We are testing a dietary supplement for its ability to enhance immune response in fish, upon challenge with a specific pathogen. The mechanism we chose to monitor this effect is through up- or down regulation of the genes for selected pro- and anti-inflammatory cytokines. However, to target the specific time at which the genes for these cytokines are at the highest (or lowest, as the case may be), we are thinking of conducting a preliminary experiment, where we would simply challenge some fish with the pathogen, then at different time courses (0, 1.5, 3, 6, 12, 24, 48 and 72 hrs) we would collect liver samples, extract the total RNA, and hopefully, find out the time that is most suitable to sample our main experimental fish.
My questions are;
1. Does this seem a reasonable course of action and
2. Has this been done before in fish (using the liver?), if so could any one throw more light on what we need to do or what we need to avoid (or watch out for)?
I agree that head kidney will give you better results. Have a look at the paper by Falco et al 2012 "Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with β-glucan supplements" Fish & Shellfish Immunology Volume 32, Issue 6, June 2012, Pages 1051–1057 to get some ideas of the time frame you are looking at.
The time frame will also depend on the pathogen you are using. If it is very virulent you probably want to look at a shorter time frame.
Is there a reason why you want to look at liver?
The study by Pionnier et al 2013 "Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection" in Fish and Shellfish Immunology looked at liver in a similar context, however studying complement.
I hope this is of some help for you
Jo as always I will slightly disagree with you ;P the selection of the tissue should be based on the pathogen tissue specificity - sometimes it is better to look at the responses in the mucosa tissues as long they are affected by the pathogen because responses there are stronger than immunocompetent tissues. Of course if you are going to inject the bacterium i.p.. than liver spleen kidney head kidney will be responding more-less similarly.
Recently I was working in two isoforms of proinflammatory cytokine, IL8 in a fish and found that their transcript distribution significantly varied in healthy tissues. And also, different immune challenges conducted with Edwardsiella tarda, Streptococcus iniae, rock bream irido virus, LPS and poly I:C exhibited differential effect in their transcriptional expression in tissues including liver, gills, spleen and liver. For a brief account you may refer the following proceeding:
https://www.researchgate.net/publication/259051823_Two_distinct_interleukin_8_homologs_in_terms_of_sequence_genomic_structure_and_mRNA_expression_from_rock_bream
Conference Paper Two distinct interleukin 8 homologs in terms of sequence, ge...
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