National Anti-vivisection Society

 

National Antivisection Society

Case Study: research using mice, rats and zebrafish funded by the British Heart Foundation

Posted: 13 December 2011. Updated: 17 April 2012

1

Stone, O.A. et al. (2009) “Critical Role of Tissue Kallikrein in Vessel Formation and Maturation Implications for Therapeutic Revascularization”, Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 29, pp: 657 – 664

British Heart Foundation

This study used mice, rats and zebrafish embryos to study the effect of a human gene (hKLK1) which is involved in vessel formation and maturation. The paper states that, in humans “function-defective variants of the hKLK1 gene are frequent in the general population” one variant, they explain, leads to “a new form of arterial dysfunction”.

Mice in the “ischemia model” experiment underwent surgery where the femoral artery in their left hind leg was blocked by “electro-coagulation”. They were then injected with a virus containing various genetic information, depending on which experimental group they were in.

Rats underwent surgery during which their intestines were lifted out of the body cavity; the fat pads were injected with the same substances injected into the mice and were replaced inside the animals. On day 6 they were re-anaesthetised, the virus injected “panel” of the intestine located and preserved for observation. The rats were killed by having their necks broken.

In Zebrafish, embryo’s were injected with mRNA (a substance which transfers information from genes to the machinery in the cell which makes proteins) at the one-two cell stage, and they were killed at two different stages post fertilisation – 24 hours and 48 hours. Embryos were fixed and stained for analysis.

Repetition

The authors acknowledge that, despite potential in “a large number of preclinical studies around the world…clinical studies have shown disappointing results”. They explain that previous research has mainly been carried out in single laboratories, whereas in the present study, “different groups were engaged in validating the arteriogenic action of hKLK1”. However, it is not stated which laboratories carried out work on which species, or if species work was repeated in the different groups studies.

After explaining the affect which hKLK1 had on mice stem and progenitor cells in this experiment, the authors refer to their previous work which examined the same enzyme in mice and humans. They also cite unpublished data - “We also know from pilot studies that human EPCs express hKLK1”. This demonstrates repetition and also that the researchers are aware of and have carried out human research.

Species differences and awareness of them

The authors note that it would be of interest to find out whether human patients with arterial disease and a mutation in this gene, suffer a worse prognosis as a result of impairments “similar to that observed in KLK1-/- mice”. This acknowledges that their results still require confirmation in humans

There are clear and obvious differences between humans and zebra fish. Genetically, humans and zebrafish last shared a common ancestor around 450 million years ago (1). However, there are also more specific structural differences between human and zebrafish hearts such as the zebrafish heart having only two chambers and the human heart having four. This means the human heart has two valves and the zebrafish’s has one.

Current available treatments

Risk factors for developing peripheral arterial disease (PAD) include those which are modifiable, such as smoking and diabetes and those which are non-modifiable, such as age and race (2)

Treatment decisions are based on each patient’s risk factors and include supervised exercise and medication to relieve symptoms. The medication includes drugs with metabolic and vasodilatory (widening of blood vessels) activity (3)

Animal suffering

Rodents undergoing surgery are likely to have experienced pain and/or discomfort afterwards as a result. It is worrying that no mention of analgesia is made in relation to the rats, which, during surgery, had their intestines lifted out of their body cavity and then replaced. Information on analgesics is important as the ethical and regulatory considerations of pain in laboratory animals has recently been highlighted as an important aspect when animals are used for experimentation (4)

Alternative advanced techniques

A human study, referred to in this paper, involved the systematic search for variants of this gene. It concluded that identification of people with mutated versions of the gene which reduce the enzymes activity “could facilitate analysis of the role of kallikrein-kinin system in renal and vascular diseases”. The study also highlighted complex environmental factors which it could be argued are much more difficult to account for in laboratory animal models. “The effects of kallikrein polymorphisms [variations] on BP [blood pressure] may be more subtle and may depend on the environment, especially sodium and potassium levels in the diet, which affect kallikrein synthesis” (5) This shows how complex the issue is in humans and reiterates the paucity of the animal model.

Poor experimental planning

The major problem of the reporting of this research was the poor indication exactly how many animals were used. The supplementary material supplies some information such as noting “Samples from 8 individual mice per group were analysed” and noting groups of 6 and 8 animals on the graphs. However, the methods section which describes the animal procedures, does not mention how many animals of each species were used in each experiment. This information is vital to ascertain the statistical robustness of the work.

Disease context

PAD is known to affect around 27 million people in Europe and North America (2) Intermittent claudation (IC), a limp which is symptomatic of PAD, “increases from around 3% in patients aged 40 to 6% in patients aged 60 years”. IC does not always predict its absence of presence of PAD (3)

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References:

1.Kumar, S and Hedges, S. B (1998) “A molecular timescale for vertebrate evolution” Nature, vol. 392, pp: 917 – 920.
2.Harris, K (Ed) (2008), The worldwide burden of peripheral artery disease, The role of the physician Based on the Inter-Society Consensus.
3.Norgren, L et al (2006) “Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II)” The Society for Vascular Surgery, doi:10.1016/j.jvs.2006.12.037.
4.Carbone, L (2011) “Pain in Laboratory Animals: The Ethical and Regulatory Imperatives” PLoS One, vol. 6, issue. 9, e21578
5.Slim, R et al (2002) “Loss-of-Function Polymorphism of the Human Kallikrein Gene with Reduced Urinary Kallekrein Activity” Journal of the American Society of Nephrology, vol. 13, pp: 968 - 976.

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