© Borgis - Postępy Nauk Medycznych 6/2014, s. 395-399
*Barbara Sobala-Szczygieł1, Brygida Adamek2, Michał Kukla3, Anna Żurek1, Jerzy Ihnatowicz4, Barbara Oczko-Grzesik1, Lucjan Kępa1, Grażyna Spausta2, Andrzej Wiczkowski2, Marek Hartleb3
Czy interferon a2b wywiera wpływ na różnicę zawartości DNA w proliferujących hepatocytach strefy okołocentralnej i okołowrotnej u szczurów po częściowym usunięciu wątroby?
Does Interferon a2b administration exert an effect on DNA content difference in proliferating hepatocytes of perivenular and periportal zones in rats after partial hepatectomy?
1Department of Infectious Diseases, Medical University of Silesia, Katowice
Head of Department: prof. Anna Boroń-Kaczmarska, MD, PhD
2Department of General Biology, Medical University of Silesia, Katowice
Head of Department: prof. Andrzej Wiczkowski, MD, PhD
3Department of Gastroenterology and Hepatology, Medical University of Silesia, Katowice
Head of Department: prof. Marek Hartleb, MD, PhD
4Institute of Electronics Silesian University of Technology, Gliwice
Head of Institute: prof. Jacek Łęski, MD, PhD
Streszczenie
Wstęp. W związku z możliwością naprawy wątroba jest unikalnym narządem organizmu ssaków. Eksperymentalnym modelem zwierzęcym do badania regeneracji wątroby jest zabieg częściowej hepatektomii u szczura. Model ten pozwala także na odrębną ocenę komórek przestrzeni okołowrotnej i okołocentralnej. Barwienie tkanek metodą Feulgena jest powszechnie przyjętym procesem w celu oceny DNA.
Cel pracy. Celem pracy jest ocena długotrwałego wpływu IFN na zawartości DNA w jądrach hepatocytów i replikację komórek pod wpływem interferonu w heterogennych obszarach okołowrotnym i okołocentralnym.
Materiał i metody. Sześćdziesiąt samców szczurów rasy Wistar, zostało podzielonych na trzy grupy, po 20 zwierząt każda. Zwierzętom pierwszej grupy (A) wstrzyknięto roztwór soli fizjologicznej, po 24 godzinach przeprowadzono 70% hepatektomię, po następnych 24 godzinach podano drugą dawkę soli fizjologicznej. Podobny schemat zastosowano w drugiej grupie (B), jednak zamiast soli fizjologicznej podano interferon (INF)-α2b. Trzecią grupę (grupa C) poddano zabiegowi pozorowanemu i podano IFN. Następnie szczury zabijano po 48, 72, 96 i 120 godzinach od zabiegu. Wszystkie skrawki barwiono metodą Feulgena i oceniano uwzględniając położenie hepatocytów względem naczyń (strefa okołowrotna i okołocentralna).
Wyniki. Uzyskane wyniki wykazały wyższą zawartość DNA w jądrach hepatocytów replikujących pod wpływem interferonu w strefie okołowrotnej w porównaniu do komórek strefy okołocentralnej. Takie samo zjawisko obserwowano w komórkach niereplikujących.
Wnioski. Wyższa zawartość DNA w jądrach hepatocytów strefy okołowrotnej replikujących pod wpływem interferonu w stosunku do komórek strefy okołocentralnej może mieć wpływ na niektóre funkcje wątroby podczas terapii interferonem oraz zwiększenie toksyczności leków stosowanych podczas leczenia przeciwwirusowego, które są metabolizowane przez cytochrom P-450. Może to odgrywać szczególną rolę w przypadku zaawansowanej choroby wątroby i małej objętości funkcjonalnej tkanki.
Summary
Introduction. The liver is almost unique amongst the tissues of the body in its capacity for regeneration. Multiple studies have been conducted to study the liver regeneration in rodents. The experimental animal model for the study of liver regeneration is a 70% partial hepatectomy in the rat. In the study periportal and perivenular zones are observed separately. The Feulgen reaction is generally accepted as a stoichiometric DNA staining method.
Aim. The aim of the study is the evaluation of the long-term influence of two successive doses of IFN on DNA content in nuclei of replicating hepatocytes and cells under the influence of interferon in periportal zones compared to perivenular zones.
Material and methods. Sixty male Wistar rats, three-month-old were divided into three groups of 20 animals each. The first group (group A) was injected with normal saline, 24 hours later 70% hepatectomy was performed and after the next 24 hours the second dose of normal saline was administered. A similar schedule was applied in the second group (group B) injected with interferon (INF)-α2b instead. The third group (group C) was injected with IFN-α2b and sham operated. Then the rats were killed at 48, 72, 96 and 120 hours after the surgery. All Feulgen-stained sections were divided into anatomical zones: perivenular and periportal. Using morphometric technique, in sequential pairs of sections around each central vein and each portal triad, the area of around 200 000 μm2 was determined, which defined perivenular and periportal zones, respectively.
Results. This study revealed a higher DNA content in nuclei of replicating hepatocytes and cells under the influence of interferon in periportal zones compared to perivenular zones. The same phenomenon is observed in non-replicating cells.
Conclusions. This study revealed a higher DNA content in nuclei of replicating hepatocytes and cells under the influence of interferon in periportal zones compared to perivenular zones. It may affect some of the liver functions while on interferon therapy as well as increase toxicity of drugs used during antiviral treatment which are metabolised by cytochrome P-45. It is particularly important in cirrhotic patients with the small volume of functional liver tissue.
Introduction
The liver is almost unique amongst the tissues of the body in its capacity for regeneration. It is likely to believe that studies on liver regeneration will contribute to our understanding of pathways governing mammalian organogenesis. The hepatocytes of adult liver divide only rarely under normal conditions. The loss of liver functional mass, leads to rapid proliferation and restoration of functional liver tissue (1). Not only is this knowledge crucial for clinical medicine, but also for the use of interferon and stem cells for therapy of liver disorders. In addition, an increased understanding of the liver regeneration cascade in humans could lead to improved therapies for the treatment of patients with acute or chronic liver pathologies, where the ability to specifically stimulate liver cells would be valuable. These patients suffer from profound liver insufficiency because of the small volume of residual or transplanted liver tissue. Since Higgins and Anderson pioneered the experimental model for the study of liver regeneration in 1931, multiple studies have been conducted to study the liver regeneration in rodents (2). The experimental animal model for the study of liver regeneration is a 70% partial hepatectomy in the rat, because more than 90% of the hepatocytes in the remnant 30% cells are in the process of dividing and the liver mass if fully restored some 7 to 10 days later.
From a metabolic perspective, the functional unit is the hepatic acinus, each of which is centered on the line connecting two portal triads and extends outwards to the two adjacent central veins. The blood flows directly from the portal triad towards the central vein, while in between the portal triads and the central vein, rows of hepatocytes are arranged in single lines with a sinusoid on one side, and a bile canaliculi on the other. Bile is emptied from the hepatocytes into the bile canaliculi and flows in the opposite direction of the blood, i.e. towards the portal bile duct for drainage. The periportal zone I is nearest to the entering vascular supply and receives the most oxygenated blood, making it least sensitive to ischemic injury while making it very susceptible to viral hepatitis. Conversely, the centrilobular zone III has the poorest oxygenation, and will be most affected during a time of ischemia.
Functionally, zone I hepatocytes are specialized for oxidative liver functions such as gluconeogenesis, β-oxidation of fatty acids and cholesterol synthesis, while zone III cells are more important for glycolysis, lipogenesis and cytochrome P-450 and thus most sensitive to toxicity (3). New hepatocytes arise in the periportal area, and then gradually migrate towards the perivenular area. In the study periportal area and perivenular area are observed separately.
Several methods have been employed to quantify nuclear DNA. Some of the studies involved bulk biochemical DNA extraction techniques to estimate the total DNA content of a preparation.
The Feulgen reaction is generally accepted as a stoichiometric DNA staining method, although the chemical processes are only partly understood. Feulgen densitometry relies on the simple premise that the amount of stain bound is directly proportional to the amount of DNA present. The quantity of stain is itself determined based on the amount of light it absorbs. In brief, in a first step, the DNA is submitted to mild acid hydrolysis to split off the purine bases from the double-stranded DNA lobes (4). The result is an apurinic acid presenting reactable aldehyde groups at the C1-position. In a second step, a Schiff’s base binds stoichiometrically to these aldehyde groups and produces a reddish to blueish-violet colour with an absorption maximum of 545 nm (5, 6).
The preparations stained DNA content by Feulgen calculated the integrated optical density (IOD) nuclei. The optical density of the transmitted material is a physical quantity equal to the logarithm ratio of the intensity of light falling on the test material (lp) to the intensity of light after passing the material (Ik) otherwise called absorbance:
Dn = \ log (I_p1/I_k1).
This volume is characterized by the amount of radiation passing through the transmittance material (slide or negative) or reflected from the material is not transmittance (photography, copy printing). The higher the optical density D, the more absorbing material. In order to measure the optical density of the cell nuclei stained by the Feulgen further assess the background optical density (laboratory slides):
Dt = \ log (I_p2/I_k2).
Calculating the difference between the optical density of cell nuclei studied (Dn) and background (Dt), we obtain the value of the integrated optical density of nuclei:
Do = Dn-Dt.
Instead, absorbance (optical density – OD) must be calculated indirectly from measurements of the amount of light passing through the object (transmittance – T). Transmittance, in turn, is measured as the difference between the intensity of incident light entering the object and that of the transmitted light leaving it. In Feulgen DNA densitometry, measurements are taken both within the nucleus and outside the nucleus in a clear area of the slide. The difference in light intensity between the two areas represents the transmittance. It is necessary to take a series of point densities covering the entire nuclear area. The sum of these individual optical densities is the integrated optical density (IOD) of the nucleus.
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Piśmiennictwo
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