Cellular death by necrosis

Necrosis is a type of cell death, but in fact the term necrosis from a general pathology point of view indicates not only cell death but all the morphological changes that dead cells of a tissue manifest, so yes, it is a type of cell death, which includes all morphological changes that dead cells manifest.

The common characteristics that all dead cells by necrosis exhibit are:

• the plasma membrane discontinuous and cytoplasmic components degraded by enzymes in lysosomes.  
• the cytoplasm corroded because some of the components have been degraded; it will exhibit vacuoles and a decrease in the number of organelles.

With histological staining, you notice the cytoplasm is much more eosinophilic, so it stains much more pink. If you do a histological staining with hemallumen-eosin (or hematoxin-eosin), which is the simplest histological staining, the hemallumen colors the nucleus, while the other dye colors the cytoplasm, and on the slide you will see blue-purple nuclei and pink cytoplasm, in the case of an intact cell. In the cytoplasm of a cell that has gone into necrosis there is a much more pronounced pink staining because this dye, emallume-eosin, binds to degraded proteins and degraded RNA. This shows that there are already degraded proteins present in that cytoplasm.  

Alterations at the level of the nucleus: when a cell dies by necrosis, it undergoes changes that affect mainly the non-nuclear component. In death by necrosis, the nucleus is the part of the cell that undergoes changes later, unlike death by apoptosis. The nucleus of a cell that dies by necrosis undergoes a late random fragmentation of the DNA. Random because nuclease are activated that cut the DNA where it happens, so it will form smaller fragments, larger fragments, of random size. This fragmentation is called nonspecific fragmentation.

In cell death by apoptosis, on the other hand, we have specific fragmentation with the activation of endonucleases that cut the DNA at specific points.

Morphological appearance of the nucleus of a cell dead from necrosis:

• cariolysis: the components of the nucleus are immediately degraded without condensation; nothing remains of the nucleus, only small fragments, all the rest has already been lysed. Depending on the type of damaging agent, different enzyme systems are activated and lysed;
• pycnosis: there is a phenomenon of condensation of the nucleus, which becomes less basophilic and pycnotic (small condensed nucleus), then it shrinks; afterwards, the pycnotic nucleus goes against karyorrhexis, that is it fragments and is digested in its components
• karyorrhexis: a cell dead from necrosis with a pycnotic nucleus, at a somewhat later stage (24-48h), fragments randomly. It is thus the phase following pycnosis.

Necrosis types

• Coagulative. A blood clot is something "stuck"; it is called coagulative necrosis because the cell after two/three days at most, has an appearance of an empty shell. You can still see the outline of the cells, but inside, the cellular components are absent. It is like a blockage: in these situations the damaging agent has also determined the denaturation of enzymatic proteins and blocks autolysis. The cell will be eliminated as part of the inflammatory process. When all tissues (except central nervous tissue or brain tissue within which cells die as a result of ischemia/hypoxia) die as a result of ischemia/hypoxia (=no more blood coming in, =infarction), they take on the appearance of coagulative necrosis. In a slide obtained from a myocardium in which there has been an event of infarction (logically within the first few days) these cardiomyocytes appear as empty shells, the same for a preparation of a columnar infarction, or a preparation of an infarction of the intestine. The speech just made does not apply to a preparation of a cerebral infarction because here the lytic enzymes are present in very high concentration, so there is autolysis even without enzymatic proteins. In coagulative necrosis the basic architecture of the tissue is retained, but not the cellular components, so I see empty shells.
• Colliquative: is characterized by the liquefied appearance that dead cells assume when all cellular components are degraded, lysed immediately. The colliquated appearance occurs when the injurious agent is a bacterial type agent. Bacterial infections attract many lymphocytes that release lytic enzymes. If the leukocytes release so many lytic enzymes, all the components are completely degraded, they are colliquated. Thus, colliquative necrosis is associated with bacterial infections and the exception of death by cerebral infarction.
• Caseous: name indicative of macroscopic appearance; in caseous necrosis the dead cells take on a soft, yellowish-white appearance. It is defined as a subtype of coagulative necrosis and occurs in the type of death we find in tuberculosis granuloma (the bacterium has tropism for the lung, which is the first site of colonization). Tuberculosis, in addition to being an infectious type of pathology, is a chronic type of pathology with granulomatous form, that is, in the course of tuberculosis, these new formations are formed that are called granulomas at the level of the lung. If the bacterium disseminates, they also form in other districts. In this form of chronic inflammatory process, in the center of the tuberculous granuloma, there is an area of caseous necrosis. In tuberculosis, which is localized at the level of the lung, the organism can eliminate the injurious agent, but the system against tuberculosis is not effective. Therefore, the tuberculosis microbacterium cannot be eliminated and attempts are made to circumscribe it. In the central necrosis zone, the cells are dead from the presence of the agent; around it, when the process becomes chronic, cells and collagen fibers are organized to prevent distribution to other areas.
• Gum-like: it occurs at the center of another form of granuloma: that of syphilis. Syphilis is a chronic inflammatory disease in which granulomas form in which the dying cells take on a gum-like appearance. In fact, syphilis used to be called gum disease because in the tertiary phase, subjects develop granulomas in different areas of the body that, when cut, have a white appearance and a gummy consistency. In appearance, gummy and caseous necrosis appear similar. The difference can be felt to the touch: caseous is soft; gummy is like gum or thick glue.
• Hemorrhagic: erythrocytes are also present in the area of dead cells. The cause of necrosis is a stagnation of erythrocytes, so a blockage of flow, a congestion of blood.
• Fibrinoid: occurs in a particular situation, in malignant hypertension. In this case there is an elevation of blood pressure and there is a particularly significant damage that sets in quickly.  This type of necrosis occurs in the vessel walls and is called fibrinoid because, in addition to dead cells, substances that are colored like fibrin accumulate in the vessel wall. The vessel loses its selective permeability and protein components of the blood leak out. If fibrin also comes out, its precursor which is fibrinogen also comes out.
• Steatonecrosis: when cell death is associated with marked lipid degradation leading the cell to take on a chalk-like appearance because saponification of fatty acids liberated by phospholipases occurs. Example: adipocytes due to pancreatic cell death (acute pancreatitis) releasing lipase.
• Gangrene: does not refer to a type of cell death but to a particular situation of death that affects all cells in a tissue, organ or portion of the body. This is a significant cell death phenomenon that can even affect a portion of a limb or an entire limb. When the gangrene episode occurs, we have the superimposition of coagulative necrosis and colliquative necrosis (at first it may have been a phenomenon of ischemia and hypoxia, then coagulative necrosis, followed by colliquative necrosis due to the superimposition of an infection). Usually, gangrene events to portions of the limbs, especially the lower limbs, are the consequence of a perfusion blocking event due to infarct-type events or, in the diabetic, due to a decrease in perfusion in general, to which an infectious-type event is superimposed.

           There are three variants of gangrene (macroscopic reference): 

            - Dry 

            - Wet: the necrotic area has a liquid component; it is the variant observed in diabetic subjects; 

            - Gaseous: superimposition of a Clostridium infection that causes the emission of gaseous type                material; it is accompanied by putrefaction of the tissue.

Markers of cellular damage/necrosis

Necrosis involves disruption of the plasmamembrane and dispersal of cellular components outside the cell, and this event always triggers an inflammatory process. From this unavoidable dispersion, it is possible to diagnose a cell death event in a given tissue.

The knowledge of the molecules that are released from the different tissues are used to analyze the levels of these in the blood and, if these appear in increased amounts, one can hypothesize a massive necrosis event in a given body district. In order to use this as painlessly as possible, a method should be used that allows for a non-invasive blood draw so that the right information can be obtained (not a biopsy of the infarcted tissue because that would be too invasive).

Overview considerations of markers of irreversible damage and cellular necrosis: 

• The markers of cell death are different depending on the type of cell that has died. In a trauma, even a mechanical one, to muscle tissue, the cells of striated tissue die and in the blood there will be proteins typical of that specific tissue (actin and myosin, for example).
• They have different timelines for increasing in the circulation. If there are different types of markers for an injury, the one with the fastest onset should be evaluated to speed up the process.
•  They must be used in association with the evaluation of the history, that is, used to confirm or not a diagnosis made with other tests (for example, if there is a heart attack, in addition to the analysis of markers, also the electrocardiogram).
•  Most markers are proteins with enzyme activity. If there is a suspicion of infarction, an initial sampling on arrival at the hospital is necessary to analyze basal enzyme levels; after a specific time, a second sampling is done. The period between the first and second sampling corresponds to the minimum time for marker elevation. We then exploit the dispersion of cellular components resulting from necrosis to arrive at a diagnosis.

Lactic dehydrogenase (LDH) is an enzyme that catalyzes the transformation of lactate to pyruvate. It is an enzyme expressed in different cell types, but it is expressed as different isoenzymes.

There are 5 different cytoplasmic isoenzymes expressed in different tissues:

- LDH1 mainly in the myocardium;

- LDH2 and LDH3 mainly in the lung;

- LDH4 not majorly produced by a specific tissue;

- LDH5 expressed primarily in the liver.

For a myocardial infarction, the LDH1 assay can be used as a marker, which increases. If, on the other hand, the suspicion is for liver necrosis, LDH5 can be used. Therefore, by measuring the amount of blood of a particular isoenzyme, it is possible to assess whether there has actually been a death by necrosis in that district where it is most expressed.

The increase in LDH, to be significant, requires 10-12h. So, you take a sample at the time of admission and one after 10-12h, and if there is an increase in LDH there is an indication in favor of necrosis.

However, the increase of these enzymes occurs also in other pathological conditions, such as megaloblastic anemia, muscle injury and renal infarction. In fact, the result must be added to the history and other evaluations.