Aorta - Science topic

1. Magnification and Working Distance: Look for a macro lens that offers a suitable magnification level for capturing the details of the oil red-O staining on the aorta. Additionally, consider the working distance of the lens, which should allow you to maintain a comfortable distance from the subject while still achieving the desired magnification.

2. Focal Length: For shooting aortas, you'll likely need a lens with a moderate focal length. A focal length between 60mm and 100mm can be a good starting point. This range offers a balance between working distance and magnification.

3. Image Quality: Ensure that the macro lens you choose delivers high image quality and sharpness across the frame. Look for lenses with good optical performance and minimal distortion.

4. Compatibility: Check if the macro lens you are considering is compatible with your SONY camera model. Verify that the lens mount matches your camera's mount type.

5. Aperture: Consider the lens's maximum aperture. A wider aperture, such as f/2.8, can help in low-light situations and provide better background separation.

6. Stabilization: If your stereomicroscope setup offers image stabilization, it may not be a significant concern. However, if you plan to shoot handheld or without stabilization assistance, you may want to consider a lens with built-in optical stabilization to minimize camera shake.

7. Budget: Determine your budget for the macro lens. There are a variety of options available at different price points. Consider the overall quality and features you need within your budget.

The time it takes for a blood vessel to heal after suturing can vary depending on several factors, including the size and location of the vessel, the extent of the injury, and the individual's overall health and immune function. In general, small blood vessels that are properly sutured can begin to heal within a few days, while larger vessels may take several weeks to fully heal.

The healing process of a blood vessel involves the formation of a blood clot to stop bleeding, followed by the migration of cells to the site of injury to rebuild the damaged tissue. New blood vessels will also begin to grow and connect with existing vessels to restore blood flow to the area. The sutures hold the edges of the vessel together to aid in the healing process and are typically removed after a period of time once the vessel has sufficiently healed.

It is important to note that proper wound care and follow-up with a healthcare provider is crucial for optimal healing of blood vessels after suturing. Any signs of infection or delayed healing should be promptly addressed by a medical professional.

Thank you very much for your reccomendation

Best wishes

Tomek

Alexander Oberhuber Thank you,

You can denude or injury the endothelium using a balloon catheter, like in the rabbit carotid model.

The attached file is only one slcie of the CT in an arterial phase. Also the window of contrast is not optimal. Normally in this patients bleeding comes directly from the fistula (esophagus wall).

Normally we perform aortic resection and iyenograft interposition, followed by an esophagus resection or endovac therapy, depending of the fistula size

Hi.

I don't know, which scissors dou you use. I would recommend a Castroviejo micro scissor. They must be very sharp. If you cut the aorta after explantation it's more difficult, and you can try to fill the lumen with saline solution/medium/buffer, if it doesn't interfere with your experiments. In the case that you open the aorta before explantation, it should be easier.

I presume you use a microscope

Best regards

AO

Seeing multiple bands when fishing for phosphorylations is a frequent observation. All it takes is a common motif, e.g. when several proteins are targets of the same kinase.

If you need to demonstrate specificity, you can immunoprecipitate your protein of interest first or do co-localization with a protein-specific antibody.

Hi! Not sure if ImageJ has an equivalent plugin, but I find the 'Incremental Distance' tool on Image-Pro Premier very useful for measuring vessel wall thickness between two irregular shaped lines or polygons. Drawback though is that it's a paid software... a free version will definitely be useful to the general community. Good luck!

Video on its use on YouTube:

Hi Lara

Easy simple protocol:

Weight aorta sample : e.g. 100mg

Add tissue to FastPrep24 tube with Lysis Matrix D

Best will be if you can snap freeze the tissue in liquid N2 before taking the weight

Work always on ice (dry ice if possible)

Add Lysis Buffer (8M urea/100mM Tris-HCl pH 8.00/protease Inhibitors)

1mL Buffer/100mg tissue

Homogenize following Instruction on FastPrep24 Instrument

Centrifuge and transfer SN into new tube.

You can wash the beads once more with 1/2 Vol. of buffer and pool with first SN

With this lysate you can do what ever you want.

Protein Assay, run a gel, direct In solution digestion ...

Best wishes and good luck

Greatings

Natasha

Atef Erasha

Thank you for your reply.

I'll try my best!

I was staining lipids in atherosclerotic plaques.

IMH or IgG4 (is IgG/IgG4 ratio known?)related Aortitis is probable. Please upload a snapshot.

dou you have PET/CT?

see these papers:

Hi Keri,

I have the same problems. I try to isolate RNA from mouse aorta and used different kits (Qiagen and Zymo Research). Unfortunately I get very low concentrations too and a very low A260/A230 ratio. Please let me know if you made any improvement or have any suggestions.

Thanks

The answer to your question depends on the type of data that you have. There are ways of estimating ED50 values with error limits for continuous data and also for discrete data (eg an all or none response). One way to start would be to learn to use the excellent statistical package called R. It will provide you with methods for analyzing your data and with methods for plotting your data. There is useful information about dose response relationships in the following reference.

https://static-curis.ku.dk/portal/files/153304510/journal.pone.0146021.pdf

It gives examples using R.

For all or none effects an author to look for from way back when is D.J. Finney. I also seem to remember from way back when a statistical book by Snedecor and Cochrane (spelling?) and a statistical book by Steele and Torrie.

Dear colleaque the question clear,need the alternative of sod.citrate to suspend chicken blood,so the answer the following two alternatives are :

1 _Acid citrare dextroseACD Solution ,allow 1 part of ACD Solution to 3 parts of blood.

2_Alsevars solution,allow 1 part of Alsevar,s solution to 1 part of blood.

Best regards

Yes, dear Fa Ro they can present in lymph node.

If you plan on isolating the cells, you cannot freeze the tissue. The ice crystals will destroy the cell membranes.

Hi Emma,

I would check the reactivity of my antibody.

Thanks

You can store the tissue cold in your sucrose solution prior to embedding in OCT, or embed it in the OCT and store frozed blocks. Performing the oil red O as a whole mount tissue first mau affect the texture of the tissue for cryosectioning. There are other histological stains that will work well after Oil red O. I do not know how antibody staing would work after oil red O because oil red O fluoresces, so immunohistochemistry may not work

Hi Patrick,

I use 10% limit when I want to see if I successfully removed the endothelium or not. If it shows a response greater than 10% that removal wasn't complete. I use 80% (some use 60%) value to see if the endothelium is at full health. If the relaxation response is 60-80% of PE that means endothelium integrity is intact. Just be careful while determining this and use Ach atleast 10 times higher than PE. Like I contract aorta with 10^-7 PE and check integrity with 10^-6 Ach. Hope that helps.

It is not a contraindication, but needs repair asap.

Alex

Daniel Carneiro Moreira Hi Daniel! Thanks for the reply.

The transcardial perfusion was done manually by inserting 22G needle attached to a 10ml syringe into left ventricle and snipping the right aorta.

As such the perfusion pressure and time was not taken into consideration. It usually take me about 5 min to inject 10mL of fluid.

10mL of Heparin/PBS was first injected followed by 10mL of 4% PFA.

Hello, I suppose that you did heparinize the animal just before taking out the heart? It sounds as if you have massive clotting in the coronaries.

This link gives you more information regarding your work

https://bmjopen.bmj.com/content/bmjopen/9/Suppl_3/118.full.pdf

Detained info seen above. I would add simply that peripheral vasoconstriction occurs during trauma when blood is shunted towards the key organs. Also of note when measuring blood pressure keep in mind that in order to externally compress an artery all the surrounding soft tissue needs to be compressed too and hence readings from the thigh can be difficult to interpret.

Himanshu, what system are you using? We use wire myography, which measures isometric tension of aortic rings. With that system we pre-contract the vessel to 20mN with PE, then we add Ach at increasing concentrations. As Bruno noted in his response we do not use set time before we give the next dose. We monitor the vessel response. At low doses there is often no response at all. If there is no response, we wait at least 3 minutes and we give the next dose. When you do start seeing relaxation it is initially noisy. The vessel relaxes, then it contracts again, often to a level equal to the previous dose. You must take this into account when calculating the percent relaxation for a given dose of Ach (we use a weighed average relaxation). Eventually, as Ach dose becomes high enough, the relaxation response elicited by Ach becomes very robust and you get clear step-like curve. At that point it's easy to determine when there is a plateau and you should give the next dose. One final note, this is what we see with healthy vessels. If you denuded the endothelium during mounting of the vessel or you have a diseased endothelium your response may differ. You must always include a healthy control vessel in your preparation to ensure that your system is working properly. Hope this helps.

A special dye of dethidfiocycine can be used to distinguish fiber.

See this linkA

V Node Ablation in Langendorff-Perfused Porcine Hearts Usin...

Some may use the term extra-pleural space, the definition with examples of processes that may affect this space. On imaging, many would call these processes pleural-based or the extra-pleural fluid pleural effusions. While these descriptors may be anatomically incorrect, the distinction is often not clinically relevant.

Esophageal perforation is probably more similar to descending thoracic aortic aneurysm rupture (or traumatic rupture) than dissection. In perforated esophagus or ruptured aorta, the extraluminal fluid needs somewhere to go. As the linked article describes above, maybe these or extrapleural rather than pleural or maybe the insult caused a tear or communication with the adjacent pleura. The processes and where ruptured content would communicate are different for the ascending aorta and the upper to mid esophagus (mediastinal).

Pleural effusions associated with dissection may be an inflammatory mediated process (as Marcel C Machado excellently explains) and third spacing of the false lumen. If it results in pleural or extrapleural blood, it may be related to tiny microruptures.

Did you use immunofluorescence or brightfield visualisation?

By using HE stain, trichrome stain, Verhoeff's or wigert;s elastica stains as conventional histological stains of paraffin sections.

reference for stain procedure:

S.K. Suvarna, C. Layton, Bancroft, J.D., (2013) Bancroft’s Theory and Practice of Histological Techniques, ed 7. Elsevier, Churchill Livingstone.

For immunohistochemistry you should follow the data sheet of antibody.You should select suitable antibody for the structure you need to observe or study it.

For example: 1–Prashanthi Menon, and Edward A. Fisher(2016)

Immunostaining of macrophages, endothelial cells and smooth muscle cells in the atherosclerotic mouse aorta.Methods. Mol Biol. 2015; 1339: 131-148

May be this article help you in staining procedure.

Hi Maria,

Yes, the left coronary artery is there which connects with aortic sinus (middle up), and the pulmonary artery is at right side.

Good luck!

Yong-Xiang

Hello!

This article can help you

Biology is rather complex subject withi each species. We already know that about man.

We already know that about humans!

There so much we do not know

ALP

I have never used PW V for the evaluation of aPWA. My method uses the invasive radial PW and locates the true aortic SP as the second pea. It can be applied to find the aortic SP when acquired by tonometry but it does not identify the DP In older hypertensives

Alfredo L Pauca MD

Thanks a lot Marie !

We will try this protocol as other researchers suggested the same methodology as you. Do you use this protocol each session or when you start a new study ? to be sure your model is still valid ?

We did hundred experiments on rats, no problem at all... not the same story with our mice model.

Thanks again.

Thank you, Carlos.

hope this link is useful

https://www.cfd-online.com/.../fluent-udf/200460-udf-inlet-aorta-3d-...

regards

Dear Misbah.

So you killed some mice, prepared aorta slices and you don´t know why you did that??

It would be interesting to mention what your aim is. What do you expect to see? Is this a stained slide? FFPE, or frozen?

If you still work in the hypertension field (as I assume after visiting your profile site), then you could look for aorta thickness using the ImageJ tool. For this you should record a better image without the grid and the lines and with a magnification bar.

Maybe a PubMed search could provide some ideas: What other scientists looked at and what methods they used.

Please provide more information in order to help you out.

Kind regards

Dear Nina,

The information that your fibroblast are from human aorta is a bit too sparse. From which histological region were they isolated, adventitia?

What type of activation are you looking for?

Would you like to know if they are in a quiescent or resting stadium after isolation, purification and re-culturing on TCPS (tissue-culture treated polystyrene) in what? Are you using a commonly used media supplemented with FCS and etc. or are you following a "better" or "more suitable" protocol for "specialized" human aortic adventitia-derived fibroblasts from a healthy young donor??

If the fibroblasts are normal activated fibroblasts (NAFs) they should or will show an increase in alpha-SMA and vimentin expression which goes along with a change in morphology: spindle-to-stellate-shape transformation;

If the cells were isolated from a fibrosis or ….diseased patient/donor you may/will have differently activated fibroblasts, e.g. fibrosis-associated fibroblasts (FAFs) or cancer-associated Fibros (CAFs).

The problem is that the different activated fibros as such are usually identified by investigating/analysing histological material. The whole process of isolation and culturing by applying common in vitro conditions leads/ may lead to stadium changes (or de-/transdifferentiation). That is the reason why we and many others (e.g. in the cancer research field, lung-fibrosis research field etc.) started longer ago to work on 3D culture methods.:-) Hope my questions, thoughts and notes will help you.

Kind regards,

Jochen

Take a look at this article. I isolated porcine aortic endothelial and smooth muscle cells and I found it very very useful.

Hey Ivan,

Lipofectamine 3000 is of course a possibility, but please be aware of the potential cytotoxicity issues as a function of a) the concentration of Lipofectamine used, b) the time of exposure to the Lipofectamine:DNA complexes, c) the cell type (not sure whether those cells are quite sensitive to Lipofectamine treatment). If I were you, and if you had a good stock of Lipofectamine, I would try to first optimise the amount of Lipofectamine and time of exposure so that cell viability is not dramatically affected (e.g. via MTS or MTT), then go for your experimental set up (you'd need to optimise the amount of DNA per well, but you can find some good approximations online for specific cell types).

Good luck!

Julio.

I am not sure what you mean by "hexagonal mesh"...

If you mean irregular polygonal volume mesh with prismatic near-wall cells, then you can do this in Fluent Meshing.

If you mean structured hexahedral mesh, then you will need ICEM or some other structured mesher. Though it is very difficult to create structured meshes that conform to complex surfaces.

Another possibility is to use a cut-cell mesh, which is possible in both Fluent Meshing and ICEM, though I have personally had little success with this approach.

Hi,

Generally, upto 4-5 passages you should follow 20% FBS supplemented DMEM and from 6 passage you can also use 10 % DMEM as per the previous reports. But from my experience when I followed 10% DMEM cells usually changed their morphology. So I would recommend 20 % DMEM only.

Yes, but for conducting the apoptosis related experiments cells, should be starved in serum free media minimum for 24 hr or maximum upto 48 h or you can use 1-2% FBS supplemented DMEM for serum starvation, cells will not detach neither they will die and morphology will also be sustained.

May be they are not different cells, could be same cells but undergoing mitosis. Most cells get round when they are dividing

Please let me recommend you the following article we have published in 2006:

Life Sciences 79 (2006) 854-860.

I attached a file for you here.

May you find out some answers for your questions within.

Hopefully,

Wilson

I have Isolated the Mouse EC cells from the lungs of B57 Mouse successfully and following the protocol for isolation Of EC cells from Fresh tissue In the Nature Protocols 2005. They are growing fine Morphology is like EC cells But When I did the Tube assay They did not form Tubes Like EC Cells Form in DMEM Media With Supplements But same cells If I grow in EBM complete media They form Beautiful tubes I am thinking they are lacking some kind of growth Factors in The Media which help them to make tube formation which is one of the characteristic of EC cell line .

We have isolated monocytes from different species and always use Ficoll Hypaque gradient to separate out the PBMCs, followed by at least 4 washes with HBSS at very low speed (180 g) to get rid of thrombocytes. We then adhere the cells overnight and remove all the non adherent cells. To differentiate into macrophages, I believe every species is a bit different, but mostly adding M-CSF and IL-1 is most common. We use RPMI 10% FBS.

here is the reference "Evolution of surgical therapy for Stanford acute type a aortic dissection Annals of cardiothoracic surgery 2016;5(4):275-295". This is almost a statement of standard of practice

Yes, it will eventully end in bone for the most part.

terima kasih...thanks

An ultrasound of the abdominal aorta is a non-invasive, painless test that uses high-frequency sound waves to image the "aorta," the main blood vessel leading away from the heart.

When the walls of the abdominal aorta become weak, they may balloon outward If the aorta reaches over 3 centimeters in diameter, it is then called an abdominal aortic aneurysm (AAA). As the aneurysm gets larger, the risk of rupture increases.

Ultrasound imaging of the aorta is useful for measuring its size to screen for AAA. Screening is particularly recommended for men over the age of 60 who have ever smoked and for anyone with a family history of AAA. In addition to screening, ultrasound is also a useful tool after the diagnosis of AAA to monitor its size on a regular basis to see if it needs to be repaired.

Maybe this is helpful:

Wada H, Sakata N, Tashiro T.Clinicopathological study on penetrating atherosclerotic ulcers and aortic dissection: distinct pattern of development of initial event. Heart Vessels. 2016 Nov;31(11):1855-1861. Epub 2016 Feb 18.

I want to compare with wild type picture :)

Anyway, it looks like medial hyperplasia (between elastin lamina). 

Trichrome stain is helpful to detect elastin lamina.

I guess it depends indeed more on symptoms, on functional capacity of a patient and maybe on NT-proBNP levels than specific echocardiographic findings. But Sir you are right, at the moment it is still a very subjective decision. 

A combination of esophageal self-expansible stent with aggressive  antimicrobial therapy might be useful, since early surgery might not be of success. If pseudo-aneurisma of the aorta is present , again endovascular prosthesis would help to prevent further complications.

Hi,

you definitely need a control. On the other hand, there is always some background level of ROS. 

Good luck

Hello,

You can thread a flexible tube or catheter(OD of tube<ID of aorta) through aorta before detaching it from the body. after you make sure you are all the way through, pin the two ends of the tube and start working on your aorta. That helps.

Veramapil is not a pure L channel agonist (it interferes with Na channels too), go back to the eighties litterature and you'll find the products and recipes.

Thank you Shuai,

I think I can try by soaking the tissue pieces for a half an hour or so, in order to improve their homogenization; since the left over I obtain after centrifugation it's quite considerable. The buffer I use contains a cocktail of inhibitors from roche, TRIS, TRITON and EDTA, shall I add anything else? Any suggestion regarding this step?

Oi Daisy, 

obrigado pela resposta. 

O tecido esta dentro de um tubo falcon (50ml) e preciso corta-lo em pedacos. A ideia eh fazer immunostaining. Neste caso o pistilo nao me ajudaria muito. 

Abraco.

Dear Nasibah,

after preparation of whole mouse aorta we fix it in formalin by shaking over night at 4°C. Next day wash in PBS, also at 4°C. Afterwards incubation for 3 mins in 100% propyleneglykol , then staining in 0.6% ORO for 3 h at 37°C. Wash aorta in 3 steps with 85% propyleneglykol and finally store it in PBS until doing the en face preparation. In case your not satisfied with the intensity of ORO its also possible to again stain the pinned aorta in a 6cm dish.

Best wishes,

Ann

Dear Méry,

Please read the following:

1. Euthanatize mice by CO2 asphyxiation followed by cervical dislocation.

2. Wipe off skin with 70% isopropanol, and fix mice on an operation table.

3. Cut off tissue to open chest and abdomen, and expose aorta.

4. Dissect thoracic aorta out, and put it into PBS washing solution in 35-mm petri dishes.

5. Under a stereomicroscope, remove connective tissue around adventitia.

6. Transfer aorta to new petri dishes, and cut it to 1~2 mm cross-sectional slices (rings) using an iris scissors.

1. Coat new petri dishes with matrix by adding 50 µl diluted Matrigel solution.

2. Allow Matrigel to polymerize at room temperature for 20 min.

3. Place aorta rings on the Matrigel layer, and cover the aorta tissue with a few drops of Matrigel.

4. Allow Matrigel to polymerize at room temperature for 20 min.

5. Wash once with pre-warmed PBS.

6. Add pre-warmed culture medium, and allow endothelial cells (EC) to sprout and grow for 5~7 days at 37 °C in a humid incubator chamber with 5% CO2.

1. When a large number of cells sprouting from aorta are observed, remove aorta rings from Matrigel with a sterile fine-tip forceps under a stereomicroscope.

2. Wash remaining Matrigel and cells with PBS twice.

3. Add dispase solution (0.1 ml/cm2

), and incubate for 20 min at 37 °C in a humid incubator chamber.

4. Pipette solution up and down, and transfer solution into a 15-ml tube.

5. Add pre-chilled 10 ml culture medium, and centrifuge at 500 X g for 10 min at 4 °C.

6. Discard supernate, and suspend the cell pellets with 1 ml culture medium.

1. Coat a dish/flask with fibronectin (0.2 ml/cm2) at 4 °C overnight, and wash twice with PBS.

2. Add EC suspension and culture medium to the dish/flask.

3. Change medium every 3 days and split cells when reaching ~90% confluence.

Appendix PBS washing buffer PBS with antibiotics (50 µg/ml penicillin and strptomycin) Matrigel solution Add pre-chilled PBS into Matrigel (BD Biosciences, Cat# 356231) at 1:1 on ice. Dispase solution Dispase I (Sigma Cat# D4818, 2 U/ml) in PBS. Fibronectin solution Fibronectin (Sigma Cat# F1141, 10 µg/ml) in PBS.

Culture medium Ham’s F12/DMEM medium with 5~10% FBS (5% for long-term culture), 50 µg/ml ECGS (endothelial cell growth supplement, made from bovine hypothalami or Sigma Cat# E2759), 10 U/ml heparin (Sigma Cat# 9399), 50 µg/ml penicillin, 50 µg/ml streptomycin.

Hoping this will be helpful,

Rafik

This article goes into a great deal of detail about the truncus and aortic sac.  My (simplistic!) understanding is that the dorsal aortic sac would remain connected to the 6th arch, and thus contribute to formation of the proximal pulmonary trunk, while the ventral sac remains connected to the 3rd & 4th arches, and so contributes to the definitive aorta.

 Thank you all for the replies. Appreciated!

If your question pertains to identifiying non normal structures, i suggest that you develop a normal template which accounts for all the normal structures. There are different techniques to do that. A combination of image processing and learning techniques can sort out the problem. Classifiying all else as abnormal would be a starting point then.

As I mentioned before, you could analyze the variability of the tested loading controls between the samples. The one which is the most stable through all conditions should be actually the best choice.

Have you considered using a full-protein stain like SYPRO Ruby for membranes as an alternative approach for normalization of the protein of interested?

Hi, 

10 minutes seems a little long for it to respond.  In my experience you should see a contraction within a couple of minutes.

i am facing problem the flourimeter is in other lab. and organ bath is automatic in my own lab.

so i am confused that after exposing tissue to the dye , intact ring can be put into the cuvette and enter the range for measuring fluorescence.  

Even protected from light and in 4 degree the signal to noise ratio slowly degenerates overtime probably due to reaction to atmospheric oxygen. I was able to resolve usefull pictures up to two weeks after staining and signal have dissapered (all the section produced uniform red signal) after one month. It's a guesswork (I never tried it with stained sections) but the storage life of DHE staining slides can be prolonged by putting it into Nitrogen or CO2 atmosphere i.e. in a sealed bag. This is the way I used to store the DHE after opening. 

Thank you Abhishek.

Something else we used to do was file the forceps to toughen them up a little. This makes them more grippy... Particularly handy when gripping adipose tissue, or adventitia by the very top of the forceps. 

Sorry Dragan... You did also mention holding the tissue by the outer layer away from the smooth muscle. 

I Have seen people use up to 15mN for mouse aorta which I thought was ridiculously high. Theoretically it should be lower in smaller blood vessels because of course MAP is lower. I think for mesenteric you should use 5mN.

As a general rule all malignant tumors that do not have significant radio/ chemo sensititivity should taken for debulking. This is beneficial to the patient asit leads to cytoreduction, decreased tumor load and better management of the residual disease.

However it may not always be possible to debulk the malignant tumors especially the thymic , due high vascularity, and being adherent/ invading the surrounding vital structures especially the pulmonary artery and aorta.