Protective Effects Of PPARγ Agonist Pioglitazone On TBI-induced Deficits Of Learning Memory Ability And Damages Of Hippocampus Neurons In Rats

Traumatic brain injury is a common disease in the department of neurology and it has a high incidence rate. Two different pathologic phasses are include in the brain injury:the primary injury and the secondary injury.The mechanisms of the secondary injury in the injury of the central nervous system includs the edema,ischemia and hypoxia of the locial tissue,the peoduction of the free radial and peroxidation lipid, the effect of excitatory amino acids,inflammatory factor, nitrogen monoxidum and the effect of apoptosis. inflammatory reaction plays an important role in it which displays as the activation of microglia and astrocyte. The activation of microglia can produce some cell toxicants such as free radicals, superoxide anion,No, tumor necrosis factor, interleukinl PGE which can induce neuron degeneration.The activation of astrocyte can translate imto iNOS Organelles which produces NO and aggratates secondary brain injury.researchs confirms that the mechanism takes effect in TBI-induced inflammatory reaction in hippocampus.Neurons in CA1 responsible for discrimination study and neurons in CA3 for long term memory.Neuron loss in hippocampal CA1,CA3 areas and dentate gyrus and synaptic transmission change are the reasons which cause the deficit of learning and memory ability. Peroxisome-proliferator-activated receptor-y is a ligand-activated transcription factor of nuclear hormone receptor superfamily.PPARy plays an important role in cell multiplication differentiation and inflammatory reaction. Now the function of PPARy agonist Pioglitazone in degenerative diseas or nerve injury such as ebrain ischemia, spinal cord injuries draws lots attention. PPARy can inhibit cytokines, adhesion molecules and metalloproteinases so as to inhibit inflammatory cells getting into the central nervous system from the periphery, and it can inhibit inflammation and oxidative stress in the central nervous system injury accordding to inhibiting NF-kB, JAK-STAT signal pathway.PPARy agonists inhibited the cytokine signaling inhibitor SOCS1,3 expression in primary cultured astrocytes and microglia, the latter inhibits JAK-STAT signal pathway through reducing the phosphorylation of JAK.Through the covalent modification of an inhibitor protein namely IκB kinase it can inhibit the expression of lower NF-κB and NF-κB signal pathway. In addition, pioglitazone inhibits the glutamate and low potassium induced neurotoxicity. PPARy may be the therapeutic target of acute and chronic central nervous system injury. In our work we suppose that PPARy agonist Pioglitazone can reatrain TBI-induced activation of microglia and astrocyte and decrease the neuron loss thereby to improve the cognitive behavior which will help to interpret the mechanisms and will conduce to find the effective drugs and methods.In our work we used Morris Water Maze to observe the changes of their learning memory behavior. On 15th day we secrificed the rats to make brain coronal frozen sections then use NeuN, OX-42, GFAP immunohistochemisty to stain neurons,microglias, astrocytes in hippocampus CA1 and CA3 areas and counted the number.We also made the modle of cortex and hippocampus neurons which were injuried by adding LPS.The results turned out that according to PPARy pathway pioglitazone can alleviate the TBI-induced inflammatory reaction in cortex and hippocampus and plays an important role in neuron protection and learning memory ability.The results are as follows:1. The effects of PPARy agonist Pioglitazone on TBI-induced deficits of learning memory abilityTo prove that pioglitazone can improve the deficits of learning memory ability we used the Morris water maze experiment to test the learning memory ability according to the studying ability of the sense of space and direction. We divided them into four groups namely Sham, Vehicle, Pio, Pio+Ant.With the MWM training,the latency and swimming distances of every group shortened which indicated the rats had learning and memory ability. Statistical analysis showed that Vehicle rats MWM latency and swimming distance were40.07±10.31s,851.58±209.95 cm the difference was significant compared with Sham rats latency (19.59±6.42s) and swimming distence (477.27±190.12cm)(P<0.01, P<0.01); Pio rats latency and swimming distance were 21.82±6.90s and 629.30±203.37cm,this was significantly shorter compared with Vehicle group (P<0.05, P<0.05);With the application of antagonist T0070907, the latency and swimming distance were 39.81±10.64s,888.51±279.29cm, which was higher than Pio rats (P<0.05, P<0.05). The latency and swimming distance of Pio rats with the sham rats, as well as the Vehicle group and Pio+Ant comparison between the two was no significant difference. Swimming speed of rats in each shows no differences which indicated that differences of latency and swimming distance were not caused by swimming ability.The results suggest that parietal cortex, the medial part of impact injury can cause a decline in cognitive function, pioglitazone can be mediated through the PPARy receptor, improve learning and memory ability in TBI rats.2.The effects of Pioglitazone on hippocampus microglia after TBIWe used OX-42 immunohistochemistry and cell counting method to study the effects of pioglitazone on TBI induced ac tivation of microglia inflammatory reponse on the experimental model of TBI in rats. The results showed that in the sham group hippocampal microglia were branch-like, small-sized, thin and short processes, and OX-42 immunohistochemical staining was light; when the Vehicle group the microglia cells were stained deep, cell body increased like amoeba with prominent long and thick synapic,which rendered the characterization of activated microglia; Pio added group microglia activation significantly decreased, the effect was T0070907 flipped.Cell counting results showed in hippocampal CA1 and CA3 areas microglia number was327±7.0 in Vehicle group, comparied with Sham group 206.6±8.9, the difference was very significant (P<0.01); Number of Pio group was 218±9.02 compaired with the Vehicle group and Pio+Ant(324.2±4.91)group the difference was significant (P<0.01). The results suggested that TBI can cause the proliferation and activation of hippocampal microglia, pioglitazone reduced microglia response in rat hippocampus after TBI by PPARy receptor.3. The effects of Pioglitazone on hippocampus astrocyte after TBIWe used GFAP immunohistochemistry and cell counting method to study the effects of pioglitazone on TBI induced activation of astrocyte inflammatory reponse on the experimental model of TBI in rats. The result showed that in Sham group normal hippocampal astrocytes was Astral-like, thin and less branching processes; and inVehicle group astrocytes stained deeper and protruding branches increased, thickening, variable-length, distribution-intensive, in the activated state; Pio treatment group the activatied hippocampal astrocyte significantly reduced compared with Vehicle group; Pio+Ant rats still showed moderate hippocampal astrocytes respond. Cell counting showed that the number of GFAP-positive cells in hippocampus CA1 and CA3 areas in Vehicle group is 216.44±33.94which significantly increased (P<0.01) compaired with Sham (152.33±15.66); The number of Pio group was 172.44±29.78 an apparent decrease as compared with Vehicle group, or Pio+Ant group 201.16±30.51 (P<0.05, P<0.05). The results suggested that pioglitazone can reduce the TBI hippocampal astrocytes proliferator-activated, and PPARy receptor mediated the effect.4. The effects of Pioglitazone on hippocampus neurons after TBIIn this study we used NeuN immunohistochemistry and cell counting method to disscuss whether pioglitazone could protect hippocampal neurons by reducing microglia and astrocyte in the inflammatory reponse on the experimental model of TBI in rats.The result showed that in Sham normal hippocampal neurons arranged in neat rows, structured, the cell bodies and apophysis were visible, NeuN protein expression was higher; Vehicle rats hippocampal neurons significantly reduced,they were irregularly arranged, ill-defined,and the number was lower than normal, and NeuN stained lighter; while in Pio treatment group neurons were of a clear outline of plump, NeuN protein expression increased. Neurons number of Sham group in hippocampal CA1 and CA3 areas was 253.50±9.33,while Veh group was 199.66±3.87, the difference very significant (P<0.01); Pio neurons number was 158.00± 8.92,compared with Vehicle group and Pio+Ant group (160.66±7.75)the increase was significant (P<0.05); The difference is not significant between Pio+Ant group and Vehicle group.The results suggested that pioglitazone can enhance hippocampal neurons NeuN protein expression, and reduce cell damage and death in TBI rats.5. The effects of Pioglitazone on cholinergic neurons cultured in vitroWe used ChAT immunocytochemistry and cell counting method to study the effects of pioglitazone on the LPS-induced inflammatory response of cortical and hippocampal cholinergic neurons cultured in vitro. We divided them into four groups namely Control, LPS, Pio+LPS.With pioglitazone for treatment we conducted ChAT immunohistochemistry staining. The result showed that the cell bodies and apophysis of the hippocampal neurons in Control group were visible and clear,ChAT protein expression was high; in group LPS hippocampal neurons significantly reduced,cells were swelling and ill-defined;ChAT stained lighter.While in Pio treatment group neurons were of a clear outline; apophysis increased and the connection between cell enhanced; ChAT expression were higher, cell counting showed the cell number of Control group was 65.16±3.18/Vf compared with the LPS group 33.83±3.43/Vf difference were significantl(P<0.01); number of positive cells of Pio+LPS group is 59.50±7.06/Vf which significantly increased than the LPS group (P<0.01).This suggested that pioglitazone can directly reduce LPS-induced inflammatory injury of cortical and hippocampal cholinergic neurons.Conclusion:1. Pioglitazone can enhance the learning and memory ability in TBI rats in MWM according to PPARy pathway.2. Pioglitazone can reduce the proliferation and activation of microglia in hippocampal CA1 and CA3 areas according to PPARy pathway.3.Pioglitazone can reduce the proliferation and activation of astrocyte in hippocampal CA1 and CA3 areas according to PPARy pathway.4. Pioglitazone can enhance NeuN protein expression in hippocampal CA1 and CA3 areas and reduce cell damage and death in TBI rats.5. Pioglitazone can reduce the LPS-induced inflammatory injury and death of cortical and hippocampal cholinergic neurons cultured in vitro,and play a role in neuroprotection.