I have tried the procedure with suggested protocol in 24 hrs Transient Focal cerebral ischemia in rats. Im not getting proper results even the animal shows severe lesion symptoms on 24hrs. Any suggestions or modification in the available protocol will be helpful for me.
Thank U in advance
We are working on BCCAo and MCAo models of Cerebral ischemia. we are following the under given protocol for assessment of Evans blue.
Protocol:
At 24 h after reperfusion, 2% EB solution in 0.1M PBS was slowly injected into the tail vein of adult rats (0.8ml/200gr rat) and let circulated for 20–22 hours. Brain tissue from injured and matching contralateral regions were collected and flash-frozen. EB was extracted by incubating tissue samples with 50% TCA solution (1:3), spinning at 10,000× and incubating in ethanol (1:3). Fluorescence was measured using (590/645).
You can also see the following attached article for EB estimation.
Uyama, O., Okamura, N., Yanase, M., Narita, M., Kawabata, K. and
Sugita, M., Quantitative evaluation of vascular permeability in the
gerbil brain after transient ischemia using Evans blue fluorescence, J.
Cerebral Blood Flow Metab., 8 (1988) 282–284.
The biphasic opening of the blood–brain barrier in the cortex and
hippocampus after traumatic brain injury in rats
Mustafa K. Baskaya, A. Muralikrishna Rao Neurosciience letters 226(1997) 33-36
I would suggest to check work from, or contact him self, Erwin Van Vliet ( [email protected] ) from the Gorter group at University of Amsterdam, who is an expert on this and who I believe has used this compound in their epilepsy studies.
Gorter JA, van Vliet EA, Aronica E. Status epilepticus, blood-brain barrier
disruption, inflammation, and epileptogenesis. Epilepsy Behav. 2015 May 6. pii:
S1525-5050(15)00216-4. doi: 10.1016/j.yebeh.2015.04.047. [Epub ahead of print]
Review. PubMed PMID: 25958228.
van Vliet EA, Aronica E, Gorter JA. Blood-brain barrier dysfunction, seizures
and epilepsy. Semin Cell Dev Biol. 2015 Feb;38:26-34. doi:
10.1016/j.semcdb.2014.10.003. Epub 2014 Nov 6. Review. PubMed PMID: 25444846.
van Vliet EA, Aronica E, Gorter JA. Role of blood-brain barrier in temporal
lobe epilepsy and pharmacoresistance. Neuroscience. 2014 Sep 26;277:455-73. doi:
10.1016/j.neuroscience.2014.07.030. Epub 2014 Jul 28. Review. PubMed PMID:
25080160.
van Vliet EA, Otte WM, Gorter JA, Dijkhuizen RM, Wadman WJ. Longitudinal
assessment of blood-brain barrier leakage during epileptogenesis in rats. A
quantitative MRI study. Neurobiol Dis. 2014 Mar;63:74-84. doi:
10.1016/j.nbd.2013.11.019. Epub 2013 Dec 7. PubMed PMID: 24321435.
van Vliet EA, Forte G, Holtman L, den Burger JC, Sinjewel A, de Vries HE,
Aronica E, Gorter JA. Inhibition of mammalian target of rapamycin reduces
epileptogenesis and blood-brain barrier leakage but not microglia activation.
Epilepsia. 2012 Jul;53(7):1254-63.
success !
best,
Paul Lucassen
Dear Kathiravan Kaliappan,
I found this Video-Paper quite helpful, I have used a simplar method for SAH BBB break-down quantification, which is much lower than in ischemic stroke. I am not sure, if you have access to the video (link can be found in the paper); but even without watching the video the paper is good and easy to understand.
I hope you will have success.
Greetings from Toronto, Cananda!
Best regards, Bert
Dear Kathiravan Kaliappan,
The following article may be helpful for you and it is available on my RG profile.
Nagakannan P, Shivasharan BD, Thippeswamy BS, Veerapur VP. Protective effect of Mimusops elengi Linn. flowers against middle cerebral artery occlusion induced brain injury in rats. Journal of Ethnopharmacology 2012;140: 247–254
There is a good video paper on that issue published in 2015.
Rudu M. et al.
Please find at: https://www.jove.com/video/50062/ein?language=German
Kind regards,
Bert Bosche
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Article Santaguida S, Janigro D, Hossain M, Oby E, Rapp E, Cucullo L...
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