METHODSAnimalmodel selection for Perinatal Ischemia and Experimental GroupsRice-Vannucci model of rat will be selected. 5 Thehemorrhagic model will be used which is characterized by periventricularhemorrhages for mother rats. Laparotomy will be applied for 20 minutes toinduce IUI. (IUI is induced by clamping the uterine and ovarian vascularate.)4 After 2-3 days, mechanical ventilation will be applied by using facemask for 20 minutes for rat pups. 6 It will be done for spreadinghemorrhages in periventricular tissues. There will be also different groups ofrats which are control,sham,IUI, sham+ventilation and IUI+ventilationto determine differences.

Control group will be normal rats will give birthnaturally (without any IUI and ventilation.) Sham group indicates the ratswhich have surgery but not induced IUI. They are anesthetized etc. Allexperiments are done according to ethical authorization. As shown in Table 1, there are not any usage of inhibitors forcontrol 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. Treatmentof inhibitorsGlibenclamide should be dissolved in DMSO. (as 25mg ofglibenclamide into 10mL of DMSO) Then, single dose which is 10µg/kg should begiven as soon as at the end of laparotomy. (before closing) Then, treatment iscontinued by using osmatic pump as soon as the end of operation. It should lastfor 1 week. The delivery amount should be 400ng/h. Additionally, vehicletreatment is applied in the same manner which provides just DMSO (dissolvent ofglibenclamide) treatment.

5TIMP which is the inhibitor of MMP9 should be upregulated inother word overexpressed to increase effect in treatment. This increment shouldbe in long term so adenoviral gene transfer can be applied. It provides 250ng/ml in plasma at eight weeks after injection. 9Determination of relationship between SUR1 and MMP-9 by usingco-immunolabelingCyrosection of brains are obtained from each group. Then, theyshould be labelled by using specific primary and fluorescent secondaryantibodies of SUR1 and MMP-9. Fluorescent microscopy will be enough tovisualize relationship between SUR1 and MMP-9.

Quantification ofimmunofluorescence is also used to determine intensity of SUR1 and MMP-9 byusing NIS-element AR software. It is useful when the inhibitors of these proteinsare used. TIMP is used as inhibitor of MMP-9 and glibenclamide is knowninhibitor of SUR1 is used.  Thus, twogroup of cyrosections are collected.

One of them is not treated with inhibitorsand other one is treated with inhibitors of proteins. However, inhibitorsshould be used separately to understand how SUR1 inhibitor or MMP-9 inhibitoraffects the other protein. 4FRET is another technique to determine protein associationdepending on fluorescence resonance energy transfer. In this method, suitableautofluorescent GFP spectral mutants are used to label our targets which areSUR1 and MMP-9 instead of antibodies. These determinants are also calledfluorophores. 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 andMMP-9 interaction. Other processes are similar with above explanation. The samemanner is followed.10Determination of relationship between SUR1 and MMP-9 by using immunoblotting(Western blot) and zymographyAddition to quantification of immunofluorescent samples,immunoblotting can be also used to determine definite amount of proteins ifthey have relationship with each other or not. Also, inhibitory effect ofproteins between each other can be also determined by immunoblotting andestimated amount of proteins with and without inhibitor usage. 2Zymography can be also used to quantify activity of only MMP-9because it is used for detection of hydrolytic enzymes.

This technique helps tounderstand there is any relationship between SUR1 and MMP-9 by usingquantification of this proteins. Also, inhibitory effect of proteins betweeneach other can be determined. 2Determinationof relationship between SUR1 and MMP-9 by using coimmunoprecipitation The primary target, SUR1 and secondary target in wordinteracting protein, MMP-9 and vice versa are captured and purified tounderstand if there is any interaction between these proteins. Primary antibodyis used for the primary target. Also, inhibitory effect is determined by thistechnique.

The result of this is quantified by using western blot to detectsecondary target is interacted with primary target or not. 8  Neurobehavioral assessments tounderstand is there any effect of inhibition of proteins SUR1 and MMP-9Morris water maze can be used on the 5th week ofrat pups to determine is there any effect of inhibition to ability inaccelerating rotarod, beam walk ability, rapid learning.  In accelerating rotarod, the parameter is thereaming time (as second) of rat on the apparatus. In beam walk, performance ofrats is determined by considering gliding. Depending on this, rank points aredetermined with respect to groups.

In rapid learning, Morris water maze(MWM) isused. The aim is to determine time to find the platform location. Thus, thespent time in the correct quadrant is determined. 1


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