Endothelin1Overexpression In Astrocytes Impact On Motor Function Recovery After Spinal Cord Injury In Mice

The injury of central nervous system mainly occur on the brain and spinal cord. It canbe divided into primary spinal cord injury and seco ndary spinal cord injury, the latteroccurs after primary spinal cord injury. Primary spinal cord injury refers to the mechanicaldamage of the spinal tissue, such as extrusion, tear. The secondary injury is caused by thedestruction of blood spinal cord barrier, vascular damage, hypoxia, tissue necrosis afterspinal cord injury. It can lead to the destruction of the balance of the microenvironmentand larger spinal cord injury.The occurrence of secondary injury is a very complicated process including manycomprehensive factors which is relative to the uncontrollable factors of primary injury ofthe spinal cord. The main goal of scientific study is to prevent secondary injury. But manycells and molecules after spinal cord injury come from organization of the circulation.These cells and molecules can enter into the spinal cord blood when the spinal cord tissueis damaged. Thus, to prevent further blood spinal cord barrier damage or repair thedamaged of spinal cord barrier in time is an important strategy to fight against secondaryspinal cord injury.Endothelin belongs to vasoconstrictor substance family, there are three subtypes whichare endothelin1(ET-1), endothelin2(ET-2), endothelin3(ET-3) and two receptor A (ETA) and the receptor B (ETB). Endothelin is found as the most powerful vasoconstrictorsubstance, ETAis mainly distributed in vascular smooth muscle cell, ETBis distributed inmany tissue cells. ETBis found in many tissue cells of the central nervous systemespecially astrocytes. There have existed more and more research on the ETB.Blood spinal cord barrier consists of vascular endothelial cells, pericyte and astrocytes.Endothelial cells and astrocytes have endothelin receptor, too. Our experiment mainlystudied the influence of overexpression of endothelin1(ET-1) in astrocytes on therecovery of motor function after spinal cord injury in mice and its main possiblemechanisms.Experime nt1:The screening of GET-1homozygous mice and the difference on therecovery after spinal cord injury between GET-1transgenic mice and wild-typemice.In this part, we screen GET1homozygous mice first. We produced offspring matingbetween genetic testing positive GET-1mice for three generations to improve thehomozygous probability of the transgenic mice, then produced offspring mating betweenthe third generation of GET-1transgenic mice which was positive in genetic identificationand wild type mice. If all the offspring was identified as positive for the GET-1, then wecould determine his father/mother identified GET-1positive was homozygous GET-1mice. After screening the homozygous GET-1mice and wild type mice, we establishedthe model of the spinal cord injury which showed that the recovery of the spinal cordinjured GET-1mice was worse than wild type mice through the BMS mice sports functionscores. Our experiment had showed that the overexpression of ET-1in astrocytes mightinhibit the regeneration of the spinal cord neuron in mice or cause more severe damageafter spinal cord injury.Experime nt2: The influence of the blood spinal cord barrier on the overexpressionof endothelin1in astrocytes after spinal cord injury.We mainly research the damage mechanism of the overexpression of endothelin1inastrocytes and its influence on the recovery of motor function after spinal cord injury inmice. First of all, we tested the expression of endothelin1after spinal cord injury during1 d,3d,7d and14d in the GET-1transgenic mice and wild-type mice. The resultsshowed that the expression of endothelin1in wild type mice first declined in3d, thenrised expression，but never higher than in the physiological conditions, while theexpression of endothelin1rised in GET-1transgenic mice high after spinal cord injury7d. This may be related to the activation of astrocytes.Then we tested the damage of the blood spinal cord barrier. The damage of the bloodspinal cord barrier often occurred after the spinal cord injury in24h. We observed theEvens blue perfusion of mice tail vein in GET-1transgenic mice and wild-type mice afterspinal cord injury in24h. Then we observed the seepage quantity of the Evens blue in theinjured spinal cord part which was significantly higher than the quantity of wild mice.What’s next, we confirmed the previous observed results on the Evens blue seepagequantity by measuring0.5cm on both ends at the centre of the spinal cord injury usingenzymes standard instrument. This experiment had proved that the overexpression of theendothelin1in astrocytes after spinal cord injury had serious impact on the integrity ofblood spinal cord barrier, thus differences on the functional recovery after spinal cordinjury between two kinds of mice could be made.Experiment3: The influence on astrocytes after the overexpression of endothelin1inastrocytes and the damage of the blood vessels nearby the spinal cord injury center.Astrocytes and vascular endothelial cells are important elements of blood spinal cordbarrier, both of them are distributed with endothelin receptors. This experiment observedthe form of the blood vessels and the astrocytes nearby injury center after spinal cordinjury at7d. It shows that astrocytes are activated obviously in GET-1transgenic mice;The existed numbers of blood vessels nearby injury center are far lower than which inwild type mice. This suggests that the overexpression of endothelin1has importantinfluence on astrocyte and blood vessels through its receptors.