Science topic

Ischemia - Science topic

A hypoperfusion of the BLOOD through an organ or tissue caused by a PATHOLOGIC CONSTRICTION or obstruction of its BLOOD VESSELS, or an absence of BLOOD CIRCULATION.
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Hi Everyone, I want to study the GPCR pathways and their role in ischemic heart disease. Which cardiac cell line would be best for such a study? I was hoping to use H9C2 cells, but people suggested it's challenging to transfect them. Please suggest if people use cell lines for such a study or if there is some other model to study the signaling pathways involved in ischemia. Thank you so much
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You may try using HL-1, which is a cardiac muscle cell line, derived from AT-1 mouse artial cardiomyocyte tumor lineage.
Please refer to the attached articles for the use of HL-1 cell line to study the signaling pathways involved in ischemia heart disease.
Best.
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In the study of myocardial ischemia-reperfusion mechanism, is it necessary to establish a separate ischemia group to compare with the ischemia-reperfusion group? Or just compare the control group to the ischemia-reperfusion group?
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I feel it depends on what you are studying, however, employing an ischemic group may help you establish that the ischemia actually did take (check for HIF1alpha etc.), keep in mind, using a control group in my view simply acts as a baseline, if you simply want to see the effect of an intervention on maintaining baseline function post reperfusion, then you may not need to do this for every experiment, but it may be nice to have atleast one figure where you do this to show that the IRI model was valid, just stuff it in the supplements if it breaks your flow.
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Hello,
I am doing research on ischemia stroke, and I am going to administer 60 mg/kg of Edaravone, which is 3-Methyl-1-phenyl-2-pyrazoline-5-one, to 6 to 7-week-old male ICR mice intraperitoneally. The injection volume is 300 uL.
However, Edaravone (EDRV) has not been dissolved in 0.9% NaCl solution and 100 mM DMSO (Dimethyl sulfoxide) solution.
I prepared 60 mg EDRV in 10 mL of saline and 60 mg EDRV in 10 mL of 100 mM DMSO.
I tried 20-minute sonication, but the sediment is still in the 15 mL conical tube.
Is the concentration of two EDRV solutions too high?
I have photos on them.
Please see my problems, and I am looking forward to your help and advice.
Thank you so much,
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Hi,
Please go through the following link to understand the solubility of your drug in DMSO.
You should prepare a concentrated stock of the drug by dissolving it in 100 % DMSO. Then dilute in 0.9% saline to desired working concentration.
I hope this helps.
Good luck,
Alpana
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My project involves inducing intestinal IRI in mice via occlusion of the superior mesenteric artery (where it branches off the abdominal aorta). After scouring the literature I’ve finally gotten a fairly detailed protocol written up, but cannot find the best approach to isolating and occluding the superior mesenteric artery. If you can provide any resources that may be helpful (tips, papers, and especially videos) I would be very grateful.
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This article includes a video of the surgical procedure. It doesn't show the isolation of the intestines, but it should be fairly simple to evert them through the abdominal incision and then isolate the artery. Their approach uses multiple surgical clips on the various branches of the mesenteric artery to induce ischemia, which I would think is safer and more reliable than ligating closer to the aorta.
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Hello,
I am planing an experiment to figure out the effect of an compound for recovery after stroke.
I am wondering whether sham mice group is necessary for this study.
Some reference didn't use sham or others did it.
In my opinion, only two groups (compound-treated mice and compound-non treated mice after stroke) are enough to demonstrate the effect of the compound. 
Will reviewer ask the sham result during paper submission process?
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Certainly, A Sham group is very helpful to rule out the actual effect of any drug in the current experiment. Must include.
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Repurposing of Sodium Thiosulfate (STS) in the treatment of COVID 19 or SARS Cov-2 Viral pandemic of 2019-2020
Repurposing or repositioning of medications to treat emerging diseases is not a new concept; however, I wish to share the story of utilizing sodium thiosulfate (STS) as a repurposed drug in the treatment of calciphylaxis and how even now this drug could possibly assist in the treatment of COVID 19 viral pandemic of 2019-2020 [1].
Repurposing of STS started out with a paper on vascular ossification calcification that included calciphylaxis in 2005 [2] and subsequent multiple papers regarding the use of STS in the treatment of calciphylaxis [3-7]. Importantly, there have been many other papers dealing with the use of STS in the treatment of calciphylaxis over the years (202 papers in pub med with search terms for sodium thiosulfate and calciphylaxis) and STS is often a part of the multimodal treatment strategy. Recently, 2 days ago, a repurposing of an older malaria drug, hydroxychloroquine, to treat patients with COVID-19 gave me the idea or concept to share this question with an international group of researchers on Research Gate.
Here is a list of other approved medications that are also being considered for repurposing as follows: Tocilizumab - sold under the name Actemra, Kaletra/Aluvia HIV drugs, Hydroxychloroquine as previously mentioned and Remdesivir is a broad spectrum anti-viral medication. There are undoubtedly many more under consideration and are also important in this pandemic crisis.
Sodium Thiosulfate (STS) is a potent antioxidant.
Instead of one unpaired electron as in most antioxidants, STS has (2) unpaired electrons that can be readily donated to sequester reactive oxygen/nitrogen species (RONS). During this chemical reaction process of sequestering RONS, STS also is capable of generating glutathione (GSH) a naturally occurring intracellular antioxidant and thus increases the cellular antioxidant strength in a region actively being damaged (Fig. 1 and 2).
Figure 1. This figure demonstrates the chemical structure of sodium thiosulfate, which is an anti-browning, reducing, and antioxidant agent: Capable of donating electrons to re-pair unpaired damaging electrons to be an effective antioxidant as well as a chelator of cations such as the calcium excess in calcific uremic arteriolopathy – CPLX [2].
Figure 2. Possible chemical reaction of STS to generate glutathione (GSH) [2]
Known side effects of sodium thiosulfate (STS) used to treat calciphylaxis
Here is what we know since sodium thiosulfate has become the standard of care in patients with calciphylaxis globally. Obviously, any known allergy to this or a similar sulfur-containing product would preclude its use see references [2-7]. STS may induce an acidosis early-on and with prolonged use could induce osteoporosis see references [2-7].
Sodium Thiosulfate Protective à To Detrimental Cytokine and Reactive Oxygen/Nitrogen Species Storm and Vascular Collapse
Upon initial infection with COVID 19 there is a cauldron of redox chemistry with excessive RONS (the first line of defense against the COVID 19 virus produced by the body’s protective inflammatory response to infection. The cells of immune response include the 1st responder’s neutrophils, mast cells and chronic persistent macrophages - with monocyte migration and monocyte to macrophage transformation at the infectious site of the nasopharyngeal bronchial lining epithelial cells, pneumocytes and capillary endothelial cells.
As these inflammatory cells attempt to clear the virus from these passageway epithelial cell’s they generate RONS with a staggering amount of unpaired reactive oxygen and nitrogen unpaired electrons and toxic cytokines, which over time creates a cytokine storm and RONS beget RONS and cytokines evoke more inflammatory invasion and thus create the cytokine storm. The healthy surrounding cells of the upper and lower pulmonary epithelial, pneumocytes and capillary (blood-oxygen barrier) endothelial cells become a source of collateral damage that originally was intended to result in deleting the invasion of viral envelope DAMPs and PAMPs recognized by these cells in a response to injury and wound healing mechanism to the invading COVID 19 viruses in the pulmonary tissues.
In turn, this RONS storm and cytokine storm leak into the systemic circulation and the protective lining of the endothelial cells (endothelial glycocalyx (ecGCx)) may become damaged and are attenuated and/or lost, which results in a marked increased vascular permeability and loss of intravascular contents into the surrounding extravascular interstitial capillary space. Unfortunately, this loss of intravascular volume due to a ‘capillary leak syndrome’ [8-10] may result in vascular collapse with decreased cardiac output and decreased perfusion of the brain with relative ischemia over time and result in the death of the host or COVID 19 infected patients that have progressed to being on ventilator life support.
Indeed, the COVID 19 virus infection-induced this pulmonary infection with bilateral pneumonia’s requiring ventilator treatment an acute respiratory distress syndrome (ARDS) syndrome (ARDS is an acute inflammatory lung injury, associated with increased pulmonary vascular permeability, increased lung weight, and loss of aerated lung tissue) but these patients actually may be dying of vascular collapse due to a loss of their local and systemic endothelial glycocalyx due to a cytokine and RONS STORM!
In conclusion sodium thiosulfate (STS) would have to be given intravenously; however, these critically ill patients with all have an intravenous established intravenous port to administer medication so it should not be viewed as invasive since these lines are already available. This antioxidant effect might possibly restore some effective endothelial cell function and even re-establish the endothelial glycocalyx or assist in its restoration. The increase in venous or arterial Syndecans may be a key laboratory readout of endothelial glycocalyx loss and systemic vascular collapse [10]. Additionally, patients on ventilators with COVID 19 certainly apply to these thoughts and concepts. The concept of increased pulmonary and possible systemic microvascular permeability is currently a possibility that is not being discussed a great deal at the moment but should be. Further, novel ideas for the prevention of this are of great importance during the COVID 19 pandemic. Drugs that may help to eliminate the excessive RONS with excessive over-reactive cytokines and cytokine storm (such as sodium thiosulfate STS) need to be considered as it relates to capillary leak syndrome with vascular collapse due to viral sepsis of the COVID 19 virus.
References
1. Xue H, Li J, Xie H, Wang Y. Review of Drug Repositioning Approaches and Resources. Int J Biol Sci. 2018 Jul 13;14(10):1232-1244. doi: 10.7150/ijbs.24612
2. Hayden MR, Tyagi SC, Kolb L, Sowers JR, Khanna R. Vascular ossification-calcification in metabolic syndrome, type 2 diabetes mellitus, chronic kidney disease, and calciphylaxis-calcific uremic arteriolopathy: the emerging role of sodium thiosulfate. Cardiovasc Diabetol. 2005 Mar 18;4:4. Review
3. Hayden MR, Kolb LG, Khanna R. Calciphylaxis and the cardiometabolic syndrome. J Cardiometab Syndr. 2006 Winter;1(1):76-9.
4. Hayden MR. Calciphylaxis and the cardiometabolic syndrome: the emerging role of sodium thiosulfate as a novel treatment option. J Cardiometab Syndr. 2008 Winter;3(1):55-9
5. Hayden MR, Goldsmith D, Sowers JR, Khanna R. Calciphylaxis: calcific uremic arteriolopathy and the emerging role of sodium thiosulfate. Int Urol Nephrol. 2008;40(2):443-51. doi: 10.1007/s11255-008-9373-4. Review.
6. Hayden MR, Goldsmith DJ. Sodium thiosulfate: new hope for the treatment of calciphylaxis. Semin Dial. 2010 May-Jun;23(3):258-62. doi: 10.1111/j.1525-139X.2010.00738.x
7. Sowers KM, Hayden MR. Calcific uremic arteriolopathy: pathophysiology, reactive oxygen species and therapeutic approaches. Oxid Med Cell Longev. 2010 Mar-Apr;3(2):109-21. doi: 10.4161/oxim.3.2.11354. Review.
8. Siddall E, Khatri M, Radhakrishnan J. Capillary leak syndrome: etiologies, pathophysiology, and management. Kidney Int. 2017 Jul;92(1):37-46. doi: 10.1016/j.kint.2016.11.029
9. Colombo R, Wu MA, Castelli A, Fossali T, Rech R, Ottolina D, Cogliati C, Catena E. The effects of severe hemoconcentration on acid-base equilibrium in critically ill patients: the forgotten role of buffers in whole blood. J Crit Care. 2020 Mar 4;57:177-184. doi: 10.1016/j.jcrc.2020.02.016.
10. Bøe OW, Sveen K, Børset M, Druey KM. Raised Serum Levels of Syndecan-1 (CD138), in a Case of Acute Idiopathic Systemic Capillary Leak Syndrome (SCLS) (Clarkson's Disease). Am J Case Rep. 2018 Feb 16;19:176-182. doi:10.12659/ajcr.906514
11. Ranieri VM, Rubenfeld GD, Thompson BT, et al; ARDS Definition Task Force. Acute respiratory distress syndrome: the Berlin Definition. JAMA. 2012;307(23):2526-2533.
THIS CONCEPT OF REPURPOSING – REPOSITIONING OF MEDICATIONS THAT ARE ALREADY AVAILABLE IS OPEN TO DISCUSSION AND COMMENTS
Sincerely with gratitude,
Melvin R Hayden, MD
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Interesting.. following
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Defects in the brain tissue of a rat model I am working with look like small ischemic (not hemorrhagic) strokes when I do H&E stains. I would like to confirm this suspicion by staining the (paraffin-embedded) tissue with some markers. So far I am thinking of a panel using the following: fibrinogen, fibronectin, GFAP for astrocytes. As I'm not familiar with stroke research I'm not sure if there's any common ones I've missed! I would be grateful for any advice.
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H&E is actually sufficient for a neuropathologist.
It shows you the state of the stroke, depending on the time after the insult.
You can see granulocytes coming into the stroke area latest 12-24 hours, later you see macrophages and finally the scar formation (from 1 week).
Macrophages you can stain with CD68, but they are easily detectable in H&E: white foamy cells.
Reactive astrocytes around the stroke region appear blown up and can show strange shapes (immuno-positive for GFAP).
Proliferation markers (BrdU, ki67 etc.) are not used in routine pathology and are actually not really helpful. NeuN labels neurons ..... lack means that the neurons are gone, but this you see in H&E already, or use a Nissl stain. White matter distruction you can stain with Luxol or MBP-immuno. Most of the old stains (LuxolFastBlue+Eosin, Nissl) are cheap and really suffcient.
Paraffin sections 4µm and you are done with best morphology.
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I am working on ischemic model of hDPSCs. I want to study apoptosis in the cells challenged for different periods of ischemia (3, 6, 12, 24, 48, and 72 h). I am following standard protocols of PI and AO/EtBr staining for the study however not getting the expected results. The bright field imaging showed toxicity in the cells but not in PI and AO/EtBr stained cells. Kindly give your valuable suggestions.
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I have done on tissues paraffin sections by Apoptag kit. Since you have cells and if you have flow cytometry facility, that kind of procedure will suit you.
Let us hear more from others
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Acute mesenteric ischemia is a syndrome in which inadequate blood flow through the mesenteric circulation causes ischemia and eventual gangrene of the bowel wall .
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Acute and severe low outflow states
emboli to mesenteric vessels
stenoses of SMA and coeliac axis (need complete occlusion of one and 80% stenosis of the other)
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This seems a bit nonsense question, however I am confused with this. I am doing the middle cerebral artery occlusion experiment using mice using intramural suture (filament) method. As we know the mice face towards the operator and when operator occlude the MCA (of mice) belonging his right side (operator) then it should be the left MCAo of mice, is not it? So the mice should circle (neurological behavior) to the right (left hemisphere impairment affects the function of left body parts). However, in my case when I occlude the right MCAo (my left side mice MCA while operating), the mice circles to the right. So could you explain where the problem is? Either I am doing left MCAo of mice (my left side while operating) or anything else?
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Dear Bhakta and others, I know it has been 5 years since this was posted, but I Bhakta is right. There must be more to the direction than just the side of the paralysis. Here, I have attached a video of left MCAO (confirmed by other scientist, at 24h post MCAO) the mice all circle left. The right side is partially paralyzed, but they indeed all circle left. Perhaps there is some form of neglect going to the other side, that explains this?
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What is you take on this statement ?
FFR estimation using CTangiography is a reliable and efficacious noninvasiveimaging modality, as it demonstrates high accuracy in the determination of anatomy and lesion-specific ischemia, which justifies the performance of additional randomized controlled trials to evaluate the clinical benefits of FFRCT guided coronary revascularization.
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The technology is rapidly becoming established, and certain centres already do referrals using FFR CT. It is of relevance to thoroughly test the measurements and calculations involved to impart further robustness or otherwise to FFR.
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I have just started using the intraluminal transient MCAO (30min) method in c57BL6 mice. I am thinking of including rotarod testing in my experiments, but have some questions about the protocol. In a paper "Assessing post-stroke behavior in mouse models of focal ischemia" from M. Balkaya et al (2013) they emphasis that pre operative training is important, but they also mention that "if mice are left untrained for several days their score will decline". How often do you need to, or can you test the mice after the surgery? As I understand it, this is most often used for short-term evaluation, but in mentioned paper they say that a difference could be seen as late as 28 days post surgery. Would the results be reliable if the mice were tested once a week (average of 3 trials per time point) for a 4 week period?
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Hi,
I had been working with this test for 5 years. Idealy, train the animals during 1 week pre-stroke. How to train them? There may be different approaches. In my experience (assuming I train them for three days), the first two days I place the mice in the Rod repeatedly. They fall, and I place them again immediately. After a while I give them a break, and then again. They must learn that dropping off the rod doesn't mean having a break. In the ideal scenario, on the third day, the mice should exceed 250 seconds. I recommend that you include in the experiment only those mice that pass this time. There are very bad mice, which never learn and do not provide reliable data. Now, in my experience, rotarod is not a good test to discriminate long-term damage if the injury is as small as yours (30 min). Secondly, you will see mice that are neurologically worse than others (barrel rolling for example), but they will last longer in the rotarod than much healthier animals. From my point of view, the rotarod can be a complementary test, but never the main one because it won't give you reliable data (especially with such small lesions). Rod time is an unpredictable variable that does not reflect the neurological state of the animals. There is a paper (that I couldn't find) where they show a protocol to evaluate the rotarod by video and explore other issues like posture, hand position, etc. If you want a complete and reliable evaluation, you should use this type of approach, not just time.
On the other hand, I recommend you this:
Is a better approach.
Regards,
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Using a mouse model, I will be collecting atrial and ventricular tissues at various ages. I will block the tissues in gelatin and generate 10um sections for immunohistochemistry.
Does anyone have suggestions for what is typically used in the field to identify cardiac dysfunction/ischemia immunohistologically?
I have seen reports that increased levels of Inosine and hypoxanthine occur following acute ischemia preceding increased troponin levels. Additionally, I have seen reports that proNGF increases in ateriols following myocardial infarct. I am new to this area of research and want to proceed properly.
Thank you everyone,
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you are welcome
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From the above article I have come to know that during Brain Stroke there are ECG changes in T-wave and QTc interval. Ischemia like ECG changes are seen in Stroke patients, though they don't have any previous heart diseases. I suppose that is just a case but not as supporting for a decision.
Can there be some other way which can be suggested ?
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ECG will not help in the diagnosis of stroke or brain attack. Diagnosis of stroke is based on history, clinical examination and imaging. However a variety of ECG changes can occur in stroke, mostly involving the ST segment and T waves Arrhythmias, both tachy as well as brady can also occur infrequently.
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Link for detailed procedure is required.
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Hi Wasim,
The LAD ligation technique is a well established model, yields reproducible rat model for MI and cardiac ischemia. The trick to succeeding in bringing post -p mortality rate to a min, is a good surgical skills and a volume controlled Rodent Ventilator.
Please check this article:
Best of Luck,
Ghanim.
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Inflamation is asociated with higher BP
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I use Lagendorff ischemia-reperfusion for rat hears 30 min ischemia then 90 min reprfusion. Half of the hearts did not come back (died) after ischemia. Any reasons for that?
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Hi...
How to stain Myofibroblasts in myocardial infarcted hearts? What about Alpha- Smooth muscle actin in the detection of myofibroblasts? Is there any other specific markers for the detection?
Thank you.
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Dear Budharaju,
I have used CD140a (PDGFRa) for mouse fibroblasts and I am planning to work with CD90 in human fibroblasts. However this is not sufficient if you need to separate fibroblasts from myofibroblasts, I suppose.
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I am currently working on ischemia- reperfusion-induced brain injury in Wistar rats, and as of now we are using Ketamin-Xylazine or low dose of Urethane for inducing anesthesia. I have over heard that these agents could greatly influence the ischemia-reperfusion-induced brain injury and hence looking for better and safe anesthetic agent.
Based on your experience and domain knowledge can any one refer better anesthetic agents for my experiments.
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Buenas noches
La pregunta parece va dirigida a anestésicos IV, indudablemente la ketamina lleva ventaja sobre otros anestésicos; en mi experiencia el segundo lugar sería para el propofol, luego el tiopental.
Para mejor opinión deberiamos conocer el objetivo de la investigación.
Gracias
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There is currently an issue with ECMO cannulae on the market and limb ischemia. My senior capstone group is curious as to what our target flow should be to prevent this issue. We understand that returning to homeostatic levels with a single insertion point is a lofty goal, so the minimum level gives us a starting point before beginning our concepts.
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Oh. I may have misunderstood the question. 8F, inserted in the SFA, is used to antegrade perfuse the leg when the ECMO inflow cannula - 15F-25F, is obstructing the flow to that leg causing the limb ischemia. 8F is NOT the primary inflow cannula.
The catch 22 in fem-fem ECMO is - if you want to flow more, you will need a bigger cannula to minimize the blood trauma caused by the high pressure drop. Also, you may require very high RPM to flow through the smaller cannula. The bigger cannula on the other hand, like you mentioned, may obstruct the flow to the limb causing ischemia. I understood that your group is trying to tackle this very conundrum.
Sorry if I muddied this up for you...
Sanjay
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Recently I am investigating the therapeutic effects of miRNA in ithe cell model of ischemia/re-perfusion injuries. I would like to quantify the levels of target miRNA isolated from ADSCs. Now I have to choose the control group so that the levels of target miRNA could be compared.
Does anyone have any suggestion for how to choose the control group?
Many thanks.
Best
Gabi
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Some approaches exist
1. External spike-in C. elegans miR-39 miRNA mimic. Add it into lyzate
2. Compare with stable miRNA level. Use Genevestigator or similar tool for such miRNA identification
3. Use total blood miRNA for comparison cause a lot of studies - it will be easier to find comparable group in dbGaP\GEO or to collect samples form the same individuals
4. Use an aliquote of the same cells without exposure as a self control
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Hello
i use h2O2 (100uM) for 24 hrs to mimic ischemia model in the cells (cardiomyocytes) . so when i do ROS measurement using DCFH-DA, do i need to add a positive control like ROSUP(solarbio kit) or h2o2 again in my positive control sample? because i have already treated the cells with h2o2 for 24hrs.
(i will use confocal microscope)
please comment.
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I have published 2 papers using DCF to detect ROS that might help you.
The first, a very simple one. Using an old fluorescent microscope, was done on a cell line I "created" that had high ROS production and it was possible to detect the difference. I still remember the wonder of seing the result so clearly. So I disagree with Dr Warnes answer, you should be able to detect the difference using a microscope.
The second paper uses a fluorescent plate reader to measure ROS in cells growing on 96 well plate which allowed me to test many different conditions. I only treated the cells with 500 uM H2O2 for 30 min before the measurement. This are HUVEC cells, also primary cultures. It is always nice to have a good positive control. I suggest you try at least in your first assay to have such a control in an extra well (or plate). I also found that the best positive control was to live the cells out of the incubator at RT for a few minutes. This stress increases ROS production very fast.
Conclusion: I recommend you include one or 2 more positive controls. And of course also 1 or 2 negative controls. You should also have a control with a good antioxidant to show that you can revert the effect, this will strongly suggest that the changes you see are due to ROS increase.
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I have collected samples from a novel chronic kidney injury model that i am currently studying (as well as control groups).
Rats were sacrificed after several weeks, and kidney tissue samples collected. On the other hand, urine samples were collected weekly.
I will analyse these samples using western blot and other techniques but before this i am considering which loading marker to use.
I have seen that levels of:
- GAPDH can increase with hypoxia
- Ischemia also strongly increases beta-actin and alpha-tubulin expression (PMID: 15464724)
- B-tubulin expression changes with the administration of antineoplastic drugs.
Has anyone got an idea?
Thank you to everyone.
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Could try beta-2-microglobulin or HPRT1
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Given that there is no definitive treatment for ischemia, is there a need for positive control in ischemic studies?
Can you use a drug such as Trimetazidine?
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I fully agree with Dominik, thinking that a positive control is necessary, simply to observe the depth of ischemia and to compare from one experiment to another
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i want to make model of global cereberal ischemia reperfusion in rat
so my question is ligation of common carotid artery is enough for induce global cerebal ischemia or i should induce hypotension with bilateral common carotid artery ligation
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You welcome!
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Hello everyone (my first question on this site)
I am inducing ischemia in H9C2 cardiomyocyte cell line. The overwhelming majority of publications use 95% N2, 5% CO2 gas concentrations for their incubation. Is there a specific reason why CO2 is not used as the main constituent of the gas mixture? Is it due to the fact that CO2 may cause excessive acidity of the media? Would CO2 elicit a different cell response?
Any help would be greatly appreciated.
Regards,
Sergey
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Hey Sergey,
inducing ischemia (or rather simple hypoxia because I leave nutrients out of the game) is based on the "displacement" of oxygen by nitrogen. As a thing to remember: That is what will happen if a container with liquid nitrogen will leak in a small storage room (that is why you always should have an oxygen sensor next to it): it will lead to asphyxiation.
The CO2 should stay at 5% since it is comparable to the body CO2 content (and even more important: keeps the right pH in your media). So you are right: It would cause a ridiculous pH and cells would die so you would not have any response at all.
Glad I could help and good luck with you experiments
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i came by a research paper the author of which attributed the death of a rat in their experimental group to the re-perfusion method . I myself have been having a very high rat of mortality in my experimental group after MCAO (1 hour ischemia). The mortility is so high thatthe animal did not even qualify the minimum 4 hour reperfusion period for TTC staining.The question is how to minimize the mortility and also that how is the method of reperfusion related to the mortility?
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From my own experience, if animal weight is too small, mortality will increase. Also, if the operating temperature around the animal is not well-controlled or anaesthesic is not properly given, the mortality could increase. In addition, if your experiment includes additional pathology e.g. diabetes, mortality will be high.
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Im doing a I/R model with C2C12 cells and I need to measure ROS production. First I will induce ischemia with the ischemia chamber and then I will measure ROS after 4,6, 8 and 12 hours. In the same timepoints I will induce reperfusion, but I dont know after how many hours after reperfusion its  recomendable to measure ROS. 
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Thank you so much !
I have done a trial, and yes, for example after 4 hours reperfusion the ROS production decreased. Now I wil repeat it, measuring ROS production 15, 30, 45 and 60 minutes after reperfusion.
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A young male 30 years old healthy BMI 29 ; no comorbid condition presents with superior mesenteric ischemia- what is left is T-colon and Duodenal stump;
On laparotomy; tube duodenostomy is being done; how to manage such a case ??
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Dear Ahmad.
You can get information on AMI here 
It is unclear which type of AMI you have met in that patient. Please, pay attention to NOMI and necessity of pharmacological correction. In any case, now you have to manage full duodenal external fistula and a short bowel syndrome. If so, then: 1. TPN and 2. anastomize asap. 
Sincerely Vladimir M.Khokha.
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I want to know about how to measure the heart infraction area after ischemia injury.
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Hi Jameel,
if you need step by step model please see this article.
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Prostatic artery embolism is an interventional radiological procedure which can be done on TURP-NON eligible patient for BPH, it involves entrance to Prostatic Artery through the femoral artery- and embolizing w/small bubbly material.
My question is- Due to ischemic necrosis of Hyperplasic prostatic tissue there will be some kind of erosion of that tissue from rest of prostate--is it possible to see that necrotic tissue can block flow of urine- leading an obstruction of urine outflow?
My other question is-- perfusion of pelvic structures are quite complicated and involves lots of small anastomosis'-- after disabling flow of the Prostatic artery, what are the chances of getting bladder neck into an ischemic necrosis--due to possible anastomosis'? 
I thank you all for your answers and your comments.
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Hi Sal,
PAE for symptomatic patients with BPH has been widely performed with low complication rates, however, there is a potential for severe complications, including technical and clinical treatment failures. Long catheter time after the procedure and repeated catheterization are seen frequently due to failed trials of voiding without catheter. We have experienced such worse outcome during conducting a comparative RCT between PAE and green light laser prostate ablation.
Furthermore, PAE has many limitations, including  lack of significant improvement in IPSS or Qmax in 25% of patients,  unknown long-term durability,  need for high dose ionizing radiation dose and contrast material for procedural guidance, can not be technically achieved on one or both sides as a result of  atherosclerosis, small artery size, and/or tortuosity and  collateral circulation may be present, and maintains vascularity.
To date, no high-quality multi-center RCTs have been published on safety, efficacy, and cost-effectiveness of PAE for BPH.  Most of the studies are of low quality due to their case series design. Therefore, advantages of PAE must be weighed against the risk of technical and clinical failures requiring a second intervention. 
Regarding your question, bladder neck will not be affected by a selective technique done by an expertise.
Regards
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I need to mark mitophagy in spinal cord, muscle and cortex
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I'm looking at microglial activity, and in some of my sections there is no H&E evidence of neuronal death but there is still microglial activation. I want to stain for neuronal stress to elucidate whether the microglial cells might be responding to a pre-apoptotic environment or if they are activated by the ischemia directly, what stain could I use for paraffin-embedded tissue using dab or fluoro IHC?
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Superoxide dismutase 2 (SOD2) is up-regulated in response to oxidative stress. We have used http://www.abcam.com/sod2mnsod-antibody-ab13534.html to successfully demonstrate this up-regulation on sections of FFPE rat and mouse tissue.
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Please help me. Is anybody working on Cardiac Ischemia using the rat as a model?
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I am working on in-vivo model for myocardial ischemia and reperfusion. If you would like to add your e-mail address I can send you some videos too. 
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Perfusion MRI shows more detailed area of infarct bone
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Perfusion MRI is the best way to evaluate hip at children with LCP between 4 and 8 years. Before 4 years old or after 8 years (age of onset) the treatment don't change very much the final result (after Salter and Harring opinion) and perfusion MRI isn't really necessary at first visit.
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i use ketamine/xylazine (110mg/kg and 25mg/kg) for sedation, buprenorphine administered at 0.1mg/kg before surgery and body temp maintained at 37*C during surgery. Most of the mice are dead within 20-30min of ischemia.
All the protocols that i have looked into recommend 2% isoflurane in oxygen, is it a good idea to completely shift to that.
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hello,
We had the same problem but  with Livers ischemia-reperfusion, everything change when we made inhalation  anesthesia ( isoflurano) in the special device (SomnoSuite)....
Good luck for you
Regards
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I am working with kidney
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Apoptosis is an ATP dependent process. It is caspase dependent: If you detect caspase cleavage (e.g. caspase 3), caspase activity (cleavage is not always increase in activity) and the cell death can be blocked by (more or less) specific Inhibitors (e.g. zVAD) it is likely that you see apoptosis. In addition, you can analyze DNA fragmentation (e.g. Nicoletti assay). Further, Annexin V/7AAD staining will help. Apoptotic cells will become Annexin V positive (7AAD neg.). Only at later time points you will detect double positive cells (late apoptotic/necrotic).
Necrosis is ATP independent. You should see cell swelling, no caspase involvement and finaly the cell will disrupt. The cells are AnnexinV/7AAD positive (necrotic).
Necroptosis is a programmed necrosis. It is controlled by the ripoptosome. Cells undergo apoptosis, but if apoptosis is blocked (zVAD) cells shifts towards necroptosis. Necroptosis is Rip3, MLKL dependent.
Ferroptosis is Gpx dependent and iron dependent. It can be inhibited by use of (non redoxcycling) iron chelators.
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Looking for a way to assess neuroinflammation in the brains of healthy older adults.
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Shalom Tamir,
Diffusion imaging can be used to detect markers of neuroinflammation. For example, low b-value dwi/dti can be used by looking at radial diffusivity (a marker of myelin integrity). Also, there are other potential markers of neuroinflammation which can be gauged from ihMTand MTR, for example. See the recent papers on this (e.g. https://www.ncbi.nlm.nih.gov/pubmed/26048294  , https://www.ncbi.nlm.nih.gov/pubmed/24769473https://www.ncbi.nlm.nih.gov/pubmed/25724201).
Regards,
Jerome
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I'm looking for venous structure divided the liver in to right and left lobes.
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he functional segmental anatomy of the liver is based on the distribution of the three major hepatic veins. The middle hepatic vein divides the liver into left and right lobes. The left hepatic vein divides the left lobe into medial and lateral segments. The right hepatic vein divides the right lobe into anterior and posterior segments. In addition, the four sections are further subdivided in a transverse plane by an imaginary line drawn between the right and left portal veins. Segments run in a clockwise fashion, with segments III, IV(b), V and VI lying below the portal veins and segments VII, VIII, IV(a) and II lying above. The caudate lobe (segment I) lies posterior and to the right of the inferior vena cav
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Hi,
I am currently trying to establish a model of intestinal ischaemia/reperfusion injury in mice, in my lab, with a goal to producing local injury and acute lung injury as a result. However, I am currently having issues with mortality in my I/R animals. My protocol is as follows:
1. Anaesthetise male mice (8 weeks old) with ketamine/xylazine i.p. and transfer to a heated platform.
2. Perform midline incision and laparotomy.
3. Identify superior mesenteric artery. I/R animals have the SMA clamped with a non-crushing surgical occluder for 30-60 minutes (have tried both). Sham animals are treated identically, except for clamping. Ischaemia is confirmed by pallor of the intestines and reflex tachycardia.
4. Gauze is soaked in warm PBS to cover the surgical site for the extent of ischaemia.
5. Clamp is removed to reperfuse the intestines and 1mL of warm PBS is administered i.p. before suturing the muscle and skin walls closed.
6. 30 mg/kg Evans Blue is administered to assess inflammatory oedema secondary to injury.
Mice that underwent 60' ischaemia died at 75 and 90 minutes reperfusion, whilst the sham counterparts survived. Mice that underwent 30' ischaemia died at 45 minutes reperfusion, whilst sham counterparts survived.
Clearly, this is an issue related to the severity of the model, yet so many publications use similar ischaemia/reperfusion timepoints - does anyone have any ideas of where I might be going wrong? I could try and reduce the ischaemic time further, but I risk not inducing a severe enough injury to give me the readouts I need.
Any/all input would be greatly appreciated!
Thanks
Ross
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Ross, I guess this is inadequate resuscitation. 1ml PBS sounds like a lot but so is fluid loss after total bowel IRI. Try to slowly inject fluid in the spleen (30G needle and then compress with earbuds 30 sec)... that is iv... besides leaving 2-3 ml ip. Then use warm pad or lamp.
Then, Look at the bright side: you have a mortality that will make the effect of an intervention pretty visible. 
Good luck!
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In limb ischemia, after revascularization, some patients suffer from SIRS and MODS. Who plays the major role, ischemia or reperfusion? 
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This is question without a clear black or white answer. Each organ has its own susceptibility for ischemia (e.g. brain vs leg). The other point is that a systemic reaction like MOF can both result from ischemic damage as from reperfusion damage (we measured Arterial Venous concentration differences over the reperfused kidney during organ transplant and found prominent cytokine release that appears to reflect a response to ischemia rather than reperfusion PMID:19459788 ; whereas reperfusion injury appears to relate to a metabolic defect PMID: 27188504 it is unclear to me how this would result in MOF).Note that we did not find indications for neutrophil, thrombocyte or complement activation in the reperfused organ.
Besides we do not find indications for gross oxidative stress during clinical reperfusion; as such a putative central role for ROS in human I/R is questionable. PMID: 23305329; 28031169; 26999803. This notion is supported by the consistent failure of clinical trials studying antioxidant-based therapy.
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2 month old baby after irreversible ischemia of hand waiting for line of demarcation on anti coagulation developed swollen hand with blisters and blackish discoloration of skin to wrist where as dry gangrene of distal fingers,How to differentiate Line of demarcation?
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I will palpate to find good pulsation and circulation of the area
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I want to do a research about the the protecting fuction of Nuclear factor-erythroid 2-related factor 2 (Nrf2) in ischemia brain disease.
However, I do not konw what kind of Nrf2 activator and inhibitor we always use?
Can they pass through blood brain barrier and regulating the Nrf2 protein level of brain tissues?
Would you mind telling me which article is the most authoritative one?
Thanks for your great answer!
              A new comer of ischemia brain disease.
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This is one of the inhibitor of keap1-Nrf2 interaction. There are several of them. But this has good computational and in vitro results. Hope that helps. MG
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Some time in case of extensive PAD and critical ischemia we need to perform the simultaneous reconstruction of aorto-iliac and femoral-popliteal segment. What is your opinion about the optimal site of proximal anastomosis for femoral-poplieal bypass with reversed saphenous vein? Should vein takes-off from the synthetic graft or from the common femoral (femoral) artery? Some surgeons claimed against the anastomosis between synthetic material and vein.
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I favored to anastomose the aorto-femoral graft limb end-to-side to the common femoral artery with a tongue extending for about 1-2 cm in the profunda femoris artery, and the venous bypass (preferentially over non-autologous graft) end-to-side implanted in the graft limb. I used to implant a venous patch in the graft limb for anastomosis with the vein graft in case the vein graft appeared rather small (< 4 mm in diameter). In skinny patients and patients in whom skin healing might be jeopardized (for instance a redo) I would cover the graft and its anastomic area with a sartorial muscle flap over which the skin is closed with an continuous intracutaneous absorbable suture.
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There appears to be a low dose therapeutic window for alpha lipoic acid in both schizophrenia and ischemia reperfusion.  Could disturbances in glutathione homeostasis, either through increased synthesis or reduced degradation be a factor?  The half-life for both alpha lipoic acid and glutathione is very short.
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Thank you very much for suggesting the article, an interesting read with many promising paths to explore. 
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There was already a significant difference in the infarct size. The ischemia time was 30 min, followed by 24 hours of reperfusion.
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MRI may be tricky in detecting the difference since the cardiac remodeling is not that obvious only 24h after reperfusion, instead, Echo is better than MRI regarding the cardiac function post surgery. 
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I am having trouble keeping them alive after the clamp is removed.
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 Thank you Matt,
I git the model up and running with 90% success.  I was making it harder than was.
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I want to compile a list of acute pathological or physiological events that last no more than 5 min. Currently, I can only think of cortical spreading depression (CSD) and seizure that qualify for this standard (very short, duration less than 5 min). Status epilepticus maybe OK, but I still think it lasts too long. Ischemia is not qualified for the same reason.
Can some one give me more examples? Any physiological and pathological events will be fine, as long as they occur acutely and are short-lived.
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Do you go for pathophysiologically or clinically defined events? I didn't get it quite clear when reading your question.
As a clinician, I would add hemifacial spasm and trigeminal neuralgia. Status epilepticus is defined by either 5 or 30 minutes ongoing seizures activity, so it won't qualify. Neither does spreading depression, at least when you translate it into complicated migraine, where the corresponding aura can last a lot longer. Ischemia is qualified if you consider TIA or syncope, which is finally transient global cerebral ischemia.
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Hi everyone,
I am operating mice to induce ischemia in the left hindlimb by ligating the femoral artery. The procedure is well established i our laboratory and after three years of working on the project I might say we got quite experienced with it.
However, recently we see that mice of one certain genotype develop swelling of the right (=not operated) hindlimb. Infection is not likely, as the rest of the mice (operate leg, eyes, etc) are not inflammated, red, or swollen...
Does anyone has an idea what might be the reason of this swelling?
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Bart,
Perhaps the ligation of the left femoral artery is causing a blood clot to form near the ligation.  The clot may be released and flushed into the common iliac artery or aorta and into the right femoral artery.  This could occur during the surgery (since blood flow in the right femoral is maintained during surgery) or just after the ligation is released.  Such a clot would cause an obstruction in the blood flow and swelling in the affected limb.  Don't know why this would be genotype related.
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ST segment in ECG signals 
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As already mentioned, we know the "pericarditis poststenocardiaca", also called Dressler Syndrome, which is different from the "pericarditis epistenocardiaca", occuring localized  together with and not after a myocardial infarction. The ECG signs may mimic an ischemia, but are, especially in case of the Dressler Syndrome, more generalized in nearly all ECG leads, often accompanied by an also generalized low voltage.
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Recanilization of the occluded vessel is guided by the state of perffused territory. In acute state recanalization is warrented within limited theraputic window. What about in cases of chronic ischemia or acute ischamia in setting of chronic occlusion?
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Hi Dr Mansour,
You may find the experience of my colleagues in Pittsburgh interesting. They reported the revascularization of chronically occluded carotid arteries. 
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I'm having trouble finding papers that clearly point out what exactly induces apoptosis in macrophages after ischemia and its effects, later on, on the failing heart.
Any links to papers would be greatly appreciated.
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Dear Mr. Chokski,
I think the publications suggested by Rueter are interested for you.
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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
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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
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In particular, may be nitrergic neurons are more sensitive to ischemia than other types?
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Hello, do you know any paper reference assessing a potential differences of sensitivity of pyramidal vs granular neurons to ischaemia or hypoxia ? Thank you.
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When I expose my primary cortical neurons to hypoxia(1%) in a professional hypoxic workstation, only about 10% of the cells die at 24 hrs. Even at 48 hrs, it just reaches up to 20%-30%. Is it normal? Should it be that neurons are more susceptible to hypoxia? Thanks~
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Hi, Helder André ,I am wondering what do you mean that  neurons are naturally exposed to hypoxia? Do you have some literature about this topic?Thank you~
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Two years ago, Zhang et al. found mitophagy or autophagy for mitochondria is protective in the reperfusion phase, while deleterious in the earlier ischemia phase. But at that time, seemingly they didn't give explicit explanation for that discrepency. So recently I wonder, to date is there any attempt to unravel the puzzle?
Thank you in advance.
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Hi Nai-Kei!
Thank you for the answer. But I still wonder why in the stage of ischemia, unlike the case in reperfusion, mitophagy exacerbates the injury?
Hao
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Dear members,
Can anyone kindly suggest me about suitable in-vitro model to study ischemia. I tried with neuro-2a cell lines by inducing apoptosis by various concentration of cocl2. it induced apoptosis but i could not found up-regulation of any related gene (TNF-a, Fas). I want to make a in-vitro ischemia model to study Fas mediated apoptosis and want to check my developed fas targeting peptide. Kindly suggest me about cell line and developed protocol.
Regards
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Did you try Oxygen-glucose deprivation (OGD ), which is widely used as an in vitro model for ischemia? In order to perform OGD, cell or tissue cultures are usually incubated in a glucose-free medium under a deoxygenated atmosphere.
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I've found various different paper which used I/R protocols in H9c2 cells to bring those cells into apoptosis, but none worked in my lab so far. Additionally, I've found none where they tested the anti-apoptotic effect of insulin after/during I/R in vitro.
Hope you can help me
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Main reasons of unsucessfull I/R protocols are: insufficient hypoxia, additional acidosis (which is per se protective), non physiologial external calcium, presence of serum in medium. My advice is to measure the actual oxygen partial pressure in the experimental medium et to evalutae the response to lack of oxygen via methylene blue test.
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i would like to know the best way to track down neurogenesis spatiotemporally in my samples, please suggest the best method? which is the best method?
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As said Christopher, you may specify what do you want really, analyzing your results of neurogenesis or to perform neurogenesis. If the second option, you want, some answers have been given above, notably concerning the use of BrdU and if you are interested I can give you more informations. 
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When intestinal tissue is analyzed for IFABP it appears to us as a very nice band at 15kDa, but always shows an "exactly" 30kDa band when serum is analyzed for western blot (and nothing else below this size). Do you have any idea what this means? the IFABP should be released into serum after intestinal ischemia (normally evaluated by ELISA), but in serum we find this higher band. Thank you!
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Hey,
latest the addition of and SDS excess in the loading buffer as well as in the gel will disrupt residual dimers. Comparing a native and a reducing page is not optimal, because there are more points that influence migration beside the size of the protein ( acidic groups, polarity...). So you could think about adding more DTT (200mM should work), or if you really want to know which kind of PTM ( I just assume it is not due to dimerisation because of denaturing conditions) is it, you could start with deglycosilation and then analysing the sample
Regards Sven
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I am having TP, TN, FP and FN for detected and classified ST segments for detection of ischemia in ECG signals
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A ROC curve is a useful way to study and illustrate the way a parameter affects a classifier. Say you want to study the effect of parameter A. Choose a value for A and perform the classification. Count correct decisions and incorrect decisions and obtain sensitivity and specificity. This gives you ONE point for the ROC curve. Now change A and repeat the procedure till you have enough points to plot the ROC curve. If your ROC curve has a sharp 'knee' towards the left-hand top corner it means your paramenter is good. The closer the ROC curve is to the main diagonal the worse the parameter is. One way of quantifying this is measuring the area under the ROC curve (AUROC): Area close to 0.5 meaning a poor parameter and area close to 1 meaning a good parameter.
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Is right hemicolon ischemia post Ivor Lewis Esophagectomy a well noted complication?
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No - its not a well recognised complication.  What was the mechanism of ischaemia?   Dissection is normally well away from the colonic mesentery and ileocolic vessels.   Any previous surgery / intra-operative difficulties etc etc........
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Would you advise against use of cortical or whole brain slices for in vitro I/R studies? Why?
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Yes.... the larger is your explant, worst is the media (and gas) perfusion/exchange. So, as you increase the size of your explant, higher is the risk of cell death by hipoperfusion, specially those cells located in the middle of the tissue (central area). 
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Failed endopyelotomy can be due to irreparable pelvi-caliceal system,puj ischemia with restenosis, anastomotic leak with urinoma and fibrosis.
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If you had anastomotic leak with urinoma and fibrosis the best option is open pyeloplastic otherwise perform laproscopic pyeloplastic 
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My understanding is, when the myocadium cells lack of blood supply, the cell will be damaged but to some degree, it's still reversible. When the blood flow recovered, the cell can become alive again. When the ischemia is serious to a certain stage, the cell will die completely. Thus even the blood flow recover, the cell cannot gain live again, call irresversible. (if these are not true, please kindly correct me)
And my question is, for a specific patient, how to judge if his ischemia is reversible, irreversible? E.g. by using some imaging machines??
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How to determine if ischemic hearts have irreversible ischemia_And How to treat them? Answer from a Clinical and Physiology investigator
First, assuming that severe ischemia or infarction occur as consequence of coronary obstruction or occlusion (clot) usually shows large number of coronary collateral anastomosis (inter/Intra artery anastomosis) along with precise natural bypass in the site of the major obstruction (Baroldi G et al, Circulation Res. 1956, Cardiovasc. Ultrasound 2005. His book is available from Internet, 2002): Macrovascular CHD. In these patients is better to assume that myocardial in “bypassed artery” has reversible options. In effect, ECG are reversed or returned to normal, under a novel therapeutic approach (Brief discussed at the end).
Second, assuming that severe ischemia or infarction occur in presence of “normal selective coronary arteriogram” (Likoff W et al, NEJM 1967), “a changing Philosophy“(Bugiardini R, Mertz BCN, JAMA 2005) implies that other critical factors, as the microvascular blood flow and O2 delivery are missing: Microvascular CHD.
However, in any scenario the incontrovertible fact is that O2 delivery occurs at the cellular level and involves a critical role of red-cell K-dependent ATP synthesis and release in presence of low pH or low PO2 (Ellsworth ML. Physiology, Bethesda 2009). Therefore, impaired capillary vasodilation instead of blood flow of the conductive coronary arteries) appears to be a major factor in the pathogenesis of CHD (Research Gate: Delgado-Almeida. Am Coll Cardiology Dec 2014, Figure).
This critical role in red-cell K-dependent function includes the K-activation of O2 binding by human hemoglobin (Delgado-Almeida A. FASEB J, 2012), suggesting that an enhanced hemoglobin O2-binding in the lung capillary bed is a required step for anti-anginal effects of nitrates or any other drugs, while explaining the failure of intracoronary administration of nitroglycerin to relieve angina induced by pacing, rapidly reversed by intravenous or sublingual doses (Ganz W, Marcus HS. Circulation 1972).
Third, how to determine if ischemia is irreversible or reversible damage? And How to treat them
a) Clinical and ECG serial evaluations and others as Echoc.
b) BOLD analysis (intra capillary levels of deoxyhemoglobin, MRI) documenting that reduced coronary perfusion in CHD does not always implies deoxygenation, opening a new way to assess myocardial ischemia (Karamitsos TD, Circ. Cardiovasc. Imaging 2010).
c) Improving the inherited effect in Red-Blood-Cell Potassium Content recorded in hypertensive and half of their normotensive offspring, as reported by our laboratory, confirmed to be a critical factor in vascular, renal function and total body water and K content (Delgado-Almeida A. Circulation. Abstract, AHA 2013).
d) Preserving the impaired RBC-K uptake related to drugs, particularly Diuretics and β-blockers (Oski FA et al, Science 1972, Agostoni A et al, Science 1973, both with propranolol) inducing abrupt K-efflux from RBC-K and disturbed oxygen affinity to hemoglobin.
e) Finally, a Physiological and Therapeutic approach addressing the defective RBC-K content and functions in CHD, by a novel composition of Amiloride HCl Dihydrate, allowing to improve Central Aortic BP and systolic pulse waveform reflection (type II-IV), reversing ischemic ECG with normal ECG in half of angina patients (in 6-months) and inducing electrical regeneration in previous areas of old infarcts (Delgado-Almeida A et al. Recent Pat on Cardiovasc Drug Discov.2010 and 2012).
Although these paragraphs might support myocytes regeneration of adult human heart by resident or bone marrow stems cells, the fact is that collateral anastomosis recorded by angiography are clear angiogenesis evidences in ischemic hearts. These peripheral and capillary support in CHD may explain innate regeneration of the heart and isolated living cells surrounded by large infarction (Anversa P, Leri A. Mayo Clin Proc. 2013).
Of note, that the role of stem cells in human biology might be recognized and traced as far as two centuries ago in different tissues and organs: Bones (bone reparation in fractures, cited in NEJM 1800’s), Liver (Donor and receptor of hepatic lobule, leading to almost normal anatomy by MRI and function in 3-4 months), Heart (collateral anastomosis and electrical regeneration of the heart in CHD). References for Bone and Liver Regeneration in PubMed.
FIGURE: Erythrocyte K-dependent ATP Synthesis-Function (See Delgado-Almeida A. β-blockers in Angina. J Am Coll Cardiol. Dec 2004; 64:2710-12).
FIGURE LEGEND: Electron microscopy views at the myocardial capillary net, in which diameters and blood flow is tightly controlled by a RBC-K dependent enzyme (pyruvate kinase activity) required for ATP synthesis and ATP release in the presence of low pH or PO2 (See, References 11-13, 38).
In myocardial cell, RBC release 1 mmol of O2 and 0.7 mmol of K from Oxyhemoglobin, in exchange for protons and CO2 from the myocardium; the reverse of K and O2-binding occurs at the lung capillary beds, as documented in our laboratory by in vitro studies in human venous blood samples (FASEB J 2012, Delgado-Almeida A).
An anatomical aspect is well evident in this picture, the larger RBC diameter (7±0.5 µ) versus 3.5-5.0 µ for most microvascular lumen (purple arrows). However, despite narrower capillary diameters, the flow velocity of these cells in healthy subject is 300-400 µ/sec, critically dependent of RBC-K dependent Pyruvate Kinase activity for ATP synthesis and release, the most powerful regulator of capillary vasodilatation and blood flow. Therefore, impaired vasodilatation and longer RBC transit time, instead of arterial vasoconstriction appears to be the most critical factor in essential hypertension and CHD, and probably represent a novel paradigm in the therapeutic management of these cardiovascular diseases.
See References in RG.net
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This situation has been plaguing our lab for some time. When exposing hippocampal slices to ischemia either they recover fully or develop an injury potential and do not recover at all? 
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Thanks Mark for taking the time to reply and my apologies for not providing enough detail. I use a submerged chamber. Flow rate is between 3 and 4 ml/min. To induce ischemia we use 0mM Glucose bibbled with 95%N2/5%C02. After 6-8 mins of this procedure and injury potential appears and then there is no recovery, even if we switch perfusate immediately. If we increase flow rate when recovery does occur it is greater than 90%.
So the basic problem is when to turn off the ischemic conditions to produce a partial recovery of function?
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There is very few data on how to manage these lesions, but the thromboembolic potential seems to be high, namely visceral and renal artery ischemia. Do you think we should preemptive treat these patients?
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I think the first option is aggressive anticoagulation if patient is asymptomatic with close monitoring with TEE. I' ve seen 2 patient treated in this way with good result. TEVAR shows a theoretical embolic potential larger than a conservative approach. Open surgery is only for peculiar cases of associated lesion. Of course we ' ve to consider a second stage of treatment if the thrombus is due to an atherosclerotic burden of the aorta rather then to a coagulation disorder.
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Intravenous thrombolysis is an effective treatment of ischemic stroke, if administered within 4.5 hours from outcome. Since sudden hearing loss is probably due to cochlear ischemia, intravenous thrombolysis within 4.5 hours from onset of deafness should be effective in this condition, too.
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Still, if it happened to me I would greatly appreciate having a cure...
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As there are three definition available in literature for ischemia.
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Dear Anit Manocha, surely ischemia produces some sort of ST segment elevation. You can find some guidelines in:
Cardiovasc Res (2000) 45 (1): 111-118.
doi: 10.1016/S0008-6363(99)00301-6)
"ST-segment elevation in the electrocardiogram: a sign of myocardial ischemia"
by Andrè G Kléber*
Department of Physiology, University of Bern, Bern, Switzerland (* Tel.: 41-31-631-8740; fax: 41-31-631-8785 kleber@pyl.unibe.ch).
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Simple question: Are there any published studies regarding the clinical benefit of I.v. tPA in ischemia stroke in a population based on proven vascular occlusion (e.g. By CTA). All the initial placebo control studies needed only a non contrast CT and therefore the site or the mere existence of a vascular occlusion was not shown. Thank you
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consistent with Lahoti s Stroke 2014, which focuse on "no occlusion" papers
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I am studying gender differences following cerebral ischemia, and i have surprisingly noted that NSPCs donot express BDNF after 7 days of ischemia. I performed IHC and co localization studies with Brdu . Has anybody seen the same expression pattern of BDNF? 
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I am working in cerebral ischemic models. I generally used in vivo models in mice or rat for the ischemia induction. I am also interested in in vitro ischemic models such as OGD-R. Would be very grateful if anybody could provide me the detail of the ogd protocol.
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This surely can help you!
thanks
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As it is obvious that the level of TNF-alpha is increased following cerebral ischemia. I am going to perform the immunostaining for TNF-alpha positive cells in mice brain sections which is challenged with MCAo induced focal cerebral ischemia. I am going to use the DAB substrate for this purpose. Could anybody provide me the detailed protocol for this purpose?
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Hello I have done it to the spleen, and i have to confess that it does not look great with DAB9 (IHC), It looks better if you use IF: Here it is the protocol:
Wash 3X with TBS ( tris buffer saline) ph: 7.4
Incubate in SSC ( sodium citrate)(1 Molar concentration in TBS), ph :6 for 40 min at 80 Celsius.
Remove section from water bath, and let cool for 5 min.
Wash 3 X with TBS
Incubate in block for 60 mins ( TBS-TS) 0.1% tx-100 + 10 % goat serum
incubate in primary PBS-TS + primary ( 0.1% tx-100 + 3% goat serum + 1:50 Rabbit anti-rat TNF-α (NOVUS NBP1-19532) over night at 4 C
Day 2
wash 3X with PBS-TS ( 0.1% tx-100 + 3% goat serum)
Secondary Ab: incubate in 2Ab for 2 hours ( anti-rabbit from goat jackson cat# 111-065-144 for DAB staining ) or any Alexa fluoro anti rabbit secondary for IF.
For DAB: wash 3 x with TBS, then incubate in ABC ( Abivin complex) solution for 1 hour ( 2 drops of A and 2 drops of B).
wash 3X with TBS, and add DAB, then wash 3 X with TBS, then mount onto slide.
Ford IF: after alexa secondary wash 3X with TBS, and mount onto slide using vecta shield wet mount with dapi.
I hope this can help. kindly let me know the look of your staining. 
Best wishes
sandra
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Recently, I take use of microdialysis to measure glutamate in ischemia, the probe is put in the center of the striatum of mice, but no rise is seen after ischemia as papers have showed. I did not clear the oxygen from the ACSF, which is used in electrophysiology.
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Thank you very much.
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I found a protocol that uses a single dose heparin in hepatic ischemia reperfusion injury induction, I wonder if it`s applicable in the small intestine.
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In my lab we do not use the heparin. However, it is desirable to use a non traumatic surgical clip. Heparin could also prevent the formation of blood clots that would occur at the microvessels, distally to the occluded area; It is not good to avoid that, since that would be more like the real acute mesenteric occlusion.
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Iba 1 is used for the immunohistochemical analysis of activated microglial cells. I am doing stroke animal research and frequently encounter the staining with the Iba 1 antibody. My problem is Iba 1 stains both MCAo operated slices (suggesting the over expression of microglia in ischemia) and the sham operated slices also (though the morphology of the iba 1 positive cells from sham and MCAo are completely different.) I used both DAB substrate or immunofluoroscence for the Iba cells and got the same result. How can I eliminate the Iba 1 positive cells in sham operated (or normal) mice brain slices?
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Iba-1 is constitutively expressed in the normal healthy brain. However, you should find that the level of Iba-1 is greatly enhanced following MCAO, particularly in the peri-infarct territories. In the healthy rat brain CD11b and CD68 are often less abundant that Iba-1 and you may often observe a greater relative change in these markers following injury.
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ST segment wrt iso-electric line
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