I am using the standard protocol for Paracetamol (PCM: Tablet from Cipla: 250 and 500 mg/kg oral) and CCL4 (in Olive oil; 0.5 and 1.5 ml/kg i.p.) induced Acute Hepatotoxicy. Blood was collected after 24 hours of the PCM and CCL4 treatment. AST and ALT level of Blood Serum shows normal (80-90 U/L) in Normal Control group. In PCM and CCL4 treated group, shows 10-20 U/L which is very low. It should increases, but not. Is it necessary to use Active Pharmaceutical Ingredients (API) of Paracetamol instead of Paracetamol Tablet.
you can do a pilot study first. Try sacrificing 2 mice at different time intervals (24 hr, 36 hr, 48 hr) and compare enzyme levels with negative control. Pick that time period for your study which gives you statistically significant increase in ALTand AST levels. You may also try intraperitoneal route of administration for paracetamol
Yes more animals shoud be used in order to obtaine better and more certan results. However it is very strange that in PCM and CCL4 groups the AST and ALT are so low?! 24h after application the liver function is starting to recover but it is not near the compleat recovery.
We are dowing the experiments with CCL4 in Wistar rats r!outinely and the levels of AST and ALT in this group are huge! Like 10x more than those in control group! One sugestion it is also possible that you mixed up serums!
Dear Nikola M Stojanovic Sir, Which protocol (for CCL4) you are following. I am following the given bellow, but unable to get the appropriate results. I am using CCL4 (Solvent) from Fisher Scientific.
Serum levels of this enzymes are generally high and some times these biochemical kits are unable to detect (or show loer values) due to above the kinetic range of the kits. Dilute the serum samples with saline (1:1 or 1:2) then you will be able to appreciate changes.
How many animals do you have per group? Are the results similar in the group?
My advice is to double check every step once again. What dilution are you using for CCL4? We are using 1:1 for acute toxicity. Try finding a paper about CCL4 acute liver damage in balb/c mice. And follow that protocol. I see that you followed a protocol for wistar rats.
Dear Ashu
Did you check other parameters than AST and ALT ? I mean, did you check hepatotoxicity or hepatic stellate cells activation in your CCl4 -treated mice by histology / immunohistochemistry ?
If you cannot see any effect of the CCl4 in AST, ALT, hepatocytes necrosis or aSMA expression may be your procedure is not optimal.
The kinetics could be useful, but 24 hours post CCl4 should be sufficient to detect hepatic suffering.
May be you should try another way of administration of the CCl4 ... per os works well too.
Good luck
Benoit
The response to liver injury and the wound healing process varies between different mouse strains. I would suggest that you run a trial using minimum number of mice and harvest your samples at different time points to determinewhich time point best suits your strain. Please see this reference: Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10663–10668, September 1997. Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. ZENGDUN SHI, ADIL E. WAKIL, AND DON C. ROCKEY. When harvested 7 days after the last CCl4 injection, Balb/C mice showed maximum fibrosis as compared to other strains. Its possible that your observation time point needs to be shifted. I've attached the reference.
Ashu, there can be many reasons and there is lot to explore. Check the expiry of the reagents/chemicals you are using, double check the kits/details/expiry/compatibility with the species. These are proven hepatotoxicants and usually used as damage control groups, which I feel tells us that there is, may be some experimental error somewhere. May be u can also check for the centrilobular necrosis in liver histology to confirm if the toxicants are really working.
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