| Objective:Amyotrophic lateral sclerosis (ALS) is a degenerative disease of nervous system that injurys upper and lower motor neurons, reflecting progressive muscle weaknessand atrophy. The mechanism of motor neuron degeneration in ALS remains unkonwn, oxidative, mitochondria disfunction, excitotoxicity, neuroinflammatory, calcium overload, abnormal protein aggregation may be involved in. The poor prognosis of ALS, patients with the disease, died in3to5years after the onset of it.At present, Riluzole is the first successful drug to extend the lifespan of patients, and to delay the tracheotomy. Riluzole can not reverse the disease course, and it so expensive that many patients can not adhere to run out a full course of treatmentButylphthalide used in basic research and clinical treatment of acute cerebral infarction, and achieved good results. It improved the micro-circulation of ischemic area and protected the structure and function of mitochondrial. It also can block the ischemic brain damage in multiple pathological aspects, so as to achieve the neuroprotective role.To observe the effects of NBP and Riluzole on hSOD1G93A transgenic mice, and to compare the combinative effects of the two drugs with the single one.Methods:1Animal and treatmentThe96hSOD1G93A mice were randomly divided into four groups, NBP group, Riluzole group, and the Combination group. Every group include12males and12femals. NBP group was administered by oral gavage at a dose of180mg/kg/day, Riluzole group was administered by water that the drug was in accordance with100ug/ml dissolved in it, the Combination group was administered both of the two durgs, while the control group received corn oil.2Behavioral observation2.1Rotarod testEach group of mice were90days and120days, adaptive training of five days before the rotarod test. Mice on a diameter of the rollers of30mm, set parameters rollers start rotating,180s within rotational speed is gradually increased from lrpm30rpm, each mouse was measured three times, the recording the mice on rollers under on continuous movement does not fall time (in seconds count), take the longest time record.2.2Step lengthThe mice were placed in long50cm,5cm wide wooden grooves inside the groove bottom surface covered with the length and width of the equivalent paper tape, licking and biting in mice with a different color ink painting, so that it ran on the tape when moving left around the palm blot of two different colors. The mice were then placed in one end of the recess, repelling mice forward its movement to the other end, and then find the continuous running time of mice to stay in the groove bottom on the tape3footprints (fledgling footprints elected to the measurement), Measuring the distance between the three footprints, then record the mean of the left and right.2.3Grip strength test Hold the back of the mouse, so each side of hindlimb caught the grasping force flatbed, repeated measurements three times on each side, to take the highest numerical records, and then take the the bilateral average values as statistics data entry.2.4The time of onsetThe mice were placed on rollers, each mouse was measured three times, If the mouse falling down of the rollers all the time, recorded on that day for the onset, the time of onset in mice from birth to the onset of the number of days.2.5The lifespanPlaced the mice on not smooth plate for the lateral position, if the mouse is not flip within20seconds, the mouse is judged to death, the day recorded as the time of death, the survival time of the mice from birth to death the number of days.3HistopathologyThe mice were anesthetized with10%chloral hydrate, cardiac perfusion with4%paraformaldehyde20min, take the lumbar spinal cord of mice with4%paraformaldehyde48h,5μm thick paraffin sections with HE staining, then observed the survival neurons in the spinal cord, to count the neurons at the edge of the ventral anterior horn of the range in the central canal of the spinal cord, in line with diameter>25microns, clear nucleoli and polygons.4Statistical analysisThe data representation are the Mean±SEM, all experiment data were treated with SPSS13.0statistical software, and repeated measures ANOVA test was used for assessment of rotation test comparisons. Biochemical and pathological results were analyzed using ANOVA or Nonparametric test. P <0.05prompted a statistically significant difference.Results:1SOD1gene identificationThe DNA extracted from the tail of the progeny of transgenic mice, after PCR amplification, after agarose gel electrophoresis:the visible brightness different strip positive SOD1mice The PCR product (236bp), shown in Figure of the strip (Fig.1) can see the bright bands, this entry with corresponding mice shall SOD1G93A transgenic positive mice.2The time of onset and survival of the mice in each groupThe onset time of the combination group, NBP, and the riluzole group mice:127.625±2.741days,118.261±2.420days,123.593±1.898days, compared with the control group106.875±2.298days, P=0.000,0.003,0.000<0.05, were significant difference. Combination groups compared with NBP group,P=0.0110.05, has statistically significant difference; compared with riluzole group, P=0.116>0.05, there is no statistically significant; the NBP group compared with riluzole group,P=0.273>0.05, there is no statistically significant.Lifespan:The survival time of the combined group mice was146.375±2.796days, the NBP group was138.870±2.235days, and the Riluzole group was141.542±2.105days, the control group was124.500±2.354days. The three treatment groups has showed an visiable advantage compared with the control group of the Lifespan. The Combination group had no statistically difference compared with the Riluzole group, but it showed significant difference compared with the NBP group, while there were no significant difference between the Riluzole group and the NBP group.3Behavioral observation3.1Rotarod testAt90day, There were no statistically significant difference between the four groups;At120day, The three treatment groups showed visiable significant difference compared with the vehicle control group; the Combination group had statistically difference compared with the NBP group, but it had nostatistically difference with the Riluzole group, while there was significant differences between the Riluzole and the NBP group.3.2Step lengthAt90day, There were statistically significant difference between the Combination group and the control group, the NBP group and the control group, Riluzole group had no difference compared with the control group. Between the three treatment group, the Combination group had statistically difference compared with Riluzole group, but it had no difference compared with NBP group, as the Riluzole group also showed no significant difference compared with the NBP group.At120day, The three treatment groups showed visiable significant difference compared with the control group. The Combination group had statistically difference compared with the the Riluzole group, as it also showed significant difference compared with the NBP group, but there were no significant difference between the Riluzole group and the NBP group. 3.3Grip strength testAt90day, There were no statistically significant difference between the four groups.At120day, The three treatment groups showed significant significant difference compared with the vehicle control group. The combined group had statistically difference compared with the Riluzole group, as it also showed significant difference compared with the NBP group, but there were no significant difference between the Riluzole group and the NBP group.4HistopathologyHE staining showed that, At120day, the combination group(16.53±2.264)retained more neurons than the Riluzole group(12.87±2.031) or the NBP group (11.40±2.197),P=0.000<0.05, there was no significant difference between the Riluzole group and the NBP group,P=0.068. The three treatment groups showed significant significant difference compared with the vehicle control group(3.67±1.397),P=0.000<0.05.Conclusions:The results show that the butylphthalide in prolong survival and improve the motor function hSOD1G93A transgenic mice having a substantially identical with riluzole.The Butylphthalide in combination with Riluzole is not superior to prolong survival, but have some advantages in improving athletic ability. |
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