the digestive tract motility , secretion , and blood flow is regulated by a division of the autonomic nervous system - the enteric nervous system ( ens ) , that detect the physiological condition of the gastrointestinal tract , integrate information about it and provide outputs to control gut movement .
the ens is organized into two ganglionated nerve networks : the submucosal plexus ( smp ) and the myenteric plexus ( mp ) .
the smaller of them , smp ( the meissner 's plexus with autonomic ganglia of the parasympathetic system ) stimulates secretion from the epithelial cells of intestinal glands or the lieberkhn 's crypt in the gut lumen and contains sensory cells that communicate with motor neurons of the mp .
the larger one , mp ( the auerbach 's plexus ) is situated between the inner circular and outer longitudinal layer of tunica muscularis and contains multipolar visceral neurons responsible for motility and for mediating the enzyme output of adjacent organs .
severe disturbances of the ens function can significantly influence on the life quality or can have acute life - threatening effects .
the nitric oxide ( no ) signalling pathway is a major nonadrenergic noncholinergic inhibitory transmitter mechanism in ens and is aimed to be an endothelium - derived relaxant - type factor in the mammalian gut .
no is a presumed neurotransmitter of the gastrointestinal tract , it can play an important role in the relaxation of the smooth muscles and is involved in many physiological and pathological processes .
the production of no is indicated immunohistochemically by the demonstration of nitric oxide synthase ( nos ) , the enzyme responsible for no generation .
the presence of the constitutive nos ( neuronal and endothelial nos ) may be inferred by positive nadph - d staining , which is specific for the depiction of nerve structures and blood vessels .
the staining of other structures is a non - specific nadph - d stain that is not equivalent to the presence of nos .
nos is occurred in 29% of all nerve cell types of the mp in the small intestine and it selectively regulates the intestinal perfusion .
ninety percent of these nos neurons are inhibitory motor neurons to the muscle and 10% are interneurons . the nadph - diaphorase ( nadph - d ) staining method is widely used in the investigation of the nervous system .
the nitrergic myenteric neurons are especially susceptible to the development of neuropathy in functional gastrointestinal disorders .
previous studies have demonstrated the localization and morphology of the nadph - d positive neural elements of ens in different areas of the gastrointestinal tract and in different mammalian species , thereby implying participation of no in ens innervations . according to that , there was a correlation between nos immunoreactivity and nadph - d staining in all neurons examined . here , as in many other anatomical tissues ,
nadph - d has been demonstrated to be a neuronal nos . in order to detect the plasticity processes in the ens after
ischemia / reperfusion ( i / r ) , this study investigates the presence of nadph - d activity in the rat jejunum at three various stages after i / r .
this histochemical staining technique was used to investigate the distribution of the constitutive nos ( cnos ) in the jejunum of adult rat , to provide the morphological basis of i / r injury in the small intestine .
this injury is of obvious relevance in such situations where there is an interruption of blood supply to the gut , as in a vascular surgery , strangulated hernias or in the construction of free intestinal graft .
the experimental animals , 60 wistar male rats , weighting 300400 g , were housed two to a cage under standard conditions of temperature and illumination , and had free access to food pellets and water .
all experiments were conducted in accordance with the committee for ethics on animal experiments at the faculty of medicine , pavol jozef safarik university , kosice , slovakia , and the experimental protocol was approved by the state veterinary and food administration of the slovak republic no .
all the experiments were made at the same daily hour and season of the year .
the animals divided into four experimental groups were previously fasted overnight and were anesthetized with the intraperitoneal injection of ketamine ( narketan 10% a.u.v . , vtoquinol , 6080 mg / kg ) and xylazine ( rometar 2% a.u.v .
animals from groups r1 , r24 and r30 ( each group with 14 animals ) were subjected after a midline abdominal laparotomy to 1-hour ischemia by total occlusion of the cranial mesenteric artery by atraumatic vascular clamp .
this artery is anatomically the major artery supplying the jejunum in the laboratory rat , without any other collateral branches .
immediately after the artery was clamped , the arterial supply and pulsation were completely stopped . during the clamping ,
the oedematous jejunal intestinal segment was changed in colour from the pale anaemic to haemorrhagic dark reddish , indicating ischemia , clearly demarcated from the intestine with normal blood flow .
one hour after the ischemic insult , the vascular clamp was removed , and the affected jejunal segment was reperfused .
the immediate restoration of the blood flow was examined visually in changing of intestinal colour , together with subsequent arterial pulsation of the intestinal wall .
the laparotomy was closed and the intestine was reperfused for different periods : 1 hour ( r1 ) , 24 hours ( r24 ) or 30 days ( r30 ) after ischemia , respectively . in the sham - operated control group k ( n=18 ) ,
the animals were treated in an identical manner except that they did not undergo clamping of the artery . after relevant time periods
( 1 h , 24 h or 30 d ) after ischemia , the abdominal incision was performed and a proximal segment ( 1 cm in length ) of jejunum 8 cm distal to the ligament of treitz was isolated in and placed immediately in the fixative consisting of 4% paraformaldehyde and 0.1% glutaraldehyde buffered with 1 m sodium phosphate , ph 7.4 .
jejunal samples were stayed in the above mentioned solution for 24 h , then 1 h in ascending concentrations of sucrose ( 1530% ) and stored overnight at 4c .
the sections of the jejunum were cut transversally in a freezing microtome into 3035 m slides and processed by nadph - d histochemistry using a technique according to scherer - singler et al .
the sections were incubated for 1 h at 37c in a solution containing 1.5 mm nitroblue tetrazolium ( nbt , n6876 , sigma - aldrich , st .
louis , md , usa ) , 1.0 mm  - nadph ( nadph , n1630 , sigma - aldrich ) , 10.0 mm monosodium malate ( malic acid , m1125 , sigma - aldrich ) and 0.5% triton x-100 dissolved in 0.1 m phosphate buffer ( ph 7.4 ) . as a control for nadph - d we carried out the processing with the omission of the substrate -nadph in the reaction medium .
the sections were rinsed in 0.1 m phosphate buffer ( ph 7.4 ) , mounted on glass slides , air - dried overnight and finally coverslipped with the mounting medium entellan ( merck 100869 , whitehouse station , nj , usa ) .
the morphologic appearance of the nadph - d positive structures in the jejunum ens was evaluated histologically by the light microscope olympus ch20 .
images were obtained with the moticam 2300 camera mounted on the microscope optika b600 ti coupled to a capture board in a computer .
morphological changes of nadph - d positive neurons were evaluated in order to compare the control and i / r groups .
the experimental animals , 60 wistar male rats , weighting 300400 g , were housed two to a cage under standard conditions of temperature and illumination , and had free access to food pellets and water .
all experiments were conducted in accordance with the committee for ethics on animal experiments at the faculty of medicine , pavol jozef safarik university , kosice , slovakia , and the experimental protocol was approved by the state veterinary and food administration of the slovak republic no .
all the experiments were made at the same daily hour and season of the year .
the animals divided into four experimental groups were previously fasted overnight and were anesthetized with the intraperitoneal injection of ketamine ( narketan 10% a.u.v . , vtoquinol , 6080 mg / kg ) and xylazine ( rometar 2% a.u.v .
animals from groups r1 , r24 and r30 ( each group with 14 animals ) were subjected after a midline abdominal laparotomy to 1-hour ischemia by total occlusion of the cranial mesenteric artery by atraumatic vascular clamp .
this artery is anatomically the major artery supplying the jejunum in the laboratory rat , without any other collateral branches .
immediately after the artery was clamped , the arterial supply and pulsation were completely stopped . during the clamping ,
the oedematous jejunal intestinal segment was changed in colour from the pale anaemic to haemorrhagic dark reddish , indicating ischemia , clearly demarcated from the intestine with normal blood flow .
one hour after the ischemic insult , the vascular clamp was removed , and the affected jejunal segment was reperfused .
the immediate restoration of the blood flow was examined visually in changing of intestinal colour , together with subsequent arterial pulsation of the intestinal wall .
the laparotomy was closed and the intestine was reperfused for different periods : 1 hour ( r1 ) , 24 hours ( r24 ) or 30 days ( r30 ) after ischemia , respectively . in the sham - operated control group k ( n=18 ) ,
the animals were treated in an identical manner except that they did not undergo clamping of the artery . after relevant time periods
( 1 h , 24 h or 30 d ) after ischemia , the abdominal incision was performed and a proximal segment ( 1 cm in length ) of jejunum 8 cm distal to the ligament of treitz was isolated in and placed immediately in the fixative consisting of 4% paraformaldehyde and 0.1% glutaraldehyde buffered with 1 m sodium phosphate , ph 7.4 .
jejunal samples were stayed in the above mentioned solution for 24 h , then 1 h in ascending concentrations of sucrose ( 1530% ) and stored overnight at 4c .
the sections of the jejunum were cut transversally in a freezing microtome into 3035 m slides and processed by nadph - d histochemistry using a technique according to scherer - singler et al .
the sections were incubated for 1 h at 37c in a solution containing 1.5 mm nitroblue tetrazolium ( nbt , n6876 , sigma - aldrich , st .
louis , md , usa ) , 1.0 mm  - nadph ( nadph , n1630 , sigma - aldrich ) , 10.0 mm monosodium malate ( malic acid , m1125 , sigma - aldrich ) and 0.5% triton x-100 dissolved in 0.1 m phosphate buffer ( ph 7.4 ) .
as a control for nadph - d we carried out the processing with the omission of the substrate -nadph in the reaction medium .
the sections were rinsed in 0.1 m phosphate buffer ( ph 7.4 ) , mounted on glass slides , air - dried overnight and finally coverslipped with the mounting medium entellan ( merck 100869 , whitehouse station , nj , usa ) .
the morphologic appearance of the nadph - d positive structures in the jejunum ens was evaluated histologically by the light microscope olympus ch20 .
images were obtained with the moticam 2300 camera mounted on the microscope optika b600 ti coupled to a capture board in a computer .
morphological changes of nadph - d positive neurons were evaluated in order to compare the control and i / r groups .
nadph - diaphorase histochemical procedure results in the deposition of a dark blue formazan reaction product insoluble in water .
the cell body , dendritic trees and nerve fibre networks of positive neurons in the rat jejunum were stained in their entirety .
nadph - d activity was generally present in the nerve fibres running throughout the muscular layer and in numerous ganglion cells and processes in the mp . in the smp
, only a few positive nerve fibers were running in the vicinity of the blood vessels , whereas in the muscular layer they were seen quite abundant ( figures 14 ) . 

figure 1histological section through jejunum in group k. the nadph - diaphorase positive neurons in the myenteric plexus of jejunum in the control group ; tunica mucosa ( m ) , tela submucosa ( sm ) , tunica muscularis ( tm ) .
the myenteric plexus is intensely nadph - d stained , neurons ( n ) are formed into small ganglions . rounded or oval shaped neurons are seen located along the nerve fibres ( thin arrows ) running throughout the tunica muscularis .
the mucosa of the jejunum is arranged into regular darkly stained intestinal glands or lieberkhn 's crypts ( lc ) , which are bordered by nadph - d positive nerve fibres ( thick arrow ) .
histological section through jejunum in group k. the nadph - diaphorase positive neurons in the myenteric plexus of jejunum in the control group ; tunica mucosa ( m ) , tela submucosa ( sm ) , tunica muscularis ( tm ) .
the myenteric plexus is intensely nadph - d stained , neurons ( n ) are formed into small ganglions . rounded or oval shaped neurons are seen located along the nerve fibres ( thin arrows ) running throughout the tunica muscularis .
the mucosa of the jejunum is arranged into regular darkly stained intestinal glands or lieberkhn 's crypts ( lc ) , which are bordered by nadph - d positive nerve fibres ( thick arrow ) .
one h reperfusion after the ischemia causes a decrease of nadph - d positivity in the tunica muscularis of the jejunum ; tunica mucosa ( m ) tela submucosa ( sm ) , tunica muscularis ( tm ) .
the neurons of myenteric plexus ( n ) are weakly nadph - d stained , variable in their shape , size and in the arrangement into the ganglion .
the nerve fibres ( thin arrows ) running throughout the tunica muscularis are interrupted , the neurons are seen with unclear contours of their cell bodies , nuclei , and cytoplasmic processes .
the intestinal lieberkhn 's crypts ( lc ) of the mucosa do not show the nadph - d positivity , only nerve fibres bordering them are present ( thick arrow ) .
one h reperfusion after the ischemia causes a decrease of nadph - d positivity in the tunica muscularis of the jejunum ; tunica mucosa ( m ) tela submucosa ( sm ) , tunica muscularis ( tm ) .
the neurons of myenteric plexus ( n ) are weakly nadph - d stained , variable in their shape , size and in the arrangement into the ganglion .
the nerve fibres ( thin arrows ) running throughout the tunica muscularis are interrupted , the neurons are seen with unclear contours of their cell bodies , nuclei , and cytoplasmic processes .
the intestinal lieberkhn 's crypts ( lc ) of the mucosa do not show the nadph - d positivity , only nerve fibres bordering them are present ( thick arrow ) .
twenty - four hours reperfusion following ischemia makes the increase of nadph - d positivity in the myenteric plexus of the jejunum compared with group r1 ; tunica mucosa ( m ) , tela submucosa ( sm ) , tunica muscularis ( tm ) . the neurons ( n ) in myenteric plexus are clearly contoured and show more regular shape of their cell bodies , stained nerve fibres ( thin arrows ) are seen running throughout the muscular layer .
the lieberkhn 's crypts ( lc ) are inside moderately nadph - d stained , bordered by nadph - d - positive nerve fibres ( thick arrows ) .
twenty - four hours reperfusion following ischemia makes the increase of nadph - d positivity in the myenteric plexus of the jejunum compared with group r1 ; tunica mucosa ( m ) , tela submucosa ( sm ) , tunica muscularis ( tm ) .
the neurons ( n ) in myenteric plexus are clearly contoured and show more regular shape of their cell bodies , stained nerve fibres ( thin arrows ) are seen running throughout the muscular layer .
the lieberkhn 's crypts ( lc ) are inside moderately nadph - d stained , bordered by nadph - d - positive nerve fibres ( thick arrows ) .
the nadph - d positivity in the myenteric plexus of jejunum after ischemia and 30 d reperfusion is seen with no differences in nadph - d positivity compared with k group ; tunica mucosa ( m ) , tela submucosa ( sm ) , tunica muscularis ( tm ) .
nadph - d activity is present mainly in numerous ganglion cells ( n ) and nerve fibres ( thin arrows ) in the tunica muscularis and is also visible in the lieberkhn 's crypts ( lc ) , which are bordered by the nadph - d - positive nerve fibres ( thick arrows ) .
the nadph - d positivity in the myenteric plexus of jejunum after ischemia and 30 d reperfusion is seen with no differences in nadph - d positivity compared with k group ; tunica mucosa ( m ) , tela submucosa ( sm ) , tunica muscularis ( tm ) .
nadph - d activity is present mainly in numerous ganglion cells ( n ) and nerve fibres ( thin arrows ) in the tunica muscularis and is also visible in the lieberkhn 's crypts ( lc ) , which are bordered by the nadph - d - positive nerve fibres ( thick arrows ) .
scale bar = 40 m . in the control group , sham - operated animals ( k ) ,
the higher nadph - d activity was found in the mp located in the muscular layer .
the nerve fibres were seen running surrounding blood vessels both in the muscular layer and lamina propria .
the mucosa of the jejunum was arranged into regular intestinal villi and lieberkhn 's crypts ( lc ) .
neurons of the mp were found in ganglia , which were predominantly elongated with the major axis oriented longitudinally in relation to the circular smooth muscle .
these rounded or oval shaped neurons were located uniformly with nerve fibres running mainly throughout the circular muscle layer ( figure 1 ) .
the most marked differences in jejunal histomorphology were observed in animals after ischemia and 1 h of reperfusion ( r1 ) .
the mucosa of the jejunum has sloughed off compared with the control and irregularly arranged with apparent differences between the intestinal villi ; some of them were visible with a total ischemic dissolution in the vicinity to the near - normal villi .
nadph - d activity of both ens plexuses from the r1 group compared with the k group has been rapidly decreased in neurons .
nadph - d positivity was distributed mainly in the mp , less in the smp .
compared with the k group , differences were predominantly detected in neurons of the mp , which were very variable in a neuronal size , shape and in their arrangement into the ganglion .
the neurons had unclearly seen contours of their cell bodies , nuclei , and processes .
nadph - d positivity was absent in the lc , but was abundant in a new vascularised lamina propria surrounded blood vessel ( figure 2 ) . in animals exposed to ischemia and 24 h reperfusion ( r24 ) ,
the nadph - d activity was markedly increased in comparison with the r1 group , but it was still lower than in the k group .
this increasing was noticed in both ens plexuses as well as and in tunica propria around the blood vessel .
the neurons in the mp compared with the r1 group become swollen ; they were more intensively nadph - d stained with greater regularity in shape of cells bodies than those of the k group .
the positive staining was not identified at the base of the intestinal villi and the lc ( figure 3 ) . on the 30th day following i / r ( r30 ) all changes noticed in previous experimental groups ( r1 and r24 ) disappeared and the histochemical picture of the jejunum returned to the control level with no differences in nadph - d positivity compared to the k group .
nadph - d activity was present mainly in neuronal cell bodies and processes in the mp .
the positive nadph - d staining was again identified at the base of the villi and in the depth of the lc ( figure 4 ) .
these results indicated that the jejunal i / r has impaired nos neurons of the ens and makes an influence on early decrease in nadph - d positivity 1 hour following reperfusion .
histomorphological analyses confirmed that 24 h reperfusion after the ischemia resulted in increasing of nadph - d staining in the ens plexuses .
finally , 30 days following the reperfusion , the nadph - d picture of the jejunal sample has been returned to the control level .
this gradual increasing of nadph - d activity in the r24 group could be considered as a result of the plasticity process .
the presence of nadph - d staining in the proximal jejunum of the adult rat during i / r was studied in this experiment .
no as an intercellular and endocellular signal molecule has an important role in the physiological processes of the intestine , it regulates muscular contraction and blood circulation of the intestinal endothelia .
the cnos is widely distributed in the intestine and the presence of the neuronal ( nnos ) and endothelial ( enos ) are in accordance with nadph - d staining . in other tissues ,
the cnos is critical to normal physiology and development , and inhibition of this enzyme causes a tissue injury .
according to the current investigations , except of the cnos there is an inducible nos isoform ( inos ) expressed in myenteric neurons , enteric smooth muscle cells , and in the capillary endothelium .
the presence of different isoforms with similar functions in the same cells might reflect a functional plasticity , in which one nos isoform can replace another under different pathophysiological conditions .
the nadph - d reactive neurons , which are inhibitory motor neurons and interneurons , are generally present mostly in the ganglions of the mp , and they are intermingled with many other unstained neurons in the small intestine .
the nadph - d neurons represent a subpopulation of myenteric neurons , which is the only one third of the total number of a myenteric population .
the ganglia of the mp are elongated and oriented longitudinally in relation to the circular layer of tunica muscularis . as for the ganglion orientation , a similar distribution was found in the jejunum - ileum in other animals : of mice , guinea pigs , sheep , cats or rabbits . labelled ganglion cells and nerve fibres
were observed in the smp as well , although they were less numerous than in the mp . in the submucosa , the positive staining
the nadph - d positive neuronal elements of the smp were located around blood vessels and can regulate the blood flow and secretion of the glands , or it is also possible that they belong to the sensory neurons . in the mucosa ,
the nerve fibres were arranged sparsely at the base of the intestinal villi and the lc .
in addition to the neuronal staining , there were seen submucosal arterioles with a pattern of small patches of staining unrelated to any perivascular innervation .
these findings indicate an extensive neuronal and vascular localization of no generation potential throughout the wall of the rat intestine , thus providing a structural basis for the functional diversity of no .
the occlusion and reperfusion of the cranial mesenteric artery is a useful animal model to elucidate the mechanism of gastrointestinal injury induced by i / r .
paradoxically , the restoration of the blood supply causes an additional cell injury that is referred to as a reperfusion injury . in the intestine ,
the intestine appears to be the most sensitive to i / r injury comparing to other internal organs due to the high susceptibility of enterocytes that are easily injured by episodes of ischemia , and subsequent reperfusion . neurons that survive show an evidence of damage for several weeks , but the mucosa quickly repairs .
the small intestinal mucosa is a vulnerable system whose epithelium turns over every 23 day .
the mucosa of the rat jejunum after ischemia and 1 hour of reperfusion were sloughed off compared to the control .
this attribute especially at early reperfusion time points have been well documented also in mice .
the nadph - d positive neurons of the rat jejunum become swollen following 1 h ischemia and 24 hreperfusion . in two different studies , rivera et al .
showed that the dendrites of nos immunoreactive neurons distorted and the neurons become swollen following 1 h ischemia and 24 h reperfusion also in the guinea pig and mouse ileum .
morphologies of no neurons were altered early and persisted up to 4 weeks following i / r , so it is suggests that no could be involved in the changes .
the cell - specific changes occurred without there being significant changes in the relative numbers of neurons of different subtypes . at 24 h after the initial ischemic insult , the mucosa was as a result of plasticity the most resistant to i / r injury .
the mp is also a highly dynamic and adaptive structure and increasing of nadph - d activity in 24 h following ischemia ( r24 ) in our experiment confirms this process of plasticity as well .
the i / r injury may contribute to the increasing of intestinal mucosa permeability and may lead to the development of a multiple organ failure .
the endotoxins , bacteria and inflammatory mediators are transported particularly by the portal blood and by lymphatic vessels and can cause damage of distant vital organs .
the present results demonstrate that the nitrergic neurons and their processes are widespread in the rat jejunum and they can react in the plasticity and regeneration processes during i / r .
this supports the hypothesis that no in this organ plays an important role not only as a neurotransmitter in the smooth - muscle relaxation or vasodilation in the periphery .
it also could be involved in neuronal communication within the enteric nerve plexuses and may influence on different digestive functions .
 the purpose of this study was to analyze the nicotinamide adenine dinucleotide phosphate - diaphorase ( nadph - d ) activity in the rat jejunum after a mesenteric ischemia / reperfusion injury . 
 nitric oxide , synthetised from l - arginine by the enzyme nitric oxide synthase , is a nonadrenergic noncholinergic relaxant neurotransmitter of the intestinal smooth muscle . 
 it plays an important role in the process of plasticity after the ischemia / reperfusion injury . 
 experimental animals were divided in two groups : the control group and the ischemic / reperfusion group , with different period of the reperfusion . the nadph - d histochemical method has been used as a marker for the nitric oxide synthase . 
 nadph - d activity has been rapidly decreased in the neurons of both enteric nervous systems in plexuses of the jejunum after 1 h mesenteric ischemia and 1 h reperfusion . 
 differences were predominantly detected in the myenteric plexus ; they were seen in change of the neuronal shape , in the arrangement of neurons and in intensity of their staining . 
 the nadph - d positivity was absent in the intestinal crypts . 
 after 1 h ischemia and 24 h reperfusion , the nadph - d activity was gradually increased , but it was lower in comparison with the control group . on the 30th day 
 following the ischemia / reperfusion there were no changes in nadph - d positivity compared with the control animals . 
 these results indicated that the jejunal ischemia / reperfusion has affected the neurons of the enteric nervous system of adult rats and resulted in the early decrease of nadph - d positivity 1 h of the reperfusion insult . 
 the gradual increasing of nadph - d activity in 24 h following the reperfusion could be considered as a result of the plasticity process . on the 30th day after the ischemia / reperfusion all histochemical changes 
 were returned to the control levels . 
Introduction
Materials and Methods
Animal treatment and tissue preparation
Histochemical procedure
Assessment
Results
Discussion