The effect of excess nitric oxide in the morpho-functional state of the pancreas in rats


  • A. Babiy
Keywords: nitric oxide, sodium nitroprusside, pancreas, experimental pancreatitis, fibrosis

Abstract

Background. Nitric oxide (NO) is a fat-soluble gas, a high-reactive and unstable compound, which is formed from L-arginine under the action of NO-synthase. NO easily inpours cell membranes, exists only for a few seconds and being subjected to oxidation transforms to nitrites and nitrates. NO acts in all directions as a universal regulator of physiological functions and transmission of nerve impulses, a potent peripheral vasodilator and regulator of motor control and secretion. NO is a potent mediator of inflammation release in response to bacteria, viruses, proinflammatory cytokines.

Sodium nitroprusside, which is well known as simple NO-donor, can not be synthesized in cells and is an exogenous source of NO, which is traditionally used for experimental studies.

Purpose: Determine the morphofunctional characteristics of the rat pancreas under conditions of NO excess caused by the administration of sodium nitroprusside as a simple NO-donor.

Material and methods. The study was conducted in 40 male Wistar rats with weight 180-230 g. Nitric oxide (NO) was administrated in the form of sodium nitroprusside "Reahim" (Ukraine), at 1.5 mg / kg for 1 day (n = 6), 2 days (n = 6), 6 days (n = 6), 12 days (n = 6), and 30 days (n = 6). Rats were sacrificed by ketamine introduction in lethal dose of 200 mg/kg. The control group (n = 10) was formed of intact rats and recieved 0,9% NaCl for 1, 2, 6, 12 and 30 days.

Sections were deparaffinized and stained with hematoxylin-eosin or Mallory Trichrom.

Biochemical process of fibrosis was evaluated on the content in the serum of free and protein-bonded hydroxyproline and hexosamines. NO production was determined by the total content of nitrite / nitrate in serum using Gris test. To estimate the exocrine function activity of pancreatic enzymes were measured in serum - a-amylase using set of ”Filisit-diagnosis” and trypsin - using Erlanger test with modifications of Shaternikov. State of the endocrine pancreatic function was evaluated by determining the serum levels of glucose set by "Phyllis-diagnosis" (glucose oxidase method).

Results. Аfter 1 and 2 days of sodium nitroprusside administration there was a significant increase of NO metabolites in rat blood (p <0,001), upward tendency in the activity of pancreatic enzymes - α-amylase and trypsin. Morphologically dilation of blood vessels and ducts were observed, alongside with accumulation of secretion in the pancreas.

After 6 days the maximum increase in serum enzyme activity - α-amylase (p <0,05) and trypsin (p<0,05), and of the NO metabolites concentration (p <0,05) was noted, in addition to gradual increase of the HA concentration (p <0,001). Morphological signs of acute pancreatitis were shown, the structural basis of which was inflammation - stromal infiltration by lymphocytes and leukocytes, dilation of blood vessels and intralobular ducts, stasis of blood cells, presence of hypersecretory acinar cells and isolated small foci of necrosis.

After 12 days there was a downward tendency in the serum activity of α-amylase and trypsin and significant reduction of the NO metabolites concentration compared to day 6 (p <0,01). There was the significant increase of the protein-bonded hydroxyproline and free hydroxyproline concentration (p <0,01) along with HA (p<0,001). Morphological signs included stromal infiltration and vasodilatation. Some acinar cells was in state of focal adipose degeneration or segmental apoptosis.

After 30 days the maximum levels of collagen synthesis was observed - highest concentration of protein-bonded hydroxyproline in the serum (p <0,01), in parallel with catabolism - highest concentration of free hydroxyproline (p <0,05) and HA (p <0,001). Against this background functional failure of the pancreas was developing, which made itself evident in the sharp decrease of the pancreatic enzymes activity - namely α-amylase (p <0,05) and trypsin (p<0,05). Reduced concentration of NO metabolites (nitrite / nitrate) was observed after 1 (p <0,001) and 2 (p <0,001) days, with gradual increase occurred afterwards - maximum concentration after 6 days (p <0,05), followed by a gradual decrease - minimum concentration after 30 days (p <0,001). Following morphological changes after 30 days of sodium nitroprusside administration were observed: compensation of previously affected microcirculation (diameter of blood vessels did not differ from the comparison group); mild dilatation of the interlobular ducts and development of intralobular connective tissue. The tiny bands of fibrous tissue enveloped main ducts, major blood vessels and penetrates into the interlobular space zone, which is typical for chronic pancreatitis.

Conclusions.

NO excess caused by the intraperitoneal administration of sodium nitroprusside leads to following morphological changes in the pancreas: vasodilation with stasis of blood cells after 1 and 2 days; focal necrosis, destruction of acinar tissue, ductal dilatation and excessive accumulation of secretion after 6 days; adipose degeneration and segmental apoptosis after 12 days and morphologically compensated microcirculatory changes which accompanied the development of fibrous tissue around main ducts, major blood vessels and in the interlobular space after 30 days – changes typical for chronic pancreatitis.

Exocrine pancreatic function of rats responded to the excess of NO in ways of increasing pancreatic enzymes levels in blood serum – α-amylase and trypsin, which than significantly decrease after 30 days of experiment. The biochemical markers of fibrosis (free and protein-bonded hydroxyproline, hexosamine) also showed increase levels, accompanied with endocrine insufficiency. All this changes is peculiar to development of chronic experimental pancreatitis.

Published
2017-10-27
How to Cite
Babiy, A. (2017). The effect of excess nitric oxide in the morpho-functional state of the pancreas in rats. Reports of Morphology, 21(2), 312-317. Retrieved from https://morphology-journal.com/index.php/journal/article/view/157
Section
ORIGINAL INVESTIGATIONS