Abstract mice, IP3R, vasodilators and other reagents are

Abstract

The research
question is clearly stated in the abstract which is: “to investigate the mechanism of
peroxynitrite-induced relaxation in the mouse aorta, the effect of
atherosclerosis on relaxation to peroxynitrite and other vasodilators, and the
effect of atherosclerosis on the expression and function of the IP3 receptor.”
abstract, p69.  These questions asked in
the article is further work on a subject the authors had previously worked on
and seems to be novel as it does not test the hypothesis with the same model or
with a different drug. However the abstract is brief and does not contain
reasons on why the questions are worth asking or how important it is that the
subject questions are answered.

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Introduction

The role of IP3R in maintaining
relaxation to ONOO – in this pathology is not well understood and
the authors wanted to further examine IP3R function in atherosclerosis. There
is a sufficient body of relevant literature cited in the introduction and adds
to the context of the study. Furthermore there is background information on the
work on Peroxynitrite, IP3R and atherosclerosis. However there is no
identification of missing research to the subject that shows that findings of
the paper are novel. Finally the aims of the experiment are clearly shown at
the end of the introduction.

 

Methods

The methods are detailed enough which
allows for the experiment to be repeated. The suppliers of mice, IP3R,
vasodilators and other reagents are stated, this allows for ease repeatability
of the experiment. For the mice used the sex, age, weight, diet and strain were
stated in the methods. The following 
declaration “Procedures conformed to the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of Health (NIH
publication No. 85-23, revised in 1996) and Directive 2010/63/EU of the
European Parliament.” Methods, p70, shows that the experiment involved ethical
and licenced conditions in order to proceed. The termination of the mice was
recorded with the anaesthetic: sodium pentobarbital and the dose: 200 mg/mL.
There were citations in the methods but the full methods were not published
elsewhere. Group sizes are not clearly stated but are however shown in the
figure legends such as Fig 1.a. The n refers to the number of mice used for an
independent experiment with the standard error of the mean taken. The authors
did not explain the group sizes and they were not stated in the methods, they
were latter shown in figure legends to be greater than 5 however were not
stated to be equal in size. Histograms were assessed blindly by 2 independent
spectator and the staining was calculated using ImageJ software. The standard
error of the mean was calculated and n was the number of mice used for each
experiment, this allows for sufficient statistical tests to be performed by the
GraphPad system. The P value had to be < 0.05 to be considered statistically significant. All of this increases the repeatability and validity of the experiment, which in turn increases the degree of believability and lack of bias in the results of the study. With this in mind it is assumed that results does not have misrepresented results. The investigators used multiple groups with multiple mice within each group, three different vasodilators used which allows the experiment to test more variables and address the aims. The methods allow a repeatable but also valid way for the investigators to answer the research question. The methods only use one type of experimentation to reach an answer: pharmacological. Had they used more approaches such as genetic manipulation or disease models then the results of such experiments could have been compared.   Results Fig.1.a shows that the antagonist 2-APB (60 ?M) has significantly reduced relaxation to peroxynitrite in the group C57BL/6. Fig.1.b Shows xestospongin C blocking relaxation to peroxynitrite at 5 ?M. Fig.1.c shows that 4-AP significantly reduced relaxation to peroxynitrite, none of the other potassium channel blockers caused significant results. Results Figure.1, p 71. Fig.2.a Shows that 2-APB (60 ? M) caused a significant relaxation to peroxynitrite In ApoE –/– (high-fat diet for 2 months) Fig.2.b Shows that 2-APB caused a significant relaxation to peroxynitrite after 4 months of a high-fat diet. Fig.2.c Compares C57BL/6 and ApoE – /– , from it can be seen that 2-APB had a significantly larger inhibitor in ApoE –/–compared to C57BL/6 mice at both 2 and 4 months.  Fig.2.d Shows that 2-APB reduced contraction of aortic rings to U46619, this was less effective in ApoE –/– mice. Results Figure.2, p 72. Fig.3.a Shows that 4 months of high-fat diet caused significant reduction in cromakalim. Fig.3.b Shows vasodilation in response to agent A769662 was caused by xestospongin C (5?M) In C57BL/6 mice. In Fig.3.c it can be seen that for ApoE –/– mice on a diet for 4 months, relaxation to A769662 was also reduced by 5?M xestospongin C. Results Figure.3, p73. Fig. 4. a Shows 1 mM of the calciumactivated potassium channel blocker TEA did not cause significant relaxation to peroxynitrite in C57BL/6 mice. In Fig. 4. b ApoE–/– mice, TEA caused a shift to the right of the dose-response curve. In Fig. 4. c a shift to the right in response was also observed in ApoE–/– 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 relaxation but in C57BL/6 mice it was comparably less. Results Figure.4, 74. Fig. 5. Shows immunostaining 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 receptor expression in the high fat ApoE –/– mice (d). Staining was not visible if 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 in BKCa expression in ApoE –/– on the diet for 2, with no change at 4 months. b Histogram shows a reduction of IP3 receptor presence after 4 months of a high-fat diet c , d  Results Figure.6, p75. The figure legends in the results have enough detail to allow detailed understanding of the figures and graphs, which were of an appropriate format. The legends contained brief description of the figure with the n numbers, symbols and the abbreviations. The results are ordered in a way that allows a logical conclusion to be achieved from the aims. The figures were clear and were not too complex or crowded with data. Data was set up in histograms, immunostaining, western blotting, bar and line graphs. In the western blots a molecular weight marker was shown however, the contrasting panels in the western blotting was unclear and did not line up in Fig.6.c this is not convincing enough evidence to show author manipulation or just poor set up of the ?-Actin and the BKCa.   Conclusion The conclusion was that relaxation to peroxynitrite in C57BL/6 mouse aorta is partially moderated by opening of Kv potassium channels caused by IP3R activation. Atherosclerosis weakens the response to vasodilators. The response to ONOO – is maintained, even though expression of IP3R is reduced. The activity of potassium channels during the formation of atherosclerosis and increases in calcium expulsion through the plasma membrane which maintains relaxation to peroxynitrite. These changes in vascular smooth muscle cells limit negative changes in responsiveness under high lipid concentration conditions. This conclusion adequately tested their hypothesis and achieved the aim of the investigation. The conclusion is reasonable as it is supported by the results of the presented figures which were achieved by repeatable methods. References are used throughout the introduction to the discussion that supports the author in their speculation. The author did not make a note of the clinical relevance of the findings which is necessary for some papers. The authors stated that some areas were not studied such as channel activity or measurements of intracellular calcium. This means that intracellular calcium from outside sources that are not IP3R in the mouse aorta may cause changes. With this information questions can be answered on mechanisms of peroxynitrite-induced relaxation in mouse aorta, the effect of atherosclerosis in relaxation to peroxynitrite and other vasodilators, and the effect of atherosclerosis on the expression and function of the IP3 receptor. The study was significant enough quality to allow a conclusion to form from the methods used, which will improve research in this area. The techniques used in the methods were adequate and logical for the data the authors wanted to show, and so these techniques can be used in my own research in similar circumstances. 

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