nne$ )ons#sts o" on$y one type o" s'b'n#t5* 1ypothalamus/ The &entr $ p rt o" the &ertebr te "orebr #n* It #s .no6n to "'n)t#on #n ! #nt #n#n% ho!eost s#s nd )oord#n t#n% the endo)r#ne nd ner&o's syste!s3 s 6e$$ s be#n% respons#b$e "or 'to! t#) "ee$#n%s s')h s h'n%er* 1ypoxia/ Ins'""#)#ent $e&e$s o" o7y%en #n the t#ss'e* Immunocytochemistry/ A te)hn#('e th t $$o6s "or the dete)t#on o" )he!#) $ )o!ponents o" )e$$s by !e ns o" nt#bod#es )o'p$ed to s'bst n)es th t ) n be ! de &#s#b$e3 e*%* "$'ores)ent nt#bod#es* In vitro/ O))'rr#n% #n n rt#"#)# $ en&#ron!ent o'ts#de the $#&#n% or% n#s!* The rt#"#)# $ en&#ron!ent #s typ#) $$y prep red to s#!'$ te the #ntern $ en&#ron!ent o" the $#&#n% or% n#s! be#n% st'd#ed* It $$o6s )ontro$$ed sett#n% "or e7per#!ents* In vivo/ O))'rr#n% 6#th#n $#&#n% or% n#s!s or n t'r $ sett#n%* Inertial loading/ Inert# #s ! ss !'$t#p$#ed by ))e$er t#on* The po#nt o" the NSI !ode$ #s th t FO! 3 nd be) 'se the ! ss o" the r t h#ppo) !p's #s so s! $$3 #t t .es $ r%er ))e$er t#on to ) 'se the s !e "or)e* A$$ "or)es )t#n% on the h#ppo) !p's re #nert# $ r ther th n other types o" "or)es* Integrity/ The st te o" be#n% 'n#!p #red3 6ho$e3 'nred')ed nd 'nbro.en* For th#s thes#s3 #nte%r#ty re"ers to ret #n#n% the %ross str')t're o" the s$#)e "o$$o6#n% TBI 4e*%* the s$#)e #s not !#ssh pen nd s!e red )ross the M#$$#pore5* Interstitial luid/ F$'#d o'ts#de o" the ) rd#o& s)'$ r syste!3 thro'%ho't the #ntern $ en&#ron!ent o" &ertebr tes* It "#$$s the sp )es bet6een )e$$s* Ion/ An e$e)tron#) $$y )h r%ed to! 4pos#t#&e or ne% t#&e5* Ion channel/ A tr ns!e!br ne pore th t $$o6s "or p ss %e o" #ons thro'%h the )e$$ !e!br ne nd do6n the#r e$e)tro)he!#) $ %r d#ents* They re 's' $$y spe)#"#) to one or "e6 #ons* There re se&er $ types o" #on )h nne$s3 #n)$'d#n% $#% ndA% ted #on )h nne$s s')h s ASICs* Ischemia/ Ins'""#)#en)y o" b$ood s'pp$y 6h#)h depr#&es t#ss'e o" o7y%en3 %$')ose3 nd other n'tr#ents th t re d#str#b'ted by the b$ood* In dd#t#on3 #s)he!# pre&ents the

-C re!o& $ o" potent# $$y to7#) s'bst n)es s')h s )#d* =hen #s)he!# #s s'""#)#ent$y se&ere nd pro$on%ed3 ne'rons nd other )e$$'$ r e$e!ents d#e3 .no6n s #n" r)t#on* 3abel/ To tt )h ! r.er to #n order to $$o6 "or dete)t#on nd so th t the ! r.er #s tr )e b$e* 3igand/ A !o$e)'$e th t b#nds spe)#"#) $$y to re)eptor s#te o" nother !o$e)'$e* 3igand%gated ion channel/ A s#%n $ re)eptor prote#n #n )e$$ !e!br ne th t ) n )t s )h nne$ "or the p ss %e o" spe)#"#) #on )ross the !e!br ne* =hen )t#& ted by spe)#"#) s#%n $ !o$e)'$e3 the re)eptor e#ther $$o6s or b$o).s p ss %e o" the #on3 res'$t#n% #n )h n%e #n #on )on)entr t#on th t 's' $$y ""e)ts )e$$ "'n)t#on#n%* ASICs re $#% ndA % ted #on )h nne$s3 6#th one !o$e)'$e o" )#d be#n% the $#% nd th t )t#& tes the )h nne$s* 3ipophilic/ A ! ter# $ h &#n% h#%h ""#n#ty "or $#p#ds 4e*%* #nso$'b$e to 6 ter3 $#.e " ts5* 'ass/ The (' nt#ty o" ! tter #n n ob0e)t* 'C!/ An )rony! th t st nds "or !#7t're o" three dr'%s/ M<AE,3 CNR@3 nd N#!od#p#ne* Th#s !#7t're b$o).s $$ re$e& nt )h nne$s e7)ept "or the ASIC )h nne$s3 thereby #so$ t#n% )e$$ de th res'$ts to )e$$ de th o))'rr#n% by )#dos#s* 'ental health/ Psy)ho$o%#) $ 6e$$Abe#n% nd s t#s" )tory d0'st!ent to the de! nds o" $#"e* M ny d#sorders o))'r "ro! d#st'rb n)es #n !ent $ he $th3 s')h s depress#on3 n7#ety3 person $#ty )h n%es3 %%ress#on3 )t#n% o't3 nd so)# $ #n ppropr# teness* 'etabolic imbalance/ A )h n%e "ro! the nor! $ r t#os o" !o$e)'$es #n&o$&ed #n !et bo$#s! 4ener%y prod')t#on5 #n n or% n#s!* 'illicell/ A )'$t're p$ te #nsert 6#th !#)roporo's !e!br ne ! n'" )t'red by M#$$#pore* It $$o6s "or s$#)es p$ )ed on #t to e7)h n%e n'tr#ents 6#th the )'$t're !ed#'! 'nderne th the !e!br ne 6#tho't be#n% s'b!er%ed #n the "$'#d #tse$"* 'olecular Weight/ The &er %e 6e#%ht o" one !o$e)'$e o" s'bst n)e* Typ#) $$y3 $ r%er s'bst n)e 6#$$ h &e $ r%er !o$e)'$ r 6e#%ht* !/ S !p$e s#2e & $'e 4e*%* n'!ber o" s$#)es p rt#)#p t#n% #n the e7per#!ent5* !euron/ The ne'ron #s the d#s)rete "'n)t#on $ 'n#t o" the br #n* It #s the type o" )e$$ "o'nd #n the ner&o's syste! o" or% n#s!s* There re se&er $ types o" ne'rons3 spe)# $#2ed to per"or! spe)#"#) t s.s* !euronal Shear Injury/ See NSI* !ondeclarative/ One type o" #n"or! t#on th t #s stored by the h#ppo) !p's* It #s #!p$#)#t #n"or! t#on3 the Kho63L e*%* pro)ed'r $ se('en)es nd e!ot#on $ responses* !onspeci ic/ No p rt#)'$ r or des#%n ted types o" ASIC )h nne$ b$o). %e* !SI (!euronal Shear Injury+/ The !ode$ 'sed "or ) 's#n% TBI #n th#s thes#s* It ) 'ses 'n#"or! she r st #n #n0'ry by )re t#n% )h n%e #n ))e$er t#on o" the )'$t're* 4pen state/ The st te o" n #on )h nne$ th t $$o6s "or the p ss %e o" #ons thro'%h the !e!br ne* Th#s #s opposed to the )$osed st te3 6h#)h pre&ents #ons "ro! p ss#n% thro'%h the )h nne$* 4rganotypic/ T#ss'e th t #s typ#) $ o" the or% n* It h s s#!#$ r )e$$'$ r r)h#te)t're3 )onne)t#ons3 nd str')t're to the in "i"o t#ss'e* PcT)( (Psalmotoxin25enom+/ A dr'% th t #s obt #ned "ro! the &eno! o" the t r nt'$ &salmopoeus cambridgei* It #s spe)#"#) ASIC8 b$o).er* Permeable/ C p b$e o" be#n% penetr ted by p rt#)'$ r s'bst n)e3 #on3 or !o$e)'$e* A$$o6#n% p ss %e o" th t %#&en s'bst n)e thro'%h the )e$$ !e!br ne 4e#ther o't or #n5*

-Physiological/ A tr #t 6h#)h #s )h r )ter#st#) o" the nor! $ "'n)t#on#n% o" n or% n#s!* Typ#) $ h'! n nd r t phys#o$o%#) $ pH #s ppro7#! te$y G*;3 nd th's the ! 0or#ty o" )e$$s "'n)t#on opt#! $$y t th#s spe)#"#) pH* PI (Propidium Iodide+/ A )he!#) $ th t enters )e$$s 6#th )o!pro!#sed )e$$'$ r !e!br ne* O"ten 'sed s ! r.er o" )e$$ de th* Plasticity/ The ) p b#$#ty o" be#n% !o$ded nd )h n%ed* In b#o$o%#) $ ter!s3 ) p b$e o" b'#$d#n% t#ss'e3 d pt#n%3 nd "or! t#&e pro)ess* Primary injury/ The "#rst #n0'ry* In th#s thes#s3 the TBI #s the pr#! ry #n0'ry* Th's3 the pr#! ry #n0'ry #s the )e$$ de th th t o))'rs s d#re)t res'$t o" the phys#) $3 n to!#) $ #n0'ry t the #n#t# $ t#!e o" #!p )t* Probe/ The %ener $ ter! "or $ be$ th t dete)ts spe)#"#ed !o$e)'$e3 s')h s prote#n $#.e ASICs* Propidium Iodide/ See PI* Psalmotoxin/ See P)T@8* Secondary injury/ The se)ond #n0'ry #n se('en)e* In th#s thes#s3 the )#d #n0'ry #s the se)ond ry #n0'ry* The se)ond ry #n0'ry #s typ#) $$y !ore 6#despre d nd de$ yed* Other e7 !p$es o" se)ond ry #n0'ry bes#des )#dos#s #n)$'de !et bo$#) #!b $ n)es3 no7# 3 hypo7# 3 ede! 3 nd #s)he!# * Semipermeable/ P rt# $$y per!e b$e:per!e b$e on$y to )ert #n !o$e)'$es3 not to $$* In b#o$o%#) $ ter!s3 $$o6#n% the p ss %e o" spe)#"#) !o$e)'$es b't )t#n% s b rr#er to others3 e*%* the )e$$ !e!br ne* Sensory processing/ The oper t#ons by 6h#)h n or% n#s! #s b$e to dete)t nd n $y2e #ts s'rro'nd#n%s* In)$'des s#%ht3 he r#n%3 to')h3 t ste3 nd s!e$$* Sham/ A Ksh !L e7per#!ent $ )ond#t#on #s one th t "o$$o6s the pro)ed're th t the #n0'red )'$t'res 'nder%o 4the !o&e!ents o" the !#$$#pores3 re!o& $ "ro! the #n)'b tor3 p$ )e!ent #nto ECF3 et)*53 b't #s not )t' $$y #n0'red* Spine (dendritic+/ See dendr#t#) sp#ne* Shear strain/ Me)h n#) $ de"or! t#on nd d#sp$ )e!ent o" the t#ss'e b sed on the ! ter# $ th t the t#ss'e #s )o!posed o"* D#sp$ )e!ent )ts p r $$e$ to the s'r" )e o" the t#ss'e* D#sp$ )e!ent ) n be !e s'red 6#th the NSI !ode$3 nd th's & $'es o" she r str #n 6ere 'sed to (' nt#"y the $e&e$ o" #n0'ry %#&en to the t#ss'e* Spine imaging/ The pro)ess o" obt #n#n% &ery )$ose p#)t'res o" dendr#tes th t #s tho'%ht to be sens#t#&e !e s're o" )e$$ he $th* Strain/ See she r str #n* Subunit/ A d#s)rete s'bd#&#s#on o" $ r%er3 !ore )o!prehens#&e 'n#t* Synaptic transmission/ In"or! t#on tr ns"er bet6een ne'rons* TBI (Traumatic Brain Injury+/ An e7tern $3 r p#d onset #n0'ry to the br #n res'$t#n% "ro! !e)h n#) $ )o!ponent* It ) n #n&o$&e s.'$$ "r )t're3 sh .#n%3 penetr t#on3 nd ! ny other " )tors* The NSI !ode$ 't#$#2es )$osedAhe d TBI to ) 'se #n0'ry* The )onse('en)es o" TBI depend on se&er $ )o!ponents* Traumatic Brain Injury/ See TBI* 5aricosity/ An en$ r%e!ent or s6e$$#n% on the dendr#te* O"ten re"erred to s Kpe r$sL or Kbe dsL on str#n%* Tho'%ht to #nd#) te $ ). o" )e$$ he $th* 5enom/ See P)T@8* Western Blotting/ A h#%h$y sens#t#&e te)hn#('e "or #dent#"y#n% nd !e s'r#n% the !o'nt o" spe)#"#) prote#n #n !#7ed e7tr )t* Prote#ns re sep r ted by %e$

-G e$e)trophores#s3 tr ns"erred to spe)# $ "#$ter p per3 nd probed "or the spe)#"#) prote#n 'nder #n&est#% t#on*

-E

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G, Med#)#ne* 883 CA88* >enn re$$#3 Tho! s A*3 8HH;* An#! te Mode$s o" H'! n He d In0'ry* Jo'rn $ o" Ne'rotr '! * 883 9CGA9-E* >o"orth3 P '$ette B*3 E r$ F* E$$#s3 nd Les$#e S* S t#n3 8HHH* Enh n)e!ent o" AMPAA Med# ted C'rrent "ter Tr '! t#) In0'ry #n Cort#) $ Ne'rons* The Jo'rn $ o" Ne'ros)#en)e* 8H48G53 G9-GAG9G;* H r!s3 <#!ber$y J* nd Ann D'n e&s.y3 +,,-* Dendr#t#) sp#ne p$ st#)#ty/ Loo.#n% beyond de&e$op!ent* Br #n Rese r)h* Art#)$e #n press3 & #$ b$e t 666*s)#en)ed#re)t*)o!* Her#n%3 He#.e nd Mor% n Shen%3 +,,8* Dendr#t#) sp#nes/ str')t're3 dyn !#)s nd re%'$ t#on* N t're* +3 EE,AEEE* H#ppo) !p's $o) t#on #n the h'! n br #n* =ebs#te* A))essed 8,A8,A,-* 666*!orphon#7*)o!* H#ppo) !p's pos#t#on #n the r t br #n* =ebs#te* A))essed 8,A8,A,-* http/::666*d'.e*ed':N !6h#te:Ado$es)en)e:h#ppo) !p $s$#)e*0p%* Jere!#ts.y3 E$ n3 L 're$ O!ert3 M#)h e$ D'nh !3 J ). Protet)h3 A're$#o Rodr#%'e23 +,,9* H rb#n%ers o" Poor O't)o!e the D y "ter Se&ere Br #n In0'ry/ Hypother!# 3 Hypo7# 3 nd Hyperper"'s#on* The Jo'rn $ o" Tr '! In0'ry3 In"e)t#on3 nd Cr#t#) $ C re* C;3 98+A98H* < .'3 D &#d A*3 Ron >* >#"" rd3 Denn# =* Cho#3 8HH9* Ne'roprote)t#&e E""e)ts o" >$'t ! te Ant %on#sts nd E7tr )e$$'$ r A)#d#ty* S)#en)e* +-,3 8C8-A8C8E* < nde$3 Er#) R*3 J !es H* S)h6 rt23 nd Tho! s M* Jesse$$* Pr#n)#p$es o" Ne'r $ S)#en)e3 ;th Ed* Ne6 Yor./ M)>r 6AH#$$ P'b$#shers3 +,,,*

G8 <o'r 3 S* S*3 E* M* Doppenber%3 A* M r! ro'3 S* Cho#3 H* F* Yo'n%3 nd R* B'$$o).3 8HHE* Re$ t#onsh#p bet6een e7)#t tory !#no )#d re$e se nd o't)o!e "ter se&ere h'! n he d #n0'ry* A)t Ne'ro)h#r'r%#) S'pp$e!ent* G83 +;;A+;-* L#pp! n3 Jo)e$yn nd Ann D'n e&s.y3 +,,C* Dendr#t#) Sp#ne Morpho%enes#s nd P$ st#)#ty* Jo'rn $ o" Ne'rob#o$o%y* -;3 ;GACG* Morr#son3 B r)$ y3 9rd3 < thryn E* S t! n3 D &#d F* Me ney3 nd Tr )y <* M)Intosh3 8HHE* In &#tro )entr $ ner&o's syste! !ode$s o" !e)h n#) $$y #nd')ed tr '! / re&#e6* Jo'rn $ o" Ne'rotr '! 8C3 H88AH+E* Neder% rd3 M*3 R* P* <r #%3 J* T n be3 nd =* A* P'$s#ne$$#3 8HH8* Dyn !#)s o" #nterst#t# $ nd #ntr )e$$'$ r pH #n e&o$&#n% br #n #n" r)t* A!er#) n Jo'rn $ o" Phys#o$o%y* +-,3 RCE8ARCEE* NIH 6ebs#te 8* A))essed 8,A;A,-* http/::666*n#nds*n#h*%o&:d#sorders:tb#:det #$1tb#*ht! NIH 6ebs#te +* A))essed 8,A;A,-* http/::)onsens's*n#h*%o&:8HHE:8HHETr '! t#)Br #nIn0'ry8,HPro%r !*pd" No& ).3 Tho! s A*3 A!y L* A$derson3 Be&er$y A* B'sh3 J y M* Meyth $er3 nd < y C n'pp3 +,,,* Co%n#t#&e nd "'n)t#on $ re)o&ery t - nd 8+ !onths postATBI* Br #n In0'ry* 8;48853 HEGAHH-* Opt#!#2 t#on %r ph* =ebs#te* A))essed 8,A8+A,-* http/::666*b#o$o%y)orner*)o!:reso'r)es:en2y!e1ph1%r ph*%#"* pH s) $e* =ebs#te* A))essed 8,A8,A,-* 666*ep *%o&:***:s#te1st'dents:#! %es:phs) $e*%#"* Pr#)e3 M r% ret P*3 > ry R* Le6#n3 S br#n L* M)I$6r th3 Ch'n Chen%3 J#n%h'# @#e3 P '$

G+ A* Heppenst $$3 Chery$ L* St').y3 Anne >* M nns"e$dt3 T#!othy J* Brenn n3 He ther A* Dr'!!ond3 J'n% R# o3 Chr#stopher J* Benson3 De#rdre E* T rr3 Ron F* Hrst. 3 B o$# Y n%3 Ro%er A* =#$$# !son3 nd M#)h e$ J* =e$sh3 +,,,* The ! !! $# n sod#'! )h nne$ BNC8 #s re('#red "or nor! $ to')h sens t#on* N t're* ;,G3 8,,GA8,88* R t br #n )rossAse)t#on* =ebs#te* A))essed 8,A8,A,-* http/::666*b#o*d &#dson*ed':)o'rses:%eno!#)s:!ethod:br #n9*0p%* R t h#ppo) !p's #! %e* A))essed 8,A8+A,-* http/::)ontent* ns6ers*)o!:! #n:)ontent:6p:en: : b:H#ppo) !p $Re%#ons*0p%* Se $3 R* P*3 nd S* >* A! r 3 8HHH* E7)#t tory !#no )#d tr nsporters/ " !#$y #n "$'7* Ann' $ Re&#e6 o" Ph r! )o$o%y [ To7#)o$o%y* 9H3 ;98A;C-* S#$&erthorn3 Dee Un%$ 'b* H'! n Phys#o$o%y/ An Inte%r ted Appro )h3 9rd Ed* S n Fr n)#s)o/ Pe rson Ed') t#on3 In)* p'b$#sh#n% s Ben0 !#n C'!!#n%s3 +,,;* Stopp#n#3 L*3 P* A* B')hs3 nd D* M'$$er3 8HH8* A s#!p$e !ethod "or or% notyp#) )'$t'res o" ner&o's t#ss'e* Jo'rn $ o" Ne'ros)#en)e Methods* 9G3 8G9A8E+* Tho!pson3 R#)h rd F* The Br #n/ A Ne'ros)#en)e Pr#!er3 9rd Ed* Ne6 Yor. C#ty/ =orth P'b$#shers3 +,,,* Unterber%3 A* =*3 J* Sto&er3 B* <ress3 nd <* L* <#en#n%3 +,,;* Ede! Tr '! * Ne'ros)#en)e* 8+H3 8,+8A8,+H* Fesp 3 P '$3 M r&#n Ber%sne#der3 N yo H ttor#3 Hs# oAM#n% ='3 S'n%AChen% H' n%3 Ne#$ A* M rt#n3 Tho! s C* >$enn3 D &#d L* M)Arth'r3 nd D &#d A* Ho&d 3 +,,C* Met bo$#) )r#s#s 6#tho't br #n #s)he!# #s )o!!on "ter tr '! t#) br #n nd Br #n

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