• Nghien CLFU - Ky thuat
Nghien ciru mo hinh danh gia hieu qua tac dung ciia cac chat ma tiiy thong qua ndng do
dopamin dya vao ky thuat tham tach micro
Pham Thu Ha', Nguyen Thanh Hai' Bruno Guiard^, Thai Nguyen Hiing Thu*
' Truang Dai hoc Duac Hd Ndi. Viet Nam
^ Truang Dai hgc Paris Sud IJ. Cdng hod Pkdp
Summary
Narcotic drugs always raise serious social problems; especially, nowadays many new synthetic narcotics are created and illegally circulated, of which the chemical structures and compositions are difficult to determine by structural analysis. Nevertheless, spectrometry scanning or high performance liquid chromatography, for example, demand refined machines, but leave true effects of the drugs unclear, especially for new synthetic drugs. Microdialysis has come to current use to offer an easier solution to these in great part. This paper presents an overview on the microdialysis. as a new method of current use. to evaluate the effects of various drug types, such as those cause hallucination, exilement and stimulation. Microdialysis enables the determination of drug effects through an intermediate value, i.e. the dopamine concentration in the brain, neglecting their chemical structures.
The experimental results of microdialysis are of significant use not only in detecting the drugs but also in comparing effect powers of different drugs.
Keywords: fi/ticrodialysis, determine, effects, dopamine concentration.
Dat van de
Nghien ma tuy tip x u a den nay ludn la van dd nhipc nhoi cua cdng ddng. Vdn de trdm frgng hon khi hang logt cac logi ma tuy tong hgp m d i xuat hien, gay khd khan cho cac c a quan chdc nang trong viec khdng dinh chiing la mgt chiit ma tuy. Viec tien hanh xac djnh cau true hoa hgc cua cac thanh phan trong mot mdu bgt trdng thu dipgc se mdt rat nhidu thdi gian, d i n cd may mdc hien dai. Trong khi do viec khang djnh hieu qua tac dung cua chung Igi vdn chua ro rang, dac bigt la cac chat ma tiiy fdng hgp logi mdi, cd cau tnic phdc tgp, chua cd chdt chuan de so sanh'^'.
Trong ndo n g u d i , dopamin (DA) la chat ddn truyan than kinh d u g c giai phong ti> cuoi cac fd bao than kinh dopaminergic de truyen tin hieu thdng tin tdi cac neuron thdn kinh tiep theo va cuoi cung la cac viing cd bieu hien hanh vi (hu'ng phan, ao giac, sang khoai...). Cac te bao th^n kinh dopaminergic cd mat chu yeu d vung VTA (ventral tegmental area), vung sNPC
(substantia nigra pars compacta) va dan tmy^n ddn cac vung nhu striatum (th^ vdn), vd nao hay nhan accumben. Tai cac vung ndy, dopamin c6 vai trd lien quan ddn bieu hign hanh vi tren dpng vat CO vu n h u dieu khien cam xiic, khodi cam hay ao giac. Vi vay mgi hanh vi, cam xuc nhu sang khoai, vui ve, kich thfeh deu cd s g lien h^
den m u c do tang ndng dd dopamin tai cac viing tren, v i du n h u cac cam giac thich an ngon, hogt dgng tinh due, hut thuoc la hay s i i dgng thu6c ma tuy ddu cd kha nang tang gidi phong dopamin'^'^'^.
Tham tach micro Id mgt ky thudt cho phep xac dinh nong do cdc phdn ti> ndi sinh (nhu serotonin, dopamin..) hay phdn t d ngogi sinh (nhu thudc) tai md dich tac dgng, dga tren nguyen tac cac phan lit d u g c k h u l c h tdn theo gradient ndng dg tren mang bdn tham ciia kim tham do d u g c cay vdo to chdc d i W " ' .
T i f cac nghien c d u ve ky thuat thdm tdch micro, chiing toi xay d y n g m^t phuong phdp mdi cd the dp dung 6k danh gid hi^u qua tdc
TAP CHi Dirpc HQC - 09/2013 (SO 449 NAM 53)
• Nghien CCPU - Ky thuat
dgng cua cac chat ma tuy gay ra cac trgng thdi do gidc, kich thich h u n g phan, sang khodi. Ky thudt ndy cho phep cd t h i xac djnh d u g c hidu qud tdc dgng cue chdt ma tuy thdng qua mi>c do tang nong dg dopamin tai cac vung nao tren chupt t h u c nghiem, ma khdng can den p h u a n g phap phdn tich xac djnh cdu true hoa hgc hay so sdnh t h d i gian l u u tren sdc ky do. H o n nua ky thugt nay con cd t h i so sanh higu qud va c u d n g dg tac dgng giua cac chat ma tuy vdi nhau, t u dd tgo CO s d cho vide xdy d u n g cac bang tham chieu mang tinh phdp ly, hd t r g cho hoat dgng phdng chong Id ngn ma tuy.
N g u y e n v a t l i e u v a phu'O'ng p h a p n g h i e n CLPU
D g n g vdt thi nghiem
Dgng vgt thi nghiem la chugt nhSt, khoang 8 tuan t u l i , can ngng t u 20 - 25 gram. Dieu kign thdc dn, n u d c u I n g ( b l sung libitum) theo c h u i n , nhidt do phdng 23°C, do I m 60%, vdng sang/t6i Id 12/12h/ngay.
Thuoc SU' d g n g t r o n g nghien c u u Thdng thudng de danh gia muc dg mgnh yeu cua cac c h i t ma tuy, morphin 10 mg/kg (tiem dudi da) d i u d u g c lay lam tieu chuan.
Cac c h i t ma tuy cdn lgi d u g c danh gia so sanh higu qud vdi morphin thdng qua muc do tang nong dd dopamin. Khi nghien cdu, chung se d u g c hda tan vdo dung mdi thfeh hgp tuy thugc vdo dg tan cua tung thuoc.
Bdng 1 : C^c chSt ma tuy va lieu nghien cuv Lo^i ma tiiy
Tg" nhifin Ting hp'p
Ten Morphin
Cocain Amphetamin
Lieu thtf&ng dung 10 mg/kg 5,0 mg/kg 2,5 mg/kg K i m t h a m d d
Kim tham dd d u g c coi la trai tim cua ky thugt t h i m tdch micro, vai tro chinh la de thu hoi ndng dd dopamin trong nao chugt thuc nghidm v l n d trgng thdi hogt ddng binh thudng. O l lam thi nghidm t h i m tdch micro thu hoi dopamin trong nao chudt thge nghigm, qua h l u h i t cdc nghien c d u d i u Iga chgn kim thdm dd dgng ddng tam vdi d i u kim Id mdng bdn tham cho phdn ti>
trung binh 20000 Dalton dl qua, c h i l u ddi cdy vdo vung nhdn accumben Id 5 mm, c h i t lidu sgi
AN69 (sodium methallyl sulphat copolymer);
d u d n g kinh ngoai 310 [im, dudng kfnh trong 220 ^im (hinh 1)
V
r.M,
Hinh 1 : Cau tao kim tham do
Ket qua
0 a n h gia hieu qua tac dung cua cac chat ma tuy dua vao ky thual tham lach micro trong nao chugt thge nghigm dugc t i l n hdnh qua 5 budc nhu sau:
B u ^ c 1 : Phau thudt cay k i m t h a m do vao nao chugt
Chudt nhat d u g c gay me bdng dung djch cloral hydrat vdi lieu 400 mg/kg, tiem mang bung Sau dd 10 phut, chugt d u g c dua vao phdng md va giO co djnh tren hg thong may mo, phdi dam bao vd trung cho tat cd cdc dung cu mo. Rgeh Idp da a dinh dau va rua sach bdng n u d c oxy gia d l co t h i nhin thdy cac vj tri xac djnh tren mang cung bao gom d i l m thdp (hay cdn ggi la bregma) va d i l m lambda (hinh 2).
Hinh 2: Hpp sp cua chuot nhin tie tren xuong Tiep theo, di c h u y i n may phau thugt theo vj tri tga dg cac vung nao (nhan accumben, t h i van, VTA) (bdng 2), r l i khoan mdt Id trdn mang cung cua v d ndo theo vj trl toa d$ da xdc djnh.
Tgi vj tri nay, c i y kim thdm dd x u I n g dg sau tuy vdo tdng vdng nao cdn nghien cuu d l cd t h i thu h l i DA. Nguyen tdc chlnh cua vigc thu hdi
rAP CHi Di/gfc H Q C - 09/2013 ( s 6 449 N^^IM 53)
• Nghien CIFU - Ky thuat
DA la d y a vao s u khuech tan thu ddng cua cac phan t u cd trgng lugng thap qua mgf mang ban tham tai phan dau cua kim tham dd d u g c cay trong vung nao. Moi kim tham dd cd mgt d i u ddy de t r u y i n djch vao va dau con lai de thu djch di ra cd chua dopamin. D l cd dinh kim tham dd ta can do mgt Idp keo gdn kim vdi mang cung cua dau chugt. Sau do d u a chugt vao phong hdu phau, cham soc cho d i n khi linh dgy. Chugt se d u g c d l qua dem cho mgi hoat dgng d u g c trd Igi binh t h u d n g .
Bang 2: Tpa dp ciy kim tham do theo atlas cua Paxinos va Watson (1998)
Toa do ciy kim tham do tinh tir vj tri bregma (mm) VCing nao
Tien lui , - . . . . , , , Bo s§u Trai phai (L) . (AP)
Nhan Accumben T h i Vein (striatum)
VTA (ventral tegmental area)
+1.2 +1.2 -6,7
±1,0
±1,5
±0,6 -5,0 -3,5 -8,0 Bud'c 2: T i l n h a n h lay m a u t h u d u g ' c c o c h u a n o n g d o d o p a m i n
Khoang 24 h sau p h l u thugt, kim tham dd d u g c k i t noi vdi mgt bom truyen va t r u y i n djch gia nao tuy (artificial CSF) vdi too do I n djnh 1,0 p l / p h u t ' . Djch gia nao tuy cd thanh phan NaCI 147 mM, KCI 4 mM, CaCb 1,5 mM d i l u chinh pH vd 6-6,5 (bang KCI)^.
Sau 1-2 h on ^ n h ( d l cdn bang nong do thge tgi synap), b i t dau lay mau dich thu d u g c d i u tidn va 20 phut lay cac mau tiep theo cho den khi thu d u g c 4 mau ( d l tinh nong dg DA trung binh c a ban hoac cac chat chuyen hda DOPAC, HAV) cho mdi chudt thuc nghiem. Hieu qua tac dung cua cac chat ma tuy nghien c u u d u g c tiem theo d u d n g tiem d u d i da va d u g c so sanh vdi chdl gia d u g c placebo (trong thi nghigm nay NaCI 0,9% t h u d n g d u g c s u dgng), cijng chinh la dung mdi pha cac chat ma tuy.
Djch thu d u g c chda DA cd t h i tfch 20 pi d u g c chuyen sang b u d c djnh l u g n g .
B u v c 3: Cat b o p h a n n a o de xac d j n h vj t r i cay k i m t h a m d o
Sau thf nghigm, chugt d u g c gay chet bdng cdch tiem mang bgng dung djch natri pentobarbital vdi l i l u 65 mg/kg hogc tidm mdng bgng 1 ml dung djch gay me cloral hydrat. Kim thdm dd va nao
chugt d u g c lay tdch ra. Nao d u g c bao quan trong dung dich fomialdehyd 4 % '^'.
Oe xac dinh tinh chinh xac cua k i t qud thu d u g c , ta can b i l t rd kim tham dd da dugc cay vao dung vj tri hay c h u a . Cac lat cat vung nao cd vet kim tham dd d u g c nhudm mau, dgt l§n p h i l n kmh va soi d u d i kfnh h i l n vi di$n t u d l xac dinh vung cay kim tham d d . D l phep do dgt do tin cgy cao, yeu cau 75% kim tham dd dugc cay trong vung nao c a y ' ^ .
B u d v 4 : D j n h l u ^ n g DA t r o n g mSu thu 6MOC va xir ly t h o n g ke ket q u a
Sac ky long hieu nang cao (HPLC) S u dung HPLC cd detector dien hda, cOt pha dao (LC-18 DB, 15 cm, 5 pm, Supeico). D l tang do nhay va thdi gian l u u , cd t h i s u dgng cOt microbore hoac cgt mao quan. Pha dgng aS thanh phdn Na2HP04 50 m M , NaH2P04 5 mM, Na2EDTA 0,1 mM, natri octyl sulphat 0,5 mM, methanol 15%, pH 5,5 ( d u g c d i l u chlnh bang acid acetic) ^^' vdl toe dg dong la 1 ml/phut.
Xf> ly thong ke
Nong do nen trung binh cCia dopamin dipgc xac dinh bdi 4 m l u thu d u g c trudc khi tiSm thuoc hay gia d u g c . T i t cd cac gia tri thu dug'c sau khi liem thuoc se d u g c trinh bay dudi dgng gia trj n i n trung binh vd sai so trung binh (t SEMf'^.
De phan tich thong ke tren s u b i l n doi ning dg dopamin, can phan tich ANOVA 1 bien (one- way) hoac 2 bien (two-way) vdi s6 lidu nong dO dopamin thu d u g c sau moi l l n do thong qua c^c gid tri dien tfch d u d i d u d n g cong nong dij dopamin theo thdi gian (AUG), d u g c tinh trong khoang thdi gian 120 phdt sau khi tiem dudi da bdng placebo hoac c h i t ma tuy. Sau dd, si>
dgng lest n g l u nhien Turkey d l xac djnh si/ sai khac long t h i rd rdt cua cac phep do, sai khSc cd y nghTa thong ke khi p<0,05. Hieu qud cua cac chat ma tuy d u g c I h l hign d u a tren % thay doi dopamin so vdi nong dO nln^^.
Bud'c 5: Bdo cao ket q u d t h u du'Q'c Hieu qua cua morphin tren ndng d^
dopamin (DA)
Vdi cac b u d c lien hanh n h u tren, chiing tfli da t i l n hanh t h u nghigm trdn 2 nhdm chuOt nhdi. Nhdm 1 (n=9) chugt nhdt d u g c tiem di/*"
da morphin vdi l i l u 10 mg/kg va nhdm 2 (n=81 tiem gia d u g c (NaCI 0,9%). Djnh lugng s g thay
TAP CHI Dirqc HQC - 09/2013 (S6 449 NAM 53)
' Nghien ciru - Ky thuat
ddi nong dd DA trong vOng nhdn accumben thu d u g c k i t qua n h u sau '"^:
1. C6 s g khdc bi^t cd y nghTa thong ke ve nong 30 DA trong vi$c s d dgng morphin {F^^,^5] = 8,54; p< 0,001), lang trung binh 295% so vdi nong d$ c a ban.
2. N u d c muoi sinh ly khdng lam anh h u d n g d i n n i n g dO DA.
It:
Si
Pi
D I
B
Hinh 3: Tdc dung cua morphin tren nong do DA trong vdng nhiin accumben & chugt thirc nghidm: A: Dt/ang cong dong hgc. B: Gia tri di$n tich dir&i duang cong (AUC) (***: p <0,001)
So sinh hi^u qua cua cocain, amphetamin va morphin tren nong tfp DA
T u a n g l g thge hign cac budc lien hanh nhu d tren, v d i nhom 3 (n=8), chugt nhdt d u g c tiem dirdi da cocain vdi l i l u 5 mg/kg vd nhdm 4 (n=7) chu^l nhat tidm dudi da amphetamin vdi l i l u 2,5 mg/kg. Djnh lugng s g thay doi nong do DA trong vung nhan accumben cua nao chugt nhal thu d u g c k i t qua nhu sau:
Bdng 3: Hi$u qud ciJa c^c chiit ma tdy thong qua ndng d0 DA
Chit ma tuy Placebo Morphin 10 mg/kg
Cocain 5 mg/kg Amphetamin 2,5 mg/kg
% ting trung binh nong dp DA trong vOng nhan
accumben 101%
295%
380%
763%
Ket lu$n
N h u vdy vdi vi$c s u dgng ky thudt I h i m tach micro d l ddnh gid hi$u qud cua cac c h i t ma tuy s§ m d ra mgt hudng di mdi trong vi^c xdc djnh cdc c h i t ma luy. Trdn co s d ndy, chung ldi xay d g n g d u g c mgt phuong phdp mdi c6 the ung
dgng xac djnh d u g c mgl c h i t bgt t r i n g cd phai la ma tuy hay khong dua vao ItFgng DA ma nd kich thi'ch giai phong tai vung nhan accumben tren nao chugt nhdt thuc nghiem. Thuan lai cua phuang phdp nay la khdng cdn phai lien hanh cac phep t h u phuc lap, chay sac ky Idng tim Ihdi gian luu hay quel pho d l xac ^ n h c l u Irtic hoa hgc cua chat bgt t r i n g . Han nua, phuang phap nay cdn cho phep lgp d u g c mgl bang tham c h i l u chuan lgi don vj danh gia, lam ca s d cho viec kel iuan hieu qua va cudng do mgnh y i u cua cac chal ma tuy can t h u .
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Bbbehav. Rev.. 27(7), 653-9, PubMed PMID:
14624809
4. Di Chiara G., Imperato A. (1988). "Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats", Proc. Natl. Acad. Sci USA.
85(14), 5274-8, PubMed PMID: 2899326 5. Di Chiara G., Tanda G., Frau R , Carboni E.
(1993), "On the preferential release of dopamine in the nucleus accumbens by amphetamine, further evidence obtained by vertically implanted concentric dialysis probes", Psychopharmacology (Berl), 112(2- 3), 398-402, PubMed PMID: 7871048
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