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

2. 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

3. 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

4. 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.

5. 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.

6. 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

7. 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.

8. 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 .

9. 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

10. 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.

11. 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%.

12. 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

13. 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

15. 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

16. 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.

17. 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.

18. 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.

19. Mapa conceptual: Verónica Peréz.

20. Mapa conceptual : Laura Rodríguez Duque.

21. GRACIAS!!