pimonidazole is good. Also HIF and HIF specific target genes such as VEGF, Glut1 or NDUFA4L2..

I do not work with animals, however I've worked on hypoxia and stem cells for a few years now. A marker of hypoxia that will be around for approx 24 hours after hypoxia is induced is HIF1A, which you can detect by western blot (and probably ELISA), the best antibodies for HIF1A are from BD. You can also use PGK1 as a marker of hypoxia, I've looked at this by qPCR and seen big increases up to 3 days after hypoxia is induced.

Hi Giovana,

I am working with HIF and we detect HIF-1a in mouse tissue as a marker for hypoxia. I have never tried to do that in rat tissue but I know that there are antibodies that are able to detect also rat HIF in immunohistochemistry. Another more direct approach is the use of "hypoxy probe" This is a compound you inject into your animal and afterwards detect the product only forming in hypoxic areas by immunehistochemistry. This works in all species.

Best luck

Nathalie

I assume you mean tissue hypoxia - apart from the obvious (arterial and venous PO2 and saturations). If the infusions are over a sufficient time, you could most easily look at plasma erythropoietin. Otherwise markers of oxidant stress, such as urine or plasma isoprostanes or red cell carbonylation, though this is indirect and would not prove hypoxia, just oxidant stress. You could as suggested below look at HIF1alpha - but I think this would require rapid snap freezing of tissues. It is more difficult in animals than cells.

In You question You need to detect the time of development of acute strong hypoxia becouse in short-term acute hypoxia (less than 30-50min) HIF not a marker yet. Next point - severity of Hypoxia - it strongly important for determining of hypoxia markers.

Trayhurn P et al. Cellular hypoxia: a key modulator of adipocyte function in obesity? Adipobiology 2009; 1: 19-26.

Hi Geovana,

As most colleagues have suggested and as you have asked for, a good molecular marker will be the different HIF. Yet, I wonder why not starting for something easier, cheaper and actually generated in the tissue itself, such as Lactate?. You have several other candidates such as LDH, glucose, glycogen, even pH. Hypoxia will be evident in most of those parameters. If you were tempted with those, you can have a look at:

http://link.springer.com/article/10.1007%2Fs00360-012-0678-3

Good look!

Two ways to look at this-

1) since you are using an in vivo rat model I am assuming, you could get arterial blood gasses before and after your infusion. A significant decrease in arterial PO2 would clearly mean you are systemically hypoxic. No molecular markers, this clearly defines aystemic hypoxia.

2) molecular markers as others are saying, Hif1 alpha is likely a good standard- stress markers could also help support this such as heat shock proteins or p-MAPK (ERK, JNK, p38), Will likely need to show this 2 ways

The hypoxia definition is a relative range of oxygen tension in tissue. Because most enviroment of tissue is below 10% of oxygen tension and also its oxygen tension is dependent on the distance to the blood vessel . Thus I suggest that you need to use the oxygen electrode to measure the region of interesting part to prove your hypothesis.

We also measure HIF-1a for hypoxia. Be aware that it is rapidly ubiquitinated, and hypoxia stabilizes it, but timing is important

Pimo staining is the most reliable method in my expirience. People report mixed success with HIF based methods. Ideally you should use phosphorescent dyes, but that would mean purchasing new equipment. Second best choice is electrodes, but that's tricky experimentally.

Why not a real and direct measurement of hypoxia level? i.e. a direct pO2 microelectrode or a NIRS technique?

A very sensitive means of detecting tissue hypoxia is to look at what happens to ATP. A reduction in local oxygen supply naturally leads to consumption of local ATP stores, with concomitant rise in adenosine and subsequent purine degradation products such as Hypoxanthine, Xanthine and Uric acid. Subsequent oxidative stress may further produce modified lipids (MDA, isoprostanes), nucleotides (8-oxoguanosine) and amino acids (nitrotyrosine). Perhaps some of the following may be of help:

2011.08.24.j.vis.exp.54.2948

2008.08.01.pediatr.health.2(4).485

Hi Geovana,

I can recommend using a pulse oximeter to measure the extent of haemoglobin saturation with oxygen. They use these devices routinely in the clinic, but there are also ones for animals (we have used these too). http://www.adinstruments.com/solutions/research/applications/oximetry#overview

Although they don't give you the actual partial pressure of O2 in the blood, they give you % saturation of Hb. The benefit, is that the recordings are continuous, which is good if your stimulus is transient. Also, they occur in real time, so you can time the duration and magnitude of hypoxia !

I hope this helps.

Kindest Regards,

Andrew

I would also try pimonidazole http://www.ncbi.nlm.nih.gov/pubmed/9826471

I would suggest eIF5 alfa (hypusination) because it come to be a significant marker of hypoxic tolerance (also few good patents issued) for cardiac and cancer tissues.

I was measuring system hypoxia by using special designed adenoviral system.

Here is link for publication.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716210/

Latic acid

I think of lactic acid as well. Thanks to Katerina, very useful link btw.

Endothelin-1 might be a good option, as there are ELISA kits available, and is sensitive to hypoxia (See work by Nancy Kanagy - KANAGY NL, Walker BR, Nelin LD. Intermittent hypoxia induces endothelin-dependent hypertension. Hypertension 37:511-515, 2001). Is vascular conductance different between saline and ANGII? Indeed, ANGII is a potent vasoconstrictor, but will also increase pressure and thus perfusion pressure, which may overcome the downstream resistance. In any event, another option is physiological measurement, such as Near Infrared Spectroscopy (NIRS), which is essentially a glorified pulse oximeter, which also would work if they make rat pulse oximeters.....This would tell you blood/tissue oxygen saturation which you could compare between saline and ANGII and help confirm or reject your hypothesis. If you only have post hoc samples in the freezer, look into transcription factors such as HIF1a, and HSPs, these stress response proteins have little translation time, some within 30 minutes. But, if possible ad hoc physiological measures combined with some post hoc assessment (i.e. ET-1) would be a good approach. Good luck!

Measurement of carboxy hemoglobin and lactate would be useful

Blood lactate is a specific biomarker of systemic tissue hypohia.

Hypoxia is a relative term. In a "normoxic" organism breathing air, many organs are "hypoxic". Moreover, ischemia implies local (not always systemic) hypoxia but the reverse is not rue. So, there is no established marker of hypoxia, but many markers of the response to hypoxia are available: most if not all of them have been listed above. So my suggestion would be to use the marker related to the "hypoxic" response you are investigating in your specific research. Good luck!

You could readily monitor the heart and look for changes in heart rate and heart rate variability. This would provide you a physiological manifestation of the systemic hypoxia. The ECGenie [ http://unbouncepages.com/aln-ad/ ] from Mouse Specifics, Inc. will let you do this fast and non-invasively. Presumably your "hypoxic" rats will show significant differences in HR relative to normoxic.

I am just thinking its beter to use lactate to pyruvate ratio to confirm more for hyperlactatemia with ischemia or low perfussion,

What does systemic mean? It is a summation of effects which arise in different tissues and organs. Therefore plasmatic levels of whatever dont nessesarily show an effect to the whole system. I recommend to measure systemic (lactate) and organ specific markers (NIRS is a good idea) in combination with histology of the different tissues and different concentrations of ATII for a long duration of infusion.

Intravital microscopy would be a good option as well, but if it is a completely new method for you its too complex and time-consuming.

Hi Moritz, what do you think about pyruvate to differentiate hyperlactatemia without hypoxia?

Hi everybody, thank you very much for your answers/help. Moritz, differently from patients, the rats should be anesthesized for blood collection, and anesthesia is maintained with isofluran and oxygen using a commercial circle saystem. This constant oxygen flow (2-2.5% for at least 15 min) would interfere with blood gas analysis, wouldn’t it? I’ve tried lactate and got very satisfying results (2-3 fold increase in comparison to baseline). But the colleagues are right and maybe I have to use lactate-to-pyruvate ratio. I will try it in my next experiment.

Thank you again!

Physiologic vs Hypoxic there in lies the problem. Most don't understand the difference, I am truly astonished people are doing research in hyperoxic 21% (TOXIC) oxygen. Stem cells are not special... all cells require physiologic oxygen.

You could just simply use a paediatric oxometer pad taped onto the rat's foot/paw. A little crude but will give you some fair estimate. Venous blood gases would be another invasive measure but is easily compromised by other confounding parameters. Arterial blood gases would be the most accurate.

Giovana, I would firstly ask if the spoken vasoconstriction was chronic... If it was then you may benefit from defining markers of angiogenesis up-regulated on systemic level. For instance I think about VEGF level (ELISA kits for detection are widely present). Hope it will be helpful for you.

Kawwdia et al have shown that acute hypoxia causes a significant decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.

Furthermore, changes in the levels of three other neurotransmitters, notably increases in gamma-aminobutyric acid (GABA), decreases in taurine, and decreases in β-phenylethylamine (PEA) in patients with obstructive sleep apnea and intermittent acute hypoxia.

Complex significant associations emerged between endothelial function, the AHI, and CD41a MPs. IL-6 level also found to be correlated with acute intermittent hypoxia.

The protein biomarkers may include alpha-1 B-glycoprotein; kallikrein, laminin, aldosterone-binding protein and/or urocortin-2 precursor. The presence of other protein biomarkers may be detected using antibodies. These antibodies may be provided in an array for detecting the presence of the at least one protein biomarker or a pattern of protein biomarkers. (David Gozal from University of Chicago has done a lot of studies on pediatric patients also mouse model to study different biomarkers for acute hypoxic injury.

The most sensitive marker of fetal systemic hypoxia is vasopressin. The reproduction physiology (fetus) suggests the essentilal substance for your study.