METHODSAnimal
model selection for Perinatal Ischemia and Experimental Groups

Rice-Vannucci model of rat will be selected. 5 The
hemorrhagic model will be used which is characterized by periventricular
hemorrhages for mother rats. Laparotomy will be applied for 20 minutes to
induce IUI. (IUI is induced by clamping the uterine and ovarian vascularate.)
4 After 2-3 days, mechanical ventilation will be applied by using face
mask for 20 minutes for rat pups. 6 It will be done for spreading
hemorrhages in periventricular tissues. There will be also different groups of
rats which are control,sham,IUI, sham+ventilation and IUI+ventilation
to determine differences. Control group will be normal rats will give birth
naturally (without any IUI and ventilation.) Sham group indicates the rats
which have surgery but not induced IUI. They are anesthetized etc. All
experiments are done according to ethical authorization. As shown in Table 1, there are not any usage of inhibitors for
control group. On the other hand, each inhibitor is used for other groups.
However, half of each group is treated, and the other half is not treated.
Additionally, half of the treated ones are vehicle treated for glibenclamide.
Enough number of pups are used for each experiment indicated above.

Treatment
of inhibitors

Glibenclamide should be dissolved in DMSO. (as 25mg of
glibenclamide into 10mL of DMSO) Then, single dose which is 10µg/kg should be
given as soon as at the end of laparotomy. (before closing) Then, treatment is
continued by using osmatic pump as soon as the end of operation. It should last
for 1 week. The delivery amount should be 400ng/h. Additionally, vehicle
treatment is applied in the same manner which provides just DMSO (dissolvent of
glibenclamide) treatment. 5

TIMP which is the inhibitor of MMP9 should be upregulated in
other word overexpressed to increase effect in treatment. This increment should
be in long term so adenoviral gene transfer can be applied. It provides 250
ng/ml in plasma at eight weeks after injection. 9

Determination of relationship between SUR1 and MMP-9 by using
co-immunolabeling

Cyrosection of brains are obtained from each group. Then, they
should be labelled by using specific primary and fluorescent secondary
antibodies of SUR1 and MMP-9. Fluorescent microscopy will be enough to
visualize relationship between SUR1 and MMP-9. Quantification of
immunofluorescence is also used to determine intensity of SUR1 and MMP-9 by
using NIS-element AR software. It is useful when the inhibitors of these proteins
are used. TIMP is used as inhibitor of MMP-9 and glibenclamide is known
inhibitor of SUR1 is used.  Thus, two
group of cyrosections are collected. One of them is not treated with inhibitors
and other one is treated with inhibitors of proteins. However, inhibitors
should be used separately to understand how SUR1 inhibitor or MMP-9 inhibitor
affects the other protein. 4

FRET is another technique to determine protein association
depending on fluorescence resonance energy transfer. In this method, suitable
autofluorescent GFP spectral mutants are used to label our targets which are
SUR1 and MMP-9 instead of antibodies. These determinants are also called
fluorophores. There is one acceptor and one donor. CFP-YFP pair can be used.
There should be a certain distance between two of them to determine SUR-1 and
MMP-9 interaction. Other processes are similar with above explanation. The same
manner is followed.10

Determination of relationship between SUR1 and MMP-9 by using immunoblotting
(Western blot) and zymography

Addition to quantification of immunofluorescent samples,
immunoblotting can be also used to determine definite amount of proteins if
they have relationship with each other or not. Also, inhibitory effect of
proteins between each other can be also determined by immunoblotting and
estimated amount of proteins with and without inhibitor usage. 2

Zymography can be also used to quantify activity of only MMP-9
because it is used for detection of hydrolytic enzymes. This technique helps to
understand there is any relationship between SUR1 and MMP-9 by using
quantification of this proteins. Also, inhibitory effect of proteins between
each other can be determined. 2

Determination
of relationship between SUR1 and MMP-9 by using coimmunoprecipitation

The primary target, SUR1 and secondary target in word
interacting protein, MMP-9 and vice versa are captured and purified to
understand if there is any interaction between these proteins. Primary antibody
is used for the primary target. Also, inhibitory effect is determined by this
technique. The result of this is quantified by using western blot to detect
secondary target is interacted with primary target or not. 8

 

 

Neurobehavioral assessments to
understand is there any effect of inhibition of proteins SUR1 and MMP-9

Morris water maze can be used on the 5th week of
rat pups to determine is there any effect of inhibition to ability in
accelerating rotarod, beam walk ability, rapid learning.  In accelerating rotarod, the parameter is the
reaming time (as second) of rat on the apparatus. In beam walk, performance of
rats is determined by considering gliding. Depending on this, rank points are
determined with respect to groups. In rapid learning, Morris water maze(MWM) is
used. The aim is to determine time to find the platform location. Thus, the
spent time in the correct quadrant is determined. 1