B Y T

TR!NG I HC D"C H N I

PHM THU H

TNG QUAN V PHNG PHP NH GI TC D#NG C$A CC

CHT MA TU D%A VO K& THUT THM TCH MICRO

KHA LUN TT NGHIP D"C S

H N I 2013

B( Y T! TR*NG I H%C D,C H N(I

PHM THU H

T'NG QUAN V" PHNG PHP NH GI TC D-NG C.A CC

CHT MA TU D/A VO K1 THU T THM TCH MICRO

KHA LU N T&T NGHI#P D,C S

Ng+i h)ng dn: 1. PGS.TS. Thi Nguyn Hng Thu

2. TS. Nguyn Thnh Hi Ni th0c hi$n

1. B mn Ho phn tch v c cht Trng i hc Dc H Ni

2. B mn Dc lm sng Trng i hc Dc H Ni.

H N(I - 2013

LI CM N

V.i lng bi$t n su s"c, ti xin g6i l/i cm n chn thnh nht $n:

PGS. TS. Thi Nguyn Hng Thu

TS. Nguyn Thnh Hi

l nh7ng ng/i thy tr8c ti$p h.ng d n v ch( bo t!n tnh & ti hon thnh

kho lu!n ny.

,ng th/i, hai thy cng d n d"t v chia s# cho ti nh7ng kinh nghi'm v cng

qu bu & hon thnh cc bi bo "Nghin c5u hi'u qu tc d3ng c4a morphin trn n,ng - dopamin v serotonin d8a vo k thm tch micro" v Nghin c5u m hnh nh gi

hi'u qu tc d3ng c4a cc cht ma ty thng qua n,ng - dopamin d8a vo k9 thu!t thm tch micro.

Ti cng xin chn thnh cm n nh7ng gp , ch(nh s6a qu bu c4a TS. Phm Hng Ln, Vi'n K9 thu!t Ho sinh v Ti li'u nghi'p v3 - B- Cng An, & hon ch(nh

nghin c5u ny c th& 5ng d3ng vo th8c t$ ti Vi't Nam.

Ti xin cm n b- mn Ho phn tch v -c cht v b- mn D2c lm sng to

i%u ki'n cung cp cho ti cc ti li'u cn thi$t & hon thnh kho lu!n ny.

Ti xin cm n Ban gim hi'u, cc phng ban, cc thy c gio v cn b- nhn vin tr/ng i h*c D2c H N-i nh7ng ng/i dy bo v trang b) cho ti nh7ng ki$n

th5c khoa h*c n%n tng su+t 5 nm h*c d.i mi tr/ng ny.

Cu+i cng, ti xin g6i nh7ng l/i thn thng nht $n gia nh, bn b, t!p th& l.p

A2K63 lun 0 bn -ng vin, gip 1 ti v2t qua m*i kh khn trong qu trnh h*c t!p v hon thi'n kho lu!n.

H ni, ngy 21 thng 5 nm 2013 Sinh vin

Phm Thu H

MC LC

Danh m@c cc k hi4u vi/t t.t

Danh m@c cc b(ng

Danh m@c cc hnh v bi3u 9

T VN ..................................................................................................................... 1

PHN 1. TNG QUAN V K THUT THM TCH MICRO V NG DNG . 3

1.1. TM T-T V0 L5CH SC PHT TRI2N V BNG D?NG CAA KF THU,T TH+M

TCH MICRO ............................................................................................................................ 3

1.2. KHI QUT V0 KF THU,T TH+M TCH MICRO ...................................................... 5

1.3. NGUYN L V C)U T&O CAA KF THU,T TH+M TCH MICRO ........................ 61.3.1. Nguyn l ...................................................................................................................... 61.3.2. Kim thm d .................................................................................................................. 71.3.3. D6ch truy1n .................................................................................................................... 91.3.4. Phng php 6nh l>ng cc phn tD thu8c ................................................................ 10

1.3.4.1. S.c k l7ng hi4u nng cao (HPLC) .................................................................................. 101.3.4.2. S.c k l7ng kh8i ph; (LC-MS) ........................................................................................ 12

1.3.5. Hi4u su*t kim thm d ................................................................................................ 181.3.5.1. Hi4u su*t tng 8i in vitro .............................................................................................. 181.3.5.2. Hi4u su*t thEc nghi4m in vivo .......................................................................................... 20

1.4. U NH=C I2M CAA KF THU,T TH+M TCH MICRO ...................................... 251.4.1. u i3m ....................................................................................................................... 251.4.2. Nh>c i3m v m

2.2. DOPAMIN .......................................................................................................................... 312.2.1. 9nh ngha ................................................................................................................... 312.2.2. Cc con Cng d,n truy5n dopamin trong no ........................................................... 312.2.3. Vai tr cHa dopamin .................................................................................................... 33

2.3. MNG HDP K2T QU% TK CC NGHIN CIU CNG B< V4 NH GI HI7U

QU% TC DFNG CH'T MA TU TRN NO CHU@T DOA VO KP THU-T TH*M TCH MICRO .......................................................................................................................... 50

K#T LU"N V $ XUT ............................................................................................. 53

1. K2T LU-N ........................................................................................................................... 53

2. 4 XU'T .............................................................................................................................. 54

TI LI&U THAM KHO ............................................................................................... 55

PH. L.C: Cc bi bo khoa h(c -c cng b) c0a % ti .................................... 67

DANH MC CC K HIU VIT TT

aCSF Dung d ch gi no tu( (artificial cerebrospinal fluid) AMP Amphetamin C N#ng % (concentration) CID Va chm phn ly (collision-induced disociation) CNS H th"ng thn kinh trung ng (central nervous system) CSF Dung d ch no tu( (cerebrospinal fluid) DA Dopamin Da Dalton DC Dng in m%t chiu (direct current) DRE Chng trnh nhn bit ma tu (drug recognition expert) ECF D ch ngoi bo (extracellular fluid) EF T l chit xut (extraction fraction) ESI Ion ho bng tia in (electrospray ionisation) HPLC Sc k l!ng hiu nng cao kDa Kilo Dalton LC Sc k l!ng (liquid chromatography) LC-MS Sc k l!ng kh"i ph$ LC-MS/MS Sc k( l!ng kh"i ph$ ghp LSD Acid lysergic MDMA 3,4-methylenedioxy-N-methylamphetamin MS Kh"i ph$ (mass spectrometry) MRM Ch % a nhim (multiple reaction monitoring) NAc Nhn accumben (nucleus accumbens) NNF No-net flux PCP Phencyclidine PET Positron emission tomography QQQ T& c'c chp ba g#m 3 t& c'c (quadrupole) RF Tn s" (radio frequency) RR Hiu sut tng "i (relative recovery) sNPC Substantia nigra par compacta THC Tetrahydrocannabinol TLTK Ti liu tham kho VTA Ventral tegmental area ZNF Zero-net-flux

DANH MC CC BNG

Bng 1.1. Thnh phn n%ng ' cc d"ch truyn )c s- d*ng trong k0 thut thm tch micro .................................................................................................................................. 10

Bng 1.2. So snh nh h(ng c+a m't s$ yu t$ th.c nghi!m n hi!u sut in vitro v in

vivo ..................................................................................................................................... 20

Bng 2.1. M,c ' tng DA trong vng no trn chu't nht th.c nghi!m khi nghin c,u

trn cc loi tc 'ng kch thch ........................................................................................ 33

Bng 2.2. So snh v kh nng gii phng DA ( 2 nhm $i t)ng: liu n $i t)ng

khng nghi!n, liu ko di $i t)ng nghi!n ................................................................. 37

Bng 3.1. c i m c+a chu't th.c nghi!m dng trong nghin c,u thm tch micro ...... 38

Bng 3.2. M't s$ cht ma tu )c s- d*ng trong cc nghin c,u thm tch micro ... 39

Bng 3.3. Thnh phn d"ch gi no tu/ ............................................................................. 39

Bng 3.4. T#a ' cy kim thm d ti m't s$ vng trong no .......................................... 40

Bng 3.5. Ci t detector kh$i ph& p d*ng cho "nh l)ng DA ..................................... 47

Bng 3.6. Thnh phn pha 'ng ........................................................................................ 48

Bng 3.7. T&ng h)p cc kt qu v % tng DA trong no chu't c$ng v chu't nht ti m't

s$ vng trong no (nhn accumben, th vn, caudate) khi s- d*ng morphin, amphetamin v cocain ............................................................................................................................ 51

DANH MC CC HNH V BIU

Hnh 1.1. Kt qu tm kim trn medline v(i t/ kho microdialysis v microdialysis dopamine ............................................................................................................................ 4

Hnh 1.2. Cc m ch tc d,ng c-a thu$c trn ng)i +c th1c hi"n nghin c.u lm

sng b*i k2 thut thm tch micro ....................................................................................... 4

Hnh 1.3. Khi qut v k2 thut thm tch micro ................................................................ 5

Hnh 1.4. S1 khuch tn th, 'ng c-a cc phn t0 c tr#ng l+ng thp khi i qua cc l&

c-a mng bn thm ti m ch tc d,ng ............................................................................ 6

Hnh 1.5. Cu to c-a kim thm d dng %ng tm ............................................................ 7

Hnh 1.6. Qu trnh cy kim thm d vo m s0 d,ng kim dn .......................................... 9

Hnh 1.7. S % ly mu bng k2 thut thm tch micro online ....................................... 12

Hnh 1.8. Nguyn tc v cu to c-a my MS ................................................................... 13

Hnh 1.9. C ch to ion c-a ESI g%m 3 giai on chnh .................................................. 15

Hnh 1.10. Cu to b' phn tch t. c1c ............................................................................. 16

Hnh 1.11. Cu to b' t. c1c chp ba ................................................................................ 16

Hnh 1.12. Cu to c-a b' phn tch by ion ..................................................................... 17

Hnh 1.13. M hnh th0 nghi"m nh gi hi"u sut kim thm d in vitro ........................ 18

Hnh 1.14. Phng php tnh ton NNF ............................................................................ 21

Hnh 1.15. Phng php ngoi suy t$c ' dng chy ....................................................... 23

Hnh 1.16. 'ng vt th1c nghi"m c th! di chuy!n v sinh hot bnh th)ng trong qu

trnh ly mu ...................................................................................................................... 25

Hnh 1.17. Kim thm d +c cy vo m c (tri) v m no (phi) .............................. 26

Hnh 1.18. B' thu tn hi"u thm tch micro online bn gi)ng b"nh ............................... 27

Hnh 2.1. Cc con )ng dn truy n v c ch gii phng DA trong no ........................ 32

Hnh 2.2. Cc con )ng dn truy n phn th*ng trong no khi s0 d,ng m't s$ cht

i!n hnh nh: opioids, ethanol, barbiturat, AMP, cocain, cannabioids, PCP cho thy dn truy n DA tp trung ch- yu v vng nhn accumben ...................................................... 34

Hnh 2.3. C ch lm tng gii phng DA trong no c,a amphetamin ............................. 35

Hnh 2.4. C ch kch thch gii phng DA c,a opiat ....................................................... 36

Hnh 2.5. C ch tng gii phng DA c,a cocain ............................................................. 36

Hnh 2.6. So snh v mt & th+ th DA ) ng(i bnh th(ng v ng(i nghi!n .............. 37

Hnh 3.1. H&p s# c,a chu&t nhn t. trn xu$ng ................................................................. 40

Hnh 3.2. Kim thm d v kim dn ................................................................................... 41

Hnh 3.3. My c$ "nh 3 chiu dng phu thut ........................................................... 42

Hnh 3.4. My ct lt m no ............................................................................................ 42

Hnh 3.5. H! th$ng sc k LC-MS v'i detector t- c/c chp ba (tri) v HPLC (phi) .... 42

Hnh 3.6. Phu thut cy kim thm d ln no chu&t ........................................................ 43

Hnh 3.7. Chu&t *c chm sc sau phu thut ................................................................ 44

Hnh 3.8. Bi u % vt kim thm d ) nhn accumben ....................................................... 45

Hnh 3.9. M hnh thm tch micro offline trong th nghi!m ........................................... 46

Hnh 3.10. Hi!u qu c,a morphin trn n%ng & DA ti vng nhn accumben ) chu&t nht

th/c nghi!m ....................................................................................................................... 50

-1-

T VN

Ma tu t: lu tr4 thnh vn $ nh9c nh,i c7a x h0i v phng ch,ng t' nn ma tu lun l m0t trong nh thu t thm tch micro (Microdialysis - MD) l m0t k> thu t v1i nguyn t!c n

gin, 5c p d6ng m0t cch hi'u qu trn th# gi1i trong cc nghin c9u ti$n lm sng v lm sng v$ nh gi hi'u qu tc d6ng c7a cc cht ma tu. Thng qua vi'c o n-ng

0 c7a m0t cht trung gian trong no l dopamin (cht to ni$m vui) ti cc vng no gii phng dopamin, hon ton c th% nh gi 5c m0t cht c phi l ma tu hay khng m

khng cn xc )nh cu trc ho h*c c6 th% c7a cht . Hn th# n thu t), cho php thu 5c mu sch, khng ln protein hay

cc enzym ti d)ch k" m, do khu lm sch mu c th% 5c b+ qua. 8ng d6ng v5t tr0i c7a k> thu t thm tch micro chnh l m hnh thm tch micro online xc )nh tr=c

ti#p cc bi#n .i n-ng 0 c7a cht cn phn tch ti m ch khi ,i t5ng nghin c9u vn c th% di chuy%n v hot 0ng bnh th2ng, cho k#t qu khch quan v1i 0 tin c y rt cao.

-2-

Vi nhng trin vng ca k thut thm tch micro v xy dng mt m hnh chun cho phng php nh gi hiu qu tc dng ca cc cht ma ty, chng ti tin

hnh thc hin ti Tng quan v phng php nh gi tc dng ca cc cht ma tu da vo k thut thm tch micro vi cc mc tiu:

1. Tng quan v k% thut thm tch micro v !ng dng.

2. Tng quan v ma tu, dopamin v mi lin h gi#a dopamin vi vic s" dng

cc cht ma tu.

3. Tng quan v phng php nh gi hiu qu tc dng c a cc cht ma tu thng qua vic xc nh nng dopamin trong no d$a vo k% thut thm tch

micro.

-3-

1. PHN 1. TNG QUAN V K THUT THM TCH MICRO

V NG DNG

1.1. TM T,T V2 L8CH SM PHT TRI4N V JNG DFNG CHA KQ THU*T

TH(M TCH MICRO

KR thu+t th)m tch micro (Microdialysis - MD) Ec bi1t 1n tL 'u th+p nin 60

nhB vo cc nghin cKu khi /t trPc ti1p cc ti th)m tch c m@t 'u truy3n d9ch vo v

m@t 'u truy3n d9ch ra vo m @ng v+t thPc nghi6m, chI y1u t$i no chu@t, nh.m mGc ch nghin cKu chKc nng sinh ho t1 bo th'n kinh trn @ng v+t s

-4-

cho thy vi!c ,ng d*ng k0 thut thm tch micro cng ngy cng pht tri n trong nh.ng

nm gn y.

Hnh 1.1. Kt qu tm kim trn medline vi t kho microdialysis v microdialysis dopamine

Cc nghin c,u m h#c, t- rt s'm, cho thy tnh u vi!t v gi tr" lm sng so

v'i cc d. li!u ly )c t- mu. C* th hn, d"ch k m l ni gn nht v'i v" tr phn ,ng c+a thu$c (m ch), do kt qu o )c s phn nh )c chnh xc m$i quan h! d)c l/c h#c - d)c &ng h#c c+a thu$c [80]. Thm tch micro l k0 thut duy nht cho

php xc "nh )c cc d. li!u ny [26, 48]. Hn n.a, thm tch micro cn l cng c* t$t nht nh gi sinh kh d*ng (bioavaiablitily) v tng ng sinh h#c

(bioequivalence) c+a thu$c ti m ch [80]. n nay, hu ht cc m trong c th con ng(i u )c nghin c,u d)c &ng h#c p d*ng bng k0 thut thm tch micro v

cho kt qu rt kh quan, trong cc m ch )c nghin c,u nhiu nht l: m no, m v, m d'i da, m ph%i, m gan... (Hnh 1.2).

Hnh 1.2. Cc m ch tc dng ca

thuc trn ngi c thc hin nghin cu lm sng bi k thut thm

tch micro [66]

-5-

1.2. KHI QUT V( K= THU"T TH M TCH MICRO

Thm tch micro (Microdialysis) l k> thu#t cho php xc -nh n1ng 3 cc cht n3i

sinh (endogen) v ngoi sinh (exogen) ti m ch tc d7ng, d

-6-

1.3. NGUYN L V CU TO C7A K< THU$T TH!M TCH MICRO

1.3.1. Nguyn l

Th"m tch micro l k= thu%t cho php xc .nh n1ng 2 cc cht n2i sinh (cc cht

d#n truy*n th n kinh nh dopamin, serotonin, noradrenalin hay phn t: glucose, glutamat, lactat) v cc cht ngoi sinh (phn t: thu0c) ti m ch tc d6ng, d;a vo

s; khuch tn th ng theo gradient nng c8a cc phn t: c tr/ng l5ng thp qua m2t mng bn thm 4 u kim thm d 5c cy vo m ch (Hnh 1.4) [7, 84, 106].

Th"m tch micro c th+ p d6ng + a cc phn t: thu0c )n t%n cc m ch tc

d6ng [71], 5c g/i l thm tch ngc (retrodialysis). N)u ti m i*u tr., n1ng 2 thu0c

b&ng 0 ho'c thp hn n1ng 2 trong d.ch truy*n vo, cc phn t: thu0c s( i qua mng bn thm v vo m ch tc d6ng [13, 80].

Hnh 1.4. S khuch tn th ng ca cc phn t c trng lng thp khi i qua cc l ca mng bn thm ti m ch tc dng [64]

No l c quan 5c p d6ng nhi*u nht v s3m nht trong cc nghin c9u lm sng

c8a k= thu%t th"m tch micro. Ngy nay, h u h)t cc m khc trn c th+ *u 5c nghin c9u p d6ng k= thu%t ny. Tuy nhin, vi-c th;c hi-n trn no di,n ra ph9c tp hn,

-7-

do m no d$ b' t+n thng, hng ro mu no cng rt d$ b' nh h/ng, v c bi%t l

d'ch no tu7 c thnh phn khc v-i d'ch ngoi bo / cc m thng th.ng [26, 66, 67].

# th6c hi%n cc nghin c3u 3ng d1ng k8 thut thm tch micro, cn chun b': (1)

kim thm d, (2) d'ch truy"n, (3) phng php 'nh l0ng cc phn t5 thu)c v (4) xc

'nh hi%u sut in vitro v in vivo c2a kim thm d.

1.3.2. Kim thm d

Kim thm d l phn c)t li c2a k8 thut thm tch micro. N 0c m ph(ng nh m,t mch mu trong c th# [67, 95].

# th6c hi%n nghin c3u thm tch micro trong no, kim thm d dng *ng tm l

ph+ bi!n v an ton nht [71, 76] (Hnh 1.5), kim 0c lm t4 thp khng g&, thch anh hoc polyethylen [67]. Thm tch micro trong no yu cu phi cy cn thn kim thm d vo m,t vng c bi%t c2a no, kim thm d s ng vai tr nh mch mu nh( v-i .ng

knh khong 200 m [13].

Cu to m,t kim thm d bao g*m 3 b, phn: (1) m,t dy truy"n d'ch vo, (2) mng

bn thm / u kim v (3) m,t dy # ly d'ch ra (Hnh 1.5).

Hnh 1.5. Cu to ca kim

thm d dng ng tm [21]

(1) Dy truyn dch vo (inlet tube)

Dy 0c n)i v-i thi!t b' bm thm tch, truy"n vo c th# d'ch truy"n trung gian

hoc d'ch c ch3a thu)c v-i t)c , xc 'nh [25].

-8-

Th nghi%m ti no yu cu d'ch truy#n vo phi c thnh phn v p sut thm thu

tng ng v/i d'ch no tu9, v/i t*c . t6 0,1 20 l/pht. Thnh phn c4a d'ch truy#n s! 2c m t chi ti"t 1 phn 1.3.3.

(2) Mng bn thm (semi-permeable membrane)

c i$m quan tr(ng nht l kch th/c c4a l- mng, v mng bn thm s! ch& cho

php cc phn t7 c kch th/c nh) hn l- mng i qua mng theo s8 chnh l%ch

gradient n+ng ., vo d'ch truy#n v 2c a ra ngoi [25, 26, 64, 66, 80]. Tr(ng l2ng phn t7 gi/i hn trong khong t6 5 "n 100 kDa [3, 44, 71, 106] (ph, bi"n nht l loi

mng c gi/i hn tr(ng l2ng phn t7 trong khong 10 30 kDa [25]), do c th$ dng k: thut thm tch micro $ ly mu nhi#u cht khc nhau. S8 vn chuy$n cc phn t7 qua mng d8a trn s8 chnh l%ch gradient n+ng ..

Mng bn thm th0ng c 0ng knh ngoi t6 220 380 m, chi#u di c4a mng

t6 7-12 mm tu thu.c vo v' tr cy kim [80]. Vt li%u ch" to mng c . tng h2p sinh h(c cao nh cellulose acetat, polycarbonat ether, polyamid, polyacrylonitril, polyether sulfonat [3, 80]. Chng #u c kh nng ht n/c v khng c tnh ch(n l(c

ha h(c. Chng khc nhau v# . dy c4a mng, mng cng dy th hi%u sut c4a mng cng thp.

(3) Dy truyn dch ra ngoi (outlet tube)

Dy 2c n*i v/i thi"t b' ch5a mu. D'ch thu 2c s! 2c chuy$n vo cc *ng

nghi%m xoay t8 .ng trong khay ch5a *ng, sau cn 2c lm lnh $ bo qun

(trong thm tch micro offline) ho c 'nh l2ng ngay (thm tch micro online).

D'ch thu 2c rt giu cc cht cn phn tch 1 dng t8 do 2c thm tch t6 d'ch

ngoi bo i qua mng bn thm theo c ch" khu"ch tn th3 .ng c4a cc phn t7 theo gradient n+ng . t6 m ch c n+ng . cao vo d'ch truy#n. N+ng . thu*c

trong d'ch thu 2c l m.t phn t& l% c4a n+ng . thu*c trong d'ch ngoi bo (extracellular fluid-ECF). T*c . dng cng thp, hi%u sut thm tch cng cao.

-9-

V kim thm d kh mnh, d$ gy nn #

cho vi%c cy kim vo m 0c d$ dng hn,

th.ng dng m,t kim dn (guide cannula) c cu to ph h0p v-i t4ng loi kim thm d # to

.ng dn tr-c khi cy kim thm d.

Qu trnh cy kim thm d vo m 0c

m t nh trong Hnh 1.6. u tin kim dn s

0c cy vo ngay st vng m cn nghin c3u,

sau kim thm d 0c cy qua kim dn sao cho ch& c phn mng hot ,ng v0t ra ngoi chi"u di kim dn # vo m ch [104].

Vi%c cy kim thm d trong i"u ki%n

khng v khun c th# lm cho phn 3ng c2a m ti v' tr cy trm tr(ng hn v to i"u ki%n cho vi khun pht tri#n [44]. Ngoi ra, # gim cm

gic au v kh ch'u, c th# s5 d1ng thu)c gy t ti ch+ hay ch.m ln vng cy kim [48].

Hnh 1.6. Qu trnh cy kim thm d vo m s dng kim dn

1.3.3. Dch truyn

D'ch truy"n c vai tr v cng quan tr(ng trong thm tch micro trong no. i"u

thi!t y!u c2a d'ch truy"n l phi v khun, c p sut thm thu ph h0p v-i mu v

ch3a cc ion i%n gii tng ng v-i d'ch no tu7 (CSF cerebrospinal fluid) [11, 71]. N cng cn c pH v nhi%t , tng t6 trong m no [11].

Cc ion trong d'ch gi no tu7 c vai tr "%m gim s)c" cho s6 thay *i thnh phn ion trong no [27, 71]. m bo ng t& l% c2a cc thnh phn rt quan tr(ng trong cc th

nghi%m ti no, v n nh h/ng tr6c ti!p !n cn bng trong d'ch ngoi bo c2a no.

-10-

Bng 1.1. Thnh phn nng cc dch truyn c s dng trong k thut thm tch micro

Loi dch Thnh phn

D)ch truy&n tng

ng d)ch no tu8

(perfusion fluid

CNS)

NaCl 147 mM

KCl 2,7 mM

CaCl2 1,2 mM

MgCl2 0,85 mM

pH ~6

(hng sn xut CMA, Thu7 i'n)

D)ch truy&n cho m

ngoi vi

(perfusion fluid T1)

NaCl 147 mM

KCl 4 mM

CaCl2 2,3 mM

pH ~6

(hng sn xut CMA, Thu7 i'n)

1.3.4. Phng php nh lng cc phn t thuc

V l1ng m u thu 1c trong k9 thu!t thm tch micro l rt nh+ (c0 microlit) nn

i h+i cc phng php )nh l1ng phi c . nhy cao, ph- bi%n hay s5 d2ng nht l

phng php s"c k l+ng hi(u nng cao (HPLC) v s"c k l+ng k%t h1p kh,i ph- (LC - MS) [44, 80].

1.3.4.1. Sc k lng hiu nng cao (HPLC)

HPLC l m.t k9 thu!t tch (separation) trong cc cht phn tch di chuy'n qua c.t ch4a cc ht pha tnh v 1c phn tch d6a vo i l6c tng ,i c3a n v/i pha tnh

v pha .ng. Tu thu.c vo bn cht c3a cht cn phn tch, pha tnh s$ 1c ch*n cho thch h1p. Pha tnh v cht cn phn tch h1p . phn c6c v/i nhau s$ cho hi(u qu r5a

gii cao hn [2]. Tuy nhin, cc nghin c4u ch4ng minh r#ng, hon ton c th' tch cc cht d n truy&n thn kinh (cc cht phn c6c) b#ng s"c k pha o (pha tnh khng phn

-11-

c=c) nh4 vo l=c tng tc gi

-12-

Hnh 1.7. S ! ly mu bng k' thut thm tch micro online

1. H th ng bm tim 2. B" phn chnh dng 3. Thit b thu mu 4. Kim thm d 5. D$ng c$ ch%a

"ng vt th&c nghim di chuyn t& do bn trong 6. Bn # 7. Van tim 8. C"t HPLC 9. Bm pha

"ng 10. Pha "ng 11. Detector 12. My tnh.

Phng php HPLC thch h1p phn tch nhi%u loi h1p cht h4u c khc nhau nh

cc phn t3 ngoi sinh (thu+c) hay cc phn t3 n-i sinh (cc cht dn truy%n thn kinh,

cc neuropeptid). Tuy nhin, trong m-t s+ tr/ng h1p HPLC k$t h1p detector hunh quang v i'n ho cho - nhy cha 2 [70]. V v y hi'n nay cc mu th/ng 1c (nh

l1ng b"ng s!c k l*ng k$t h1p v.i kh+i ph, [70].

1.3.4.2. Sc k lng khi ph (LC-MS)

Trong hn 10 nm tr0 li y, kh+i ph, (MS - mass spectrometry) tr0 thnh m-t

detector c5c k ph, bi$n cho thm tch micro offline v online lin k$t v.i s!c k l*ng (LC - liquid chromatography).

Khi qut v% LC-MS

S!c k l*ng (LC, liquid chromatography) l m-t k6 thu t c bn dng & phn tch

cc cht. Kh+i ph, k$t h1p v.i s!c k l*ng (LC-MS) ngha l khu np mu s# l gin

ti$p, n l u ra c2a mu sau khi ti$n hnh phn chia (separation) b0i c-t s!c k, v kh+i ph, s# hot -ng nh m-t detector c2a s!c k l*ng.

K$t h1p s!c k l*ng v.i kh+i ph, (LC-MS) hay kh+i ph, ghp (tandem mass spectrometry) (LC-MS/MS) lm tng ng k& tnh ch)n l)c v - nhy c2a phng php

-13-

phn tch. Gi,i hn pht hi$n c th# !n 10-14 gam [2]. N)ng + cc cht trong mu thm

tch micro rt nh', thnh phn tng (i ph0c tp, bao g)m c thu(c, cc cht chuy#n ha c/a thu(c v cc cht khc c trong d%ch ngoi bo. S2 d.ng LC - MS gip cho k!t

qu thu -c chnh xc hn v c gi tr% hn. V vy rt nhi"u nghin c0u v" thm tch micro s2 d.ng phng php ny # %nh l-ng.

Nguyn l hot +ng c/a kh(i ph*

Khc v,i cc k6 thut quang ph*, kh(i ph* khng dng b0c x i$n t1 nhng vn c

th# xc %nh -c cc )ng v% ho h&c. y l m+t k6 thut o tr4c ti!p t s khi lng v in tch ca ion (m/z) -c to thnh trong pha kh t1 phn t2 ho c nguyn t2 c/a mu. T5 s( ny -c bi#u th% bng n v% kh(i l-ng nguyn t2 (1 n v% kh(i l-ng

nguyn t2 bng 1/12 kh(i l-ng c/a Carbon 12C) ho c bng dalton (1 dalton bng kh(i l-ng c/a nguyn t2 Hydro) [2].

Kh(i ph* cung cp nh3ng thng tin v" kh(i l-ng phn t2, cu trc ha h&c c/a h-p cht, s( l-ng h-p cht v + tinh khi!t c/a mu phn tch. Nh3ng d3 li$u ny lm chc

chn thm cc k!t qu %nh tnh v %nh l-ng [2].

My MS c nguyn tc hot +ng v cu to nh sau :

Hnh 1.8. Nguyn tc v cu to ca my MS

-14-

(1) B, np mu : ni mu 1c a vo my. N u mu / dng l(ng hoc rn

th s 1c chuy"n sang dng hi thch h1p / b, phn ho hi.

(2) B, ngu*n ion: Ion ho cc phn t6, nguyn t6 c3a mu / trng thi kh

hoc hi.

(3) B, phn tch kh)i: tch cc ion theo t8 s) m/z. Cc ion 1c gia t)c v tch

ring nh. tc d2ng c3a t5 tr.ng, i$n tr.ng, " i n detector.

(4) Detector: c nhi$m v2 chuy"n cc ion n thnh tn hi$u i$n o bng h$ i$n t6 c3a my kh)i ph+.

(5) B, thu tn hi$u.

Tt c qu trnh ny di#n ra trong h$ chn khng v-i p sut t5 10-3 n 10-6 Pa. Tn

hi$u thu 1c 1c th" hi$n d-i dng cc pic cc c.ng , khc nhau, tp h1p li to thnh m,t khi ph hoc ph khi.

B, phn quan tr'ng nht c3a my kh)i ph+ l b, ngu*n ion v b, phn tch kh)i.

B ngun ion (ionisation) c3a my kh)i ph+

Trong 2 thp k8 qua, b, ngu*n ion ho bng tia i$n (electrospray ionisation ESI)

th" hi$n tnh u vi$t v tr/ thnh cng c2 quan tr'ng trong cc phng nghin c4u lm sng. ESI MS (kh)i ph+ s6 d2ng b, ngu*n ion ho bng tia i$n) l m,t cng c2 nhy bn, mnh m v ng tin cy, n cho php pht hi$n 1c nh7ng mu v-i th" tch nh( c0

microlit, cc cht kh bay hi, cc cht b& ph hu8 b/i nhi$t (do khng th" p d2ng ion ho bng nhi$t) [47]. ESI cho php ion ho nh nhng cc phn t6, lm cho phn t6 cht

khng b& ph hu8 hay to ra nhi!u ion mnh (ion fragment) (Hnh 1.9)

Sau khi ion ho, cc ion 1c chuy"n n b, phn tch kh)i. Ti y, ion s 1c

ch'n l'c theo t% s) m/z.

-15-

Hnh 1.9. C ch to ion c a ESI gm 3 giai on chnh [47]:

(1) Gii phng cc git mn mang in t" u mi mao qun di tc dng c a in

trng.

(2) Bay hi dung mi to cc git nh.

(3) Gii phng ion t" cc git mn nh tch in.

B phn tch khi (mass analysers)

D)i tc d,ng c-a b( phn tch kh$i, ion di chuy n qua t/ tr*ng hoc i!n tr*ng

theo t" s$ m/z c-a ion.

C 3 loi b( phn tch kh$i ph& bin trong ESI MS, l (1) b( phn tch t. c0c

(quadrupole), (2) b( phn tch t. c0c chp ba (tandem quadrupole), (3) by ion (ion trap) [47]. Trong , b( phn tch t. c0c chp ba v by ion l thng d,ng nht [2].

(1) B phn tch t! c#c g%m 4 thanh kim loi t song song cch u nhau (Hnh

1.10) M'i cp $i nhau c i!n tch bng nhau. Hai u c-a kh$i t. c0c +c t vo dng i!n m(t chiu (DC) v)i tn s$ (RF) xc #nh, to nn i!n tr*ng bn trong, khin

cho cc ion i theo phng z (Hnh 1.10) h+p v)i mt phng x-y c-a n [75]. Ba phng x-

-16-

y-z to nn -ng dao +ng c trng c2a ion.

Ng-i ta c th" ch$nh cc gi tr% DC v RF " thu 0c ion mong mu(n [47]. B+ phn tch t3

c5c 0c nh gi l rt mnh m, ti t ki#m, nh' g&n v tng thch v,i u vo (inlet) n(i ti p

c2a cc k7 thut khc [47] nh sc k kh, sc k l'ng [75].

Thi t b% ny l5a ch&n cc ion theo t6 l#

m/z. Tuy nhin, gi tr% m/z ny c th" bao hm

nhi!u nhm ion khc nhau (wide band pass) hoc ch$ m+t loi ion duy nht (narrow band pass) [75].

(2) B phn tch t cc chp ba hay cn g&i l QQQ g)m 3 b+ phn tch t3 c5c li!n nhau trn m+t -ng thng (Hnh 1.11). Ion sau khi 0c ch&n l&c (rational ion) qua t3

c5c u tin (Q1) s 0c va chm v,i kh tr (Ar, N2) . Q2 v b% chia nh', y g&i l qu trnh va chm phn ly (collision-induced disociation CID). Ion con (product ions) c

kh(i l0ng phn t4 tng ng ion g(c ti p t1c 0c ch&n l&c b.i Q3 " cung cp thng tin v! cu trc phn t4 [47, 70]. T3 c5c chp ba, hay MS/MS, cho php sng l&c k t qu k7 l/ng hn khi ch$ dng m+t b+ t3 c5c, do cho cc tn hi#u c hi#u hn so v,i khi ch$

phn tch MS [47].

T3 c5c chp ba c th" hot +ng . nhi!u ch + (mode of data), tu theo m1c ch

c th" ch&n qut ph* ion g(c, ion con hoc c hai (a nhi#m) [47].

Hnh 1.11. Cu to b t cc chp ba [47]

Hnh 1.10. Cu to b phn

tch t cc [47]

-17-

(3) B phn tch by ion (ion trap

mass analyser) hot ,ng theo nguyn tc :

ion 1c by trong i$n tr.ng v-i hai i$n c8c / hai u v m,t i$n c8c vnh ai v-i tn s) v bin , lin t2c thay +i, to nn

vng xoy i$n tr.ng 3 chi"u t4 c8c, khi!n ion lin t2c di chuy#n theo t% s) m/z

c3a n [47]. Ch% nh7ng ion c m/z thch h1p m-i i qua 1c i$n tr.ng vo detector

(Hnh 1.12).

Hnh 1.12. Cu to ca b phn tch

by ion

By ion t4 c8c r v nh( hn nhi"u so v-i 2 thi!t b& trn trong khi vn c th# tc

,ng va chm phn li (CID) lin t2c ln ion l't vo i$n tr.ng, hnh thnh nn khi ni$m LC MS/MSn (n = s) ln CID). Tu vo m2c ch nghin c4u c th# l8a ch'n b, phn

tch c8c ph h1p nht, QQQ ph h1p cho &nh l1ng, cn by ion ph h1p cho phn tch &nh tnh v xc &nh cu trc cc cht cha bi!t [2].

LC-MS v LC MS/MS l k9 thut y tri#n v'ng cho cc nghin c4u ti"n lm

sng v lm sng v" sinh l h'c, d1c l h'c c3a thu)c v c th#. V-i t cch m,t detector, kh)i ph+ khc ph2c 1c nh7ng hn ch! c3a cc phng php tr-c v" , nhy, tnh ch'n l'c v , chnh xc cn thi!t # x6 l cc mu c th# tch rt nh( (c0 microlit)

v-i n*ng , rt thp (t5 10-9 !n 10-6 M) [44]. Hi$n nay, k9 thut ny 1c p d2ng r,ng ri c bi$t trong cc 4ng d2ng v" pht hi$n dng chuy#n ho c3a thu)c v cc cht n,i

sinh v-i n*ng , rt nh( nh. vo , nhy v tnh ch'n l'c cao c3a detector kh)i ph+ [75], c bi$t thm tch micro online k!t h1p LC MS to nn b-c ,t ph cho cho vi$c

xc &nh n*ng , phn t6 thu)c rt nh( trong cc m ch tc d2ng [44].

-18-

1.3.5. Hiu sut kim thm d

Hi$u sut kim thm d (recovery) 0c &nh ngha l t% l$ gi4a n*ng , thu)c thm

tch 0c trn n*ng , thu)c th5c t! / m ch, ch&u nh h/ng b/i kh nng thm tch c1a mng kim thm d [94]. Trong a s) tr.ng h0p, n*ng , o 0c ch% l m,t phn c1a

thu)c 0c trch ra t3 d&ch k m, khi hi$u sut t 0c 0c g'i l hi$u sut tng )i (relative recovery - RR) [26, 41, 48]. Hiu sut tng i c nh ngha l t l chnh

lch nng gia nng thuc trong mu thu c v trong dch xung quanh kim thm d [76].

Ng.i ta c th# xc &nh 0c hi$u sut in vitro v in vivo c1a kim thm d. M+i

hi$u sut s cho cc ngha v gi tr& cn thi!t # nh gi hi$u qu c1a kim thm d.

1.3.5.1. Hiu sut tng i in vitro

y l cch n gin nht, xc &nh 0c hi$u sut tng )i bng cch c) &nh gi tr& n*ng , d&ch xung quanh v o gi tr& n*ng , d&ch thu 0c t3 kim thm d [46].

Hnh 1.13. M hnh th nghim nh gi hiu sut

kim thm d in vitro [76]

u tin, ti!n hnh in vitro thm tch th.ng, kim thm d s 0c nhng vo m,t

l' nh( c ch2a n*ng , bi!t tr-c c1a thu)c, sau kim thm d s 0c truy"n vo m,t dung mi trung tnh. Hi$u sut c1a thu)c, cn g'i l hi$u sut tng )i (relative recovery

RR), l t% l$ gi4a n*ng , thu)c thu 0c v n*ng , thu)c trong l', theo cng th2c sau :

-19-

(1a) [21, 30, 45, 46]

trong : RR l hi&u sut tnh b ng %

Cout l n+ng . thu*c thu 2c t6 kim thm d

Csol l n+ng . thu*c bi#t tr/c trong l)

+ng th0i, ti#n hnh song song in vitro thm tch ng2c. Cc b/c tng t8 nh

trn, nhng thay dung d(ch trong l) b ng dung mi trung tnh (pH = 7,4), truy$n vo kim thm d m.t l2ng thu*c bi#t tr/c n+ng .. Hi&u sut thm tch ng2c 2c tnh nh

sau :

(1b)

trong : Cout l n+ng . thu*c thu 2c t6 kim thm d

Cin l n+ng . thu*c bi#t tr/c 2c truy$n vo kim thm d

Phng php ny ch4 y#u % nh gi . hi&u qu c4a kim thm d v/i t6ng loi

thu*c khc nhau nh hi&u qu mng bn thm, t*c . dng chy, nh h1ng c4a n+ng . thu*c #n hi&u sut, tnh ,n (nh, t6 c th% l8a ch)n 2c kim thm d ph h2p nht

v/i th nghi&m. % th8c hi&n 2c, cn s7 d3ng t*i thi%u 5 kim thm d v/i chi$u di mng bn thm khc nhau (v d3 5, 10, 20, 30 mm) ho!c t*c . dng chy khc nhau (v

d3 nh 1, 2, 3, 4, 5 pht), m-i kim thm d ly 6 mu em (nh l2ng v tnh hi&u sut [45].

Phng php ny c nhi$u hn ch# v trn c th% s*ng c rt nhi$u y#u t* sinh l

nh h1ng #n k#t qu thu 2c v d3 nh cu trc quanh co c4a d(ch k" m, kh nng hp thu, chuy%n ho, ti hp thu c4a m [46, 105], do hi&u sut tng *i in vitro th0ng

cao hn nhi$u so v/i hi&u sut in vivo [48]. B1i vy, tnh hi&u sut in vitro ch' l cng c3 cn thi#t % nh gi tnh kh thi cho nghin c5u in vivo [26, 48, 105].

-20-

1.3.5.2. Hiu sut thc nghim in vivo

Hiu sut in vivo chu nh h$ng c&a rt nhiu yu t! d%c l)c h c, d%c "ng h c,

do phng php tnh ton cng ph'c tp hn nhiu. D#i y l bng phn tch so snh nh h$ng c&a m"t s! yu t! th)c nghim n hiu sut in vitro v in vivo.

Bng 1.2. So snh nh hng ca mt s yu t thc nghim n hiu sut in

vitro v in vivo [26, 41, 48, 71]

Yu t thc nghim In vitro In vivo

La chn dng c

+ Cu to mng thm tch (gi#i hn kh!i l%ng phn t(

i qua, chiu di mng)

+ T!c " dng chy v thnh phn dch truyn

X X

c tnh l ho ca thuc

+ Tnh thn n#c/du

+ Tr ng l%ng phn t(

+ Kh nng lin kt protein

+ " tan

+ Kh nng bm vo !ng kim

X X

Thao tc th nghim

+ " su c&a kim %c cy

+ Nhit " th nghim

X

Cc yu t ni sinh

+ M ch

+ Lu l%ng mu qua m

+ Chuyn ho

+ Nhit " c th

X

-21-

T+ , ng&i ta chia thnh 4 phng php xc nh hiu sut in vivo :

Phng php no-net-flux (NNF) hay cn g!i l zero-net-flux

Phng php ny s, d)ng n#ng % thu"c ti d ch k m, n#ng % thu"c truyn vo v thu (c t+ kim thm d. im mu ch"t l n#ng % thu"c ' d ch k m phi $n nh

trong su"t qu trnh th nghim. Trong khi , cc n#ng % khc nhau c*a mu phn tch s (c truyn vo (Cin) v ng&i ta s o n#ng % mu thu (c (Cout) xc nh im

no-net-flux, chnh l giao im gi-a &ng chun c*a cc gi tr Cin v &ng biu th Cout Cin = 0. D.a vo biu # sau, ng&i ta xc nh (c gi tr c*a Cnnf (n#ng % thu"c

ti im no-net-flux), cng l n#ng % c*a thu"c trong d ch ngoi bo [48] (Hnh 1.14).

Hnh 1.14. Phng php tnh ton NNF [76] :

Khi nng c#a thuc trong dch k m bng vi nng thuc trong dch truyn, t$c l khi Cin = Cnnf, im NNF s xut hin. S% d"ng dch truyn vi nng cao

hn v thp hn nng c#a thuc trong dch k m, ng i ta c th xc nh !c Cnnf bng phng php ngoi suy [76].

Phng php no-net-flux

(NNF) (1)

Dng linh hot

c*a phng php NNF (2)

Ngoi suy dng chy

(stop-flow/flow-rate)

(3)

Thm tch ng(c

(retrodialysis)

(4)

Cc phng php tnh hiu sut in vivo hay s,

d)ng nht g#m c 4 phng php sau y [19]

-22-

Sau khi tm ra 1c Cnnf, hi%u sut in vivo c3a th nghi%m c th$ tnh 1c. - d)c

c3a /ng NNF, 1c g(i l extraction fraction (EF) chnh l hi%u sut tnh theo phng php NNF, c cng th4c t+ng qut nh sau:

(2) [26, 46]

trong : Cin l n*ng - thu)c truy#n vo

Cout l n*ng - thu)c ly ra

Cnnf l n*ng - thu)c 0 d'ch ngoi bo xung quanh kim thm d

Gi tr' EF phn nh 1c qu trnh d1c -ng h(c c3a cht cn phn tch trong c th$. N 1c p d2ng rt nhi#u trong gii thch s7 thay +i c3a n*ng - thu)c 0 d'ch

ngoi bo. c bi%t l khi c s7 tng tc c3a thu)c v.i cc cht khc trong c th$, v d2 nh r1u [46].

NNF l phng php chun vng v n thch h1p v.i c cht n-i sinh v ngoi sinh. Nh1c i$m duy nht c3a phng php ny l n yu cu - +n 'nh c3a c th$

trong th/i gian di, khng ph h1p trong cc tr/ng h1p c chuy$n bi"n [26, 67, 91].

(2) Dng linh hot ca NNF (Dynamic NNF)

Dynamic NNF c nguyn tc tng t7 nh NNF, nhng thay v thay +i Cin, ng/i

ta s! s6 d2ng t)i thi$u 3 c th$ $ ti"n hnh th nghi%m, m,i c th$ 1c truy#n vo m-t l1ng d'ch c ch4a n*ng - Cin nh nhau. /ng chun 1c xy d7ng tng t7 v suy

ra 1c Cnnf [26, 76].

Phng php ny 1c Olson v Justice 4ng d2ng trong nhi#u nghin c4u v# gii

phng dopamin trong no chu-t khi s6 d2ng cocain, amphetamin, haloperidol [69]. N hn ch" 1c nh1c i$m v# - +n 'nh c3a thu)c trong c th$ trong th/i gian di c3a NNF [26]. Tuy nhin, phng php ny c nh1c i$m l phi s6 d2ng nhi#u c th$ th nghi%m. Bn cnh cn cn phi tnh ton cho t5ng kim thm d trn m,i mu thay v ch& trn m-t kim thm d. Do vy m phng php NNF vn 1c l7a ch(n nhi#u hn [76].

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(3) Phng php ngoi suy tc dng chy (stop-flow/flow-rate)

Phng php ny ra -i t3 nm 1985 b.i Jacobson v c+ng s6 d6a trn s6 thay *i

t(c + dng chy. Hi#u sut tng (i ph0 thu+c vo t(c + dng chy (xem Bng 1.2). T(c + dng chy cng nh' th hi#u sut t /c cng cao [91]. Trn l thuy t, cn bng ch$ t /c khi t(c + dng chy bng 0, khi m,i xc %nh /c n)ng + tht s6 c1a

mu. N u ngoi suy t(c + dng chy n gi tr% bng 0 theo m+t -ng chun s thu

/c Cout tht s6 c1a mu [48]. T3 c cng th2c :

(3a) [76]

trong : Cout l n)ng + mu thu /c

Ce l n)ng + d%ch bao quanh kim thm d (d%ch k m)

r l h# s( chuy"n kh(i

A l di#n tch mng bn thm

F l t(c + dng chy

Suy ra : (3b)

Hnh 1.15. Phng php ngoi suy tc dng chy [54]:

A : mi lin h gia RR v F. Khi F = 0 l/pht th RR t 100%

B : cch xc nh Ce. Khi ngoi suy v F = 0 l/pht v RR t 100% th Ce = Cout

Phng php ny c nh/c i"m l t(n nhi!u th-i gian do t(c + dng chy nh'.

Hn n5a, " tng + tin cy c1a k t qu ngoi suy, cn phi th6c hi#n th nghi#m . t(c +

dng chy thp nht c th" [76, 91], khng v/t qu 0,3 l/pht [26]. Do , phng php ny khng /c p d0ng nhi!u trong cc th4 nghi#m d/c +ng h&c lm sng [91].

-24-

(4) Phng php thm tch ngc (retrodialysis)

Thm tch ng,c th# hi$n tnh v,t tr)i hn so v*i cc phng php trn v" th+i

gian v s' l,ng vt th nghi$m [76], do hi$n nay y l phng php thng d-ng nht [3, 44]. Trong thm tch ng,c, ng+i ta c th# s1 d-ng chnh cht phn tch hoc s1 d-ng cht chun n)i [19, 26, 54].

Khi khng s1 d-ng cht chun n)i, m)t n(ng ) bi!t tr*c c.a thu'c ,c truy"n

vo c th# thng qua u vo kim thm d. V qu trnh phn tn qua mng l %nh l,ng ,c c khi chuy#n thu'c vo c th# (delivery) hay ly thu'c t0 c th# (recovery), nn

hi$u sut thm tch ng,c (R) c th# coi nh tng ng v*i hi$u sut thm tch micro [76].

Phng trnh c.a thm tch ng,c l tr+ng h,p c bi$t c.a cng th/c (2) p d-ng

khi n(ng ) cht phn tch trong d%ch ngoi bo bng 0. i"u ny khng th# t ,c khi

o cc cht n)i sinh do chng lun t(n ti trong d%ch ngoi bo [26, 46]. Cng th/c tnh hi$u sut c.a thm tch ng,c nh sau :

(4) [19, 45, 67, 76, 91]

trong : R l hi$u sut thm tch ng,c

Cin l n(ng ) thu'c truy"n vo

Cout l n(ng ) thu'c ly ra

Tuy nhin, khi thay vo l m)t cht chun n)i (internal standard) trong d%ch

truy"n, n(ng ) cht chun n)i trong d%ch ngoi bo s bng 0 v nh+ vy c th# o ,c c cc cht n)i sinh hay n(ng ) thu'c ti m ch. Nh+ c cht chun n)i, cht cn phn tch c th# ,c ly ra ngoi # %nh l,ng, (ng th+i hi$u sut in vivo c.a th nghi$m

,c xc %nh thng qua qu trnh thm tch ng,c cht chun n)i [46].

Cht chun n)i phi c c tnh tng t2 cht phn tch nh ) phn tn, ) dn

truy"n v chuy#n ho, t't nht nn l2a ch&n cht chun n)i l (ng phn -t-ri hoc

(ng phn phng x c.a cht phn tch [26]. Hi$u sut in vivo thm tch ng,c lc ny ,c tnh nh cng th/c (4) p d-ng cho cht chun n)i [46].

-25-

Trong th@c t+, xc 1nh hi/u su"t in vivo g*p ph!i nhi,u kh khn do cc phng

php xc 1nh ,u yu c#u 5 4n 1nh c d:ng ch"t chu%n n5i gip cho vi/c quan st

cc bi+n 4i trong qu trnh l"y m&u. Thm vo , ch0 nh?ng tr7ng h9p hi/u su"t tng 2i trn 20% m6i c th. 9c ch"p nh(n, v cc th> nghi/m in vitro l b)t bu5c . xc

1nh tnh kh! thi c

-26-

K thut

Xc &nh /c n)ng + cc phn t4 thu(c . dng t6 do [3, 7, 15], v cc dng chuy$n ha c1a n ti d&ch k! c1a t" bo [48], . dng ny thu(c m,i c tc d0ng v c m(i

tng quan thun v,i hi%u qu i#u tr&, )ng th-i cung cp nh5ng thng tin v# d/c +ng h'c c1a thu(c ti m ch tc d0ng.

C th$ cy nhi#u kim thm d vo cng m+t m ch hoc cy cng lc vo nhi#u c quan khc nhau trn c th$ [71].

Kim thm d /c dng nghin c3u trn nhi#u m khc nhau [48] (Hnh 1.17).

Hnh 1.17. Kim thm d c cy vo m c (tri) v m no (phi)

V,i kch th,c l* mng thch h/p, c th$ ly /c mu sch trong cht phn tch khng b& nh h.ng b.i hot tnh c1a enzym v protein [48, 76, 80]. Mu ly /c

c th$ &nh l/ng tr6c ti"p ngay, khng mt th-i gian chun b& hay lm sch. y l m+t u th" v/t tr+i c1a thm tch micro, cht phn tch khng b& hao h0t do

qu trnh lm sch mu [44].

2ng d0ng k7 thut thm tch ng/c (retrodialysis) trong i#u tr& ti m ch [76],

khi m cc phn t4 thu(c khng th$ phn b( "n cc m b%nh; trong nghin c3u tc d0ng c hi%u c1a cc cht dn truy#n thn kinh [74]; trong so snh s6 chuy$n ho c1a cc tc nhn gy ung th ti cc u b,u v,i cc m kho mnh [7, 60].

-27-

Thm tch micro online k$t h3p v0i d' dng

v0i cc k; thut *nh l3ng khc nh HPLC, LC-MS, khi$n cho vi(c ra 1i cc thi$t b* "t

cnh gi1ng (bed-side) ph4c v4 cho vi(c xt nghi(m v theo di cc thay .i ho sinh trn

b(nh nhn m/t cch thun l3i, t7 cho php pht hi(n v ch9a tr* b(nh s0m, -ng

th1i gp phn gii thch c ch$ c5a cc b(nh l thn kinh nh Parkinson, trm cm, tm

thn phn li(t [44] (Hnh 1.18).

Hnh 1.18. B thu tn hiu thm

tch micro online bn ging bnh

1.4.2. Nhc im v mt s bin php khc phc

Thao tc cy kim thm d

Vi(c cy kim thm d gy ra nh9ng thay .i nht *nh ti m, "c bi(t nh h2ng

$n hng ro mu no khi th nghi(m ti m no, v do nh h2ng $n k$t qu nghin c6u. C th& kh c ph4c b!ng cch 3i $n khi hng ro mu no .n *nh (sau khong 2h) [15].

Qu trnh cy kim thm d i h+i s: kho lo, chnh xc v v khun [66], i h+i

ng1i lm th:c nghi(m cn hi&u c"n k# v% k; thut v 3c trang b* y 5 cc k; nng cn thi$t [48].

Mng bn thm

K; thut ny c m/t s, kh khn khi ly mu cc cht thn du ho"c cc cht c i

l:c cao v0i protein. V d*ch truy%n vo c dung mi l n0c (Ringers, aCSF), k; thut thm tch micro vn 3c quan ni(m ch) gi0i hn trong nghin c6u cc thu,c

tan trong n0c. Tuy nhin, m/t vi th8 nghi(m in vitro thnh cng trong vi(c o n-ng / c5a cc cht thn du, trong dung d*ch n0c 3c thay th$ b!ng nh

tng ho"c b. sung cht di(n hot [26].

-28-

Mng bn th"m thng th:ng b2 gi9i h n cc phn tE i qua mng v9i tr3ng l=ng

khng qu 100 kDa [44, 76]. Tuy nhin, sG h7 tr= c@a mng siu th"m v l9i kim lo i (metal meshes) c th/ tng gi9i h n ny [64].

Thi gian th nghim v th tch thu c

Th/ tch m&u r"t nh4, n6ng 8 thu5c th"p (c; microlit) nn i h4i cc phng

php phn tch ph!i c 8 nh y cao, ch*ng h n nh HPLC, LC MS [26, 44, 76].

N6ng 8 thu5c thGc t- t i m ch r"t kh c th/ xc 2nh chnh xc, hn nFa

khng th/ t =c tr ng thi cn b)ng hon ton v qu trnh truy.n v l"y m&u l

lin t?c [26], v v(y c#n c cc phng php tnh hi0u su"t in vitro v in vivo / hi0u ch1nh n6ng 8 chnh xc t i m [48].

Th:i gian l"y m&u ph!i ko di hn nhi.u so v9i cc phng php khc [13], trung

bnh qu trnh l"y m&u ko di 15 20 pht.

1.5. QUY TRNH NGHIN CAU CHUNG KHI SD D>NG KH THU'T TH$M

TCH MICRO TRONG NGHIN CAU Y D

-29-

2. PHN 2: TNG QUAN V MA TU, DOPAMIN V MI

LIN H GIA DOPAMIN VI CC CHT MA TU

2.1. T5NG QUAN V. CC CH'T MA TU

2.1.1. nh ngha ma tu

Ngha Hn Vit ca ma tu

Ma ty l t? Hn Vi1t, v8i ngha: ma l t m, ty l say sa. Nh v,y, ma ty

l ch(t a -n sB say sa, m m*n. y cng l t? ti-ng Vi1t dng 0 d2ch chA n8c ngoi khi mu4n ni t8i cc ch(t gy nghi1n thu7c lo%i nguy hi0m nh(t: thu4c phi1n,

morphin, heroin, cocain, c)n sa v m7t s4 thu4c t6ng h;p c tc d

-30-

2.1.2. Phn loi cc cht ma tu

C nhiu cch phn loi cc cht ma tu, g#m c: phn loi theo lut php, phn loi theo tc d)ng, phn loi theo ngu#n g"c sn sinh. Trong chng ta s. d)ng nhiu

nht l phn loi theo tc d)ng v phn loi theo ngu#n g"c sn sinh c*a cht ma tu.

Phn loi theo tc dng ca cht ma tu

Danh m)c phn loi theo DRE (Drug Recognition Expert) c*a M0 chia ma tu thnh

7 nhm chnh theo tri u ch,ng v tc d)ng c*a chng trn c th [53]:

+c ch thn kinh (CNS Depressants): r(u, thu"c an thn, barbiturat...

Kch thch thn kinh (CNS Stimulants): cocain, amphetamin, cafein

Cht gy o gic (Hallucinogens): peyote, psilocybin, ht gi"ng rau mu"ng, cc

cht t$ng h(p nh acid lysergic (LSD) v ectasy (MDMA)

PCP v cht tng t/.

Cht gim au thu%c nhm ma tu (Narcotic Analgesics): heroin, morphin,

codein, oxycondin, vicodin, percodan, fentanyl, dilaudid, demerol

Cht xng ht (Inhalants): gasoline, N2O (kh c&i), amyl nitrit, ether

Cannabis: THC, cannabioids.

Phn loi theo ngun gc sn sinh ca cht ma tu [35]

D/a theo ngu#n g"c sn sinh, cc cht ma tu c th (c phn loi theo 3 nhm :

t/ nhin, bn t$ng h(p v t$ng h(p [35]. Trong cc cht bn t$ng h(p v t$ng h(p l cc cht rt nguy him, c th gy nh h'ng n kh nng nhn th,c v hnh vi c*a con

ng&i.

T nhin

Cc cht ma tu c ngu#n g"c t/ nhin bt ngu#n t- th/c vt v i h!i t hoc hu

nh khng giai on ch bin. Opium, cannabis v cao cca thu%c vo nhm ny.

Bn tng hp

-31-

Cc cht ma tu bn t*ng h0p 0c to ra bng cch x5 l ho h&c cc cht ma tu

t7 nhin. Vi$c x5 l ho h&c ny 0c dng # chi!t tch hot cht chnh hay thay *i cu trc c2a chng. Heroin l v d1 i#n hnh c2a nhm ny.

Tng hp

Cc thu(c t*ng h0p 0c sn xut hon ton thng qua cc quy trnh ho h&c, bao

g)m cc cht nh methaqualon (mandrax), amphetamin, diazepam, ecstasy v.v

2.2. DOPAMIN

2.2.1. nh ngha

Dopamin (DA), hay cn g&i l cht to "ni"m vui", l m,t cht dn truy"n thn kinh

c bn cht catecholamin 0c t*ng h0p b/i cc t! bo thn kinh dopaminergic, hot

,ng thng qua cc th1 th# c hi$u [40], c kh nng lm tng hoc gim hot ,ng c2a cc t! bo thn kinh. DA c rt nhi"u nh h/ng ln no ,ng vt c v, bao g)m vai tr

trong i"u ch%nh kh nng nhn th3c, vn ,ng, khoi cm, i"u ho n,i ti!t, ghi nh- v h&c h'i [61, 72]. DA c quan h$ cht ch v-i c ch! "phn th/ng" thng qua con .ng

mesolimbic (Hnh 2.1).

Phn th/ng (reward) l m1c tiu, l khao kht m con ng.i mu(n t 0c # to

ra cm gic hng phn, sng khoi cho bn thn [86]. Phn th/ng c vai tr rt quan tr&ng

(i v-i t4ng c nhn v n h+ tr0 cho cc hot ,ng thng th/ng nht nh n u(ng v ti

sn xut. C ch! c2a phn th/ng trong hnh vi gn li"n v-i qu trnh h&c h'i v ghi nh- nh6ng kch thch, khoi cm m ta gp hng ngy.

2.2.2. Cc con ng dn truyn dopamin trong no (dopaminergic pathway)

Cc t! no thn kinh dopaminergic / vng no gi6a 0c phn b( phn l-n / 2 vng

no chnh: vng gn pha sNPC (substantia nigra par compacta) v vng gn trung tm

VTA (ventral tegmental area), chng h-ng !n no tr-c v to ra cc con .ng dn truy"n DA khc nhau [28, 33, 56, 61, 63, 72, 103] :

-32-

- Con "ng mesolimbic (mu xanh) : cc t bo thn kinh dopaminergic t'

vng VTA h!ng s$i tr%c n vng b%ng c&a th vn (striatum), bao gm nhn accumben, to thnh con "ng mesolimbic [14, 65]. N cn c tn gi khc l con "ng phn th#ng (reward), c bit trong vic lm d%ng cc cht gy

nghin [79, 90].

- Con "ng mesocortical (mu vng): cc t bo thn kinh dopaminergic t'

vng VTA h!ng n vng no tr!c s to thnh con "ng mesocortical [14, 65, 93], hay cn gi l con "ng ng l(c cm xc.

- Con "ng nigrostriatal (mu ) hay cn gi l con "ng vn ng: t' vng

sNPC, VTA v no tr!c, cc t bo thn kinh dopaminergic h!ng t!i vng b%ng th vn (striatum), bao gm amygdala v nhn accumben [14, 65].

Hnh 2.1. Cc con ng dn truyn v c ch gii phng DA trong no

-33-

2.2.3. Vai tr ca dopamin

Trong no ng-i, dopamin (DA) l cht dn truy"n thn kinh c vai tr quan tr&ng trong vi$c ghi nh, v h&c h'i [72, 85]. Qu trnh ny c lin quan mt thi!t !n c ch! "phn

th/ng" c2a DA. . con ng-i, s5 xut hi$n c2a cc "phn th/ng" khi s4 d1ng cc cht kch thch nh cocain, morphin, amphetamin, r0u hay thm ch tr chi i$n t4 0c

bo co trong cc xt nghi$m thm tch micro v PET (ch1t ct l,p pht x positron) cho thy DA thay *i r r$t v tc +ng ln cc vng nhn accumben, th# vn, no tr,c nh

m+t tc nhn i"u ch%nh tc +ng c2a "phn th/ng" [16]. DA chnh l tc nhn chnh khuy!n khch con ng-i tm ki!m li cm gic hng phn, sng khoi khi s4 d1ng chng [72].

Nh phn tch / trn, cc t! bo thn kinh dopaminergic c mt ch2 y!u / vng

VTA (ventral tegmental area), vng sNPC (substantia nigra pars compacta) v dn truy"n !n cc vng nh th# vn (striatum), v' no hay nhn accumben. Ti cc vng ny, DA

c vai tr ph3c tp lin quan !n bi#u hi$n hnh vi trn +ng vt c v nh i"u khi#n cm xc, chuy#n *i cm xc, v trong cn kch thch, khoi cm, o gic [16]. V vy m&i hnh vi, cm xc nh sng khoi, vui v , kch thch "u c s5 lin h$ !n m3c + tng

n)ng + DA ti cc vng trn, v d1 nh cc cm gic thch n ngon, hot +ng tnh d1c, ht thu(c l hay s4 d1ng thu(c ma ty "u c kh nng tng gii phng DA.

Bng 2.1. Mc tng DA trong vng no trn chut nht thc nghim khi nghin cu trn cc loi tc ng kch thch

Cc hot ng Tng DA (%) Vng no TLTK

Th3c n ngon 150% Nhn Accumben [12]

Hot +ng tnh d1c 140% Nhn Accumben [39]

Khi thu(c l (nicotin 0,13 g/kg) 160% Nhn Accumben [36]

R0u (ethanol 1,0 g/kg) 180% Nhn Accumben

[32] 130% Caudate

Cocain (1,0 mg/kg) 220% Nhn Accumben [22]

-34-

Ngoi ra, vi.c thi*u h:t DA cn gy ra h'u qu! nghim tr0ng nh ng tr hay r3i

lo n v'n 5ng 8 ng7i gi, ng7i b/ b.nh Parkinson [40, 72].

2.3. M2I LIN H- GI?A DOPAMIN V S= D9NG CC CH"T MA TU

Trong cc ch#t d&n truy+n th$n kinh, dopamin (DA) c lin quan nhi+u nh#t *n c

ch* t o hnh vi c!m xc (hng ph#n, s!ng khoi) khi s> d:ng ch#t ma ty. Hn n@a, DA

cn lin quan *n c! c ch* ph: thu5c vo cc ch#t ma ty hay cn g0i l nghi.n thu3c. Nghi.n l m5t b.nh l th$n kinh khi m vi.c l m d:ng cc ch#t gy nghi.n gy h i cho h. th3ng ph$n th8ng v !nh h8ng *n hnh vi bnh th7ng c;a no [72]. Khi s> d:ng thu3c

gy nghi.n nh cocain hay amphetamin, ng7i s> d:ng s) tr!i nghi.m vi.c tng m nh DA trong no, gy ra c!m gic "ph$n th8ng" m h0 thm mu3n [101]. Vo nh@ng nm 80,

cc nghin c

-35-

Xt v" th0 th#, cc bng ch2ng khoa h'c ch% ra rng receptor D1 khuy!n khch

vi$c c th# tng c.ng s4 d0ng thu)c kch thch, trong khi receptor D2 i"u ho qu trnh c th# b& kch thch do thu)c [58, 61, 89]. Ni cch khc, kch thch th0 th# D1 s lm tng nhu cu s4 d0ng thu)c c1a con ng.i, ng/c li, kch thch th0 th# D2 s lm gim

nhu cu s4 d0ng thu)c. Do , nhi"u " ti nghin c2u cc cht )i khng th0 th# D1 nh m,t li$u php cai nghi$n ra .i.

Tuy cc cht ma tu "u lm tng n*ng , DA, nhng c ch! c1a m+i cht ma tu l khc nhau. Hi#u r c ch! tc d0ng c1a t3ng nhm cht ma tu l m,t y!u t) quan tr'ng

trong vi$c nghin c2u v pht tri#n cc loi thu)c cai nghi$n m-i. D-i y l c ch! tc d0ng c1a m,t s) loi thu)c gy nghi$n /c bi!t !n:

Amphetamin

Khi s4 d0ng amphetamin, n*ng , DA trong no tng ln theo 3 con .ng: 1)

Amphetamin gn vo mng tr-c synap c1a cc t! bo thn kinh dopaminergic v tng

c.ng gii phng DA ti cc tn cng. 2) Amphetamin tng tc v-i cc b'c nh( ch2a DA lm cc b'c ny tng c.ng gii phng DA. 3) Amphetamin gn v-i cc receptor ti

hp thu DA trn mng synap v lm o ng/c c ch! vn hnh c1a cc receptor ny, ngha l khi!n chng nh DA thay v ti hp thu [20] (Hnh 2.3). Do n*ng , DA khi s4 d0ng amphetamin tng ln gp hng ch0c ln so v-i khi s4 d0ng cocain hay cc cht ma

ty nhm opiat.

Hnh 2.3. C ch lm tng gii phng DA trong no

ca amphetamin

-36-

Opiat

Opiat gn v,i cc receptor c

hi%u , , [20], trong receptor -opiat c vai tr quan tr'ng trong c ch" "phn th.ng" v l ch tc d0ng

chnh c1a opiat [20, 100]. Opiat s! kch thch cc t" bo thn kinh

dopaminergic thng qua vi%c 2c ch" t" bo thn kinh trung gian GABA c

ch2a cc th0 th$ -opiat. y s! l tn hi%u kch thch cc t" bo thn kinh dopaminergic gii phng DA.

Hnh 2.4. C ch kch thch gii phng DA ca opiat

Cocain

C ch" gy nghi%n c1a cocain l ngn

ch n ti hp thu DA . cc knh ti hp thu .

tn cng synap [20, 100]. Bnh th-ng, DA /c gii phng ch1 y"u t3 vng VTA. T3 y, cc s/i thn kinh /c a "n cc vng c bi%t

nh nhn accumben. M+t phn DA /c gii phng s! /c ti hp thu qua cc knh ti

hp thu DA . tn cng synap. Khi c m t cocain, cocain s! ch n cc knh ny li, dn

"n DA khng /c ti hp thu. N*ng + DA tng cao trong d&ch ngoi bo, v gn v,i cc th0 th$ DA . b# m t neuron ti"p nhn DA.

Hnh 2.5. C ch tng gii phng DA ca cocain

S4 kch thch qu m2c trong m+t th-i gian di s! lm gim s) th0 th$ DA . khe sau

synap, trong khi cc th0 th$ cn li tr. nn km nhy hn v,i DA. C th$ i h(i ti"p t0c dng thu)c $ p 2ng nhu cu b 1 cc th0 th$ km nhy cm v,i DA, gip ti to

-37-

cm gic sng khoi, hng phn. y chnh l c ch gy nghin c$a cc cht ma ty v

cc cht kch thch thn kinh khc.

Cc xt nghim PET v nh du phng x (radiotracer) cho thy, s l"ng th#

th DA ! cc i t"ng nghin thuc (cocain, AMP, heroin, morphin, r"u) gim ng k ti vng th vn (striatum) v nhn accumben, tnh trng ny c th ko di hng thng

lin sau khi cai nghin [98, 99] (Hnh 2.6).

Hnh 2.6. So snh v mt th th DA ngi bnh

thng v ngi nghin [59, 87, 88, 98]

Hn n&a, ! ng i nghin, kh nng gii phng DA cng gim i rt nhiu so vi

ng i bnh th ng khi s% d#ng cc cht kch thch nh AMP [98] (Bng 2.2).

Bng 2.2. So snh v kh nng gii phng DA 2 nhm i tng: liu n i tng khng nghin, liu ko di i tng nghin

Thi gian s dng Thuc s dng Tng DA (%) TLTK

Liu n Morphin (20 mg/kg) 192% [79]

Amphetamin (2,5 mg/kg) 5500% [87]

Liu ko di (nghin)

Morphin (20 mg/kg) 192% [79]

Amphetamin (2,5 mg/kg) 4000% [87]

-38-

3. PH$N 3: T.NG QUAN PHNG PHP NH GI HI*U QU"

TC D4NG C6A CC CH#T MA TU THNG QUA ,NH

L2NG DOPAMIN TRONG NO 0NG V'T D8A VO K9

THU'T TH&M TCH MICRO

3.1. NGUYN V!T LI(U S: D5NG

3.1.1. 1ng v(t th nghi+m

0ng v"t th nghi)m th2ng s; d6ng nht l chu0t nh#t ho$c chu0t c,ng, ty theo

m/i ch7ng loi chu0t m c th' cho p 8ng hi)u qu c7a cc cht ma ty khc nhau, t8c

l n-ng 0 tng dopamin (DA) khc nhau trn m/i loi chu0t th=c nghi)m.

Bng 3.1. c im ca chut thc nghim dng trong nghin cu thm tch micro

Lo!i chu1t S- l3ng chu1t (n) Tu%n tu/i Cn n)ng (g) TLTK

Chu0t nh#t 10 20 6 8 25 35 [38, 68]

Chu0t c,ng 10 20 6 8 200 350 [10, 17, 55, 90]

i&u ki)n th8c n, n1c u,ng theo tiu chu n trong 1 tun, nhi)t 0 phng 230C, 0

m 60%, vng sng/t,i l 12/12h/ngy [5, 10, 12, 29, 34].

Chu0t lun 4c ki'm tra m bo s8c kh+e trong th2i gian nghin c8u th=c nghi)m

theo cc tiu chu n 4c quy *nh b3i B0 Y t%, $c bi)t l gi< m thn nhi)t trong khong 37oC [104].

3.1.2. Cc thu-c 3c s7 d5ng trong th nghi+m

Cht ma tu s dng

Thng th2ng ' nh gi m8c 0 mnh y%u c7a cc cht ma ty, ng2i ta &u ly

morphin 10 mg/kg lm tiu chu n, y l li&u thp nht cho hi)u qu r r)t trn hnh vi

thay .i c7a chu0t nh#t th=c nghi)m trong khong li&u t9 5 100 mg/kg c kh nng gy ra cc tc d6ng kch thch v"n 0ng trn chu0t nh#t [38], v1i chu0t c,ng, khong li&u ny nh+ hn. Cc cht ma ty cn li hay n-ng 0 khc c7a morphin 4c nh gi so snh

-39-

hi$u qu v*i morphin 10 mg/kg thng qua m.c ) tng n'ng ) DA. Phn loi cc cht

ma tu th+ng ,c chia theo ngu'n g&c, g'm 3 nhm: t/ nhin, bn t(ng h,p v t(ng h,p. Khi nghin c.u, chng s ,c chuy"n thnh dng mu&i sulfat hay mu&i hydroclorid d# tan trong n*c v ho tan trong n*c mu&i sinh l.

Bng 3.2. Mt s cht ma tu c s dng trong cc nghin cu thm tch micro

Loi ma ty Tn Liu thng dng TLTK

T/ nhin Morphin 10 mg/kg [38]

Cocain 5,0 mg/kg [42]

Bn t(ng h,p Heroin 6,25 mg/kg [59]

T(ng h,p Amphetamin 2,5 mg/kg [23]

Cht gy m: cloral hydrat 400 mg/kg ,c ho tan trong n*c mu&i sinh l [4, 5, 12, 29], dng " gy m chu)t trong khi phu thut cy kim thm d vo no chu)t th/c

nghi$m.

Cht gy cht: natri pentobarbital 65 mg/kg [51] dng " lm ch t nhanh )ng vt sau khi k t thc th nghi$m.

Cht bo qun: formalin 10% " bo qun no chu)t.

Dung dch gi no tu (artificial cerebrospinal fluid): c th" t mua c-a cng ty CMA, Thu0 i"n hoc t/ pha ch theo cng th.c trong Bng 3.3.

Bng 3.3. Thnh phn dch gi no tu [104]

Thnh phn Nng

NaCl

Na2HPO4

KCl

CaCl2

MgCl2

pH 7,4

145,0 mM

2,0 mM

2,7 mM

1,2 mM

1,0 mM

i!u ch%nh bng NaOH 1N hoc H3PO4

-40-

D%ch gi no tu2 nn ,c pha ch ngay tr*c khi th nghi#m, phng php nh

sau: ho tan 8,470 g NaCl (145,0 mM); 0,284 g Na2HPO4 (2,0 mM); 0,201 g KCl

(2,7mM); 0,133 g CaCl2.2H20 (1,2 mM); 0,952g MgCl2 (1,0 mM) trong 1 lt n*c ct. pH

,c hi#u ch$nh v 7,4 b+i NaOH 1N hoc acid H3PO4 [104]. Sau d%ch gi no tu2 ,c

l&c qua mng cellulose 0,22 m [68].

3.1.3. Ta cy kim thm d vo cc vng no ly DA

! c th! xc %nh ,c n(ng ) DA + ng vng no nghin c/u, kim thm d cn

,c cy chnh xc vo vng no theo m)t to ) %nh sn. Atlas c.a Paxinos v Watson cung cp v% tr c.a t0ng vng trong no theo 3 to ): tr*c sau (anterior-

posterior/AP), tri phi (lateral/L) v ) su (vertical/V) t,ng trng cho 3 chi u trong khng gian. To ) ny c th! ly t0 cc i!m g'c d" xc %nh, l i!m thp (bregma)

v i!m lambda (Hnh 3.1). T0 m)t trong 2 i!m ny, d1a theo atlas, ta p d-ng to ) thch h,p c.a vng no cn th nghi#m v cy kim thm d thng t0 trn xu'ng (Bng

3.4).

Hnh 3.1. Hp s ca chut nhn t trn xung

Bng 3.4. Ta cy kim thm d ti mt s vng trong no

Vng no Ta cy kim thm d tnh t v tr bregma (mm)

TLTK Tin li (AP) Tri phi (L) su (V)

Nhn Accumben +1,2 1,0 -5,0 [38]

Th! vn (striatum) +1,2 1,5 -3,5 [21, 38]

VTA -6,7 0,6 -8,0 [90]

-41-

3.1.4. Chun b kim thm d

Kim thm d /c coi l tri tim c1a k6 thut thm tch micro, vai tr chnh l # ly cc hot cht thu(c hoc cht n+i sinh ti cc m nghin c2u. # lm th nghi$m thm

tch micro trong no, qua cc th4 nghi$m khc nhau, hu h!t cc nghin c2u "u l5a ch'n kim thm d dng )ng tm v,i mng l'c c gi,i hn phn t4 i qua mng l 20 kDa.

Ty t3ng vng no khc nhau m ng-i ta s ch'n + di c1a kim thm d cho ph

h/p, khong 10 14 mm. Chi"u di c1a mng bn thm l 5 8 mm, cht li$u s/i AN69 (sodium methallyl sulphat copolymer), -ng knh ngoi 310m, -ng knh trong 220m [12]. Hi$n nay hng sn xut kim thm d ph* bi!n nht l CMA.

Kim thm d cn /c th4 nghi$m in vitro tr,c . 37oC # nh gi tnh kh thi

hi$u sut c1a kim khi ly DA v cc dng chuy#n ho c1a n (DOPAC, HVA) [73]. Tr,c khi ti!n hnh th nghi$m, phi ki#m tra kim tht k6 # trnh b& r r% hoc b& b't kh, v phi m bo v trng tuy$t (i [104].

Ng-i ta th-ng s4 d0ng thm m+t kim dn (guide cannula) # bo v$ cho kim thm

d, v kim thm d rt mnh v m"m [48]. Kim thm d sau s /c cy qua kim dn sao cho ch% phn mng hot +ng v/t ra ngoi chi"u di kim dn (xem Hnh 1.6).

Ngoi ra, # ti!t ki$m chi ph, hon ton c th# t5 ch! to kim thm d, nh trong

nghin c2u c1a Zapata v c+ng s5 trnh by [104] hay cc nghin c2u c1a Di Chiara v

c+ng s5 [12, 34].

Hnh 3.2. Kim thm d v kim dn

-42-

3.1.5. Chun b cc thit b m v nh lng

My c nh 3 chiu (stereotaxic)

M%t my c# !nh 3 chiu s s- d*ng cc to

% gip xc !nh vng cn cy kim thm

d. To % s g$m c 3 thng s#: Tr&c sau (anterior-posterior/AP), tri phi (lateral/L)

v % su (vertical/V) t)ng trng cho 3 chiu trong khng gian. Tay gn #c vt trn my mang theo kim thm d s t kim vo

ng v! tr nh to % mong mu#n [107].

My ct lt m (vibratome)

My ct lt m cho php ct

kh#i m thnh t,ng lt m"ng

nh' vo l(i dao rung c bit c+a my. Bin %, t#c %

v gc % c+a my c th

iu ch nh )c.

Hnh 3.3. My c nh 3 chiu dng phu thut

Hnh 3.4. My ct lt m no

H thng sc k

Hnh 3.5. H thng sc k LC-MS vi detector t cc chp ba (tri) v HPLC (phi)

-43-

3.2. CC B5C TI)N HNH

D6i y l t3ng k*t cc b6c trong th= nghi-m nghin c

-44-

i&m ny n m trn cng m/t m"t ph!ng song song v0i bn c+ (nh c5a my [104],

& m bo cc b0c xc (nh to / v% sau 3c chnh xc.

4. Theo v( tr to / nhn accumben 3c xc (nh v m t b2i Paxinos v Watson

(1998), ti$n hnh khoan m/t l. trn mng c6ng (dura) c5a v* no v0i chi%u su nh nu trong Bng 3.4.

5. Ti v( tr ny "t vo m/t kim dn v sau kim dn 3c c+ (nh b ng cch - m/t l0p keo & gn kim v0i mng c6ng c5a u chu/t [25, 90].

6. Sau , cy kim thm d qua kim dn [25]. Kim thm d s# cho php thu h,i DA

ti vng no nghin c6u, 2 y l nhn accumben. Nguyn tc c5a qu trnh thu h,i 3c gii thch 2 phn 1.

7. Cu+i ca phu thut, chu/t 3c a vo phng hu phu, chm sc cho $n khi

t'nh dy. Chu/t s# 3c & qua m cho m)i hot /ng 3c tr2 li bnh th1ng

(Hnh 3.7).

Hnh 3.7. Chut c chm sc sau phu thut

3.2.2. Tin hnh ly mu thu c c cha DA

1. 24h sau phu thut, kim thm d 3c k$t n+i v0i m/t bm truy%n v truy%n

d(ch no t5y nhn to (aCSF) c thnh phn n,ng / trong Bng 3.3 v0i t+c / -n (nh 1,0 l/pht [4, 5, 23, 29, 34, 79, 107] ho"c 1,5 l/pht [21, 36, 46, 73] s7 d4ng bm

truy%n chm (CMA100, Thu9 i&n) [4, 5, 107].

2. Sau 1 - 2h -n (nh (& cn b ng n,ng / th8c ti synap), bt u ly mu d(ch

thu 3c u tin v sau 20 pht (tng 6ng v0i t+c / truy%n l 1,0 l/pht)

-45-

ho!c 15 pht (tng 4ng v.i t*c - truy$n l 1,5 l/pht) ly cc mu ti#p theo

cho #n khi thu 1c 4 mu. N+ng - c3a cc mu ny s" to /ng n$n trn + th'.

3. Sau , tim dung d'ch placebo (n.c mu*i sinh l) vo m-t nhm chu-t (nhm

mu ch4ng), nhm cn li tim dung d'ch thu*c (cht ma tu cn nghin c4u) v

bt u ly mu ngay, nh du m*c th/i gian l 0. Cc mu 1c ly cch nhau 15 ho!c 20 pht trong khong 120 pht. M,i mu c th% tch khong 20 l [4].

4. D'ch thu 1c c ch4a DA, cn 1c bo qun lnh trong t3 -40oC % trnh

DA b' phn hu6 [107].

3.2.3. Ct b phn no xc nh chnh xc v tr cy kim thm d

1. Sau khi th nghi&m k#t thc, chu-t 1c gy ch#t b ng cch tim mng b2ng

dung d'ch natri pentobarbital 65 mg/kg [51].

2. Sau , kim thm d v no chu-t 1c ly

tch ra. No 1c bo qun trong dung d'ch formaldehyd 4% [12].

3. Phu thut no xc 'nh v' tr kim thm d : % xc 'nh tnh chnh xc c3a k#t qu thu

1c, phi bi#t r kim thm d 1c cy vo ng v' tr hay cha. No chu-t 1c !t

vo my vibratome % ct thnh cc lt s( nh) 60 m [24] theo atlas c3a Paxinos v

Watson (1998) [12, 23, 29]. Sau , cc lt ct vng no c v#t kim thm d 1c c* 'nh b0i gelatin v nhu-m tm, !t ln phi#n knh

v soi d.i knh hi%n vi i&n t5 % xc 'nh vng cy kim thm d [24, 52, 73]. % php o

t - tin cy cao, yu cu 75% kim thm d Hnh 3.8. Biu vt kim thm

d nhn accumben [29]

-46-

-c cy trong vng nhn accumben [52]. Ch% mu c/a nh3ng kim thm d no

n m ng vng nhn accumben m+i -c s2 d.ng cho php &nh l-ng [23, 24, 38, 90].

3.2.4. nh lng bng HPLC hoc LC-MS/MS v tnh ton kt qu thng k.

HPLC l phng php &nh l-ng -c s2 d.ng ph) bi!n trong k5 thut thm tch

micro. V+i s4 ti!n b* c/a khoa h'c, k5 thut LC-MS ang dn chi!m u th! v -c s2 d.ng r*ng ri trong 10 nm gn y. Sc k pha o -c l4a ch'n cho nghin c0u cc

cht dn truy"n thn kinh, -c ch0ng minh b,i Molnar v c*ng s4 [62], trnh by trong phn 1 c/a kho lun. Sau y l m hnh cho thm tch micro offline:

Hnh 3.9. M hnh thm tch micro offline trong th nghim [104]

# tri#n khai sc k pha o, cn th4c hi$n cc b+c sau :

(1) Xc &nh m.c tiu c/a phn tch

(2) Ch'n k5 thut x2 l mu # tch cc cht phn tch

(3) Ch'n detector thch h-p

(4) Ch'n i"u ki$n sc k

(1) Xc nh mc tiu ca phn tch

M.c tiu c/a phn tch l &nh l-ng n(ng * DA trong mu thu -c t1 qu trnh thm tch micro.

-47-

(2) Chn k thut x l mu

Mu thu /c t3 k7 thut thm tch micro khng cn qua qu trnh x4 l mu v n /c thm tch tr5c ti!p t3 c th# m+t cch +c lp, khng ln protein v enzym [25]. Mu cn /c bo qun lnh # trnh DA b% phn hu6 b.i nhi$t [107].

(3) Chn detector thch hp

# %nh l/ng v,i s c k l'ng trong k thut thm tch micro, detector /c s4

d0ng nhi"u nht l detector i$n ha v detector kh(i ph*, trong detector kh(i ph* th# hi$n tnh u vi$t hn c v" tnh ch&n l&c, + nhy, + chnh xc v hi$n

ang l detector /c s4 d0ng nhi"u nht ti cc phng nghin c2u (xem phn 1 c1a kho lun).

Detector in ho hay /c s4 d0ng nht l ESA Coulochem II, v,i cc thng s( i$n c5c: +130 mV cho i$n c5c oxy ho v -125 mV cho i$n c5c

kh4 v,i + nhy cho php pht hi$n !n n)ng + DA t(i thi#u 5fmol/20L [23].

Detector khi ph c th# ch&n my API 3000 s4 d0ng k7 thut ion ho ESI # ion ho m"m cc phn t4 cn phn tch v ch&n detector t2 ph* ghp

(triple quadrupole - QQQ) ph h/p v,i m0c tiu %nh l/ng c1a th nghi$m.

Bng 3.5. Ci t detector khi ph p dng cho nh lng DA [49]

ESI

Ch! + ion ho dng (positive mode)

i$n th!: 5 kV

p sut kh tr (N2) va chm: 7 psi

Nhi$t + kh4 solvat: 500oC

Tc dng 1 ml/pht (b' 1ml u t3 c+t s c k)

QQQ

Ch! + lm vi$c: a nhi$m (MRM mode)

Th-i gian lu (dwell time): 40 msec

i$n th! ph kh(i (declustering potential): 20 V i$n th! trung tm (focussing potential): 80 V

-48-

i&n th# va chm u vo (collision entrance): -10 V

i&n th# va chm u ra (collision exit): 15 V

m/z 154 (ion g*c) -> 91 (ion con)

(4) Chn iu kin sc k

Ch)n c,t sc k: loi c,t ph+ bi#n nht l c,t pha o (LC-18 DB, ht 5 m,

Supelco, 4.6 x 150 mm) [12], mu /c ly v-i t*c , 1 l/pht trong 20 pht ho!c t*c , 1,5 l/pht trong 15 pht.

Ch)n pha ,ng: pha ,ng l2a ch)n l dung d(ch &m phosphat.

Pha ,ng (c thnh phn nh Bng 3.6) c t*c , dng chy 1.0 ml/pht [38] .

p sut 3000 psi km theo bm y m,t piston (SSI Inc., model 220B) [79].

Cn lu r ng c,t sc k ch' c th% ch(u /c dung d(ch &m phosphat v-i pH t1 1,9 - 7,0. pH trn 8 s" lm gim tu+i th) c0a c,t [62].

Bng 3.6. Thnh phn pha ng [23]

Thnh phn Nng

Na2HPO4

NaH2PO4

Na2EDTA

Octyl sodium sulphat

Methanol

pH 5,5 (i$u ch'nh b ng acid acetic)

50 mM

5,0 mM

0,1 mM

0,5 mM

15% (v/v)

Cch pha ch# pha ,ng nh sau: ho tan 7,0 g Na2HPO4 (50 mM); 0,6 g NaH2PO4 (5,0 mM); 0,03 g Na2EDTA (0,1 mM); 0,1 g Octyl sodium sulphat

(0,5 mM); 150 ml methanol trong 1 lt n-c ct; pH 5,5 i$u ch'nh b ng acid acetic. Sau pha ,ng /c l)c qua mng cellulose 0,2 m tr-c khi ti#n

hnh chy sc k.

Ch)n nhi&t ,: 30o [38, 43].

-49-

(5) Thng k

N(ng + n#n trung bnh c1a DA /c xc &nh b.i 4 mu thu /c tr,c khi tim thu'c hay gi d/c placebo (4 gi tr& sai khc nhau khng qu 10%) v t gi tr&

trung bnh ny l 100%. Tt c cc gi tr& thu /c sau khi tim thu'c s! /c th$ hi%n d,i dng % so v,i gi tr& n#n trung bnh SEM (sai s' chun) (%) [34, 38].

$ phn tch th'ng k trn s4 bi"n )i n(ng + DA, chng ta tnh ton qua phn tch ANOVA 1 bi"n (one-way) ho c 2 bi"n (two-way) trn s' li%u n(ng + DA thu

/c sau m*i ln o thng qua cc gi tr& di%n tch d,i -ng cong n(ng + DA theo th-i gian (AUC) /c tnh trong khong th-i gian 120 pht sau khi tim d,i

mng b0ng bng placebo ho c morphin. Ti"p theo, test ngu nhin Turkey /c s3 d0ng $ xc &nh cc sai khc t)ng th$ r r%t c1a cc php o, v,i gi tr& p < 0,05. Hi%u qu c1a cc cht ma ty /c th$ hi%n d4a trn % thay )i DA so v,i n(ng +

n#n [34].

3.2.5. Bo co kt qu thu c

V,i cc b,c nh trn, ti"n hnh tim morphin v,i hm l/ng 10mg/kg, tim mng b0ng cho nhm 1 (n = 9) v tim gi d/c (NaCl 0,9%) cho nhm 2 ch2ng (n = 8).

&nh l/ng n(ng + DA trong vng nhn accumben . no chu+t s3 d0ng morphin

thu /c k"t qu nh sau:

1/. C s4 khc bi%t c ngha th'ng k v# n(ng + DA trong vi%c s3 d0ng morphin

(F(1,15) = 8,54; p < 0,001) [6].

2/. N,c mu'i sinh l (NaCl 0,9%) dng lm gi d/c v dung mi $ pha morphin,

khng lm thay )i n(ng + DA trong vng nhn accumben.

-50-

Hnh 3.10. Hiu qu c a morphin trn nng DA ti vng nhn accumben chut nht th!c nghim [6]

A: ng cong ng hc. B: Gi tr din tch di ng cong (AUC) (***: p < 0,001)

3.3. T4NG H:P K*T QU# TA CC NGHIN C?U CNG B2 V, NH

GI HI/U QU# TC D

-51-

Bng 3.7. Tng hp cc kt qu v % tng DA trong no chut cng v chut nht ti mt

s vng trong no (nhn accumben, th vn, caudate) khi s dng morphin, amphetamin v cocain

Tn thuc Loi chut Liu Vng no Tng DA (%) TLTK

Morphin

Chut cng

1,0 mg/kg Nhn accumben 148% [12]

Th vn 137% [78]

2,5 mg/kg Nhn accumben 182% [77]

Th vn 150% [77]

3,0 mg/kg Nhn accumben 150% [17]

5,0 mg/kg Nhn accumben 210% [29]

Chut nht

5,0 mg/kg Nhn accumben 140% [9]

Caudate putamen 115% [9]

10 mg/kg Nhn accumben 295% [6]

Caudate putamen 125% [9]

18 mg/kg Th vn 142% [82]

20 mg/kg Th vn 190% [38]

Amphetamin Chut cng

0,5 mg/kg Nhn accumben 642% [92]

Th vn 608% [92]

1,5 mg/kg Nhn accumben 650% [50]

Th vn 1291% [83]

2,0 mg/kg Nhn accumben 1236% [92]

Th vn 1629% [92]

5,0 mg/kg Nhn accumben 3568% [92]

Th vn 2291% [92]

-52-

Bng 3.7. Tng hp cc kt qu v % tng DA trong no chut cng v chut nht ti mt s vng trong no (nhn accumben, th vn, caudate) khi s dng morphin, amphetamin

v cocain (Tip)

Tn thuc Loi chut Liu Vng no Tng DA (%) TLTK

Cocain

Chut cng

1,0 mg/kg Nhn accumben 220% [22]

Dorsal caudate 120% [22]

2,5 mg/kg Nhn accumben 250% [29]

5,0 mg/kg Nhn accumben 320% [22]

Dorsal caudate 220% [22]

7,5 mg/kg Nhn accumben 370% [37]

20 mg/kg Nhn accumben 420% [55]

Chut nht 20 mg/kg Th vn 280% [82]

40 mg/kg Th vn 380% [82]

-53-

KT LUN V XUT

1. K+T LU%N

1) KB thu&t th#m tch micro m: ra cho chng ta m7t h8ng i m8i trong vi0c xc

2nh n4ng 7 c=a cc ch!t n7i sinh hay ngoi sinh ti m ch trn c th. s3ng. V8i nguyn l n gi n v kh nng thu ;c ch!t phn tch : dng tA do, khng l$n protein v enzym, kB thu&t th#m tch micro c b8c ti,n v;t tr7i v8i >ng dc tp hn, )c bi0t v8i sA xu!t hi0n c=a cc ch!t ma ty d8i dng t5ng h;p m8i, c

c!u trc m8i kh c th. c ch!t chu#n . xc 2nh th9i gian lu. M7t v!n - khc ;c )t ra, l khi thu ;c m7t m$u b7t tr'ng, mu3n bi,t chng c ph i ma tu hay khng,

c"n ti,n hnh r!t nhi-u ph n >ng th nghi0m . xc 2nh. Th#m tch micro thng qua n4ng 7 DA cho php nh gi tc d

-54-

ny theo nhi)u h1ng * nh gi hi+u qu c6a thu.c trn lm sng, &c bi+t 3 y l vi+c

ti'n hnh cc nghin c7u v) nh gi hi+u qu tc d5ng cc ch!t ma tu, t8 pht hi+n 4c cc loi ma tu m1i cng nh tm ra c ch' cai nghi+n hi+u qu cho cc .i t4ng

nghi+n ma tu.

4) D9a vo chu#n so snh l morphin 10 mg/kg, ta c th* nh gi tc d5ng kch

thch tng ti't DA c6a b!t k ch!t ma tu no, k* c cc ch!t cha r c!u trc ho h-c, loi ma tu t/ng h4p m1i, cho php xy d9ng ln m0t bng tham chi'u chu#n c tnh

php l, ph5c v5 cho cng tc i)u tra v phng ch.ng t0i phm ma tu.

2. ( XU T

Kho lu$n xy d9ng t/ng quan phng php nh gi tc d5ng c6a cc ch!t ma

tu d9a vo k: thu$t th#m tch micro. Sau y, chng ti xin 4c ) xu!t ti'p t5c m0t s. h1ng pht tri*n cho k: thu$t ny:

1) Ti'n hnh cc th nghi+m trn th9c t' to c s3 xy d9ng m0t bng tham chi'u chu#n, c tnh php l, p d5ng cho vi+c nh gi tc d5ng kch thch c6a cc

ch!t ma tu ti Vi+t Nam.

2) Tri*n khai cc nghin c7u nh%m tm hi*u thm c ch' tc d5ng c6a cc ch!t

ma tu, cng nh tm ra cc gii php cho ra 2i cc ch!t cai nghi+n m1i c hi+u qu cao hn.

3) Nghin c7u cc 7ng d5ng khc c6a k: thu$t th#m tch micro v1i cc nhm

thu.c tc d5ng trn th"n kinh trung ng nh thu.c ch.ng tr"m cm, Alzheimer, Parkinson, v &c bi+t l trong nh gi sinh kh d5ng v tng ng sinh h-c c6a thu.c.

4) Chu#n b, c s3 v$t ch!t, thi't b, v nhn l9c 4c trang b, cc ki'n th7c c"n

thi't * tri*n khai k: thu$t th#m tch micro trong nghin c7u pht tri*n d4c ph#m 3 Vi+t Nam.

-55-

TI LIU THAM KHO

Ti liu bng ting Vit: 1. B' Lao 'ng - Thng binh v X h'i, (2011), Bo co v cng tc cai

nghin ma tu ti Vit Nam thi gian qua,

2. B' mn Ho phn tch, (2007), Ho phn tch II, tr(ng i h$c D)c H N'i, Nh xut bn Y h$c;

3. Nguy!n Sinh Thi, Nguy!n Thnh Hi, Thi Nguy!n Hng Thu, (2012),

"Thm tch micro v ,ng d*ng trong nghin c,u thu%c ch%ng ung th ti m ch", Tp ch dc hc, 7(435), 7-12.

4. Nguy!n Thnh Hi, Alain Gardier, Thi Nguy!n Hng Thu, (2012),

"Nghin cu ch tc d*ng c+a escitalopram trong i u tr# trm cm d-a vo k. thut

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