Tag Archives: rat

Found in Translation: Using a Personal Tragedy to Drive Innovative Research

Kathryn Henley is a doctoral candidate at the University of Alabama at Birmingham. She studies pain in animals, currently pigs, trying to understand the different and often subtle signs that animals may be in pain. In this post, she explains why her research is important – both to the development of good animal welfare and the development of better pain management in humans.

Ten years ago, my dad fell off a ladder while he was cleaning the gutters on our house. Although he only fell five feet, the position in which he fell broke vertebrae in his neck. He was taken via MedFlight to a specialty hospital, where a neurosurgeon diagnosed him with a C4/5 complete spinal cord injury. In simple terms, he was paralyzed from the chest down and could not move or feel anything below that level.

One unfortunate side effect of spinal cord injury is that while the ability to feel internal stimuli (e.g., needing to go to the bathroom) and external stimuli (e.g., someone touching your hand) is lost, the majority of people with spinal cord injury live with the feeling of pain. This pain is usually severe and can significantly affect their physical capabilities, mental and emotional health, and social activities.  For example, my dad’s pain made it extremely difficult for him to participate in rehabilitation. I would often bring him to physical therapy where he would sit with cold packs on his shoulders instead of performing exercises to help him regain function. The medications available to treat pain after an injury to the central nervous system are few. Most of the medications my dad tried failed to provide adequate relief, and the few that did were highly addictive or left him feeling “out of it.” He usually chose not to take his medication and to live with the pain rather than dealing with the side effects.

I find the lack of effective therapeutics for pain extremely frustrating. If the number of preclinical studies of pain is increasing, why haven’t they translated into pain relief for people like my dad? In general, pain is extremely hard to measure. There is no biomarker for pain and we can’t ask the animals how they are feeling or have them fill out a survey. However, we can make observations about their behavior. One way pain is assessed in animals is measuring withdrawal reflexes. This is the same reflex that humans experience when we touch a hot stove and immediately pull back our hand. However, this is a spinal reflex that occurs so quickly, it happens before the feeling of pain reaches your sensory cortex. This is problematic for the study of pain after spinal cord injury because axons in the spinal cord that carry pain signals to the cortex may be damaged. In other words, the injury may prevent pain information from reaching the brain even though the withdrawal reflexes remain intact. Therefore, we can’t assume that there is pain sensation below the level of a spinal cord injury even if there is a withdrawal reflex. Additionally, the most devastating part of living with pain is the physical, emotional, and mental effects of feeling the pain, not withdrawal responses. My research focuses on behaviors in animals that tell us when there is a “feeling” of pain that reaches the sensory cortex and then results in a behavioral reaction.

The first behavior I examine is the “pain face,” also known as a grimace. When humans are in pain, we grimace by narrowing our eyes, wrinkling our nose, and raising our upper lip. Animals also grimace by changing certain parts of their faces, including their eyes, ears, cheeks, and nose or snout. An assessment called the grimace scale was first developed in mice by Dr. Jeffrey Mogil at McGill University in Canada. The grimace scale has since been translated to rats, rabbits, cats, pigs, sheep, and horses. Researchers have found that mice with lesions to their insular cortex don’t grimace. Because the insular cortex is involved in the emotional component of pain in humans, this may indicate that grimacing reflects the emotional effect of pain.

Example of the Rat Grimace Scale. There are four action units in the rat’s face that change with pain: the eyes, ears, whiskers, and nose/cheek. Image source: K. Henley, unpublished.

I also use vocalizations to measure pain. Some animals vocalize to communicate when they are in pain because this ultimately benefits them and promotes the survival of their species. However, other animals like mice and rats may not vocalize when they feel pain because this would attract predators. Right now, I am characterizing the vocal repertoire of pigs. This means I record all the sounds that pigs make and classify them based on how they sound and look on a spectrogram. Sound analysis software enables me to analyze different components of their calls in detail, so I can determine even slight differences in duration and frequency. Knowing all their calls will allow me to better assess differences when using their vocalizations as an outcome measure. So far, I have characterized 16 different call types. Did you know that pigs bark?!

One important consideration when assessing pain is the confounding effect of other mental and emotional states, such as stress or anxiety. Animals may behave differently because of stress, regardless of whether they are in pain or not. As such, we take extreme care to ensure our animals feel safe and comfortable in their environment. We allow the animals to acclimate to their new space for three days after their arrival, without any interaction with study staff. On the following days, we slowly habituate them to our presence by offering treats and other positive reinforcement. We do not begin any study-related procedures until each animal can be calmly approached and touched by the investigators. Many prey animals will hide signs of pain from predators; therefore, it is vital that our animals do not feel threatened at any time. In fact, the pigs enjoy our presence very much (as it typically accompanies food) and I enjoy spending time getting to know each individual animal. They are also acclimated to any rooms, equipment, or procedures they will experience in the study to reduce any effects from stress or anxiety.

I love my research because it serves a dual purpose: to help both animals AND people like my dad live pain-free. The more I learn about animals and their behavior, the more information there is to guide animal welfare policies in both biomedical research and the production (farm) industry. This means that scientists, veterinarians, laboratory animal technicians, and farm personnel will have access to better tools to assess whether an animal is pain and if a pain medication is working. In addition, more accurate assessments of pain will lead to more valid results from preclinical studies. This means that people like my dad will have better options to help manage their pain and be able to achieve a better quality of life.

Kathryn Henley

Paralyzed man walks again after olfactory cell transplant, thanks to animal research

Today, almost 30 years after Prof. Geoffrey Raisman first identified their potential to repair nerve damage in mice, the BBC reports that olfactory ensheathing cell transplantation has been successfully used to enable Darek Fidyka, who was paralyzed from the chest down in a knife attack in 2010, to walk again.

The paper reporting the transplant, which was carried out by surgeons in Poland and  led by Geoffrey Raisman of the UCL Institute of Neurology, is published today in the journal Cell Transplantation (5). The technique involves taking specialized cells known as olfactory ensheathing cells (OECs) from the patient’s own patient’s olfactory bulbs, and then grafting these cells at the site of injury, where they promote nerve cell growth to bridge the gap and restore function. An added advantage in using the patient’s own cells is that it avoids the problem of rejection by their immune system.

Speaking earlier today Geoffrey Raisman described the results as “more impressive than man walking on the moon”. He’s not to far wrong, this achievement shows what is possible for regenerative medicine, and is the result of decades of basic and translational research. Indeed, whereas only 12 people have  walked on the moon, this new technique has the potential to help many thousands of people to walk again here on earth.

2014 has been an extraordinary year of progress restoring function after spinal injury, in May we saw how epidural stimulation allowed 4 paralyzed men in the US to move their legs again, while scientists at Newcastle University in the UK used closed loop electrostimulation to restore voluntary movement in temporarily paralyzed monkey arms. These techniques, and now OEC transplantation, show that many cases of paralysis are potentially reversible. Not every technique will be appropriate for every patient, and it will take much additional research before they are widely available, but together they represent a huge advance.

Darek Fidyka learns to walk again following OEC transplantation. Image BBC News.

Darek Fidyka learns to walk again following OEC transplantation. Image BBC News.

In each case it is an advance that rests on many decades of careful research in both animals and in human subjects, in particular basic research that uncovered the role of specialized cells and provided scientists with the knowledge about organization and function of the brain and spinal cord that enabled these pioneering therapies to be developed.

In a post in 2012 I discussed how Geoffery Raisman’s research led to the successful testing of olfactory ensheathing cells in injured dogs, and I’m reposting that article here:

Paralysed dogs walk again thanks to nasal cell transplants…and Professor Raisman’s rats. (published 19 November 2012)

This morning the BBC News carried a report on a medical breakthrough – and it is not a term I use lightly – that has enormous implications for people who have been paralyzed following spinal cord injuries. A team at the University of Cambridge led by Professor Robin Franklin Department of Veterinary Medicine, along with colleagues at the MRC Centre for Regenerative Medicine in Edinburgh succeeded in restoring the ability to walk with their hind legs to dogs which had been paralyzed by spinal injury. To do this they removed a special type of cell called the olfactory ensheathing cell (OEC) from the nasal passageways of the dogs, grown them in culture until a sufficient number had been produced, and then transplanted them at the site of injury. Many of the dogs which received the transplant were subsequently able to walk with their hind legs if supported by a harness, and some even able to walk without being supported by a harness, whereas dogs which received a control injection did not recover the ability to move their hind legs.

This is a major medical advance, and the first time that cell transplantation has been demonstrated to reverse paralysis in a real-life situation where the injury involves a combination of damage to the nerve fibre and to surrounding tissues, and there is a significant delay between injury and treatment, and while the therapy did not completely restore function it marks a very significant step towards a therapy that can be evaluated in a human clinical trial. It also of course is a very promising therapy for dogs that have suffered spinal injuries, for example after being hit by a car, and as such is an excellent example of the One Health concept which seeks a closer integration of human and veterinary medicine.

As with many breakthroughs this one did not happen overnight, indeed it is the result of decades of research. The story really begins in 1985 when Professor Geoffrey Raisman at University College London (for a good overview of his work see the UCL spinal Repair Group homepage) was studying the unique ability of nerve fibres in the olfactory system to grow and make the connections with central nervous system – an ability that other adult nerve cells lack and which is probably retained in the olfactory system due to the importance of preserving the ability to smell despite exposure of nerve cells in the nasal passages to toxins in the environment (a good sense of smell being crucial to survival for many mammalian species). He found that in a part of the brain termed the olfactory bulb of mice and rats a specific type of glial cell, cells that act to support and regulate the activity of the nerve cells along which nerve impulses travel , were responsible for creating the pathway along which the olfactory nerve fibres could regenerate (1).

Studies in rats were key to unlocking the potential of olfactory ensheathing cells in repairing spinal injuries. Image courtesy of Understanding Animal Research

This discovery suggested that if these specialized olfactory ensheathing cells (OECs) were transplanted at the site of spinal cord injury they might promote the growth of a bridge of nerve cells that would reconnect the severed pathway and restore function. In a series of experiments in rats Professor Raisman and colleagues demonstrated that OEC transplantation could repair a variety of different types of spinal cord injury, in order to restore function, for example to improve the ability to breath and climb following spinal cord injury (2) and to restore the ability of rat paws to grasp in order to climb following lesion of the spinal nerve that runs from the spinal cord down through the arm (3). Other scientists provided additional key information, for example scientists at the University of New South Wales in Australia demonstrated that OECs could be isolated from the nasal mucosa as well as from the olfactory bulb (4), and that these can also repair spinal cord injuries, an important step since obtaining OECs from the nasal mucosa is far more straightforward and safer than harvesting them from the brain. These discoveries, and the refinement of OEC transplant techniques over the past 2 decades by scientists such as Prof. Raisman, paved the way for the “real life” veterinary study reported today. A human clinical trial of this technique cannot be far off, though it is worth noting Prof. Raisman’s words of caution to the BBC concerning what has been achieved and what is still to be done:

“This is not a cure for spinal cord injury in humans – that could still be a long way off. But this is the most encouraging advance for some years and is a significant step on the road towards it…This procedure has enabled an injured dog to step with its hind legs, but the much harder range of higher functions lost in spinal cord injury – hand function, bladder function, temperature regulation, for example – are yet more complicated and still a long way away.”

In this respect it is worth noting the other approaches to repairing spinal cord injury, for example using other glial cell known as astrocytes and the use of electrical stimulation have produced promising outcomes in animal studies and early human clinical trials. Indeed, a clinical study of electrostimulation that we discussed last year reported “improved autonomic function in bladder, sexual and thermoregulatory activity that has been of substantial benefit to the patient”. In the future these different approaches may be combined to maximize the benefit to the patient, but it is still far too early to say which techniques will best complement each other. One thing we can be sure of is that turning these very promising technologies into effective treatments – perhaps even cures – for paralysis will require further research, both in the lab and in the clinic.

Paul Browne

1) Raisman G. “Specialized neuroglial arrangement may explain the capacity of vomeronasal axons to reinnervate central neurons.” Neuroscience. 1985 Jan;14(1):237-54. PubMed: 3974880

2) Li Y, Decherchi P, Raisman G. Transplantation of olfactory ensheathing cells into spinal cord lesions restores breathing and climbing.” J Neurosci. 2003 Feb 1;23(3):727-31. 12574399

3) Ibrahim AG, Kirkwood PA, Raisman G, Li Y. “Restoration of hand function in a rat model of repair of brachial plexus injury.” Brain. 2009 May;132(Pt 5):1268-76. Epub 2009 Mar 13. PMID: 19286693

4) Lu J, Féron F, Mackay-Sim A, Waite PM. “Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord.” Brain. 2002 Jan;125(Pt 1):14-21. PMID: 11834589

5) Tabakow P et al. “Functional regeneration of supraspinal connections in a patient with transected spinal cord following transplantation of bulbar olfactory ensheathing cells with peripheral nerve bridging” Cell Transplantation, published online 20 November 2014 http://www.ingentaconnect.com/content/cog/ct/pre-prints/content-CT-1239_Tabakow_et_al

Nobel Prizewinner John O’Keefe warns of threat to science from overly restrictive animal research and immigration rules

In an interview with the BBC yesterday 2014 Nobel laureate  John O Keefe has warned of the dangers posed by regulations that restrict animal research and the free movement of scientists across borders.

“It is an incontrovertible fact that if we want to make progress in basic areas of medicine and biology we are going to have to use animals.

“There is a worry that the whole regulatory system might begin to be too difficult, it might be constrictive.”

Professof John O'Keefe, 2014 Nobel Laureate in Medicine or Physiology. Image: David Bishop, UCL.

Professof John O’Keefe, 2014 Nobel Laureate in Medicine or Physiology. Image: David Bishop, UCL.

His concerns are well founded. Our post yesterday discussed the key role of recordings of single neuron activity in rats to the discoveries made by John O’Keefe, May-Britt Moser and Edvard Moser. The post also discusses two other advances made through basic research in animals whose impact in medicine has been recognized by awards, deep brain stimulation in Parkinson’s disease, and infant massage in preterm babies. Nevertheless in many countries around the world there is increasing pressure from animal rights groups on politicians to restrict, and even ban, animal research. Scientists have a key role to play in ensuring that important basic and translational research, and we welcome John O’Keefe’s statement,  it’s an example that scientists around the world should follow.

The issue of immigration is another important one for science, and John O’Keefe knows this better than most. Born in New York, he completed his PhD at the University on Montreal under the supervision of renowned Psychologist Ronald Melzack, before moving to the UK to undertake a postdoctoral fellowship, and credits the research environment in the UK and at UCL for giving him the opportunity to make his discoveries, and later May-Britt and Edvard Moser spent time as postdoctoral researchers at his laboratory.  For science to flourish scientists must be free to travel to centres of excellence in other countries, to learn skills and establish collaborations that are key to success in many fields of research in the 21st century. This freedom is under threat from narrow-minded isolationism in many countries, for example earlier this year Switzerland found its position as a leading scientific nation undermined by a new immigration law that threatens its ability to recruit talented scientists from abroad, and has disrupted its participation in a key EU research programmes.

John O’Keefe’s warning is a reminder that the threats to scientific research can come from many directions, and of the need for supporters of science to be ready to take action to defend the freedoms on which science is built.

Speaking of Research

Paralysis breakthrough – electrical stimulation enables four paraplegic men to voluntarily move their legs

This weeks issue of the neuroscience journal Brain carries an unusual image; against a background of nerve activity traces a man lies on the ground, and as you scan down the images he lifts his right leg off the ground. For most people this might just be a simple warm-up exercise, but for Kent Stephenson it was little short of a miracle, because he has suffered complete paralysis after suffering a mid-thoracic spinal cord injury. Speaking about his experience Kent noted that “Everything’s impossible until somebody does it”, and this is a breakthrough that is possible due to animal research.

Brain_cover image

Kent was one of four patients participating in a pilot study of epidural electrical stimulation sponsored by the Christopher and Dana Reeve foundation, which is overseen by an international team comprising of Claudia Angeli and Susan J. Harkema of the University of Louisville, Yury Gerasimenko of the St. Petersburg’s Pavlov Institute and UCLA, led by V. Reggie Edgerton of UCLA.

Before the implantation of an epidural stimulator all four participants were unable to move their lower extremities, and two had also lost all sensation below the injury. This continues a study published in the Lancet in 2011 that evaluated the effects of epidural stimulation in the first participant, Rob Summers, was able to stand again thanks to electrical stimulation, which also improved his general health and quality of life by improving bladder and sexual function, and thermoregulatory activity.

The key findings reported in an open-access article “Altering spinal cord excitability enables voluntary movements after chronic complete paralysis in humans” in Brain (and discussed in detail on the Christopher and Dana Reeve Foundation website) detail the impact of epidural stimulation in all four participants, including new tests conducted on Rob Summers. Surprisingly the 3 new participants were able to perform voluntary leg movements immediately following the implantation and activation of the stimulator, and the researchers to speculate that some pathways may be intact post-injury and therefore able to facilitate voluntary movements. For a pilot study this is extraordinarily encouraging, as it shows that many paraplegic patients, even those who are diagnosed as having complete motor and sensory injuries can benefit from this technique. V. Reggie Edgerton, who led this project remarked:

“This is a wake-up call for how we see motor complete spinal cord injury, We don’t have to necessarily rely on regrowth of nerves in order to regain function. The fact that we’ve observed this in four out of four people suggests that this is actually a common phenomenon in those diagnosed with complete paralysis.”

There should be no doubt that this is a medical advance that depended on animal research, indeed in a guest article on this blog in 2009 Professor Edgerton noted following the publication of a key Nature Neuroscience paper on epidural electrical stimulation in rats that led to this clinical trial:

It has been characterized as a major breakthrough in facilitating the level of recovery of locomotion following a severe spinal cord injury. This in itself implies that these findings were the result of a single experiment with rats. But the reality is that these experiments were based on 100s of other experiments by not only my laboratory, but many other scientists. All of the previous animal experiments relevant to our understanding of the control of movement, involving many different species ranging at least from fish to humans, have contributed to the evolution of the concepts that underly our most recent publication. This full range of animal species is essential for the continuing progress toward the development of interventions to recover all of those functions that are lost, following a severe spinal cord injury. Our particular publication only addressed the recovery of locomotion, but there are other severe functional losses such as bladder and bowel control and hand function among others that are in need of breakthroughs. It is certain that the concepts which led to the Nature Neuroscience publication would not have evolved at any time in the near future without these gradual and incremental experiments which formed the scientific basis of these concepts. There is no way that these concepts and the experimental results could have been predicted by any non-animal mechanism, for example, computer modeling.

In these videos from Professor Edgerton we see how years of careful animal research underpinned the development of this therapy.

Animal studies continue to be crucial to Professor Edgerton’s work, for example the use of rats in the evaluation of a new multi-electrode array for improving spinal cord epidural stimulation in order to enable more complete restoration of function. The success reported in Brain this week is only just the beginning!

So when you hear animal rights activists claiming that animal research is an outdated science remember these four young men who can move again, and the hundreds of scientists whose decades of careful studies in animal models of spinal cord injury made this breakthrough possible.

Paul Browne

To learn more about the role of animal research in advancing human and veterinary medicine, and the threat posed to this progress by the animal rights lobby, follow us on Facebook or Twitter.

Guest Post: Characterising high fructose corn syrup self-administration in laboratory rats

It’s January, and across the country millions of people have promised themselves that they will eat less, loose weight and become healthier. But why do some people eat more than others? No matter what they try there seems to be no way to stop their overeating. Public education is a powerful tool to combat some of these issues but what happens when it turns into an addiction? It is challenging to provide accurate information when food addiction is a little studied field. In an effort to answer these questions scientists can use laboratory rodents to explore neurobiological mechanisms involved in relapse to drug-seeking behavior, comorbid mood and substance dependence disorders, as well as perseverative reward seeking. These complex answers cannot be solely obtained though human patients because the physiological and psychological mechanisms that influence food addiction are not fully understood.

AnneMare Levy is a PhD student and Francesco Leri is an Associate Professor of Neuroscience and Applied Cognitive Science in the Department of Psychology at the University of Guelph. In the article below these scientists explain how and why the development of a new animal model to understand the addictive properties of some foods is necessary and how its use can begin to answer some of these questions. They believe that through studying rats their findings could lead to novel pharmacological interventions for obese individuals that could help them selectively reduce intake of unhealthy foods.

The views expressed below are that of the authors alone and do not necessarily reflect the views of her employer or institution.

Overconsumption of foods high in sugars and saturated fats is an important contributing factor to the modern epidemic of overweight and obesity1, which are leading causes of metabolic disorders and cardiovascular diseases2. It is therefore important to understand why patterns of excessive food intake develop and persist despite the negative health consequences. Considerable evidence supports the hypothesis that, for some people, addiction to food may motivate these behaviours3-4. In fact, behavioural and neurobiological similarities between obesity and drug dependence support the “food addiction” hypothesis5-8 and studies in both humans and laboratory animals have identified a variety of biological and behavioural indicators of “food addiction”9-12.

The food addiction hypothesis suggests that similar to drugs of abuse, particular foods should reinforce behaviours that lead to their consumption. Therefore, to assess the addictive potential of such foods, we adapted procedures commonly used for studying the reinforcing properties of drugs of abuse (i.e. operant intravenous drug self-administration) to the investigation of operant self-administration of sweet solutions delivered directly into the mouth of rats. To this end, an intraoral cannula was surgically implanted13 into the cheek of rats and the animals were subsequently trained to press a lever to voluntarily receive a test solution directly into their mouth; hence the term intraoral self-administration. The sweet solution selected for testing was high fructose corn syrup (HFCS) because, although controversial14, there is evidence that HFCS may be linked to the modern epidemic of obesity15.

corn-syrup

The disadvantage of requiring minor surgery to employ this procedure is offset by several advantages that make intraoral self-administration in rats optimal for studying the reinforcing properties of sweet solutions. First, an operant response (i.e., pressing a lever) is required to obtain an infusion and therefore it is possible to modify the schedule regulating the relationship between response requirement and delivery of intraoral infusions. Hence, by employing a progressive ratio (PR) schedule16, whereby more lever presses are required to get more sweet solution, it is possible to assess how much an animal “wants”17 the next infusion and by employing a continuous schedule of reinforcement, whereby each lever response is reinforced, it is possible to measure total intake, escalation of intake, and the development of bingeing behaviour.  Second, intraoral self-administration allows testing of any concentration and any volume of any water-soluble food additive. The importance of controlling and manipulating concentration/volume ratios is mandatory in experiments where intake can be modulated both by the caloric value of a solution (i.e., nutrient-specific satiety) and by how much of that solution can be consumed within a given period of time (i.e., fullness)18. Third, intraoral self-administration shortens the delay between the operant response and the delivery of the primary reinforcer, a factor that plays an important role in the acquisition and maintenance of operant behaviour19-21. Finally, this procedure allows for the delivery of passive intraoral infusions of controlled quantities of the test solution.  This makes it possible to measure orofacial responses of “liking” (objective hedonic reaction such as tongue protrusions)22 as well as administer priming infusions23 of the test solution prior to tests of reinstatement of sweet-seeking behaviour.

The objective of this study was to characterize HFCS self-administration behaviour in laboratory rats. It was important to establish a reliable animal model of self-administration because it will allow future studies to identify and manipulate the neurobiological substrates that are responsible for the escalation and maintenance of excessive food intake. Moreover, using this animal model, the rats will be able to self-administer solutions for extended periods of time (i.e. months) to establish how sweeteners, such as HFCS, may contribute to the development of metabolic disorders.  For all experiments, rats were surgically implanted with an intraoral cannula while under an anaesthetic. Post-operative care included administering analgesic, daily flushing of the cannula with an anti-bacterial solution as well as closely monitoring weight gain and food intake. Following recovery, rats received one 3-hour self administration session per day, whereby, rats were placed into a standard operant chamber and trained to lever press to receive intraoral infusions of different concentrations of HFCS (8%, 25%, and 50%) on either continuous or PR schedules of reinforcement, as previously described.

It was found that the behavioural profile of rats responding for HFCS is similar to the pattern of intake observed when rats self-administer drugs of abuse24-25. Using intraoral self-administration, it was established that on a continuous schedule of reinforcement, rats acquire and maintain intraoral self-administration of a wide range of HFCS concentrations (8%, 25% or 50%), and that rats adjust their self-administration behaviour according to the different concentrations (i.e., rats self-administer twice as much a 25% solution than a 50% solution)13.  Furthermore, higher concentrations of HFCS engender higher responding on the PR schedule of reinforcement, suggesting that increasing the HFCS content likewise increases the reinforcing value of the solution. The relationship between operant responding and HFCS concentration on continuous and progressive ratio schedules is similar to the dose-response relationships observed when rats self-administer drugs of abuse24.

It was further noted that total intake of 25% HFCS escalated over three weeks of testing, possibly reflecting the development of “bingeing” behaviour9,13. In fact, after a week of self-administration, rats displayed a clear period of elevated intake during the initial 90 minutes of each self-administration session and this “loading” increased in magnitude over the weeks of training. This effect is reminiscent of escalation of drug intake and increased loading that are observed when rats have prolonged and/or repeated access to drugs of abuse26.

The results of these experiments also indicated that HFCS is reinforcing because of its caloric content. Even though 0.1% saccharin (a non-caloric sweetener)27 and 25% HFCS produce similar hedonic reactions (i.e. the perceived palatability of the two solutions is similar in tests of taste reactivity17), 0.1% saccharin could not maintain self-administration at the same level that 25% HFCS. Moreover, when substituted for HFCS, a wide range of saccharin concentrations (0.01%, 1.0%, and 10%) significantly reduced self-administration behaviour, indicating that HFCS reinforcement is largely determined by its caloric content rather than its palatability.

Taken together, these experiments indicate that intraoral infusion of HFCS reinforces lever-pressing in rats, and this behaviour was maintained primarily by the caloric content and not the palatability of the solution made available for self-administration.  In these rats, stable self-administration was maintained for up to three weeks, it was concentration-dependent, and rats developed a tendency to “binge” on HFCS at the start of sessions. Using intraoral self-administration, future studies should investigate the possibility that HFCS engenders other “addictive-like” behaviors, and whether escalation of HFCS self-administration can be causally linked to the development of metabolic changes (i.e., weight gain, insulin resistance) associated with obesity and type-2 diabetes.

AnneMarie Levy & Francesco Leri

University of Guelph

University of Guelph

Department of Psychology, NACS

References

1. Barry D, Clarke M, Petry NM (2009) Obesity and Its Relationship to Addictions: Is Overeating a Form of Addictive Behavior? Am J Addict 18: 439-451.

2. World Health Organization (2013) Obesity and overweight Fact sheet N°311. Available: http://www.who.int/mediacentre/factsheets/fs311/en/index.html#. Accessed 20 June 2013.

3. Davis C, Carter JC (2009) Compulsive overeating as an addiction disorder: a review of theory and evidence. Appetite 53: 1-8.

4. Ifland JR, Preuss HG, Marcus MT, Rourke KM, Taylor WC, Burau K, Jacobs WS, Kadish W, Manso G (2009) Refined food addiction: a classic substance use disorder. Med Hypotheses 72: 518-526.

5. Avena NM, Bocarsly ME, Hoebel BG, Gold MS (2011) Overlaps in the nosology of substance abuse and overeating: the translational implications of “food addiction”. Curr Drug Abuse Rev 4: 133-139.

6. Volkow N, Wise RA (2005) How can drug addiction help us understand obesity? Nat Neurosci 8: 555-560.

7. Fortuna J (2012) The Obesity Epidemic and Food Addiction: Clinical Similarities to Drug Dependence. J Psychoactive Drugs 44: 56.

8. Levy AM, Salamon A, Tucci M, Limebeer CL, Parker LA, Leri F (2012) Co-sensitivity to the incentive properties of palatable food and cocaine in rats; implications for co-morbid addictions. Addict Biol:  doi: 10.1111/j.1369-1600.2011.00433.

9. Avena NM, Rada P, Hoebel BG (2008) Evidence for sugar addiction: behavioural and neurochemical effects of intermittent, excessive sugar intake. Neurosci Biobehav Rev 32, 20-39.

10. Gearhardt AN, Davis C, Kuschner R, Brownell KD (2011) The addiction potential of hyperpalatable foods. Curr Drug Abuse Rev 4: 140-145.

12. Johnson PM, Kenny PJ (2010) Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats. Nat Neurosci 13:  635-644.

13. Levy AM, Limebeer CL, Ferdinand J, Shillingford U, Parker LA, et al. (2014) A novel procedure for evaluating the reinforcing properties of tastants in laboratory rats: operant intraoral self-administration. JoVE: in press.

14. White JS (2008) Straight talk about high-fructose corn syrup: what it is and what it ain’t. Am J Clin Nutr. 88: 1716S-1721S.

15. Bray GA, Nielsen SJ, Popkin BM (2004) Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity, Am J Clin Nutr 79: 537-543.

16. Richardson NR, Roberts DC (1996) Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy. J Neurosci Methods 66: 1-11.

17. Berridge, K. C., & Robinson, T. E., Parsing reward. Trends in Neuroscience 26 (11), 507-501(2003).

18. Houpt, K. A. Gastrointestinal factors in hunger and satiety. Neuroscience and Biobehavioural Reviews 6 (2), 145-164 (1982).

19. Panksepp, J. & Trowill, J. A. Intraoral self injection: I. Effects of delay of reinforcement on resistance to extinction and implications for self-stimulation. Psychonomic Sciences 9 (7), 405-406 (1967)

20. Mazur, J. E. Effects of rate of reinforcement and rate of change on choice behaviour in transition. Journal of Experimental Psychology 50 (2), 111-128 (1997).

21. Samaha, A. N., & Robinson, T. E. Why does the rapid delivery of drugs to the brain promote addiction? Trends in Pharmacological Sciences 26 (2), 82-87 (2005).

22. Berridge, K. C., & Kringelbach, M. L. Affective neuroscience of pleasure: reward in humans and animals. Psychopharmacology (Berl) 199 (3), 457-480 (2008)

23. Shaham, Y., Shalev, U., Lu, L., De Wit, H., & Stewart, J. The reinstatement model of drug relapse: history, methodology and major findings. Psychopharmacology (Berl) 168 (1-2), 3-20 (2003).

24. Deroche-Gamonet V, Belin B, Piazza PV (2004) Evidence for addiction-like behaviour in the rat. Science 305: 1014-1017.

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Trial of gene therapy in heart failure launches following success in rats and pigs.

Heart failure is a deadly condition that affects about two out of every hundred adults in the USA, and occurs when the heart is unable to provide sufficient pump action to maintain blood flow to meet the needs of the body. Among the more common causes are heart attacks and hypertension, but less frequently it can also be caused by viral infections or autoimmune diseases.

While the therapies available for heart failure have improved a lot in recent years thanks to the development of drugs such as Ivabradine, heart failure is still a major cause of death and disability, particularly among the over 65’s. As you might expect scientists around the world are developing several innovative approaches to treating heart failure – the British Heart Foundation’s “Mending Broken Hearts” appeal is an excellent example of the concerted effort now underway – and we have highlighted on this blog and our Facebook page  techniques ranging from electrical stimulation of the vagus nerve to collagen patches that stimulate tissue repair.

To those animal research has added another: Gene Therapy!

Image courtesy of Imperial College London

Image courtesy of Imperial College London

Yesterday the BBC reported the recent launch in the UK of a Clinical trial of gene therapy for heart failure, and Professor Peter Weissberg of the British Heart Foundation, who funded much of the basic and applied research leading up to this trial, noted the promise that this approach holds:

Whilst drugs can offer some relief, there is currently no way of restoring function to the heart for those suffering with heart failure. This early clinical study is the culmination of years of BHF funded laboratory research and offers real promise.

“Gene therapy is one of the new frontiers in heart science and is a great example of the cutting edge technologies that the BHF is using to fight heart failure. Gene therapy aims to improve the function of weak heart muscle cells, whereas our Mending Broken Hearts Appeal is aimed at finding ways to replace dead heart muscle cells after a heart attack. Both approaches are novel and both offer great potential for the future.””

This trial, which is being run by researchers at Imperial College London and the Royal Brompton Hospital, is part of a multinational multicentre trial of 200 patients – CUPID-2b – which seeks to assess whether injection into heart tissue of a adeno-associated virus 1-based gene therapy vector driving expression of the enzyme SERCA2a can repair damaged heart tissue and improve cardiac function. The reasoning behind this is that as a calcium transport protein SERCA2a plays a key role in maintaining the correct balance of calcium ions in heart muscle cells, and studies in both human heart failure patients and animal models of heart failure the amount of SERCA2a is lower than normal. A combination of studies in human heart muscle tissue and animal models of heart failure over several years demonstrated that this decrease is associated with calcium overload, an abnormal heart rhythm and tissue damage, suggesting that increasing the amount of SERCA2a in the injured heart tissue may reverse the damage.

In 2010 paper was published reporting on the first clinical trial of this therapy1 (available to read for free), whose primary goal was to assess the safety of the technique, and it noted that studies in animal models of heart failure provided vital evidence underpinning the decision to move it into clinical trials.

In preclinical HF models in rodents,(20) pigs,(18) and sheep,(21) increasing the level of SERCA2a using recombinant AAV vectors was well tolerated and restoration of SERCA2a levels resulted in significant improvement in cardiac function and energetics, even when the underlying pathophysiology or insult (eg, mitral valve rupture or pacing induced heart failure) was not corrected. Based on these findings, this first-in-human Phase 1/2 Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial(4) aims to restore levels of this key enzyme in HF patients via gene transfer of the SERCA2a cDNA by delivering a recombinant AAV (AAV1/SERCA2a) via percutaneous intra-coronary infusion.”

These studies, first in short- term studies in rats published in 2007 and subsequently longer duration studies in pigs and sheep published in 2008, indicated that this therapy was safe, could restore SERCA2a to normal levels, promoted heart muscle repair and improved heart function.

It’s just one more example of how animal research is contribution to the exciting field of gene therapy, and to advances in treating heart failure.

Paul Browne

1)      Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B, Borow K, Dittrich H, Zsebo KM, Hajjar RJ; Calcium Up-Regulation by Percutaneous Administration of Gene Therapy In Cardiac Disease (CUPID) Trial Investigators. “Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-in-human phase 1/2 clinical trial.” J Card Fail. 2009 Apr;15(3):171-81. doi: 10.1016/j.cardfail.2009.01.013. PMCID: PMC2752875

Animal research leads to promising results for first clinical trial of stem cell therapy for stroke

The BBC reported yesterday that a small trial of a stem cell therapy developed by the biotech firm Reneuron has produced promising results, with 5 of the 9 patients enrolled in the trial showing unexpected improvements. The improvements were unexpected because the trial was intended to assess the safety of the technique, and the scientists did not expect to observe any measurable improvement. Larger trials agianst control groups are now planned to determine if the observed effect is indeed due to the stem cell therapy, and if so how much it contributes to the improvement. Behind the statistics are the human stories, for trial participant Frank Marsh, the result was a significant improvement to his quality of life, though he hopes for further improvement over time:

I can now grip things that I couldn’t grip before, like the hand rails at the swimming baths…I’d like to get back to my piano. I’d like to walk a bit steadier and further.”

Studies in rats are playing a key role in stem cell medicine. Image courtesy of Understanding Animal Research.

Studies in rats are playing a key role in stem cell medicine. Image courtesy of Understanding Animal Research.

As the Reneuron website points out animal studies allowed this therapy (ReN001, using the CTX0E03 human neuronal stem cell line) to be evaluated and assessed prior to launching human trials.

Post stroke rehabilitation – the aim of post stroke rehabilitation is to improve both functional and cognitive recovery in the patient some weeks or months after the stroke event.

It is this third treatment stage that our ReN001 stem cell therapy seeks to address. A number of treatments exist or are in development to treat stroke patients in the acute phase. However, there are currently no therapies available for patients who have a stable and fixed neurological deficit following a stroke. Our ReN001 cell therapy for stroke consists of a neural cell line, designated CTX, which has been generated using our proprietary cell expansion and cell selection technologies and then taken through a full manufacturing scale-up and quality-testing process. As such, ReN001 is a standardised, clinical and commercial-grade cell therapy product capable of treating all eligible patients presenting.

ReN001 has been shown to reverse the functional deficits associated with stroke disability when administered several weeks after the stroke event in relevant pre-clinical models. Extensive pre-clinical testing also indicates that the therapy is safe, with no adverse safety effects arising from the administration of the cells. Clinically, the potential of the ReN001 treatment is to engender a degree of recovery of function in disabled stroke patients sufficient to give them an improved quality of life and a reduced reliance on health and social care.”

In particular a study published in 2009 – the year this trial was announced – showed that CTX0E03 cells could restore a high degree of function when injected into the brains of rats 4 weeks after experimentally induced stroke, indicating that they could aid recovery in a time frame that was likely to be achievable in the clinic, where doctors will  – at least until this therapy is more established – wish to wait and assess the degree of functional recovery in stroke patients before deciding on whether or not stem cell therapy might be beneficial.

More recently their animal studies have focused on elucidating the mechanism through which CTX0E03 cells increase neurogenesis by increasing the populations of endogenous cells in the brain rather than directly replacing lost nerve cells, fundamental discoveries that will help scientists to optimise the use of this therapy and the development of future stem cell therapies.

Speaking of Research