In the spirit of openness and transparency, I want to share what 42 animals that were involved in my 2014 research efforts experienced.
I recently blogged about Paul Henry’s interview with NZ Anti-Vivisection Society (NZAVS) executive director Stephen Mason, in which Paul Henry repeatedly referred to the 300 animals a day tortured to death in the name of science. I’m paraphrasing, but you can watch the interview for yourself here. Stephen Mason was talking about the 2014 figures for the animals used for research, teaching and testing in New Zealand, just released by the Ministry for Primary Industries. He condemned the secrecy and lack of openness by publicly funded New Zealand institutions using animals. I agree with him. I wish that we would adopt a similar concordat to the UK in which almost 100 institutions, charities and companies have committed to help the public understand more about animal research.
I contacted Paul Henry’s producers and offered to talk about the animal research I carried out in 2014 but, after a chat with one of his team, I didn’t hear from them again. But who need’s The Paul Henry Show when we have SciBlogs! So here’s one of the things the Bioluminescent Superbugs Lab were researching in 2014.
In 2013 I was approached by an orthopaedic surgeon, Simon Young, whose aim is to prevent any of his patients developing an infection from their surgery. At the moment, patients who are having knee replacement surgery are given intravenous antibiotics at the beginning of surgery to try to kill off any bacteria that might accidentally contaminate the surgical site during the surgery. Despite the best efforts of their surgeons, about 2-3% of knee surgery patients develop an infection, sometimes months to years after their surgery. Some of these patients will require the replacement knee to be removed and replaced again. Sometimes the infection can never be completely eradicated and patients can end up in severe pain, some even losing the use of their limb.
Simon wanted to test out a different way of delivering the antibiotics. It’s called intraosseous delivery. The idea is that the surgeons apply a tornique to the limb being operated on, and then inject a large dose of antibiotics directly into the bone. This puts the antibiotics exactly where they are needed and means they can get higher doses than if the antibiotics are delivered to the knee via the blood stream and the rest of the body. The idea could be trialled on his patients but because it can take months to years for patients to develop an infection, and only a small number of them will get an infection in the first place, Simon wanted to know if it was worth going ahead with such a study. And that’s where the Bioluminescent Superbugs Lab came in!
We got some money from the New Zealand Orthopaedic Association, and permission from our local animal ethics committee, to try out Simon’s idea on mice, performing knee surgery and then deliberately infecting the surgery site with a bacterium that can often be found causing infections in people after surgery. We’d be delivering a very unrealistic dose of bacteria, but testing different ways of delivering the antibiotics to see if they could reduce the amount of bacteria. If this worked, the technique would be likely to kill the small number of bacteria that manage to infect the surgical site of some patients and go on to cause an infection.
Once we had our scientific question, it was my job to apply the 3R’s (replacement, refinement and reduction) – the ethical framework that governs the use of animals in research. As we dont’t have a replacement to mice in this case, I had to think about how to refine the experiments to cause the least suffering, and how to use the minimum number of animals. Being the Bioluminescent Superbugs Lab, the first technique we decided to use was biophotonic imaging: the visualisation and measurement of light from within living animals. Using biophotonic imaging, we could infect the animals with a glowing strain of the bacterium Staphylococcus aureus and then track whether the bacteria were dead or alive for a few days after surgery and delivery of the antibiotics. We could get data on the dynamics of how the treatments were working without having to use large groups of animals and euthanizing some of them each day after surgery to grow any living bacteria. The technique would also make the experiments more humane, as it would give us a head’s up if the bacteria were moving into the bloodstream to cause a more serious infection, without us having to wait for the animals to show any physical symptoms.
Once we’d decided we were going to use glowing S. aureus, we then had to settle on a dose of bacteria that would be bright enough for us to detect with our biophotonic imaging machine. To do this we didn’t use animals at all, but laid slices of ham over different amounts of bacteria to see which doses we could still detect. Dose sorted, we moved on to the experimental design. It’s important for experiments like these to carry out the surgeries on different days, using different cohorts of mice and different batches of bacteria, just in case something behaves oddly. We settled on 6 treatments (5 different antibiotic therapies and a no-antibiotic control) and chose to use a randomised block design so that on any given day one or two mice were randomly allocated to each of the 6 treatments. This was repeated on 6 different days to give us surgeries, treatments and infections that had been independently carried out at least 6 times, using a total of 42 mice. An alternative experimental design would be to use groups of 5-6 animals for each treatment and perform the experiments on 2-3 different days, using 60-108 mice.
Having settled on an experimental design, we then met with the animal welfare officer to go through our surgery plan (you can see photos of each of the steps of the surgery on FigShare here) and discuss the best way to relieve any pain the mice might experience afterwards. We settled on giving the mice a subcutaneous injection of carprofen each day and putting paracetamol in their drinking water. The surgery was done under anaesthesia by an orthopaedic surgeon and then the animals cared for by my team. Each day the mice were visually checked to see if they were looking bright eyed and behaving in their usual inquisitive way. They were also weighed to check they were eating properly. Two of the mice started losing weight so they were euthanised, but the rest all recovered really well from their surgery, running around and climbing up and down inside their cages* like they normally would. Each day the mice were also given a light dose of gas anaesthesia and imaged to visualise any glowing S. aureus. Four days after surgery the animals were euthanised and the knee dissected out and put onto agar to grow up any remaining S. aureus which could then be counted.
You can see some of the results of our experiment below. Click on the figure to see it in better resolution. Three groups of animals are shown, the first 7 were not treated with any antibiotic. The top line is the mice imaged 24 hours after surgery and the bottom line is 96 hours after surgery. The blue/red splodges are the S. aureus glowing within the mice.
In a nutshell we found that intraosseous administration of antibiotics was more effective at reducing the number of S. aureus than the same dose of antibiotic delivered intravenously – you can see there are less glowing splodges on the those animals than any of the others. All in all a success, and good evidence from 42 mice that studies should be done in humans. Our findings were published in the journal Clinical Orthopaedics and Related Research (1) and described in an accompanying editorial as “elegant” (2). Our paper is open access so you are welcome to go and read it for yourself. A word of warning though, the target audience is surgeons so it’s a little dry….
- Young SW, Roberts T, Johnson S, Dalton J, Coleman B, Wiles S (2015). Regional intraosseous delivery of prophylactic antibiotics effective in a murine model of total knee arthroplasty. Clinical Orthopaedics and Related Research. 473(11):3573-84. doi: 10.1007/s11999-015-4464-x.
- Zalavras CG (2015). CORR Insights: Regional Intraosseous Administration of Prophylactic Antibiotics is More Effective Than Systemic Administration in a Mouse Model of TKA. Clinical Orthopaedics and Related Research. 473(11):3585–87. Doi: 10.1007/s11999-015-4521-5
*We call them cages, but that makes people think of wire bars. We use what are known as individually ventilated cages (IVCs) which are self contained houses made of clear plastic. Inside we provide corn cob, bedding material and usually either little houses of empty toilet rolls for the animals to nest inside of or play with, as well as free access to food and water.