Experimental Models to Study Gastroprotection

An experimental model for the study of gastroprotection requires the induction of mucosa injury with the less as possible suffering of the animal. Between all the experimental models the induction of injury for ethanol absolute and NSAIDs administration are the most popular. However, administration of acidified ethanol (HCl:EtOH), NaOH, stress-induced ulcer, pylorus ligation and acetic acid are used depending of the mechanism and expected results from the researcher. In this chapter, we will describe the gastric damage induce by ethanol and NSAIDs administration and the parameters that could be measured.

4.1. Ethanol Induce Gastric Damage

Oral administration of ethanol has been described to induce gastric damage; the severity of the injury is related with the doses of ethanol. Absolute ethanol induces severe histopathological changes in oxyntic mucosa of mouse and rat stomach consisting of acute erosive hemorrhagic lesions, vascular congestion, edema and necrosis [94, 95]. Furthermore, ethanol causes depletion of the gastric levels of proteins, nucleic acids, NP-SH (non-protein sulfhydryl groups) and an increment on MDA (malondialdehyde) levels and decrement of antioxidants substances [96].

Moreover, it has been described that depletion of NP-SH groups by ethanol increases the content of free radicals mediate tissue injury by stimulating lipid peroxidation and membrane damage [94].

Low doses of ethanol can induce damage as well; for example, administration of 25 % of ethanol induced a decrement on mucus secretion and an increment on the acid juice secretion [97]. However, this effect has been related lately with the termed cytoprotection adaptative.

Furthermore, administration of 50 % of ethanol induces injury by constriction of venules and this effect is reverted by prostaglandin exogenous administration [98]. Absence of blood flow develops extensive gastric mucosa damage within a short period of time after contact with absolute ethanol. In contrast, no changes in blood flow exhibits no injury after ethanol administration [99, 100].

Those results together suggests that blood flow plays an important role in the pathogenesis of ethanol-induced gastric injury; studies by intravital microscopy have shown that damage occurs first by submucosal venular constriction, followed by cessation of mucosal blood flow and later mucosa necrosis [101]. How it has been seen with NSAIDs, on ethanol-induced gastric injury the decrement on gastric blood flow appears after an increment on leukotrienes [102] which may obstruct mechanically blood flow and recruits leukocytes that make worst the damage. Besides, mieloperoxidase activity (MPO, a marker of neutrophil infiltration) increased after ethanol administration. MPO increment correlates with ethanol-induced gastric injury [103].

TNF-α has been related in ethanol-induced gastric mucosa injury due to it cause inflammation and its synthesis is inhibited by cytoprotective prostaglandins. Furthermore, inhibition of TNF- α decrease ethanol-induced gastric injury [7] and pentoxyfilline a TNF- α inhibit neutrophil migration, being this a step to produce gastric damage [104]. In addition, exposure of the gastric mucosa to 40% of ethanol caused an increment in plasma TNF- α levels [7].

In summary, the damage induced by ethanol destroys the mucosa and submucosa caused by necrosis. The low levels of prostaglandins observed after ethanol administration is not due to cyclooxygenase inhibition, this should be more related with the necrosis of epithelial cells by direct contact with ethanol; epithelial cells are the responsible for prostaglandin secretion.

4.2. NSAIDs-Induced Gastric Damage

NSAIDs induced gastric damage due to their ability to inhibit prostaglandin synthesis, and it has been established in various studies [105, 106]. There are a correlation between time and dose dependent manner of suppression of gastric prostaglandin synthesis by NSAIDs and their ability to induce gastric ulcers [105].

The most common alterations caused by NSAIDs in gastric area are hemorrhagic gastric erosions, found more often in the fundus and corpus. While gastric ulcers in the antrum, are of greater clinical importance than erosions, due to their chronicity and the potential for perforation and bleeding [107].

As it has been described previously COX-1 is the predominant form expressed in the normal gastrointestinal tract [109]; however, COX-2 is rapidly up-regulated in response to a number of stimuli, such as administration of aspirin of indomethacin or following a period of ischemia [108, 109]. COX-1 participates in the secretion of mucus and bicarbonate and in the increment on gastric blood flow, while COX-2 is enrolled in the decrement of leukocyte adherence and re-epithelization of gastric cells [32]. Furthermore, COX-2 plays a very important role in ulcer healing [36]. Then, inhibition of COX-1 by NSAIDs reduces gastric mucosal blood flow [110]. Prostaglandins are potent vasodilators that are continuously produced by the vascular endothelium. NSAIDs also produce damage to the vascular endothelium and this is a very early step to induce injury in the gastrointestinal tract [33, 111]

(Figure 3).

Figure 3. Phospholipase A2 (PLA2) synthesizes arachidonic acid (AA) from membrane phospholipids.

Cyclooxygenases metabolizes AA to different prostaglandins in gastric mucosa PGE2 and PGI2 are bioconverted to increase mucosal gastric blood flow, increase mucus and bicarbonate secretion by COX-1. While prostaglandins synthesized from COX-2 participates decreasing leukocyte adherence and in re-epithelization of gastric cells. When an NSAID is administered, prostaglandins synthesis is inhibited by NSAID-inhibit COX and then gastroprotection is disrupted.

Besides, the increment on leukocyte adherence induce by NSAIDs is another important step in the pathogenicity caused in gastric mucosa. Treatment with monoclonal antibodies that blocked neutrophil adherence to the vascular endothelium markedly attenuated the severity of NSAID gastropathy in rats and rabbits [112, 113]. The augmented leukocyte adherence in mesentery is induced in an early step by leukotrienes due to inhibitors of leukotriene synthesis or antagonist of leukotriene receptors have been shown protective effects in experimental NSAID-induced gastric damage [112, 113]. Also, it has been studied the leukotriene B4 levels after NSAID administration to rats [15] and humans [114].

Furthermore, inhibitors of leukotriene B4 exert a decrement on leukocyte adherence to venules induce by NSAID [91].

Neutrophil adherence is regulated by endothelial expression of intracellular adhesion molecule 1 (ICAM-1) and selectin expression in neutrophil, those molecules allows the attachment of neutrophil to endothelial tissue [92, 115]. Another signal molecule that mediate NSAID-induced leukocyte adherence is the increment observed in TNF-α; TNF-α levels are increased in plasma after indomethacin administration to rats, and this correlates with the accumulation of neutrophils in the gastric microcirculation and the appearance of gastric injury [93].

Traditional NSAIDs inhibit COX-1 and COX-2, and then their gastric toxicity is induced by inhibition of prostaglandins derived from both enzymes. In the nineteen decade researchers thought that just COX-1 played the role of gastroprotective enzyme and COX-2 synthesized pro-inflammatory prostaglandins. With this premise coxibs (selective COX-2 inhibitors) were synthesized to reduce gastric injury induced by NSAID treatment [116].

However, some coxibs such as rofecoxib and valdecoxib have been withdrawn from the market due to their induced cardiovascular problems [32], such as heart broken. This event occurs because COX-2 inhibitors blocks prostacyclin (PGI2) synthesis in endothelium, a vasodilator and anti-aggregative molecule; while COX-1 remains free to produce thromboxane (TXA2) a vasoconstrictor and pro-aggregative substance. Furthermore, later was found that COX-2 plays a healing role in gastric mucosa, it is highly expressed in process where COX-1 is absent, and both isoforms of COX needs to be inhibited to cause gastric injury [35].

The solution at this moment for the abolishment of NSAIDs-induce gastric injury has been the attachment of NO or H2S to the moiety of traditional NSAIDs such as naproxen, diclofenac and aspirin. Those molecules that release NO or H2S have demonstrated to reduce the severity of NSAID-induce gastric damage throw the decrement on leukocyte adherence, adhesive molecules, TNF-α and the increment on gastric blood flow [5, 13, 24, 45, 63]. The studies in new molecules of NSAIDs donors of NO or H2S could help to reduce NSAIDs gastric toxicity.

The main mechanism for NSAIDs to induce damage is the inhibition on the synthesis of prostaglandins. The absence of prostaglandins derive a decrement on mucus and bicarbonate secretion, increment on acid secretion, decreased gastric blood flow, an augment on leukocyte adherence and adhesive molecules. Those events are significant changes for the appearance of damage; the good management of them may help to diminish the pathogenecity of NSAIDs.