AbstractThe researchquestion is clearly stated in the abstract which is: “to investigate the mechanism ofperoxynitrite-induced relaxation in the mouse aorta, the effect ofatherosclerosis on relaxation to peroxynitrite and other vasodilators, and theeffect of atherosclerosis on the expression and function of the IP3 receptor.
“abstract, p69. These questions asked inthe article is further work on a subject the authors had previously worked onand seems to be novel as it does not test the hypothesis with the same model orwith a different drug. However the abstract is brief and does not containreasons on why the questions are worth asking or how important it is that thesubject questions are answered.
IntroductionThe role of IP3R in maintainingrelaxation to ONOO – in this pathology is not well understood andthe authors wanted to further examine IP3R function in atherosclerosis. Thereis a sufficient body of relevant literature cited in the introduction and addsto the context of the study. Furthermore there is background information on thework on Peroxynitrite, IP3R and atherosclerosis. However there is noidentification of missing research to the subject that shows that findings ofthe paper are novel. Finally the aims of the experiment are clearly shown atthe end of the introduction. MethodsThe methods are detailed enough whichallows for the experiment to be repeated.
The suppliers of mice, IP3R,vasodilators and other reagents are stated, this allows for ease repeatabilityof the experiment. For the mice used the sex, age, weight, diet and strain werestated in the methods. The following declaration “Procedures conformed to the Guide for the Care and Use ofLaboratory Animals published by the US National Institutes of Health (NIHpublication No.
85-23, revised in 1996) and Directive 2010/63/EU of theEuropean Parliament.” Methods, p70, shows that the experiment involved ethicaland licenced conditions in order to proceed. The termination of the mice wasrecorded with the anaesthetic: sodium pentobarbital and the dose: 200 mg/mL.There were citations in the methods but the full methods were not publishedelsewhere.
Group sizes are not clearly stated but are however shown in thefigure legends such as Fig 1.a. The n refers to the number of mice used for anindependent experiment with the standard error of the mean taken. The authorsdid not explain the group sizes and they were not stated in the methods, theywere latter shown in figure legends to be greater than 5 however were notstated to be equal in size. Histograms were assessed blindly by 2 independentspectator and the staining was calculated using ImageJ software. The standarderror of the mean was calculated and n was the number of mice used for eachexperiment, this allows for sufficient statistical tests to be performed by theGraphPad system. The P value had to be < 0.
05 to be considered statisticallysignificant. All of this increases the repeatability and validity of theexperiment, which in turn increases the degree of believability and lack ofbias in the results of the study. With this in mind it is assumed that resultsdoes not have misrepresented results. The investigators used multiple groupswith multiple mice within each group, three different vasodilators used whichallows the experiment to test more variables and address the aims. The methodsallow a repeatable but also valid way for the investigators to answer theresearch question. The methods only use one type of experimentation to reach ananswer: pharmacological.
Had they used more approaches such as geneticmanipulation or disease models then the results of such experiments could havebeen compared. ResultsFig.1.ashows that the antagonist2-APB (60 ?M) has significantly reduced relaxation to peroxynitrite in the group C57BL/6.
Fig.1.b Shows xestospongin C blocking relaxation to peroxynitriteat 5 ?M. Fig.1.cshows that 4-AP significantly reduced relaxationto peroxynitrite, none of the other potassium channel blockers causedsignificant results. Results Figure.1, p 71.
Fig.2.a Shows that 2-APB (60 ? M) caused asignificant relaxation to peroxynitrite In ApoE –/– (high-fat dietfor 2 months) Fig.2.b Shows that2-APB caused a significant relaxation to peroxynitrite after 4 months of ahigh-fat diet. Fig.2.
c Compares C57BL/6and ApoE – /– , from it can be seen that 2-APB had a significantlylarger inhibitor in ApoE –/–compared to C57BL/6 mice at both 2 and 4months. Fig.2.d Shows that 2-APB reduced contraction of aortic rings toU46619, this was less effective in ApoE –/– mice. Results Figure.2,p 72.Fig.
3.a Shows that 4 months of high-fat dietcaused significant reduction in cromakalim. Fig.
3.b Shows vasodilationin response to agent A769662 was caused by xestospongin C (5?M) In C57BL/6 mice.In Fig.3.c it can be seen that forApoE –/– mice on a diet for 4 months, relaxation to A769662 was alsoreduced by 5?M xestospongin C.
Results Figure.3, p73.Fig. 4. aShows 1 mM of the calciumactivated potassium channel blocker TEA did not causesignificant relaxation to peroxynitrite in C57BL/6 mice. In Fig. 4.
b ApoE–/– mice, TEAcaused a shift to the right of the dose-response curve. In Fig. 4.
c a shift to the right in response was also observed inApoE–/– mice on a high fat diet for 4 months. Fig. 4. D shows that Iberiotoxin had no effect on relaxation in ApoE–/–mice what were fed high fat for 4 months, while 4-AP caused significant relaxationbut in C57BL/6 mice it was comparably less. Results Figure.4, 74.Fig.
5. Showsimmunostaining for IP3 receptor expression in C57BL/6 (a,b) and ApoE –/–(c,d).IP3 receptor was present in C57BL/6 mice (b), with reduction of receptorexpression in the high fat ApoE –/– mice (d). Staining was not visibleif the major antibody was not present (a, c). Results Figure.5, p75.Fig. 6.
a Shows two blots. In the d Histogram a significant increase inBKCa expression in ApoE –/– on the diet for 2, with no change at 4months. b Histogram shows a reductionof IP3 receptor presence after 4 months of a high-fat diet c , d Results Figure.6, p75.The figurelegends in the results have enough detail to allow detailed understanding ofthe figures and graphs, which were of an appropriate format. The legendscontained brief description of the figure with the n numbers, symbols and theabbreviations.
The results are ordered in a way that allows a logicalconclusion to be achieved from the aims. The figures were clear and were nottoo complex or crowded with data. Data was set up in histograms,immunostaining, western blotting, bar and line graphs. In the western blots amolecular weight marker was shown however, the contrasting panels in thewestern blotting was unclear and did not line up in Fig.6.c this is not convincing enough evidence to show authormanipulation or just poor set up of the ?-Actin and the BKCa. ConclusionThe conclusion was that relaxation to peroxynitrite in C57BL/6 mouseaorta is partially moderatedby opening of Kv potassium channels caused by IP3R activation.
Atherosclerosisweakens the response to vasodilators. The response to ONOO – ismaintained, even though expression of IP3R is reduced. The activity ofpotassium channels during the formation of atherosclerosis and increases incalcium expulsion through the plasma membrane which maintains relaxation to peroxynitrite.These changes in vascular smooth muscle cells limit negative changes inresponsiveness under high lipid concentration conditions. This conclusion adequatelytested their hypothesis and achieved the aim of the investigation. Theconclusion is reasonable as it is supported by the results of the presentedfigures which were achieved by repeatable methods.
References are usedthroughout the introduction to the discussion that supports the author in theirspeculation. The author did not make a note of the clinical relevance of thefindings which is necessary for some papers. The authors stated that some areaswere not studied such as channel activity or measurements of intracellularcalcium. This means that intracellular calcium from outside sources that arenot IP3R in the mouse aorta may cause changes.
Withthis information questions can be answered on mechanisms of peroxynitrite-inducedrelaxation in mouse aorta, the effect of atherosclerosis in relaxation toperoxynitrite and other vasodilators, and the effect of atherosclerosis on theexpression and function of the IP3 receptor. The study was significant enoughquality to allow a conclusion to form from the methods used, which will improveresearch in this area. The techniques used in the methods were adequate andlogical for the data the authors wanted to show, and so these techniques can beused in my own research in similar circumstances.