Review for 10.1186/1471-230X-11-31 (2011)

Reviewer's report
Title:1 MAC Isoflurane Preconditioning Induce Protective Effects of Heme
oxygenase-1 on Hepatic Ischemia Reperfusion in Rats
Version: 1Date:10 May 2010
Reviewer: Greg Barritt
Reviewer's report:
Major Compulsory Revisions
1. Effects of intervention on HO-1 enzymatic expression, Results, paragraph 1
and Fig. 1. The effects of IR and IR plus isoflurane compared to sham are
relatively small. IR has induced an approximately 1.5-fold increase, and IR plus
Iso in approximately 2-fold increase, compared to Sham. Given the magnitude of
the error bars, it is not completely clear whether there should be or is a significant
difference between IR and Sham. Again, taking account of the size of the
indicated error bars, it is not completely clear whether there should be or is a
significant difference between IR plus isoflurane and IR. Since this is really the
central point of the whole paper, more convincing evidence for these changes
should be provided.
2. Relating further to the above, the effect of Znpp. The bar indicated Iso plus
Znpp. The authors claim that Znpp added prior to isoflurane pre-treatment
completely blocks the action of isoflurane. It should be made clear in this graph
that isoflurane treatment after Znpp involves both isoflurane treatment and IR.
There is no control for the effect of Znpp on the effect of IR in absence Iso.
Therefore it seems that the authors cannot conclude that Znpp has reduced the
isoflurane effect. The decrease in the bar marked Iso plus Znpp could be due to
an effect of Znpp on the IR action and not on isoflurane.
3. Related further to the above comments, the description of the groups in the
Methods under “4. Experimental groups” should be stated in list form much more
clearly than it presently is. As mentioned, it seems that it would be necessary to
add an additional group which is Znpp and IR without isoflurane pre-treatment.
4. Western blots, Fig. 2, Results, paragraph 2, effect of the different conditions on
HO-1 protein experiment analysed by Western blot. I believe two things are
required for these experiments. Firstly, data on the quality of the primary antibody
employed in Western blot should be shown with a full scale gel showing the
position of the HO-1 band and molecular weight markers. How specific is the
antibody? Secondly, the data should be quantitated by measuring the intensity of
the spots by image analysis, number of experiments given and mean values for
each condition given. The argument above related to the effects of Znpp and the
interpretation of these results also applies for Fig. 2 Western blot, Group D, Iso
plus Znpp Group compared to Group C which is IR plus isoflurane.
5. Fig. 3, immunohistochemistry results and Results text, paragraph 2. The data
should be scored quantitatively using an accepted form of scoring for injury
(grading score), quantitative results expressed and statistics undertaken. Serafin
et al. (2002) Am. J. Path. 161, 587 is one example of a grading system.
6. Fig. 4, plasma ALT and AST concentrations, Results, Section 2. The changes
in marker enzymes for liver damage, apart from the effect of IR compared to
Sham, are small. The trend is there (as for also Figs. 5 and 6), but again the
changes are very small. A confirmation on the statistical analysis is important. As
indicated by others, in other studies effects of ischemic preconditioning, for
example, or intermittent ischemia are not usually revealed as changes in liver
marker enzyme concentration until about 6 h post beginning of reperfusion. (The
same comment also may apply to histological changes.) With respect to liver
marker enzymes, there is no data for effect of Znpp alone. Since Znpp plus Iso
increases liver marker enzymes compared to IR plus Iso, it would be important to
check that Znpp alone does not increase compared to Sham.
7. Title and relationship of this study to Reference 16. As indicated by the present
authors, Schmidt et al. have previously shown that isoflurane will induce HO-1 in
liver. The potential value of the present studies is the effects of isoflurane at
clinically-relevant doses. It would be better to change the title to reflect this more
explicitly. The term “1 MAC” may not be readily understandable to many readers.
Something like “isoflurane preconditioning at clinically relevant doses induces
protective effects of HO-1 …”. (There may be better wording.) Also in this
connection a paper of Beck-Shimmer et al., Annals of Surgery, 248, December
2008, could also be cited. This refers to clinical trials and possibly induction of
HO-1.
Minor Essential Revisions
1. Methods, paragraph 2, hepatic ischemia reperfusion. Rats were anaesthetised
with sodium pentobarbital and remain under anaesthesia for at least 5 h. Some
comment on effectiveness of anaesthesia during this time period should be
included.
2. Methods, hepatic HO-1 activity assay, homogenisation. More details of
ultrasonic homogenisation should be given. Also a reference for measurement of
bilirubin, the principle.
3. Methods, Section 9, transaminases TNF and myloperoxidase. Where
appropriate, principle of the assay should be given, rather than just saying, for
example, “using a kit from ….”.
4. Results, paragraph 2, histology, Fig. 3. The statement “based on
morphological characteristics HO-1 was expressed mostly in hepatocytes”. This
requires more detail in order for the reader to be convinced that HO-1 is actually
expressed principally in hepatocytes.
5. Results, Section 3, effects of intervention on cell morphology, Fig. 7, second to
last paragraph before Discussion. The statement “integrity of the overall structure
of hepatic lobules were seriously compromised” should be clarified and explained
better.
6. In the Discussion, first three paragraphs. To a great extent these paragraphs
refer to reasons why the experiments were performed, especially relating to
Schmidt et al. reference 16 and the aim of the present study which is to use
lower clinically-relevant doses. This material should be expressed more concisely
that it is now but in the Introduction.
7. Discussion, paragraph 4, referring to results relating to “As Znpp is not the
specific inhibitor of HO-1, it completely blocked the increase of HO-1 activity”. As
mentioned, cannot really see clearly from the data the effects of Znpp. Also the
differentiation between effects of Znpp on activity of the enzyme which is present
in the cells and effects of Znpp on gene expression of the enzyme should be
more clearly described. These issues should be made more clear.
8. Later in Discussion, two paragraphs on “HO-1 ubiquitous enzyme and among
the three mammalian isoforms of HO-1 etc”. Should be expressed more
concisely in Introduction and Discussion shortened.
9. Second to last paragraph of Discussion, comment that “IR damage through
phenomenon called preconditioning”. This should be modified to say “through the
same mechanism as preconditioning”. The phenomenon, although it is a form of
preconditioning, is not the same.
10. Fig. 1, for some statistics, there is no symbol described in the legend.
Discretionary Revisions
1. Methods, Section 7, Western blot, chemiluminescence agents, source should
be indicated.
2. Immunochemistry, reference for the principle of measurement of HO-1
immunoreactivity should be given.
3. Methods, Section 8, towards the end of the paragraph, the word “polymerise”
in relation to anti-rabbit IgG is probably not correct. Possibly covalently-linked.
4. Discussion, beginning of paragraph 2, the term “hepatic flux” possibly means
blood flow?
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests.