{"paper_id":"563754a2-b6bf-45de-8b33-365dd1ef1cf4","body_text":"Prostaglandins (PGs) are members of a group of lipid compounds derived enzymatically\nfrom fatty acids ( Goodwin, 2010 ). They\nmediate many cellular functions such as cytoprotection of the gastric mucosa, renal\nfunction, gestation, parturition, neurotransmission, vasomotion, reproduction,\nmetabolism, homeostasis, and are implicated in dysmenorrhea. Dysmenorrhea is the\npain associated with menstruation and is classified as either primary or secondary\nbased on the existence of underlying disease ( Wong\n et al ., 2009 ). Primary dysmenorrhea, the most\nfrequent type of dysmenorrhea, is associated with elevated prostaglandin levels,\nuterine ischemia and painful menstrual cramps with no detectable pelvic pathology.\nIn women clinically diagnosed with dysmenorrhea, endometrial PG levels were found to\nbe significantly higher when compared with controls ( Ostad  et al ., 2001 ). Reports include accounts of\nabdominal pain, cramps, headaches, and other systemic symptoms such as facial\npaleness, cold sweats, nausea, vomiting, and bloating in primary dysmenorrheic women\n( Deligeoroglou, 2000 ;  Jia  et al ., 2006 ). The\nincreased release of uterine PG produces significant degree of myometrial\nhyperactivity, which results in uterine hypoxia and ischemia associated with\nabnormal uterine contractions. Many non-steroidal anti-inflammatory drugs (NSAIDs)\nthat are prostaglandin synthase inhibitors have been shown to be effective in the\ntreatment of primary dysmenorrhea.\nPiroxicam and nitroglycerin are common NSAIDs used in the management of dysmenorrhea\n( Wong  et al ., 2009 ;\n Marjoribanks  et al .,\n2015 ). These drugs work by reducing the activity of cyclo-oxygenase\npathways and thus inhibiting PG production ( Burian\n& Geisslinger, 2005 ). Since prostaglandins are also involved in\nplatelet plug formation, renal excretion of electrolytes, parturition and metabolism\n( Goodwin, 2010 ), the treatment of\ndysmenorrhea with NSAIDs is likely to disrupt these functional pathways. This study\ncompared the effects of piroxicam and nitroglycerin on some prostaglandin-modulated\nblood and electrolyte indices during the di-estrous phase in female Wistar rats. The\ndi-estrous phase of female Wistar rats was used as a model for dysmenorrhea because\nof the similarities it shares with the human menstrual phase.\n\nDrugs: Piroxicam (purchased under the brand name Feldene) and Nitroglycerin were\nobtained from Pfizer Pharmaceuticals, New York, USA.\nFifteen (15) female Wistar rats (120-160 g) were divided into three groups of 5\nrats. The animals were acclimatized to laboratory conditions for 14 days prior\nto experimental procedures. They were housed in well aerated cages, maintained\non standard rat pellets (Ladokun feeds, Nigeria) and allowed free access to\ndrinking water. The guidelines of Animal Care and Use of the Research Ethics\nCommittee, University of Ibadan, and of the National Research Committee, 1996\nfor the Care and Use of Laboratory Animals published by the National Academy\nPress, 2101 Constitution Ave. NW, Washington, DC 20055, USA, were followed.\nPrior to the study, estrous cycle was established in each rat using the pipette\nsmear method of  Long & Evans (1922) \nand monitored for 12 days. The different estrous phases (pro-estrous, estrous,\nmet-estrous and di-estrous) were established by the presence, absence or\nproportional numbers of epithelial cells (two types), cornified (keratinized)\ncells and leucocytes. Presence of predominance leukocytes in the vaginal smear\nwas used to classify animals in the di-estrous phase ( Long & Evans, 1922 ). Each group was then allocated 5\nanimals in the di-estrous phase as follows:\nGroup 1 served as controls and were given distilled water (0.3 mL).\nGroup 2 was treated with Piroxicam (3 mg/kg) ( Sharp & Villano, 2012 ) and\nGroup 3 was treated with Nitroglycerin (1 mg/kg) ( Sen  et al.,  2011 )\nAll treatments were administered orally for four days.\nBleeding time was assessed by manually amputating 5 mm of the rat tail tip. The\ntime it takes bleeding to cease completely from the time of amputation is taken\nas the bleeding time ( Dejana  et\nal ., 1982 ). Clotting time was assessed using blood\nsamples withdrawn into capillary tubes from the retro-orbital sinus. Appearance\nof fibrin thread every thirty seconds was used as the endpoint ( Dejana  et al ., 1982 ).\nBlood was collected daily from each rat through cardiac puncture under ether\nanesthesia into heparinized and plain bottles. Heparinized blood samples were\nsubjected to platelet count evaluation ( Harris\n et al ., 1956 ).\nBlood concentrations of sodium, potassium and urea were evaluated using serum\ncollected from plain blood samples after centrifugation at 3500rpm for 10\nminutes. Levels of sodium, potassium and urea were determined using colorimetric\nassay kits (Randox Laboratories, UK).\nData were expressed as mean ± SEM and analyzed using one-way analysis of\nvariance (ANOVA). Statistical significance was considered at\n p <0.05 using the New-man-Keuls post-hoc test.\n\nThe piroxicam-treated group showed a significant 57.14% ( p =0.05)\nincrease in clotting time compared to controls, which was 18.18% higher than the\ntime seen in the nitroglycerin-treated group during di-estrous ( Figure 1 ).\nFigure 1 Clotting time in control and treated rats during di-estrous phase Values are Mean ± SEM; p ≤0.05,\nn=5 *indicates values that are significantly different from\ncontrols.\nClotting time in control and treated rats during di-estrous phase\nPlatelet count increased in the piroxicam-treated group when compared to controls and\nthe nitroglycerin-treated group. However, this increase was not significant ( Figure 2 ).\nFigure 2 Platelet counts in control, piroxicam- and nitroglycerin-treated rats\nduring di-estrous phase Values are Mean ± SEM;  p ≤0.05,\nn=5.\nPlatelet counts in control, piroxicam- and nitroglycerin-treated rats\nduring di-estrous phase\nNitroglycerin caused a significant 13.78% ( p =0.05) increase in blood\npotassium levels when compared to controls, while piroxicam significantly\n( p =0.05) increased blood potassium (45.89%) and blood urea\n(29.17%) levels and significantly decreased ( p =0.05) sodium levels\n(26.13%) compared to controls during di-estrous ( Figures 3  to  5 ). In addition, the\nPiroxicam-treated group showed a significant ( p =0.05) increase in\npotassium (24.02%) and urea levels (22.40%) and a significant 52.59%\n( p =0.05) decrease in sodium levels when compared to the\nnitroglycerin-treated group.\nFigure 3 Blood sodium levels in control, piroxicam-and nitroglycerin-treated rats\nduring di-estrous phase Values are Mean±SEM,  p <0.05, n=5 *indicates values that are significantly different from control † indicates significant difference from\nnitroglycerin.\nBlood sodium levels in control, piroxicam-and nitroglycerin-treated rats\nduring di-estrous phase\nFigure 5 Blood urea levels in control, piroxicam-and nitroglycerin-treated rats\nduring di-estrous phase Values are Mean±SEM,  p <0.05, n=5 *indicates values that are significantly different from control † indicates significant difference from\nnitroglycerin.\nBlood urea levels in control, piroxicam-and nitroglycerin-treated rats\nduring di-estrous phase\nResults in other phases were not significant when compared to controls.\n\nAbnormal uterine contractions induced by PG production and release during the\nmenstrual cycle are suppressed with the use of drugs that inhibit prostaglandin\nproduction ( Wong  et al .,\n2009 ;  Marjoribanks  et\nal ., 2015 ). Suppression of this abnormal uterine activity\nenhances blood flow and reduces menstrual cramps and other symptoms that appear with\ndysmenorrhea. Results of this study showing increased levels of leukocytes during\nthe di-estrous phase correlate with earlier reports of human menstrual phase\ncharacterized with endometrial leukocytes and increased myometrial prostaglandins\n( Vijayakumar & Walters, 1981 ;  Salamonsen & Lathbury, 2000 ). It is\ntherefore very possible that increased production and release of PGs reported during\nthe human menstrual phase occurs during the di-estrous phase in female rats.\nLikewise, it is very possible that increased production and release of PGs\naccompanied with dysmenorrhea reported during the human menstrual phase also occurs\nduring the di-estrous phase in female Wistar rats.\nCommonly used NSAIDs such as piroxicam and nitroglycerin in managing dysmenorrheal\npain potentiate their anti-nociceptive and analgesic effects by inhibiting the\nproduction of PGs and/or activating nitric oxide synthase ( Bianchi & Panerai, 2002 ;  Miclescu & Gordh, 2009 ). Prostaglandins, especially thromboxane\nA 2  (TXA 2 ), are involved in the promotion of platelet\naggregation, vasoconstriction and smooth muscle cell proliferation during bleeding.\nIncreased clotting time is an indicator of delayed platelet activation, which may\nhave been due to the inhibition of TXA 2  production ( Tapiero  et al ., 2002 ). Increased clotting time\nrecorded in piroxicam-treated rats may be due to the inhibitory effect of piroxicam\non thromboxane A 2  production (a kind of prostaglandin in the platelets)\n( Desouza  et al ., 1979 ).\nIncreased clotting time is an indicator of delayed platelet activation ( Dejana  et al ., 1982 ), and\nconsequently may lead to hemorrhagic anemia in an individual when injured. On the\ncontrary, platelet count increased with piroxicam treatment during di-estrous,\nalthough this increase did not result in reduced clotting time. Piroxicam may have\nprevented the activation of circulating platelets, thereby inhibiting the formation\nof clots or aggregates that might have helped shorten clotting time. This side\neffect was however not observed in animals treated with nitroglycerin.\nProstaglandins also play a significant role in renal control of sodium, potassium and\nblood urea nitrogen. In the kidney tubules, prostaglandins exert an inhibitory\neffect on vasopressin-modulated activities of adenylyl cyclase in collecting duct\nepithelial cells to attenuate vasopressin dependent transtubular water movement. Its\ninhibitory effects on cAMP in the thick ascending limb provide a tonic influence on\nNa-K-2Cl cotransporter expression ( Fernández-Llama  et al ., 1999 ;  Page  et al ., 2007 ). Removal of\nthis inhibitory effect of PGs can inhibit water excretion and thereby promote the\ndevelopment of hyponatremia ( Page  et\nal ., 2007 ). Treatment with piroxicam and nitroglycerin\nshowed a hyperkalemic effect, while significant hyponatremic effect was recorded\nwith piroxicam in rats at di-estrous as earlier reported by  Kim (2008) . The hyponatremic effect of piroxicam indicates that\npiroxicam inhibited the effects of PGs on sodium homeostasis. This hyponatremic\neffect suggests risks of renal impairment, metabolic disorders, muscle spasms,\ncardiac arrhythmia and nervous system disorders ( Wharam  et al ., 2006 ;  Kim, 2008 ). According to  Kim & Joo\n(2007) , potassium regulation is also influenced by the effect of PGs on\nrenin secretion and angiotensin II-induced aldosterone secretion, among other\nfactors. Lowering the effect of PGs on renin secretion and/or fall in aldosterone\nsecretion can reduce potassium excretion and raise the extracellular levels of\npotassium. Increased extracellular potassium levels were observed in both\npiroxicam-treated and nitroglycerin-treated animals at di-estrous. Inhibitory\nactivity of piroxicam on the COX2 pathway and stimulatory effects of nitroglycerin\non Nitric Oxide (derived from iNOS) modulated proximal tubular Na-K-ATPase activity\n( Sharma, 2004 ) may have caused\nhyperkalemia. In addition to earlier reports linking use of NSAIDs to renal\nimpairment ( Kim, 2008 ;  Bennett  et al ., 1996 ;  Aprioku & Uche, 2013 ), this study also found elevated blood\nurea levels in piroxicam-treated animals in di-estrous. The positive effect of\nnitroglycerin on the renal excretion of metabolic waste might be attributed to its\nanti-oxidative activity ( Sen  et\nal ., 2011 ).\nThe study shows that Nitroglycerin may produce minimal alteration of hemostasis and\nrenal function during di-estrous compared to piroxicam.","source_license":"CC0","license_restricted":false}