| To search a way of making rats PD model which have low mortality and high chronic-PD-symptom simulation, behavioristics ? immunohistochemistry as auxiliary studies, we researched on pertinence between rotenone transdermal treatment and neurotransmitter, and compared the pharmacodynamic effects with intraperitoneal injection of rotenone. Furthermore, we research on the neuroprotection of Morinda officinalis.Using behavioral science, immunohistochemistry and biochemistry to study how damage the rats had. Behavior used weight test, posture stability study, forelimb power research, movement initiation and rearing. Immunohistochemical used brain striatum TH and alpha synaptic protein immunohistochemical, and semi-quantitative research on the optical density of TH slices. Used brain-microdialysis to collect neurotransmitters in t brain, and content determination was studied by HPLC- ECD. The water extract of Morinda officinalis rotenone were treated at the same time to study brain neurotransmitter by microdialysis.1. Behavioral scienceCompared with the rats of control group(CL), the rats of rotenone 2.5 mg/d group(rotenone low-dose group, RL), rotenone 5.0 mg/d group(rotenone middle-dose group, RM), rotenone 10.0 mg/d(rotenone high-dose group, RH), which were transdermal treated for 30 days,(RL: 357.13±21.33 g, RM: 358.73±12.20 g, RH: 339.77±27.92 g)didn't have the marked weight-change(P>0.05), but were weightier than rotenone(3.0 mg/d/kg) intraperitoneal injection group(IP)(367.7±20.56 g, P<0.01). The postural stability of RL, RM, RH(8.73±0.05 cm, 8.47±0.12 cm, 7.13±0.05 cm)were lower than CL(6.6±0.08 cm, P<0.01), and only RH were lower than IP,( 9.15±0.05 cm, P<0.01). Compared with CL(41.00±0.82°), the forelimbs strength of three rotenone groups( RL: 34.67±1.25 °, RM: 31.00±2.16 ° RH: 31.33±1.25 °) all weakened, P<0.01. And only RH were clearly lower than IP( 37.50±0.50°, P<0.01). Compared with CL movement initiation(6.60±0.57 s)), the movement initiation of RL: 10.83±2.27 s, RM: 12.90±2.22 s, RH: 14.40±0.82 s inceased obviously(P<0.01), but without IP(9.90±0.01 s, P>0.05). Rearing test show that RL: 19.67±3.77 t, RM: 21.67±4.03 t, RH: 21.67±3.40 t compared with CL(24.3±2.87 t) and IP(15.0±2.00 t) all had no difference(P>0.05). After 30 days, according to the results, transdermal treatment group, with the increase of rotenone dose, the postural stability, forelimbs strength was being weak, the time of movement initiation was increasing.2. ImmunohistochemistryThe TH immunohistochemical optical density(IOD) RL: 0.133±0.001, RM: 0.151±0.005, RH: 0.126±0.005, were decreasing distinctlier than CL(0.185±0.004), P<0.01, and RM, RH were lower than IP(P<0.01). Compare with 0 d group(0.184±0.004), 30 d goup(0.120±0.005) and 20 d group(0.147±0.003) TH expression quantity decreased obviously(P < 0.01), and 10 d group(0.172±0.005) just P < 0.05. Alpha-synaptic protein polymers were found in he rats' substantia nigra compacta of all treatment groups.3. BiochemistryUnder 10 mg rotenone transdermal treatment, we observed the change of rat neurotransmitter within 24 hours. At 240 min, neurotransmitter had some distinct change,(DOPAC+HVA)/DA decreased from 3.78±2.35 to 1.84±1.56, DOPAC decreased from 0.88±0.09 ?g/m L from 0.33±0.13 ?g/m L, DA increased from 0.40±0.11 ?g/m L to 0.46±0.24 ?g/m L,HVA decreased from 0.65±0.21 ?g/m L to 0.52±0.13 ?g/m L, and kept stabilization after then.To three rotenone groups, dopamine extracellular concentrations RL: 0.57±0.02 ?g/m L, RM: 0.49±0.06 ?g/m L, RH: 0.72±0.01?g/m L, had no significant difference with CL(0.49 ± 0.14 ?g/m L) and IP(0.47 ± 0.07 ?g/m L). DOPAC extracellular concentrations RL: 0.77±0.11 ?g/m L, RM: 0.68±0.10 ?g/m L, RH: 1.58±0.15 ?g/m L, only RH obviously higher than CL(0.58±0.11, P<0.05) and IP(0.33±0.39,P<0.01). HVA extracellular concentrations RL: 1.50±0.14 ?g/m L, RM: 0.98±0.17 ?g/m L, RH: 1.39±0.15 ?g/m L, which compared with CL(0.45±0.05 ?g/m L) and IP(0.30±0.07 ?g/m L),both RL and RH had obvious difference(P<0.01), but RM had lower difference(P<0.05). The dopamine metabolism coefficient(DOPAC + HVA)/DA of RH(4.33±0.43), compared with CL(2.28±0.74) and IP(1.43±0.16), had significant differences(P < 0.05). On time research, 10 d group(1.04±0.02 ?g/m L) had a significantly higher concentration of extracellular DA than 0 d group(1.43±0.16 ?g/m L), while no obvious difference was found between 0 d group and the 20 d(0.33±0.02 ?g/m L), 30 d(0.72±0.01?g/m L) groups.In the result of DOPAC and HVA, only 30 d group(DOPAC: 1.56±0.17 ?g/m L,HVA:1.39±0.16 ?g/m L)compared with 0 d group(0.58±0.11 ?g/m L,0.45±0.05 ?g/m L) which had obvious difference(P < 0.01). 10 d groups(DOPAC + HVA)/DA, 0.45±0.12, significantly lower than 0 d group(5.63±0.14, P<0.01), and 20 d(7.08±1.02), 30 d(7.50±0.25) group is higher than 0 d group(P < 0.05).4. The results of Morinda officinalis' neuroprotection researchDA of 10.0 g/d group, 0.94±0.02 ?g/m L,was obviously higher than that of CL(0.49±0.14 ?g/m L, P < 0.01), while the difference was not obvious between CL and others. All of Morinda officinalis test group had higher DA extracellular concentrations than 0.0 g/d group(P<0.01). In the result of DOPAC and HVA, 2.0 g/d group(DOPAC: 0.13±0.08 ?g/m L,HVA:0.23±0.01 ?g/m L) and 10.0 g/d group(DOPAC: 0.23±0.04 ?g/m L,HVA:0.28±0.016 ?g/m L) were obviously different from 0.0 g/d group(DOPAC: 1.58±0.15 ?g/m L,HVA:1.39±0.15 ?g/m L), P<0.01. And 6.0 g/d group(DOPAC: 0.40±0.01 ?g/m L, HVA : 0.38±0.04 ?g/m L) had weaker difference with CL,but stronger with 0.0 g/d group(P<0.01).10.0 g/d group and 2.0 g/d group(DOPAC + HVA)/DA, 0.55±0.14?1.06±0.17, were significantly lower than 0.0 g/d group(2.68±0.67) and CL(1.17±0.74), but 6.0 g/d group(2.11±0.36) had weaker difference with 0.0 g/d group(P < 0.05), and stronger with CL(P<0.01).This study shows that transdermal treating with rotenone 10 mg/d for 30 d can cause obvious damage to rats' neurotransmitter.This treatment had the more stable damage effect and lower mortality rate than intraperitoneal injection treatment. In addition, the rats which treated with 10.0 g/d Morindae officinalis had more DA. |
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