A small selection of highlights from the Dr Hadwen Trust’s research programmes to replace animal experiments.
- Fingerprinting the microbial culprits without animal tests
- Uncovering clues to human kidney failure
- Simulating human brain damage
- Computer models ensure safer pregnancies
- Potency testing of botulinum toxin without animals
- New tools for diabetes research
- Modelling human biomechanics in dental research
- MEG reveals a glimpse inside the human brain
- Replacing the Draize eye test on rabbits
- Rheumatism research in the test-tube
2002 – 2003
When there is an outbreak of disease, it is essential to identify the disease-causing microbe quickly, a process that often involves guinea pigs and rabbits. Dr Hadwen Trust funded research by Professor Haroun Shah at the Health Protection Agency developed hi-tech non-animal methods to precisely identify disease-causing microbes, in place of standard animal tests.
Professor Shah applied a state-of-the-art form of mass spectrometry, called MALDI-TOF MS (Matrix-assisted laser desorption/ionization - Time of Flight Mass Spectroscopy), to directly analyse the surface of intact bacterial cells. This method uses a pulse of laser light to generate unique spectral patterns from the surface of bacteria. These surface patterns, combined with sophisticated analysis of microbial DNA and proteins, produce individual profiles that can be used to ‘fingerprint’ and identify disease-causing culprits.
This new non-animal technique was successfully used to rapidly and reliably distinguish between infectious and non-infectious forms of several different types of bacteria (including those responsible for dysentery, food poisoning, pneumonia, meningitis, ear and other infections).
Notably, the new technique is able to identify different forms of bacteria that were previously only distinguishable by animal tests that involved inducing abscesses on guinea pig legs. The research also revealed important new information about infectious bacteria.
The beauty of MALDI-TOF-MS is that it can be performed in a matter of minutes with tiny samples and only minimal preparation, making it supremely cost-effective and ideal for replacing animal tests. The development of this technology is particularly important now, in a global political climate that has prompted a flurry of research into diseases such as plague, anthrax, botulism and smallpox. The widespread adoption of these methods would replace large numbers of animals in microbial research and testing.
2000 – 2004
Dr Hadwen Trust funded researchers at Guy’s Hospital London used non-animal techniques to achieve a major breakthrough in understanding human kidney failure. The research team, led by Dr Marinaki and Dr Simmonds, focused on blood samples obtained directly from volunteer patients with kidney disease instead of studying animals with artificially induced kidney damage.
Our kidneys are vital organs that filter out waste products from the blood, regulate water levels in the body and produce urine. Kidney damage caused by disease or toxins may lead to organ failure and the need for a kidney transplant.
Because the kidneys are such important organs, much medical research has focused on trying to understand the process of kidney damage and kidney failure. Experiments on animals have involved severely damaging or removing the kidneys, usually in rodents, for results that may not apply to human patients.
Chemicals and medicines are widely tested for kidney toxicity in rats, mice, rabbits, dogs and pigs, in tests that can last from a few hours to 80 days. Again, species differences mean that test findings may not apply to humans. A shortage of human organs for transplants has led to research into cross-species transplantation (xenotransplantation) being carried out on pigs and monkeys, these experiments have already inflicted horrific surgical procedures and terrible suffering on countless animals.
The researchers used test-tube studies to identify a previously unknown substance that accumulates in the blood cells of kidney failure patients and is linked to kidney damage. The researchers devised new methods of chemical analysis to confirm the identity of this compound, a unique nucleotide called 4PYTP. Nucleotides are naturally occurring substances that play a vital role in many processes within living cells.
Research is now underway to understand how this rogue nucleotide forms in red blood cells. Finding ways to intervene and prevent its build-up to high levels in the blood stream could be the key to preventing or treating kidney failure. This important new knowledge was gained without animal experiments and demonstrates that human, rather than animal approaches, are the best way to achieve progress in kidney research.
1998 – 2001
In 1998 the Dr Hadwen Trust funded Dr Amanda Ellison at Oxford University to use a then little-known technique called Transcranial Magnetic Stimulation (TMS) to investigate the human brain. TMS uses a brief magnetic pulse to safely and temporarily disrupt the functioning of parts of the human brain, enabling non-invasive investigations in human volunteers instead of severe brain damaging experiments on monkeys.
For decades scientists have studied the effects of brain damage in animals, especially monkeys. The animals are trained to carry out certain tasks, and then the results of damaging or removing parts of the brain are investigated. Monkeys may be tested for months or even years after the injury, sometimes with electrodes implanted in their brains.
This research project used TMS to mimic ‘brain damage’ in human volunteers, and investigate processes that would normally be studied in purposely brain-damaged monkeys. Using TMS in conjunction with a brain imaging technique, known as Magnetic Resonance Imaging (MRI), enabled our researcher to study the parts of the human brain involved in aspects of attention, vision, perception, reaching, intention and awareness. This work with human volunteers replaced the use of many monkeys over a period of three to five years.
TMS provides information not only about which areas of the brain are involved in an activity but also the way areas of the brain interact and the order in which they do so. The results contributed to understanding how the brain works and helped devise future treatments for brain injury.
Dr Ellison’s work and that of her supervisor Dr Vincent Walsh, firmly established the use of TMS as a replacement for animals in brain research. The technique is safe and ethical, allows relevant studies of humans and is comparatively inexpensive. This project demonstrates how innovative thinking combined with new technology can provide new ways of replacing animal experiments at the same time as improving scientific methodology.
1995 – 1998
In 1995 the Dr Hadwen Trust awarded an equipment grant to Dr David Talbert and Professor Nick Fisk of Imperial College London. They were developing computer models to help explain what can go wrong in some pregnancies. At the time, computer simulations were still fairly new in medicine and work was needed to improve them and make more realistic simulations into complications that can occur in pregnancy.
The computer models were based on information from human pregnancies gained from ethical studies using ultrasound scanning and donated blood samples. This approach contrasted with – and provided an alternative to – invasive experiments on animals. In other laboratories, for example, scientists operate on pregnant animals such as rabbits and sheep to create abnormalities. However, results from animal experiments cannot be relied upon to predict the human situation, because of species differences in maternal and foetal physiology.
The computer simulations were used to conduct ‘virtual’ experiments, humanely, cost-effectively and without the problems of species variation. This research explained the cause of a telltale abnormality in blood-flow to the womb, seen in women who go on to develop a dangerous complication (pre-eclampsia) later in pregnancy. The computer model showed that the cause was an abnormality in the elasticity of the artery walls rather than a problem with the placenta – which was the favoured theory at the time.
The computer simulations also revealed how an imbalance in some, but not all, blood vessel connections to the placenta can cause illness or death to one of a pair of twins in pregnancy. These finds led to a new clinical test to predict the likelihood of this complication and another test to predict survival, allowing the best form of treatment to be applied.
The work at Imperial College has since been built on by other scientists worldwide. The computer models, continually updated and refined, have become more realistic and contributed to progress in ensuring safer pregnancies. This has been achieved without animal experiments.
1995 – 1997
Botulinum toxin is used medically to treat certain movement disorders and muscle spasms, and is increasingly used cosmetically as an anti-wrinkle therapy. Botulinum toxin is highly poisonous and so careful quality control of products containing this toxin is essential. A grant from the Dr Hadwen Trust was instrumental in the development of a test-tube method to test the potency of batches of botulinum toxin and replace lethal tests in mice.
The standard animal test for checking the strength of batches of botulinum toxin is an LD50 (lethal dose 50%) test in which groups of mice are injected abdominally with the toxin. As a result some animals suffer a slow, distressing death by paralysis and suffocation. As thousands of animals were used in these tests each year, the development of a non-animal alternative was urgently needed.
Between 1995 and 1997 the Dr Hadwen Trust awarded two grants to Dr Dorothea Sesardic at the National Institute for Biological Standards and Control (NIBSC), the UK’s national laboratory for standardising biological medicines. Dr Sesardic’s group investigated several test-tube alternatives to the standard mouse test, based on biochemical knowledge of how the toxin works.
One non-animal method, now usually called the SNAP-25 assay, was found to be particularly suitable as an alternative to the animal test. It was more sensitive than the mouse test and it was faster and cheaper, taking less than four hours to perform, compared to four days for the animal test.
The NIBSC established the SNAP-25 assay for in-house final batch testing of botulinum toxin, replacing the use of some 5,000 mice each year. The non-animal test is now included in the European Pharmacopoeia (2005), which is the standard reference text for regulatory testing of biological medicines. At present the SNAP-25 test can be used to replace the mouse LD50 only in final batch testing of botulinum toxin products but earlier production stages still use animal tests. Nonetheless, the use of SNAP-25 test has already saved the lives of thousands of mice in the UK and co-ordinated efforts are now underway in Europe and the USA to implement the SNAP-25 in place of animal tests on an international scale.
1995 – 1997
Many diabetics suffer from problems with their blood circulation. Damage to tiny blood vessels can affect their eyes, nerves, skin and kidneys, and may lead to serious complications such as diabetic blindness and kidney failure.
To study these problems some scientists use diabetic rabbits and rats, although the diabetic condition in these animals differs from that in humans. The Dr Hadwen Trust funded work by Professor Angela Shore at Exeter University to show that a safe and non-invasive technique known as laser Doppler perfusion imaging, could easily be used to directly investigate the circulation in tiny blood vessels of human volunteers instead of conducting experiments on diabetic animals.
In animal experiments, rabbits, rats and mice are either bred to be diabetic or made artificially diabetic. Animals may have their pancreas removed surgically or damaged by injecting a harmful chemical. However, the condition in these animals differs from the human diabetic or pre-diabetic state, and makes it difficult to apply findings from animal experiments to patients. It is therefore important to develop new ways to directly investigate diabetes in patients.
A Dr Hadwen Trust grant to Professor Shore in 1995 provided a major piece of equipment, a laser Doppler perfusion imager, which was used to non-invasively study blood flow in tiny blood vessels of volunteers. The technique uses a laser beam to scan an area of skin tissue. As the laser beam penetrates the skin a fraction of light is back-scattered by moving red blood cells, generating a signal proportional to blood flow in the tiny microvasculature of the skin.
Another technique, called iontophoresis, was used to introduce substances across the skin by means of a small electric charge, and to study the effects on skin microvascular blood flow. Detailed reactions of human blood flow to chemicals were measured for the first time and differences between the responses of diabetics and healthy volunteers identified.
Changes in blood flow were traced to abnormal responses in cells that line the blood vessels, called the endothelium. Patients with non-insulin diabetes and even people at risk of developing the condition, were found to have poorly functioning endothelium lining their blood vessels. The endothelium is vital for circulatory health.
The work by Professor Shore’s group gleaned new information about microvascular changes in human diabetes and obtained results that contradicted those obtained from ‘animal models’ of diabetes. It also demonstrated how easy it is to measure microvascular function in human volunteers.
1994 – 1996
Dr Hadwen Trust funding was critical to the development of a three-dimensional computer model of human teeth and jawbone, for use in dental research in place of animals. The Dr Hadwen Trust supported Professors Malcolm Jones and John Middleton at the University of Wales Dental School in Cardiff in developing a computer simulation of tooth movement for testing orthodontic treatments without animal experiments.
Orthodontics is a speciality of dentistry that involves the movement and alignment of teeth to correct deformities. Although teeth are routinely moved in orthodontic practice, it is more art than science, because little is known about the precise effects on the surrounding tissues. The behaviour of teeth in response to dental devices and surgical techniques are often tested on animals, including dogs, cats, monkeys, pigs and ferrets.
The animals’ teeth are moved by applying large forces via a variety of contraptions, including rubber bands, springs, wires and magnets. These devices are fitted in the animals’ mouths, sometimes by surgery, and may be left in for many weeks or months. Such experimental treatments can force teeth partly out of, or up into, the jaw bone. Damage to the gums, ligaments and bone, as well as bleeding, abscesses and inflammation may occur. At the end of most studies the animals are killed and dissected to investigate the effects of the experimental treatments. However, the results of animal experiments are not easily extrapolated to the human situation
The idea for the computer model came from a system used in engineering to test out the stresses and strains of new bridge designs before building them. Our dental model was based on measurements made directly from the teeth of human volunteers. A special laser apparatus was devised that accurately measured tiny tooth movements in volunteers. This human data was used to create a computer-based finite element model of the teeth and surrounding tissues and to validate computer simulated tooth movements.
The computer model was the first to predict the responses of human teeth to dental treatments, such as surgery or braces. Scientists from Japan and Germany, some of whom had been doing animal experiments, are now working with the computer model saving many animal lives.
Until recently it was very difficult to safely study the human brain and so researchers tended to study animal brains instead. As a result, science knew a great deal about the brains of monkeys and cats but relatively little about the human brain.
Between 1992 and 1999 the Dr Hadwen Trust funded groundbreaking research at Aston University (Birmingham), by a pioneer in the field of human brain imaging, Professor Graham Harding. Professor Harding’s work demonstrated that a new type of brain scanner, called MEG (magnetoencephalography) could be used to study the human brain safely and reliably. MEG non-invasively detects electrical activity in the human brain, enabling safe and relevant research on humans, in place of experiments on animals.
Monkeys and cats are particularly used in brain research, and experiments on them may involve invasive surgery, implanting electrodes to record brain activity or inflicting brain damage. Such animal experiments are carried out to try and investigate how the brain works and to study functions such as vision, hearing, pain or brain injury. Brain research on animals is also conducted for research into neurological illnesses such as Alzheimer’s disease, epilepsy, or migraine.
The studies with MEG provided a new understanding of human vision and epilepsy in children. It also laid the foundations for subsequent MEG studies of the human brain. The MEG unit at Aston is now internationally recognised as a centre of expertise for brain imaging and research. Back in the 1990s, Aston possessed the only MEG equipment in the whole country, but now several new MEG imaging centres have opened around the UK. Studies with MEG have helped to replace experiments on monkeys strapped into restraining chairs with electrodes implanted in their brains.
The Dr Hadwen Trust continues to fund research at the Neurosciences Research Institute at Aston, which now combines a range of complementary, non-invasive brain research techniques, including MEG, fMRI (functional magnetic resonance imaging) and a method called MRS (magnetic resonance spectroscopy) to build up a composite picture of how the human brain functions.
1975 – 1976 and 1982 –1986
The replacement of the notorious Draize eye test for irritancy was one of the earliest achievements of the Dr Hadwen Trust. The test involved dripping test substances (e.g. shampoos, weed killers, household cleaners) into the eyes of conscious rabbits who were usually held in stocks and given no pain relief. These painful tests could continue for 7 days, causing ulceration, haemorrhaging, discharge, swelling and redness of the eyes. In 1982 some 19,100 experiments described as “application of substances to the eyes of rabbits” were conducted in Britain.
As well as causing animal suffering, the Draize eye test has serious scientific limitations. It is widely acknowledged to be unreliable, due to poor reproducibility and species differences between rabbits and humans.
In 1975 the Dr Hadwen Trust funded the first-ever research into replacing the Draize eye test. Our research demonstrated that it was possible to distinguish between shampoos of different irritancy using cell cultures. This work laid the foundations for the development of cell-based tests, currently in use today to replace rabbit experiments.
Subsequent research funded by the Dr Hadwen Trust in the 1980s by Dr Colin Muir at Leicester Polytechnic (now Leicester University), produced the first eye irritation test based on corneal opacity. This work used isolated bovine cornea, obtained as waste from the slaughterhouse. At that time the Dr Hadwen Trust supported work that used animal tissues with the aim of replacing experiments on living animals, but only if the tissues had been obtained from animals killed for reasons other than Dr Hadwen Trust funded research. The Dr Hadwen Trust no longer funds any research on animal cells or tissues.
Dr Muir devised an opacitometer, which used a beam of light shone through isolated cornea, to enable changes in cornea opacity to be accurately measured in response to various chemicals. This work was highly innovative and laid the groundwork for subsequent test-tube methods of predicting eye irritancy (including the BCOP test) throughout Europe.
1972 – 1981
During the 1970s, Dr Hadwen Trust funded researchers pioneered techniques to enable the culture of human cartilage tissue outside the body, in laboratory petri dishes. This enabled humane research into rheumatism without resorting to painful animal experiments.
At that time much research into rheumatism used animals, such as rabbits, rats or mice, which had chemicals or bacteria injected into their joints and paws. These experiments would have caused intense pain and swelling, but did not accurately replicate the condition of rheumatism, and yielded misleading results. Even tissue culture studies of rheumatoid arthritis relied on animal tissues, mainly pig and embryonic chick cartilage, although species differences were already known to be important.
With Dr Hadwen Trust funding, Professor Malcolm Jayson and co-workers Chris Lovell and Richard Jacoby, developed techniques for culturing human joint tissues – cartilage and synovium – in the laboratory. At an international conference held in 1973 in Cardiff (also funded by the Dr Hadwen Trust) Dr Jacoby reported the sustained organ culture of adult human cartilage had been made possible for the very first time.
The techniques were extended to include both healthy and arthritic human joint tissues from knee operations. Further work over several years included the study of human spinal cord ligament, skin, muscle and other tissues from patients, and resulted in discoveries of the chemical and structural changes, which occur in rheumatism.
By the late 1970s Professor Jayson and Dr Jacoby were looking at the effect of drugs on human joint tissues in culture. Their research revealed some of the first detailed understanding of how the drugs used to treat rheumatism at that time, actually worked. This knowledge assisted subsequent research to develop improved rheumatism treatments without the unpleasant side effects of steroids.
These non-animal approaches provided an important tool for replacing animal experiments, and produced accurate information about human rheumatism, paving the way for the new and better treatments available today.