PPARγ Agonist Pioglitazone Atttenuated Glial Inflammation And Increased Neuroprotective Effects Following Traumatic Brain Injury In Rat

Traumatic brain injury (TBI) is clinically a serious disabling disease. The rate of disabilities and mortalities of TBI increase year by year and have no effective way to cure.To study pathogenesis and effective medicine after TBI to protect brain tissue, decrease its death, promote restoration of structure and function will have great benefits to theory and medicine. The ensuing loss of neuronal tissue is believed to evolve in a biphasic manner consisting of the primary mechanical insult and a progressive secondary necrosis. Secondary traumatic injury results from several pathophysiological events in the death cascade that contribute to neuronal damage and death, including inflammation, glutamate-mediated excitotoxicity, oxidative stress and apoptosis. Neuroinflammation following secondary brain trauma is considered to play a prominent role in both the pathological and reconstructive response of the brain to injury.Microglial cells are strongly activated following injury and play an important role in injured brain tissue in central nervous system. Activation of microglia is related with the inflammatory cascade, the inflammatory cascade characterized by proinflammatory cytokines and activation of microglia induce neuronal death by releasing proinflammatory cytokines, cytotoxic proteases and reactive oxygen species, which in turn can exacerbate neuronal dysfunction. Activation of astrocytes(AST) have double effect on neuronal tissue following TBI. Reactive astrocytes serve a neuroprotective role during the early stages of injury and are responsible for maintaining physiological homeostasis through production of trophic factors and phagocytic resolution of the lesion and free radical elimination during periods of energy deprivation. Abundant activated astrocytes release oxygen radicals and inflammatory cytokines such as IL,TNF,INF.In the advanced stages of injury progression, abundant activated astrocytes form a glial scar inhibitory to neural regeneration.Peroxisome proliferator-activatedreceptor-y (PPARy) is a ligand-activated transcription factor of nuclear hormone receptor superfamily and can either initiate or suppress the transcription of the target genes. As PPARy plays a significant role in glucose and lipid homeostasis, Thiazolidinediones(TZDs) such as pioglitazone(pio) are potent synthetic agonists and are currently approved by the United States Food and Drug Administration (FDA) for type-2 diabetes treatment.PPARyagonist can reduce the express of inflammarory cytokines TNF-a, IL-1β,INF-γwith inhibition of nuclear factor kappa-B, transcription activating factor JAK-STAT signaling pathway. Recent studies showed that TZDs are extremely neuroprotective through inhibiting the expression of inflammatory cytokines, chemotactic factor, adhesion molecules and activation of astrocytes and microglia in animal models of acute CNS insults including focal ischemia. TZDs pretreatment also can reduce spinal cord lesion volumes,attenuate the expression of inflammatory and activation of gliocyte,and enhance the express of neuroprotective genes in adult mice subjected to spinal cord injury(SCI).Their efficacy was also shown in animal models of chronic CNS injuries like Parkinson's disease, Amyotrophic lateral sclerosis and Alzheimer's disease.Currently, repair mechanisms and treatment of functional recovery following brain injury is difficult and hot medical research. New research shows that pioglitazone treatment on brain ischemia injury can reduce inflammatory reaction with neuroprotective effect, so that we further study anti-inflammatory effects of pioglitazone in rats after TBI has important theoretical significance. We used controlled cortical impact (CCI) animal model in rat and studied the effects of pioglitazone on the activation and infiltration of astrocytes and microglia at the edge of cortical lesion, numbers and morphology of neuron on overall level. We further observed the LPS-induced cellular toxicity of rat cortical cells and neuroprotective effects of pio and explore its possible mechanisms.The main results of our research and discussion are as follows:1. pioglitazone significantly attenuated the cortical lesion volume following TBIControlled cortical impact results in a progressive cortical lesion, Neutral red staining and cortical injury volume detection were used to observe and analyze the effects of pio to cortical injury volume in rats post-TBI. Experiment was divided into four groups, namely sham operation group (sham group); the vehicle treatment group after TBI (vehicle+TBI group); the pio treatment group after TBI(pio+TBI group); pio plus PPARy antagonist group after TBI(pio+T0070907+TBI group). A cavitary cortical lesion was seen at the site of injury at 15 days after injury. In most cases, the hippocampus was completely intact or showed only slight deformation. Cortical lesion volume in pio group(2.21±0.94mm3) decreased obviously compared with TBI group(5.51±2.11 mm3)and antagonist group (4.54±1.72 mm3).These indicate that pioglitazone treatment decreased obviously the volume of cortical lesion of TBI rats.2. pioglitazone significantly reduced activation and infiltration of the microglia at the edge of the cortical lesion following TBIThe present study, OX-42 immunohistochemical staining was used to observe microglia activation and number changes in experimental rats after TBI.Under normal conditions microglia are in a resting state, characterized by a small cell body with thin, short processes and low expression of surface antigens OX-42. Traumatic brain injury (TBI) produces a rapid and robust inflammatory response in the brain characterized in part by activation of microglia. Activited microglia at the site of cortical lesion at 15th day following TBI showed a notable increase in total microglia numbers and very different morphology (most of the activated microglia) compared to the control group which was populated predominately by resting microglia. Activited microglia with enlarged amoeboid morphology and much longer processes were observed in a reactive state in TBI group and pio antagonist group. Pio treatment significantly reduced the number of activated microglia, indicating that pio had significant effects on microglia inflammatory responses after TBI. PPARyantagonist reserved the effects of pio. Thus, pio may represent a new therapeutic approach to secondary brain pathology after TBI.3. pioglitazone significantly reduced activation and infiltration of the astrocytes at the edge of the cortical lesion following TBITo determine whether astrocyte activation is influenced by pio treatment, immunohistochemical analysis of GFAP, was performed. Astrocytes are activated exhibiting a hallmark increase in glial fibrillary acidic protein (GFAP) expression. The expresses of GFAP proteins were few and the numbers of GFAP-positive astrocytes were sparse, the bodies of astrocytes were small, neuritis of astrocytes were thin in control group. The expresses of GFAP proteins were strengthened and the numbers of GFAP-positive astrocytes increased, the bodies of astrocytes were large, neuritis of astrocytes were thickened in TBI group and pio antagonist group at the site of cortical lesion at the 15th day after CCI injury. Pio treatment significantly reduced the number of activated astrocytes, indicating that pio had significant effects on astrocyte inflammatory responses after TBI. PPARyantagonist reserved the effects of pio. Thus, pio may represent a new therapeutic approach to secondary brain pathology after TBI through inhibiting activation and infiltration of astrocytes, especially glial scar formation.4. The protective efficacy of pioglitazone on neuron at the edge of the cortical lesion following TBIThe numbers and morphology of survival neurons after TBI are the most important factors determining the function of center nervous system following TBI. NeuN immmunohistochemical method was used to observe the changes of neurons at the 15th day after TBI. NeuN immunostaining positive neurons line up in order and were normal in morphology, and the express of NeuN protein was much in control group. In TBI group it was hard to see neurons in injuried cortical areas, the numbers of NeuN immunostaining positive neurons significantly changed at the site of cortical injury. Compared with control group the express of NeuN protein was less,cortical neurons line up were sparse, the body of neurons were less, parts of neuritis broke, the numbers of neurons were greatly reduced at the site of injury cortex in TBI group(p<0.05). The numbers of NeuN immunostaining positive neurons at the site of injury cortex at 15 days after TBI in pio group reduced greatly compared with TBI group and PPARyantagonist group(P<0.05). Treatment with pio showed neuroprotective effects at the site of injuried cortex after TBI.5. The dose-dependent toxic effect of different concentrations of LPS on cultured cerebral cortical neurons and neuronprotective effect of pioglitazone on neuronsInverted microscope and NSE immunohistochemistry staining technique were used to observe the LPS role on the morphology and NSE positive neurons of cerebral cortex cells. After treatment with LPS of different concentrations (1μg/mL,10μg/mL,20μg/mLs 100μg/mL) 48h, The morphology of cerebral cortex cells in inverted microscope were as follows:The normal cells had large and well-stacked cell bodies. The boundary of each cell was clear. There were many synapses between different cells. The morphology of the cells in 1μg/mL LPS group was almost the same as the normal cells. After treatment with 10μg/ml LPS 48h, the number of decreased and the bodies of cells were smaller. The synapses between different cells broke and the number of synapses decreased. After treatment with 20μg/ml LPS 48h, the number of cells significantly decreased and there existed clumps of dead cell debris. After treatment with 100μg/ml LPS 48h, there existed more dead cell debris and was little living cells. The living cell count in the 10,20,100μg/mL LPS group decreased significantly compared with the control group(P＜0.01). After treatment with LPS of different concentrations, the number of NSE positive neurons in the 10,20,100μg/mL LPS group decreased significantly compared to the control group(P<0.01). After 10μmol/Lpio and 10μg/mL LPS were added to cultured cerebral cortex neurons and coexisted 48h, the number of living cells and NSE positive neurons increased significantly compared with 10μg/mL LPS group(p<0.01).These results above indacated LPS can induce the death of cultured cerebral cortex neurons and it was dose-dependent effect. Pio can regulate the effects of LPS and attenuated the neuronal death induced by LPS.6. Pioglitazone reduced the LPS-induced cell apoptosis of cultured cortical neurons in neonatal ratTo study the LPS-induced toxicity mechanism in cultured neonatal rat cortical neurons, we carried out Hoechst333258 fluorescent staining. Hoechst333258 is a lipophilic substance, entering living cells across members and binding AT-rich DNA-specific binding region. UV excitation, has been labeled normal DNA to issue hazy blue fluorescence, while apoptotic cells issue bright and compact fluorescent due to chromatin condensation. Neonatal rat cortical cells were cultured to the fifth day, 10,20,100μg/ml LPS solution were added, the number of apoptotic cells increased in a dose-dependent compared with normal control group(P<0.01). After 10μmol/L pio and 10μg/mL LPS were added to cultured cerebral cortex neurons and coexisted 48h, the number of apoptosis cells increased significantly compared with 10μg/mL LPS group(p<0.01).Apoptosis was induced by LPS in cultured neonatal rat cortical neuronal, Pio blocked partially LPS-induced cultured cortical neurons apoptosis.Conclusions:1. Cortical lesion post-TBI in rat resulted in secondary neuronal death. Pio treatment significantly reduced the cortical lesion volume and the loss of cortical neurons at the edge of the cortical lesion, hence enhancing neuronal survival and repair. Treatment with T0070907 reversed partly the neuroprotective effects of pioglitazone on the brain.2. CCI injury in rat elicited activation and proliferation of astrocytes and microglia, glial scar wall formation at the edge of the cortical lesion, Pio treatment significantly reduced activation and infiltration of the astrocytes and microglia at the edge of the cortical lesion, playing important role on inhibiting brain tissue inflammation. Treatment with T0070907 reversed partly the inhibiting inflammation effects of pioglitazone on the brain. 3. LPS induced the death of cultured cerebral cortex neurons and it was dose-dependent effect. Pio regulated the effects of LPS and attenuated the neuronal death induced by LPS.