Neuroprotective Effect And Its Mechanism Of Propentofylline After Spinal Cord Injury In Rats

Spinal cord injury (SCI) is one kind of serious trauma. SCI may cause the movement function barrier, often leaves behind the serious disability. Since long ago believed that, neuron of central nervous system in the grown-up mammal is not impossible to be regenerated after the damage only can be replaced by the spongiocyte. At present each kind of cell transplantation and histological transplantation change the traditional ideas of the central nervous system damage to be unable completely to regenerate and repair, which causes treatment of the spinal cord damage into a hot spot in neuroscience now. Each kind of stem cell,the organization,the nerve nutrition factor transplanted to the damage spinal cord, substitute and fill up defect because of the neuron cell which were damaged to die. To stimulate axon regeneration, reconstruct the neuron return route, simultaneously can release the nerve to hand over the nature independently, to secrete massive medical nerve nutrition factor or the nerve protection factor, to inhibit nerve denaturation,the death, and to promote the neuranagenesis, thus can improve the spinal cord movement function barrier. Reproductive property of mature neuron is low,and becauseof changing in inner environment, the spinal cord secondary damage, like the neuron denaturation,necrosis, the neuron excitability increasing,and the changing of neurotransmitter make axon growth damped. After SCI causes the astrocyte proliferation, the reactivity astrocyte (Ast) proliferation is the response of cicatrization for the nervous tissue damaged, finally becomes the sol scar. The reactivity Ast simultaneously has the factor which each kind of suppression axon grows. Thus the research on SCI, becomes a big topic which the foundational and the clinical medicine workers face. As early as in 1911, Allen once used the heavy item to fall (Weight-Dropping, WD) for the first time to duplicate SCI model on the experimental animal. This is the start on experimental SCI research.hereafter, appeared various damage model for the spinal cord to provide the advantageous safeguard for study. According to the time, the animal models of SCI were divided into acute and the chronic damage, and divided into the incomplete damage and the complete damage model according to its pathology advancement. Believed generally, after SCI, function condition of the spinal cord commonly carries on consistent pathological change process. From the pure cell damage to the irreversibility organization necrosis as well as the sol scar forms have the time process. If can discover its change tendency which corresponds with the pathological change, block this sequential change process, then possibly can obtain the good function restoration.Propentofylline(PPF),that chemistry name is 3-Methyl-1 (5-oxohexyl) -7- propyl- xanthine, is one kind medicine which Hoechst Corporation develops can suppress the phosphoric acid diesterase and the gland glucoside ingestion xanthine derivative.The molecular formula was C15H22N4O3, molecular weight 306.36 .At present more experiments have already proved that PPF in the treatment dementia sickness function was reliable and effective. The PPF is a kind of moderator of the nerve spongiocyte. ThisⅢclinical test has demonstrated it has the accurate function also has the good security.This study in SCI ,early time want to use medicine PPF to adjust Ast activeness, improve the inner environment, reduce the spinal cord secondary damage, delay or avoid finally becoming the sol scar,which provides a time window for the axon growth after each kind of cell transplantation and the histological transplantation. This experiment altogether is divided three parts: 1. To produce an animal model of acute SCI using the modified Allen's method and to observe the effect of PPF on the BBB's motor scales, Rivlin's inclined plane scales, programmed cell death and the histopathological changes after acute SCI in rats. 2. To investigate the effect of PPF on GFAP,VIM in the rat spinal cord after SCI. 3. To investigate the effect of PPF on GFAP,VIM in vitro cultured Ast of mechanical injury.PartⅠThe Animal Model Establishment and the Spinal Cord Organize ObservationAbstract: Objective To produce an animal model of acute spinal cord injury (SCI) using the modified Allen's method and to observe the effect of propentofylline (PPF) on the BBB's motor scales, Rivlin's inclined plane scales, programmed cell death and the histopathological changes after acute SCI. Material and methods 66 healthy rats were randomly divided into 3 groups. Group A: PPF treatment group; Group B: operation group; Group C: normal group. An acute SCI model of rats was made by the modified Allen's weight dropping method. The rats in group A (test group n=30) were treated with PPF after SCI. The second group (the control group n=30) were treated with 0.9%NS after SCI. The third group (n=6) were normal group. To study BBB's scales, Rivlin's inclined plane scales,the spinal cord blocks were cut in cross section 10μm thick and stained with HE,Nissl stain,TUNEL technique was used to mark the apoptosis cells. The histopathological changes and the apoptosis cells were observed with Light microscope. Results 1 .There was significantly haemorrhage, edema, degeneration, vacuole and so on in spinal cord after SCI in rats. But the pathological and ultrastlvctural changes of spinal cord were significantly improved in PPFgroups. 2. The BBB's scales and Rivlin's inclined plane scales decreased significantly after SCI. The neurological function of rats in PPFgroups was significantly improved at 72-hour after SCI. 3. The number of apoptosis in spinal cord significantly increased after SCI but significantly decreased in PPF groups (P<0.01).Conclusion 1. According to change rule of the common organization pathology change and the neuroglia cell apoptosis in the spinal cord, the acute spinal cord damage animal model established successfully, may be applied to the experimental study of the acute spinal cord injury. 2. The neuroglia cell apoptosis possibly is one of mechanisms of the secondary spinal cord injury. 3. PPF intervention may reduce nerve cell apoptosis of the acute spinal cord injury, protect the nerve cell effectively and enhance big mouse's nerve function grading after acute SCI. In brief, PPF has the protective function to early time spinal cord of the rats after acute SCI.PartⅡEffect of Propentofylline on GFAP,VIM Gene Expression in the Rat Spinal Cord after SCIObjective To investigate the effect of propentofylline (PPF) on GFAP,VIM in the rat spinal cord after SCI Methods In the study, the mRNA expression of GFAP,VIM were measured using reverse transcription polymerase chain reaction(RT-PCR) and protein expression of GFAP,VIM were measured by western blot method and immunohistochemistry in the rat spinal cord after SCI,healthy rats were randomly divided into 3 groups. Group A: PPFtreatment group; Group B: operation group; Group C: normal group. An acute SCI model of rats was made by the modified Allen's weight dropping method. The rats in group A were treated with PPF after SCI. The second group (the control group) were treated with 0.9%NS after SCI. The third group is normal group. The expressions of GFAP,VIMmRNA were measured by RT-PCR, as well as the protein expression of GFAP,VIM quantitated using western blot and immunohistochemistry (S-P) in the rat spinal cord after SCI. Results1.The mRNA expression of GFAP was detected in non-injured spinal cord. The expression of GFAP mRNA up-regulated at 6h postinjury (P<0.01) and peaked at 5d postinjury(P<0.01,vs. the control group), and dropped after 7 d; VIM mRNA expression up-regulated at 3d after SCI(P<0.01), and remained high level until 7d postinjury(P<0.01,vs. the control group); 2. The protein expression of GFAP was detected in non-injured spinal cord. The expression of GFAP protein up-regulated at 12h postinjury (P<0.05,vs. the control group), VIM protein expression was detected at 3 d, remained high level until 7d postinjury(P<0.05,vs. the control group). Conclusions 1. The level of GFAP,VIM mRNA and protein increased significantly at the lesion area after SCI, and correlated with cell proliferation in the lesion area, which indicates that GFAP,VIM play an important role in cicatrization after SCI. 2. Nerve protective function possibility mechanism on PPF after SCI is: to suppress GFAP,VIM expression of the damage area spinal cord organization after SCI and multiply excessively of Ast, thus to promote function restoration of damage spinal cord.PartⅢEffect of Propentofylline on GFAP,VIM Expression in Astrocytes Cultured in VitroObjective: To investigate the effect of propentofylline(PPF) on GFAP,VIM expression in vitro cultured astrocytes(AS) of mechanical injury. Methods: The model of mechanical injury on in vitro cultured and purified AS was made. AS were randomly divided into three groups: PPFgroup(group A),group B.Group A were treated with propentofylline after injury. Immunohistochemical technique and imagine analysis technique were used to detect the expression of GFAP,VIM and the number of GFAP,VIM positive cell. Results: The expression of GFAP,VIM and the number of GFAP,VIM positive cell showed increased tendency after injury. AS injured had hypertrophy, proliferation, and the expression of GFAP,VIM and the number of GFAP,VIM -positive cells were increased. After treated with propentofylline, the hypertrophy of AS was attenuated and the expression of GFAP,VIM and the number of the GFAP,VIM -positive cells were decreased. Conclusion: Mechanical injury can promote responsive proliferation. PPF could modulate the hypertrophy and proliferation of AS effectively after mechanical injury.