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Biológia seminario.

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Seminario de biológia: Effect of progesterone intervention on the dynamic changes of AQP-4 in hypoxic- ischaemic brain damage.

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Biológia seminario.

  1. 1. INTRODUCTUCTION •PROGESTERONE Is a steroid hormone involved in the female menstrual cycle and pregnancy. Other functions are to increase the excretion of sodium. progesterone has antidepressant effects in some cases, by intervening directly in some intracellular receptors in the brain . It has been shown to be effective in treating some behavioral disorders. Exist some studied of progesterone in relation with brain tumors , and stopping their growth. This is because progesterone regulates the production of tumor cells by blocking the interaction with the intracellular receptor tumor
  2. 2. AQP–4 Main function is to participate in metabolic balance regulation in brain. Its expression is closely related to the occurrence and development of cerebral edema. Transmembrane transport of wáter. 13 kinds of AQPs
  3. 3. CEREBRAL ISCHEMIA • CEREBRAL ISCHEMIA INTERRUPTION OF SUPPLY BLOOD TO THE BRAIN , OCCURS WHEN BLOOD VESSELS THAT COME BRAIN BECOME NARROWED OR CLOGGED WITH FATTY DEPOSITS CALLED PLAQUE ( CAN BE CAUSED BY THROMBOSIS OR EMBOLISM) . THUS INTERRUPTING THE FLOW OF OXYGEN AND NUTRIENTS NEEDED TO MAINTAIN THE FUNCTIONING OF BRAIN CELLS . WHEN A BLOODCLOT HAS OCCLUDED VESSEL AND REDUCES CEREBRAL BLOOD FLOW TO A SPECIFIC REGION OF THEBRAIN.
  4. 4. The progesterone has the function of inhibiting de AQP-4 proteins  neuroprotector AQP-4 is the responsable of edema cerebral. That increase in a isquemia situation.
  5. 5. OBJETIVE • THIS EXPERIMENT AIMED TO FURTHER EXPLORETHE MECHANISM OF PROG IN THE TREATMENT OF CEREBRAL ISCHAEMIC INJURY FROM AQP-4 EXPRESSION ENCEPHALOEDEMA AND BBB PERMEABILITY
  6. 6. MATERIALES Y METODOS •ANIMALES : SE USARON 72 RATAS WISTAR DE 7 DÍAS DE EDAD, ESTAS SE DIVIDIERON EN 3 GRUPOS (GRUPO CONTROL, GRUPO CON HIPOXIA-ISQUEMIA, GRUPO TRATADO CON FÁRMACOS) LAS RATAS QUE FUERON TRATADAS CON FÁRMACOS SE LES INYECTO PROGESTERONA DE MANERA INTRAPERITONEAL A 0.5 G/L A DOSIS DE 8 MG/ KG. DESPUÉS DE LA HIPOXIA, LAS RATAS EN EL GRUPO CONTROL Y HI FUERON INYECTADOS CON SOLUCIÓN SALINA.
  7. 7. PERMEABILIDAD •SE DETECTO PERMEABILIDAD DE BBB ( BLOOD- BRAIN BARRIER) GRACIAS AL AZUL DE EVANS(EB) ,EL CORAZÓN FUE INYECTADO CON SOLUCIÓN SALINA 20 G/L EB ( 20 MG / KG). EL TEJIDO CEREBRAL IZQUIERDO SE PESÓ . SE DETECTÓ LA ABSORBANCIA POR ESPECTROFOTOMETRÍA. EL CONTENIDO DE EB SE CALCULÓ EN LA MUESTRA DE MEDICIÓN .
  8. 8. Posición supino + inhalación de anestesia Piel del cuello cortada. Separa carótida de el ECM 37ºc + 2-3 hrs+ 8% O2 + 92% N2 Modelos con hipoxia ESTABLECIMIENTO DEL MODELO
  9. 9. MATERIALES Y METODOS •INMUNOHISTOQUIMICA • LA INMUNOHISTOQUIMICA ES LA IDENTIFICACIÓN DE UN TEJIDO POR MEDIO DE UNA INTERACCIÓN ENTRE UN ANTÍGENO Y UN ANTICUERPO, DONDE EL ANTICUERPO HA SIDO MARCADO, LA CÉLULA SE COLOREA PARA DEMOSTRAR LA PRESENCIA Y LOCALIZACIÓN CELULAR DE UNA MOLÉCULA DE INTERÉS EN LUGAR DE UN ANTICUERPO PRIMARIO ,SE AÑADIÓ TAMPÓN DE FOSFATO AL GRUPO DE CONTROL NEGATIVO, LAS CÉLULAS POSITIVAS SE EXPRESARON EN LA PROTEÍNA AQP. El cerebro fue segmentado Se fijo durante la noche en 10 % de formalina , fue deshidratado, corteado , desparafinado , y conservados en un refrigerador a 4° C.
  10. 10. DETERMINACIÓN DEL CONTENIDO DE AGUA Muerte de la rata Incubación del cerebro 80% del tejido cortical en vidrio. Se pone electrotérmico 110 ° C caja seca a temperatura constante a hornear durante 48 h. Peso seco Contenido de agua del cerebro = [(peso húmedo peso seco) / peso húmedo] x 100%.
  11. 11. WESTERN BLOT • FUNDAMENTO: • PRUEBA UTILIZA PARA DETECTAR LA RELACIÓN ANTÍGENO-ANTICUERPO; NON USA SONDAS. • USA LA TÈNICA DE SDS-PAGE: SDS  - FILTRO INCUBACIÓN CON ACRESULTADO. Corteza Lisado de proteínas centrifugación Sobrenadante  ácido bicinconınico Electroforesis en gel de poliacrilamida Anti-rata AQP-4 y anticuerpo primario β-actina Prueba t. Análisis de correlación= P <0,05
  12. 12. RESULTADOS Los resultados inmunohistoquímicos demostraron que AQP- 4 células positivas eran redondos u ovalados . La expresión AQP- 4 en la corteza cerebral de la HI grupo fue significativamente superior a la de el grupo de control después de la hipoxia de 6 , 24 y 72h . expresión de AQP - 4 en la corteza del grupo de drogas fue significativamente menor que la del grupo de HI después de la hipoxia de 6 , 24y 72 h
  13. 13. ACTOR Wath say yes or no Zhou C, Tu J, Zhang Q, Lu D, Zhu Y, Zhang W, Yang F, Brann DW, Wang R The change in microenvironment results in the up regulation of AQP-4 expression after brain injury, changes the structure of the cell membrane, increases the permeability of BBB and enhances the water permeability yes Sun B, Chen L, Wei X, Xiang Y, Liu X, Zhang X. proved that AQP-4 gene knockout can significantly alleviate cerebral edema caused by acute water intoxication or ischaemic stroke, suggesting that AQP-4 performs a key function in brain tissue edema, restrains AQP-4 expression, and inhibits or alleviates cerebral edema induced by AQP-4 yes
  14. 14. Author What they said? Yes or not ? - Northington FJ, Chavez-Valdez R, Martin LJ. - Doi K, Sameshima H, Kodama Y, Furukawa S, Kaneko M, Ikenoue TK. BBB damage is one of the most important pathophysiological mechanisms of ischaemic brain damage. Traumatic cerebral oedema is a type of mixed oedema that is closely related to the increase in BBB permeability. Yes. - Zhu YM, Wang CC, Chen L, Qian LB, Ma LL, Yu J, Zhu MH, Wen CY, Yu LN, Yan M. - Zhang X, Zhang X, Wang C, Li Y, Dong L, Cui L, Wang L, Liu Z, Qiao H, Zhu C, Xing Y, Cao X, Ji Y, Zhao K. Studies have indicated increased AQP-4 expression in cerebral oedema caused by cerebral infarction, cerebral haemorrhage, brain tumour, inflammation, and brain injury in others’ reports. Yes.
  15. 15. CONCLUSION • PROGESTERONEDECREASES THE LIKELIHOOD OF CEREBRAL EDEMA DUE TO ISCHEMIA OCCURRED . IT CAN BE INFERRED THAT BOTH NEONATAL RATS AND THEIR MOTHERS SHARE HIGHLEVELS OF THIS HORMONE DURING PREGNANCY AND SUBSEQUENT BIRTH. A CONCLUSION MAY RESULT IN THE EVOLUTIONARY DEVELOPMENT OF NEONATAL RATS (AND OTHER ORGANISMS SHARING THE PRODUCTION OF PROGESTERONE ) REDUCE THE RISK OF DEATH IN EARLY LIFE STAGES INCLUDING DURING PREGNANCY AND IN THE FIRST WEEKS AFTER BIRTH THE SAME WAY IT CAN BE ASSUMED THAT MOTHERS MAY HAVE A SIMILAR DECREASE IN THE RISK OF DEATH FROM DAMAGE TO THE IRRIGATION SYSTEM BRAIN DURING PREGNANCY AND AFTER DELIVERY.
  16. 16. CONCLUSION • ALTHOUGH AQP - 4 EXPRESSION IS NECESSARY IN BRAIN TISSUE BUT IT CAN ALSO BE HARMFUL IN SOME CASES , SINCE THIS ( AQP -4 ) PRODUCES AN INCREASE IN CEREBRAL EDEMA AS IT INCREASES THUS CAN BE TARGETED INVESTIGATIONS TREATMENT IN DIFFERENT CASES AS IS THE ISQUEMIA TO PRE AND POST- OPERATIVE TREATMENTS. • AQP4 PARTICIPANT IS IMPORTANT CAUSE OF CEREBRAL EDEMA, ALLOWINGINCREASED BLOOD BRAIN BARRIER PERMEABILITY PROMOTING THEAGU ENTER HER. • HYPOXIA IS A PROCESS THAT PROMOTES THE CEREBRAL EDEMA AS IT INCREASES THE EXPRESSION OF AQP4 CHANNELS IN THE BB. DEPUES 6 HRS OF HYPOXIA IS THAT THIS EFFECT IS SIGNIFICANTLY INTENSIFIED.
  17. 17. Mapa conceptual: Verónica Peréz.
  18. 18. Mapa conceptual : Laura Rodríguez Duque.
  19. 19. GRACIAS!!

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