| Part I Honokiol improve the Neurological disfunction of rats after traumatic brain injuryPurpose:To investigate the appropriate dose of Honokiol treatment after traumatic brain injury, and to observe whether Honokiol could ameliorate the neurological dysfunction after traumatic brain injury, providing evidence for its potential clinical applications.Methods:1.50 S-D rats were randomly divided into 5 groups (10 in each):sham operation group (sham group), traumatic brain injury group (TBI group), Honokiol 0.5mg group, Honokiol 1.Omg group, Honokiol 2.0mg group. The rats in sham group only got sham-operation, other groups were given standard CCI moderate craniocerebral injury using TBI-0310 brain injury device; on the first day after the trauma, the sham group and TBI group were given the same volume of Vehicle (solvent for Honokiol) on the 8th and 16th hour after injury, then once daily on 8th hour in the morning for successive 5 days. All the Honokiol groups were administered appopriate dose of Honokiol via caudal vein according to the time pattern mentioned before. The mNSS was made on the 1th,3th,7th,14th,21th days to evaluate the neurological deficits in rats after TBI.2.30 S-D rats were randomly divided into 5 groups (n= 6), grouping and treatment as mentioned above. For each dose Honokiol-treated group, Honokiol was administered only twice on the 8th and 16th hour on the first day after injury, brain tissue in each group were extracted 24 hours later after injury. Brain water content was measured by dry-wet weight method.3.30 S-D rats were randomly divided into 3 groups (n=10 in each group):sham group, TBI group, Honokiol 1.Omg group (the appropriate dose of Honokiol was determined according to the dose-effect relationship discussed above), molding and dosing regimen as method one. Beam WalK Test was made on the 1th,3th, 7th,14th,21th days and the Morris Water Maze (MWM) test was made on the 15th,16th,17th,18th,19th days, respectively.Results:1. The result of mNSS score indicated that, compared with the Sham group, TBI caused obvious neurological dysfunction, on the first day after Honokiol treatment, each dose group can not reduce the severity of neurological function impairment; compare with TBI+Honokiol group, Honokiol 0.5mg group can reduce the deficits of neurological function on the 3th,7th,14th days. Neurological dysfunction can be reduced on the 3th,7th,14th,21th days in Honokiol 1.Omg group after TBI. While neuralogical function deficits can be only reduced on the 3th,7th,21th days in Honokiol 2.0mg group after injury; comparison among each dose group, Honokiol 1.Omg group in more efficient in protecting the neuralogical function than Honokiol 0.5mg group or Honokiol 2.0mg, and the difference was statistically significant.2. Compared with Sham group, TBI increased the brain water content after TBI, and Honokiol treatment could reduce the brain water content in each dose group; however, comparison among each dose group, Honokiol 1.Omg group in more efficient in reducing the brain water content than Honokiol 0.5mg group or Honokiol 2.0mg, and the difference was statistically significant.3. Results of Beam WalK test showed that, compared with Sham group, TBI caused obviously fine motor coordination, and Honokiol treatment could sifnificantly reduce the fine motor dysfunction; Compared with TBI+Vehicle group, Honokiol can improve the performance in the Beam Walk Test on the 3th,7th,14th,21th days.Results of Morris Water Maze showed that, compared with the Sham group, TBI caused remarkable spatial learning capacity impairment in the Spatial Learning test, and the Honokiol treatment could obviously improve the learning dysfunction. Compared with TBI+Vehicle group, Honokiol could reduce the spatial learning impairment of rats induced by TBI on the 16th,17th,18th days after injury; Meanwhile, compared with TBI+Vehicle group, TBI caused severe impairment of reference memory in the Probe Trial test. While Honokiol treatment could significantly improve the memory function.Conclusions:Honokiol can reduce the composite neurological deficits and brain edema after TBI in rat.1.0 mg/kg Honokiol is the optimal dose in this study. Meanwhile, Honokiol can improve the fine motor coordination function and the learning as well as memory function caused by TBI in rats.Part II Effect of Honokiol on pathological changes and apoptosis of neurons induced by traumatic brain injuryPurpose:To evaluate the effects of Honokiol on pathological changes induced by traumatic brain injury, and to observe whether Honokiol can. protect neurons form degenerating and apoptosis after brain injuryMethods:1.30 S-D rats were randomly divided into 3 groups (10 in each):sham operation group, traumatic brain injury group (TBI group), Honokiol 1.Omg group. The rats in sham group only got sham-operation, other groups were given standard CCI injury using TBI-0310 brain injury device. On the first day after the trauma, the sham group and TBI group were given the same volume of Vehicle (solvent for Honokiol) on the 8th and 16th hour after injury, then once daily on 8th hour in the morning for successive 5 days. The Honokiol group was administered 1.Omg/kg Honokiol according to the time pattern mentioned before. H-E staining was made on the 21th day after TBI/Sham operation to evaluate the brain tissue loss2.30 S-D rats were randomly divided into 3 groups (n=10 in each group):sham group, TBI group, Honokiol 1.Omg group, the molding and dosing regimen as method one. Nissl staining was made on the 21th day after TBI/Sham operation to evaluate the survival neurons in the cortex and hippocampal CA1 subregion.③ 24 S-D rats were randomly divided into 3 groups (n=8 in each): sham group, TBI group, Honokiol 1.Omg group, the molding and dosing regimen as mentioned above. But Honokiol was administered only twice on the 8th and 16th hour on the first day after injury, FJ-B staining was made 24 hours later after TBI/Sham operation to evaluate the degenerating neurons in the cortex and hippocampal CA1 subregion.4.24 S-D rats were randomly divided into 3 groups (n=8 in each):sham group, TBI group, Honokiol 1.Omg group, the molding and dosing regimen as mentioned before. But Honokiol was only administered for three days. NeuN+Tunel Double-labelling immunofluorescence technique was adopted to evaluate the neuronal apoptosis in the cortex and hippocampus on the 3th day after TBI/Sham operationResults:1.Compared to TBI+Vehicle group (36.79±1.13mm3), Honokiol treatment (21.37±1.01mm3) could significantly reduce the brain tissue loss caused by TBI (P< 0.05); Linear regression analysis indicated that the reduction of brain tissue volume was highly correlated with motor function improvement in the Beam Walk test (r2=0.93;P<0.0001).2. TBI caused severe neuron loss in the peri-contussional cortex (62.84±4.22 vs 206.39±1.91 in sham) and hippocampal CA1 subregion (76.19±4.21 vs 185.97±1.88 in sham). Compared with TBI+Vehicle group, Honokiol treatment could significantly reduce the neuron loss in the peri-contussional cortex (145.14±2.59) and CA1 of the hippocampus (136.76±2.53) in rats after TBI. Linear regression analysis indicated that the increasement of surviving neurons was highly correlated with the improvement of learning capacity in the Morris Water Maze test (r2= 0.94; P<0.0001).3. TBI caused severe neuronal degeneration in the peri-contussional cortex (110.70±4.72) and hippocampal CA1 subregion (134.95±8.63). Compared with TBI+Vehicle group, Honokiol treatment could significantly reduce the neuronal degeneration in the peri-contussional cortex (33.28±1.88) and CA1 of the hippocampus (59.74±6.50) in rats after TBI (P< 0.05 vs TBI+Vehicle).4. TBI caused obvious neuronal apoptosis in the peri-contussional cortex (37.13±1.94%) and hippocampal CA1 subregion (60.01±3.18%). Compared with TBI+Vehicle group, Honokiol treatment could significantly reduce the neuronal degeneration in the peri-contussional cortex (12.11±1.16%) and CA1 of the hippocampus (19.12±1.67%) in rats after TBI (P< 0.05 vs TBI+Vehicle).Conclusions:Honokiol can reduce the contussion volume and increase the surviving neurons in both peri-contussional cortex and hippocampal CA1 subregion after TBI in rat. Meanwhile, Honokiol can reduce the neuronal degeneration in both peri-contussional cortex and CA1 of the hippocampus, as well as the neuronal apoptosis in both peri-contussional cortex and hippocampal CA1 subregion. Furthermore, this protective effect is beneficial for motor and cognition improvement impaired by TBI.Part Ⅲ The effects of Honokiol on cell cycle activation of neurons after traumatic brain injuryPurpose:To observe the changes of cell cycle in brain tissue and neurons as well as the effects of Honokiol treatment on cell cycle activation, so as to illuminate the possible mechanism of brain injury and the protective effects of Honokiol on neurons.Methods:1. For each cell cycle protein to be studied,12 S-D rats were randomly divided into 2 groups (n=6 in each group):sham group and TBI group. The rats in sham groups only got sham-operation, while TBI groups were given standard CCI injury as mentioned before. Western-Blots were made on the 1th,3th,7th,14th,21th days to evaluate the changes of cell cycle proteins after TBI/Sham-operation.2. For each cell cycle protein to be studied,18 S-D rats were randomly divided into 3 groups (n=6 in each):sham group, TBI+Vehicle group, Honokiol 1.0mg group. The molding method as mentioned above. While the dosing regimen was choosen according to the peak time point of each cell cycle protein studied in experiment one to decide the total time of Honokiol administeration. On the first day after the trauma, the sham group and TBI group were given the same volume of Vehicle (solvent for Honokiol) on the 8th and 16th hour after injury, then once daily on 8th hour in the morning for successive 5 days. Western-Blots were made on the appropriate time point to evaluate the effects of Honokiol on the cell cycle protein changes after TBI/Sham-operation.③ 24 S-D rats were randomly divided into 3 groups (n=8 in each group): grouping and modeling as before. As to Honokiol group, drug was administered only for 3 days after injury. Brain tissue samples were obtained 3 days after TBI/Sham operation to observe the effect of Honokiol on the activation of cell cycle protein Cyclin D1 and E2F1 in neurons and the cell cycle protein-related neuronal apoptosis using Double-labelling immunofluorescence technique.Results:1. The expression of cell cycle proteins Cyclin D1, CDK4, pRb(Ser780) and E2F1 increased and the expression of p27 decreased after TBI. The expression of Cyclin D1 and E2F1 peaked on the 3 th day after brain injury and the expression of pRb (Ser780) reached the peak time on the first day. While the CDK4 expression successively increased untill the ultimate time on the 21th day. On the other hand, the expression of p27 down-regulated to the lowest valley-time on the 3th day after brain injury and didn’t recovered untill the 21th day2. Honokiol treatment can significantly reduce the expression level of cell cycle-related proteins Cyclin D1, CDK4, pRb (Ser780) and E2F1 after TBI. Meanwhile, Honokiol can increase the espression of endogenous inhibitory cell cycle protein p27.3 The expression of cell cycle proteins Cyclin D1 and E2F1 was significantly up-regulated in cortical neurons after TBI. Meanwhile, the most majority neurons that express Cyclin D1 or E2F1 were apoptotic neurons. But all these abnormal changes were obviously inhibited by Honokiol treatment after brain injury.Conclusions:Cell cycle progression was abnormally activated after TBI. Meanwhile, the aberrant changes of cell cycle proteins in neurons are closely correlated with neuronal apoptosis. But administration of Honokiol can remarkablly inhibit the unusual changes of cell cycle proteins as well as the related neuronal apoptosis. |
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