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There are no healthy people, there are under-examined: early diagnosis of coronary heart disease



Modern diagnosis of diseases through imaging has many methods: MRI, CT, FA, ultrasound, etc. Each of them is unique in its own way and provides a certain range of information about the patient’s health. However, everything has its drawbacks, and the above methods are no exception. Scientists from Stanford University decided to develop a new visualization method that will combine the strengths of several classical methods at once, but at the same time be free from their drawbacks. The basis of the invention was the contrast agent developed by them, with which you can conduct an early diagnosis of coronary heart disease in patients who still have no visible symptoms. What is the basis of the new method and how does it work? We learn about this from the report of scientists. Go.

Study basis


One of the most common causes of death in the world is coronary heart disease (CHD), when coronary circulation is disturbed, which leads to myocardial damage. The acute form of coronary heart disease is expressed by myocardial infarction, and the chronic form is manifested by periodic attacks of angina pectoris. However, before the manifestation of obvious symptoms, the disease proceeds quite secretively, from which the patient does not immediately know that he is sick.

Due to the absence of obvious symptoms in the early stages of the development of coronary heart disease, it is quite difficult to detect its symptoms even with CT (computed tomography). If the disease is at a “noticeable” stage for diagnostic equipment, then other difficulties arise: for example, MRI cannot accurately determine the load on atherosclerotic plaques and the degree of their stenosis (narrowing of any hollow structure of the body), which greatly complicates the prediction of heart attacks. Consequently, the earlier an IHD is diagnosed, the more likely it is to prevent plaque rupture.

Researchers believe that non-invasive plaque pathobiology imaging techniques may be the key to a successful early diagnosis of coronary artery disease. In nuclear imaging, the level of absorption of fluorodeoxyglucose (the most common PET label *) directly correlates with the degree of plaque infiltration in macrophages in most inflamed arteries compared with healthy arteries.
PET-label * - a radioactive preparation (radioligand) used in positron emission tomography (PET) as a marking substance.
For example, Gorton’s disease (giant cell arteritis) and Takayasu’s syndrome (nonspecific aortoarteritis) are detected and quantified using the [11C] PK11195 label in combination with PET and CT.

In turn, MRI can be used to detect the accumulation of iron oxide by macrophages, which is a sign of atherosclerotic inflammation of composite plaques. The visualization of iron oxide nanoparticles is usually carried out by means of sequences of gradient echo signals. These sequences create stable contrasts in parenchymal organs such as the myocardium (heart), liver and pancreas, generating hypo-intensity of the signal or negative contrast surrounding the region of iron oxide nanoparticles. However, with this approach, it is difficult to display the vessel walls due to the lack of a suitable background for negative contrast due to the presence of a large amount of air in the structures surrounding the vessel wall in the chest and neck.

Another diagnostic method is photoacoustic imaging. This relatively young method is used to detect accumulations of lipid-specific plaques in the structure of collagen and carotid artery thrombosis in the wavelength range of 1130–1250 nm. However, this method also has its significant drawbacks.

In this study, scientists decided to create a new diagnostic method that can compete in effectiveness with MRI, CT or photoacoustic imaging, but without their drawbacks. The basis of the method is the contrast NIR agent (NIR - near infrared range) developed by the authors of the study, called NET (NIR erythrocyte sensor). In addition to NET, photoacoustic (FA) and fluorescence imaging systems were used, which together made it possible to detect inflammation in the coronary arteries.


Image No. 1: experimental setup of a new diagnostic method.

The process of the new method can be described as follows: during FA imaging, when a non-ionizing laser pulse enters the coronary artery, it can detect stenosis or blockage due to thermoelastic expansion from NET energy absorption inside inflammatory cells. Further, these extensions will be detected by the ultrasonic transducer as an acoustic pressure wave and converted into FA signals.

At this stage of the study, all experiments were conducted on laboratory mice with ligation * of the left anterior descending coronary artery.
Ligation * - applying ligature (special thread) to the blood vessel to stop bleeding. In the case of this study, to simulate clogged arteries.

NET preparation


NET were made from red blood cells from Swiss Webster Sentinel mice. Blood was drawn using syringes coated with 3% heparin. Next, centrifugation was performed (1000 x g for 5-10 minutes at 4 ° C) to separate red blood cells.

Plasma and leukocyte membrane were removed before re-suspension of the erythrocyte sediment in physiological saline with phosphate buffer (PBS) with an osmotic concentration * of about 320 milliosmol. Then, the red blood cells were centrifuged again (1000 x g for 5–10 minutes at 4 ° C) and washed three times with PBS.
Osmotic concentration * - the total concentration of all dissolved particles.
After each wash, supernatant * was removed .
Supernatant * - liquid remaining after insoluble matter precipitates during centrifugation.
The red blood cells were then subjected to hypotonic treatment by resuspension in 0.25xPBS (≈80 milliosmol) and allowed to incubate for ≈10 minutes before centrifugation (20,000 xg for 20 minutes at 4 ° C).

The hemoglobin-containing supernatant was removed and the hypotonic treatment was repeated until the erythrocyte sediment became white, indicating their hemoglobin depletion (EG).

To form nanosized particles, an erythrocyte pellet was sieved 20 times through polycarbonate porous membranes (400 nm and 200 nm). After that, the centrifugation and washing process was repeated several more times.

Experiment Results


NET showed a peak distribution of hydrodynamic diameter within 89.92 ± 3.35 nm based on dynamic light scattering ( 2a ) and strong absorption at 600-800 nm due to the presence of ICG * ( 2b ).
ICG * (Indocyanin Green) is a cyanine dye added to NET for better visualization.

Image # 2: NET characteristic.

In response to photoexcitation at 650 nm, peaks of fluorescence were observed at ≈ 700 and 790 nm, corresponding to the H-like and monomeric forms of ICG ( 2c ).

To determine the correct NET dose concentration, the radiation efficacy of two concentration options was evaluated: 20 μM and 1 mM ( 2d ). This analysis showed that the radiation efficiency for 20 μM was 5.43 x 107 (f / s / cm 2 / sr) / (μM / cm 2 ).
f / s / cm 2 / sr * - the number of photons per second that leave a square centimeter of tissue and are emitted into a solid angle of one steradian.
At the same time, nothing was found for the concentration variant of 1 mM, which indicates aggregation suppression. Therefore, the obvious decision was made to use exclusively 20 μM NET for the planned experiments on mice.


Image No. 3: a diagram of the heart of the mouse, which shows the location of the ligation of the artery and the region of the heart attack.


Image No. 4: visualization of the artery ligation region 1 hour after NET administration.

The 1.5 mm region above the ligation point ( 4a , yellow arrow) corresponds to NET accumulation, as a result of which the photoacoustic signal of this section ( 4b ) was 256 times stronger (± 13.73) than the signal of the section without ligation.

Moreover, NET accumulation in the coronary artery, thymus, and liver provided strong fluorescence signals when imaged through the whole skin ( 5a ), i.e. without the need for invasive intervention.


Image No. 5: visualization of NET accumulation in the ligation region, confirmed by dissection.

Similar signals emanating from the heart were also observed after dissection of the chest ( 5b ).

The infarction region with fibrous tissue was also successfully identified below the ligation ( 5c , blue arrow) of the coronary artery ( 5c , green arrow).


Image 6: Comparison of visualization signals with a whole or open chest.

An increase in fluorescence signals of 1.6-2 was also observed with imaging of the open chest, than with non-invasive diagnosis. This is due to the weakening of light resulting from scattering on the bones of the chest, skin and fat layers. That is why the liver showed the same signals in both visualization options.

Comparison of signals from different regions showed that the signal of the coronary artery with ligation is 1.32 times higher than the signal of the thymus, and 1.96 times higher than the signal of the liver.


Image 7: visualization of the area of ​​myocardial infarction.

The area of ​​myocardial infarction ( 3b ), consisting of scars and tissue necrosis in the left ventricle of the heart, was directly under the coronary artery bandaged ( 7a and 7b ).

Entire viable myocytes (muscle cells) were observed only in the right ventricle. A trichrome-stained septum between the left and right ventricles was isolated by blue collagen, indicating a heart attack area with scarring, granulation, and fibrosis ( 7c ). The visualization marker Picrosirius red indicates degraded collagen ( 7d ) in the affected coronary artery. Polarized light was used to identify the type of degraded collagen ( 7e ). Oil Red O (lysochromic azo dye) was used to determine the amount of lipids near the ligation region ( 7f ). And Mac-2 and Dapi revealed a strong inflammation due to the accumulation of large macrophages in the ligation region ( 7g and 7h)

H&E stained liver showed a normal portal tract ( 7i ), lack of parenchymal necrosis, inflammation, fibrosis or other pathological changes, indicating a high level of NET biocompatibility.

For a more detailed acquaintance with the nuances of the study, I recommend a look at the report of scientists .

Epilogue


According to the researchers, their diagnostic method combines the strengths of classical methods, and these are not just big words. The combination of photoacoustic and fluorescence imaging can more accurately detect occlusions and possibly inflammatory cells, such as macrophages.

When the NET agent introduced into the body is activated by pulsed laser radiation, FA signals are generated that can be converted into images.

The reconstruction of photoacoustic images is similar to ultrasound, but with a spatial map of optical absorption by endogenous and exogenous components. The optical absorption and ultrasonic scattering of the coronary artery tissues make it optimal for high-resolution photoacoustic imaging with a depth of 3-5 cm. While fluorescence imaging is limited both in terms of resolution and in terms of depth (several millimeters).

One of the most important features of the developed contrast agent NET is that it is not absorbed by muscle cells, thereby reducing the background noise that is usually present when using standard PET agents (for example, 18F-FDG).

In the experiments, scientists considered the heart as an object for diagnosis. However, the new method can be applied to other organs, thereby allowing physicians to obtain such important information about patients, even when the symptoms are not so obvious.

The work of the diagnostician is comparable to Formula 1, but instead of the opponent’s car, the doctor competes in speed with the disease, and the prize in this race is the patient’s life. When someone’s life is at stake, every second is precious, because the creation of new methods for early diagnosis is so important.

Thank you for your attention, stay curious and have a great weekend everyone, guys! :)

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