药物的抗惊厥作用.doc

Anticonvulsant Effects of Drugs
XXX
（China Pharmaceutical University，639 Longmian Avenue, Nanjing, Jiangsu, China 210009）

ABSTRACT: OBJECTIVE: To study the anticonvulsant efficacy of Phenytoin sodium and Phenobarbital and valproate on convulsion. METHODS: The anticonvulsant activity of the drugs was evaluated by the maximal electroshock (MES) test and remeflin seizure test. Induced rats were treated by intraperitoneal injection ,then observe the incubation period (up to tails upright 45°) and degree of reaction of each mouse(spasm, falling down ,stiff or death). RESULTS: 1. In MES experiment, the degree of convulsion: The Phenobarbital group (P<0.05 vs normal saline group) was the lightest, and the NS group was the severest one. 2. In remeflin experiment, the similar result was observed between valproate group and NS group (P<0.01). CONCLUSION: Phenobarbital has better anti-convulsion effect than Phenytoin sodium on convulsion induced by electroshock, and valproate has a significant effect to anti-convulsion induced by remeflin.

KEYWORDS Phenytoin sodium; Phenobarbital; valproate; remeflin ; anti-convulsion

Introduction

Phenytoin sodium: its mechanism of action: It can block sodium channels (voltage-, frequency-, and time dependent fashion) and inhibit the generation of action potentials and It can increase the function of inhibitory transmitter GABA, inhibit nerve terminal to uptake GABA and induce the increasing of GABA receptor, thereby enhance GABA-mediated postsynaptic inhibition.
Phenobarbital: its mechanism of action: Phenobarbital can inhibit the paradoxical discharge of epilepsy focus selectively, enhance stimulation of surrounding tissues and block discharge diffuse to normal tissues and Phenobarbital facilitate GABA-mediated inhibition of neuronal activity.
Valproate is broad spectrum antiepileptic drug, which is used to all types of epilepsy; its mechanism is that enhancing the enzymatic activity of glutamate decarboxylase. And Inhibiting GABA reuptake and synapse inactivation via releasing synapse frontal membrane GABA and then enhance GABA postsynaptic inhibition
Remeflin, a central stimulate which can directly excite respiratory center, can produce convulsion with over-dose. The protective effects of drugs on convulsion produced by remeflin can be used to screen antiepileptic and anti-convulsion.
Stimulating mouse head with a strong electric current can cause general tetanic convulsion. If a drug can prevent the occurrence of general titanic convulsion, we could initially suspect that it has antagonistic effect on grand mal. Remeflin, a central stimulant which can directly excite respiratory center, can protective effects of drugs on convulsion produced by remeflin can be used to screen antiepileptics and anticonvulsants. [1]

1 Material
Equipments
Balance, syringes, pharmacological and physiological multipurpose apparatus
Reagents
Phenytoin sodium, 0.5% solution w/v, Phenobarbital sodium, 0.5% solution w/v, 0.04% Remeflin, 2% sodium valporate, normal saline
Animals
5mice, 18-22g, no restriction on sex
2 Methods
2.2 Methods of MES experiment
Adjust the stimulator firstly. Stimulating parameters: working condition---electric shock; output voltage--maximum; model of stimulating--single; frequency--32~16Hz; connect the outlet with clips and plug the connection for power. Select convulsion mice. Attach the clips moistened with saline to the mouse ears. Punch the key to starting, and a current is applied to the head of the mouse, then cause tonic convulsion that is the state of the flexing forelimb and straightening hind limb. If the animals don’t fall into convulsion with 32Hz, modulate to 16Hz, and start again. Replace another one to test if have no reaction, yet.
Select three mice in which convulsions occur typically. Mark and weigh them. Treat them by intraperitoneal injection of 5% phenytoin sodium 75mg/kg (0.15ml/10g), 5% phenobarbital sodium 75mg/kg (0.15 ml/10g) and normal saline 0.15 ml/10g separately. 40 minutes after injection, stimulate the mice again with pro-threshold. Record the response.

2.2 Methods of remeflin experiment

Mark and weigh two mice. Treat them separately by intraperitoneal injection of 2% sodium valproate 600mg/kg (0.3ml/10g) and normal saline 0.3ml/10g. 30 minutes after administration, inject them with remeflin 8mg/kg (0.2ml/10g) subcutaneously. Observe the speed and degree of reaction of each mouse (spasm, falling down, stiff or death).

2.3 Statistic method and intensity measures

Statistic methods: Experimental data in each groups were expressed by x±s in the experiment used remeflin, analysis of variance and t-test (One-way Analysis of Variance, ANOVA) in the intergroup analysis , analysis of the intensity degree (Rank-Sum-Test ) results and analysis of rate by χ2 —test (Fisher’s exact test) were applied statistical software SPSS 19.0, significant level α=0.05.
Outcome measures on behaviors in remeflin induced convulsion model: degree 0: no phenomenon; degree 1: tails upright; degree 2: tumble; degree 3: stiff; degree 4: death.

3 Results

3.1 Results of MES experiment

Phenobarbital was obvious to have the protective effects on MES convulsion (P<0.05 vs Normal saline), and it is better than phenytoin sodium which has a weak anticonvulsant effiancy that the convulsion rate was between normal saline group and Phenobarbital group.
Table 1 result of protective effects on MES convulsion of phenytoin sodium and phenobarbital.
Drugs
Dosage（mg/kg）
Convulsion/Sum
Convulsion Rate(%)
Normal saline
——
6/8
75
Phenobarbital
75
1/8
12.5*
Phenytoin sodium
75
4/8
50
*P<0.05 vs Normal saline

3.2 Result of remeflin experiment

There was highly significant difference (P<0.01) between the sodium valproate group and normal saline group in death rate, and significant difference (P<0.05) between the two groups in convulsion incubation and reaction intensity. This means that sodium valproate has the anticonvulsant efficacy to play a protective effects on the convulsant induced by remeflin.

After injection of remeflin, mice may experienced incubation period, stiff flexion phase, straighting hind limb phase, clonic phase and restoration period. The mice in normal saline group behavior as follows: walked haltingly, jumped, uplifted fore limbs and stood on two feet, ears towards the back, and tails upwards like letter C. The mice in sodium valproate group behavior as: Walked slowly and the tail was almost straight up. The whole body shaked, and after several minutes its hind limbs were stiff, and died.
Table 2 the results of effects of sodium valproate on convulsion produced by remeflin (x±s, n=8)
Group
Incubation (s)
Death Time (s)
Death Number
Death Rate (%)

Normal Saline

4.18±1.18

No death

0

0
Sodium Valproate
8.24±2.54*
7.68±0.80 #
6
75 **
*P<0.01 vs Normal Saline group; ** P<0.01 vs Normal Saline group # except 2 mice that didn’t die

Table3 the reaction intensity degree on mice convusion produced by remeflin
Group
Reaction Intensity(Degree)
Num
1
2
3
4
5
6
7
8

Normal Saline

4

3

4

4

4

1

4

4

Sodium Valproate*

1

1

2

1

1

1

1

1
*P<0.01 VS Normal Saline Group
4 Discussions

Because of individual difference of animals, stimulating parameters should be set with experiment. Don’t set it over-strong, in order to prevent animals from death. Prevent short circuit of clips carefully, otherwise the stimulator would be damaged. The convulsion of animals can be divided in five phase, latent period, stiff flexion phase, straightening hind limb phase, clonic phase and restoration period. If condition permits, pentetrazole is recommended to be substituted for remeflin because the convulsion caused by pententrazole is more typical.

5 Conclusions
Both Phenobarbital and phenytoin sodium have the protective effects on electric shock convulsion. And Phenobarbital whose effect is obvious has better anti-convulsion effect than phenytoin sodium whose effect is weak respectively on convulsion induced by electroshock. And valproate has a significant effect to anti-convulsion induced by remeflin, while remeflin can kill the most of the mice in experiment and valproate can reduce the toxicity of remeflin.

Reference
[1] Peng Xiaodong, et al Anticonvulsant efficacy of Chinese Materin Medicine Cynanchum Chinese R.Br in mice [J] J Fourth MilMed Univ 2009,30 (4):340-344