Equine Science Update

Transcription

1 Equine Science Update Reports from the world of equine research Summer 2004 Stem cells offer new hope for damaged tendons. In the normal horse, tendon growth ceases at about two-years of age and the tendon loses its ability to regenerate. Current thinking is that strained tendons usually result from accumulated lowgrade damage rather than a single injury. Even normal use may cause a strain when the tendon is already weakened. Hence, it is important to remember that similar degenerative changes will be occurring in the other limb. Many treatments have been advocated for tendon injury. Most have no effect; but some may make matters worse. No single treatment has proved more effective than others. In fact, research has shown that the outcome is most closely related to the severity of the initial injury rather than any of the currently available treatments. This is because the healing process is one of repair rather than regeneration - giving scar tissue rather than replacement tendon. Recent developments in tendon treatment aim to make the repair more like normal tissue rather than scar tissue. Various growth factors have been used to try to promote the growth of tendon tissue and reduce the amount of scar tissue. These are still at the early experimental stage. They are still largely unproven and the most appropriate dosage regime is unknown. Stem cells offer the possibility of regenerating, or re-growing, tissues that are normally incapable of doing so. Stem cells from the embryo are capable of differentiating into any type of cell. In the adult, the stem cells are more restricted in their ability to differentiate. Haematopoietic stem cells can produce a range of blood cells. They offer a possible way of treating blood cell disorders such as leukaemia. Mesenchymal stem cells (MSCs) are restricted to producing cells involved in skeletal tissues such as tendon, cartilage, and bone. In the adult, the liver and muscle contain adequate MSCs to be able to regenerate after cell Large numbers of mesenchymal stem cells are injected into the core lesion under ultrasound guidance. damage. However, tissues such as tendons and heart muscle do not, and so can only patch up the damage using scar tissue. Currently MSCs show promise as part of the treatment of ischaemic cardiomyopathy (heart attack) in human medicine, where they are being used to regenerate the muscle in the damaged part of the heart. In horses, the exploitation of the potential of stem cells began with the direct implantation of neat bone marrow into the suspensory ligament to encourage regeneration. Neat bone marrow actually contains very few stem cells, but does contain a number of growth factors. So to provide sufficient stem cells a large volume of bone marrow is required. This has raised concerns about whether it would cause damage when injected into a small structure like a tendon. The bone marrow sample may also contain bone or cartilage cells, which could lead to the production of bone or cartilage within the tendon. Professor Roger Smith, has been working with colleagues at the Royal Veterinary College, London, and VetCell Bioscience Ltd, to develop a method of treating tendon injuries by injecting MSCs grown in large numbers in the laboratory from a bone marrow sample taken a few weeks earlier. Because the cells are taken from the injured horse there is no problem with the implanted cells being rejected. A small initial study has been carried out to assess that the technique is safe. They treated six cases of severe superficial digital flexor tendonitis. All involved the mid-cannon region and had an obvious core lesion (a black hole down the centre of the tendon.) They collected a bone marrow sample within 2 months of the injury. The stem cells were cultured, usually taking about a month, and were then implanted into the damaged tendon within 3 months of the original injury. The results so far indicate that the core lesion fills rapidly. Radiography and nuclear scintigraphy (bone scanning) showed no evidence of bone or cartilage formation. The longitudinal fibre pattern in the tendon improved with a graduated exercise regime. Following on from the trial they now recommend that the bone marrow should be collected as soon as possible after the injury and at least within one month. Culturing the MSCs is not an easy procedure and in about 25% of cases the growth is insufficient and a second sample is required. The recommended size of sample usually yields enough bone marrow for two attempts at culturing MSCs. Half of the sample is

2 Equine Science Update 2 used to start the tissue culture. The remainder is stored and can be used if the first sample fails to grow. Only rarely is it necessary to take another bone marrow sample. One worry that the team had originally was that it might be difficult to grow MSCs from the bone marrow of older horses. However, that concern does not appear to have been borne out in practice. The aim is to implant at least two million MSCs.. Implantation is carried out under ultrasound guidance to ensure the cells are injected into the core lesion. Usually two or three injections is sufficient. The mechanical environment and the presence of neighbouring tenocytes (tendon cells) are believed to stimulate the MSCs to differentiate into tendon cells and not bone or cartilage). Prof. Smith explains that the optimum time for implanting the stem cells is after the initial inflammatory phase has subsided - otherwise the cells may receive the wrong cues and develop into scar tissue. The latest time for successful implantation is 3 months after the injury. After that there will have been significant fibrous tissue formation, which causes difficulty injecting, and reduces the benefit of the treatment He points out that, as yet, there is no conclusive evidence that the technique produces better results than conventional methods, but the initial results are encouraging. Independent experts have said that the appearance on the ultrasound scan after treatment was at least as good as could be expected using conservative treatment (ie rest alone). Research into the technique is continuing. In particular they are checking that the stem cells are able to survive and grow in the tendon and whether the cells do develop into tendon cells or whether they induce bone, cartilage, fat or fibrous tissue. More details are available at the VetCell website. See: Oxidative stress in chronic laminitis. Ponies suffering from chronic laminitis would benefit from dietary supplementation with anti-oxidants. New research indicates that high levels of free radicals are found in ponies with chronic laminitis. Free radicals are highly reactive particles produced as a result of normal metabolism. In addition they can result from reduced perfusion or be released as part of the inflammatory process. They can react with tissues locally (at the site of release) or throughout the body, resulting in tissue damage. They are thought to play a significant role in the ageing process. Although free radicals form a normal part of the inflammatory process, if present in excess they can cause damage to the tissue itself, resulting in a state known as oxidative stress. Oxidative stress can be quantified by measuring the products that result from free-radical activity. One way of doing this is to measure TBARS (thiobarbituric acid reactive substances) in the urine. A research team, headed by Dr Rachel Neville at the University of Lincoln, compared the concentration of TBARS in the urine of normal ponies and those with chronic laminitis. They reported their findings in a recent edition of the Equine Veterinary Journal. Two groups of native ponies from an equine sanctuary were used for the study. The first group comprised twenty ponies with physical signs of chronic laminitis (alteration in foot shape with dropped soles). The ponies in the second group were apparently normal and had no history of previous laminitis. The average age of ponies in the two groups was similar. Urine samples were collected from each pony and frozen until analysed. The concentration of substances in the urine is influenced by the amount of water present. The results were expressed as a ratio of the concentration of creatinine in the urine. The body produces creatinine at a relatively constant rate. So this provides a way of taking into account how dilute the urine is and standardising the results. ponies with chronic laminitis was three times higher than that of normal ponies. It was particularly interesting that the laminitic ponies had high levels of free radicals despite not being lame at the time. They suggest a number of possible explanations for the high levels of free radicals in the laminitic ponies. Reduced blood supply and inflammation in the laminae could be responsible, but as the ponies were not lame it seemed less likely. Inflammation due to degenerative changes in the foot could be responsible. Peripheral Cushing's disease (metabolic syndrome), in which the glucose and insulin metabolism is disturbed, can lead to laminitis and is associated with free radical production. It is likely that several factors are involved and further investigation is warranted to clarify the situation. The findings have implications for the nutrition of ponies with chronic laminitis. Teresa Hollands of Dodson and Horrell, one of the researchers, advises that all horses and ponies suffering from laminitis should be fed appropriate levels of antioxidants, such as the plant based antioxidants vitamins A, C and E. Many anti-oxidants are found in grass (one of the best sources of vitamin E and -carotene). If the grass intake is restricted because of the laminitis it is important to provide other dietary sources of antioxidants. Dr Neville adds that antioxidants will not cure laminitis, nor will they prevent it. But as free radicals are highly damaging to cells and tissues, antioxidants could perhaps ameliorate the additional stress of free radical damage. Evaluation of urinary TBARS in normal and chronic laminitic ponies. RF Neville, T Hollands, SN Collins, FV Keyte. EVJ (2004) 36, The researchers found that the concentration of TBARS in the urine of

3 Equine Science Update 3 Value of hoof disinfectant. Recent research confirms the value of the regular use of a hoof disinfectant as part of a hoof care programme. Many factors influence the quality of hoof horn. High grain diets cause problems unless the diet is properly balanced for mineral levels. Calcium deficiencies are seen especially in horses fed excessive amounts of grain, because of the phytates that make the calcium unavailable. In addition, some horses have difficulty absorbing calcium from inorganic sources such as limestone flour. Calcium deficiency leads to weakening of the bonds between the horn tubule cells. This results in unzipping of the horn cells, allowing bacteria to spread between the cells and eventually enter the cells themselves. High carbohydrate diets can also have deleterious effects on horn quality. In particular diets high in barley, maize and molasses tend to produce abnormal ( trans ) fatty acids when they are digested in the large intestine. The cell membrane requires normal ( cis ) fatty acids to function properly. The cell membranes break down and allow bacteria in. Laminitis affects the horn quality by disrupting keratinisation because of the breakdown in the basement membrane. Fluid leaks into the intercellular space leading to disruption of the intercellular lipids and their attachment, allowing bacteria to invade the horn. Over zealous supplementation with selenium and with vitamins A and D can be detrimental to hoof quality. Trauma also plays a part. Nails damage the horn and allow bacteria to penetrate beneath the surface layers. As well as causing physical damage to the horn, they have a chemical effect on the horn. Metal ions leach out of the nails and react with cell membranes. Once the hoof horn has been weakened, secondary bacterial and fungal infections can cause further damage. This prompts the question of whether disinfecting the hoof can prevent its degradation. Farrier Ruaraidh Robb worked with Dr Sue Kempson of the Edinburgh Vet School to investigate whether using a hoof disinfectant did indeed have an effect on hoof quality. Over a two-year period they studied twenty -three horses, each of which had problems with poor quality hoof horn. Typically the affected horses had crumbling horn, often around the nail holes, with or without cracks in the hoof. The more severely affected animals had white line disease. In some horses the changes followed a previous bout of laminitis. The horses included in the study ranged from 3 to 19 years old. They examined horn samples from each horse at the start of the study. All of the damaged horn samples contained bacteria, which were clearly visible on the electron microscope. About a third also had fungi present. Owners were given advice about appropriate diet and management to promote horn health. In particular the diet should be balanced for calcium and phosphorus - for example by feeding alfalfa. Bran and selenium-containing supplements should not be fed In the first year, they studied twelve horses, in second year eleven. All had long-term hoof horn problems The owners were instructed to wash and dry the hoof before applying the disinfectant. At first this was done daily. Then, when the hoof problem had resolved, the disinfectant was used only when the feet were being trimmed, rasped or shod. The active ingredients of the hoof disinfectant were an iodinecontaining compound (a poloxameriodine complex) ethylenediamine dihydriodide, isopropyl alcohol and propylene glycol. Initially it was mixed in emu oil but this was later changed to tea tree oil when the emu oil was not available. Tea tree oil itself has antiseptic properties and penetrates the hoof horn well. As well as being able to see an improvement in the feet Kempson and Robb assessed the progress by collecting hoof wall clippings at each shoeing and examining them under the electron microscope. Only one horse failed to improve dramatically, and that had metabolic problems associated with long-term selenium toxicity. Although some horses could have improved because of the correction in their diet, even those horses that were being fed correctly at the start of the study improved Kempson and Robb conclude that using the hoof disinfectant as part of the hoof care programme appeared to be successful in treating a wide range of hoof horn disorders. Reference Use of a topical disinfectant as part of a hoof care programme for horses with diseases of the hoof capsule. SA Kempson, R Robb Vet Rec. (2004) 154,

4 Equine Science Update 4 Homocysteine and laminitis. Corticosteroids and laminitis. Do food supplements containing methionine increase the risk of laminitis in susceptible animals? Research reported recently in the Equine Veterinary Journal aimed to find out. Homocysteine is a sulphur-containing amino acid formed by the breakdown of methionine. When methionine is plentiful the homocysteine is used to produce cysteine, a process that relies on a vitamin B6 dependent enzyme. If there is a deficiency of methionine, it is possible for the process to be reversed and the homocysteine turned back into methionine. The amount of homocysteine in the blood is a balance between the rate at which it is produced and the rate it is used up. Homocysteine has been associated with diseases of the blood vessels in humans. Concentrations in the blood that are just above the normal range have been found in patients with atherosclerosis, high blood pressure and coronary heart disease. Interest has turned recently to the effect of homocysteine in horses and ponies, in particular whether it could influence the blood flow in the foot and increase the risk of laminitis. Methionine is widely used as a food supplement to promote keratin production and aid hoof growth in ponies that have had laminitis. So it is important to known if the practice could actually increase the risk of laminitis by boosting the amount of homocysteine in the blood. A research team based at the Royal Veterinary College under the direction of Dr Jonathon Elliott has been conducting an investigation into the role of homocysteine in laminitis. The first part of the study involved laboratory tests to see if homocysteine has similar effects on the blood vessels of horses as it does in humans. The researchers looked at blood flow in the foot and found that homocysteine inhibited the function of the cells lining the blood vessels (endothelial cells), which control blood flow. Homocysteine sensitised the blood vessels to the effect of serotonin (5HT) which causes constriction of the digital blood vessels. Homocysteine also significantly reduced the response to carbachol and bradykinin, which normally act on the endothelial cells to inhibit the vasoconstriction caused by 5HT. In tissue culture experiments, the research team found that homocysteine inhibited the production of nitric oxide (NO), a chemical mediator, which is produced by the endothelial cells. NO plays an important role in the local control of blood flow. These results showed that homocysteine has effects on blood vessels in the foot which might potentially aggravate the changes in blood flow seen in laminitis. In a third part of the investigation they collected blood samples from twenty normal ponies and twenty-four with a history of recurrent laminitis. There was no difference in plasma homocysteine concentration between the two groups. The range of values found was similar to the normal range in man. In humans, subtle changes in the metabolism of homocysteine are measured using a methionine challenge test. A loading dose of methionine is given and homocysteine levels in the blood are measured four and eighth hours later. The researchers suggest that a similar test could be developed for use in ponies and might throw more light on the role of homocysteine in laminitis. The laboratory research demonstrated that homocysteine interferes with function of the endothelial cells that influence blood flow in the horses foot. The researchers point out that more work needs to be done to see if feeding supplements containing methionine causes prolonged elevation of the blood levels of homocysteine in ponies that are susceptible to laminitis. Reference Y Berhane, SR Bailey, PA Harris, MJ Griffiths, J Elliot. In vitro and in vivo studies of homocysteine in equine tissues: implications for the pathophysiology of laminitis. Equine Veterinary Journal (2004) 36, A recent report questions the widely held view that glucocorticoid treatment carries the risk of inducing laminitis. There are many causes of laminitis, including over-ingestion of grain or grass, toxic metritis, gastro-intestinal disease and trauma. Laminitis is also common in horses with Cushing's disease. This is thought to be due, at least in part, to long term elevation of the cortisol concentration in the blood. Cortisol (also known as hydrocortisone), a hormone produced by the adrenal gland, is the body's natural glucocorticoid. It has widespread effects throughout the body, increasing glucose production in the liver and promoting the breakdown of fat and protein. It causes sodium and fluid retention and potassium excretion, and inhibits the absorption of calcium from the intestines. Many veterinarians are reluctant to treat horses with glucocorticoids ( cortisone ) because of the perceived risk of precipitating laminitis. A study carried out in Australia suggests that the risk may not be as great as previously thought. Veterinary surgeons Michael J McCluskey and PB Kavenagh writing in a recent issue of Equine Veterinary Education reported on their experience of using the synthetic glucocorticoid triamcinolone acetonide in their equine practice at Geelong, in Victoria, Australia. Over a four-year period they treated 132 adult horses. About one-third of the horses were treated more than once, giving a total of 205 cases. Most were performance horses, comprising Thoroughbred or part-thoroughbred (nearly 90%) or Standardbreds. Other breeds of horse accounted for less than 3%. There were no ponies in the series. They used either 40mg or 80 mg of triamcinolone. This is higher than the dosage commonly used, which ranges from 5-20 mg. Even so McCluskey and Kavenagh point out that the dose they use is still relatively low. In comparison, that used in humans is often up to 60 times higher on a weight for weight basis. The main use for the triamcinolone was in treating joint inflammation. In almost all cases the treatment was given locally, either into a joint or tendon sheath, or into the lesion itself for inflammatory conditions. In only

5 Equine Science Update 5 1.5% of the cases was it given into the muscle to treat a generalised condition, such as pruritus (itching) or inflammatory airway disease. Predicting endometritis. Laminitis was reported in only four of the treated horses. In two of those, the laminitis occurred before the horses were treated with triamcinolone and did not recur afterwards. One horse developed laminitis 18 months after treatment. However, as it had toxic metritis at the same time, that was the more likely cause. Of the 202 cases that had not previously had laminitis, none developed the condition after treatment with triamcinolone. In only one horse did laminitis occur shortly after treatment (in this case after a week). This particular horse was one of three in the study that had previously been affected with laminitis. The authors point out that this is the only case of laminitis they have ever seen after using triamcinolone, despite the fact that they have been using high doses of the drug for a total of 19 years between them. The authors conclude that, at the moment, there is no conclusive evidence that glucocorticoids cause laminitis in horses. They suggest that more research is needed to ascertain the most appropriate dose of triamcinolone for local injection into joints and tendon sheaths and for intramuscular administration for treating more generalised conditions. Reference. Clinical use of triamcinolone acetonide in the horse (205 cases) and the incidence of glucocorticoid-induced laminitis associated with its use. MJ McCluskey PB Kavenagh. Equine Veterinary Education (2004) 16, A practical method for identifying mares susceptible to endometritis after mating has been described by veterinarians at Texas A&M University. It is normal for there to be some inflammation in the uterus following mating. This is called post-breeding endometritis. The uterus reacts to the sperm and the other components of the semen. When a stallion covers a mare, debris from the penis as well as the semen is deposited into the uterus. This material, which is recognised a foreign by the uterus, sets up an inflammatory response. The inflammatory reaction is less pronounced if artificial insemination is used as the debris is filtered out before use, and smaller numbers of sperm are used. However, a significant inflammatory reaction may still occur. Usually the inflammation is transient. In the healthy mare the inflammatory fluid is cleared away before the fertilised egg passes out of the oviduct into the uterus, at about five days after ovulation. However, if the endometritis is still present when the embryo reaches the uterus the embryo will be lost. The problems can be exacerbated if the mare is mated again while the inflammatory fluid is still present. This is likely only to make the inflammation worse. The inflammatory fluid may also inactivate the sperm. The earlier potential problems can be identified, the more effective treatment is likely to be. In a recent report Dr Steve Brinsko and his colleagues described how the measurement of free fluid in the uterus during oestrus could be used to predict whether the mare is at risk of developing post-breeding endometritis. Mares are susceptible to post-breeding endometritis if there is delayed clearance of fluid from the uterus. Delayed uterine clearance may be identified by free fluid within the uterus 3 days after insemination or at other times during the dioestrus phase. Tests can be performed to assess the ability of the uterus to clear fluid and bacteria. For example Streptococcus zooepidemicus, the bacteria responsible for many uterine infections, can be instilled into the uterus. If there is delayed uterine clearance, fluid and/or bacteria will still be present 4 days later. But if the uterine defences are working normally there should be no sign of fluid or bacteria. A more sophisticated test is to place a suspension of radioactive particles in the uterus and the record the clearance rate by measuring the decline in radiation from the uterus. Over a two-year period the researchers studied 27 mares. They classified the mares as susceptible or resistant to endometritis by examining biopsies of the uterus and assessing uterine function at different stages of the oestrus cycle. Thirteen of the mares were susceptible to post-breeding endometritis, and fourteen were normal. They found that if the fluid in the uterus was more than 2cm deep during oestrus the mare was very likely to be susceptible to post-breeding endometritis. Small amounts of fluid in the uterus when the mare was in season did not appear to be correlated with susceptibility to matinginduced endometritis. The researchers point out that this information will allow the early identification of mares prone to postbreeding endometritis. It will allow early treatment and improve fertility by helping to prevent persistent matinginduced endometritis. An ultrasound scan of mares before mating can identify those likely to be susceptible to mating-induced endometritis and allow early treatment. Reference. A practical method for recognizing mares susceptible to post-breeding endometritis. Steven P Brinsko, Sherry L Rigby, Dickson D Varner, Terry L Blanchard- Proc AAEP (2004) 49,

6 Equine Science Update 6 Cushings disease: a common cause of laminitis. Nerve degeneration in Cushing`s disease. Cushing`s disease was identified as the most common cause of laminitis in a study conducted at the University of Pennsylvania. Laminitis is a well known result of the excessive intake of carbohydrate - such as grain or fructans. It may also occur after severe gastro-intestinal damage, for example after colic surgery, or in septic metritis following retention of the afterbirth. Horses with Cushing`s disease (CD, also known as pituitary pars intermedia dysfunction, PPID) are also known to be at increased risk of developing laminitis. However, until recently it had not been clear just how many cases of laminitis were due to Cushing`s disease. A report in the Journal of the American Veterinary Medical Association, shows that over two-thirds of the cases of laminitis seen in a first opinion practice were associated with Cushings disease. Dr Mark Donaldson, of the University of Pennsylvania`s Veterinary School, studied horses with laminitis that were seen by the University`s ambulatory practice between 1996 and During that time he examined forty horses with laminitis. To investigate whether CD was involved, he measured plasma ACTH levels. Plasma ACTH concentrations greater than 7.7pmol/l were taken to indicate Cushing`s disease. ACTH is a hormone which is normally produced by the pituitary and acts on the adrenal glands to release cortisol. It plays a vital role in the regulation of metabolism. In horses with CD, high levels of ACTH and other related hormones are released from the intermediate lobe of the pituitary. Donaldson explained that, theoretically, a single blood sample with an elevated concentration of ACTH was not conclusive evidence of Cushing`s disease. For example, severe pain could produce an increase in ACTH level. However, he collected the samples for ACTH evaluation after the pain had been controlled. Post mortem examinations were carried out on six horses with high ACTH levels and all had enlarged pituitary glands typical of Cushing`s disease. Donaldson found that over two thirds of the horses with laminitis had CD as defined by elevated ACTH levels. Many of the horses with normal plasma ACTH levels also had signs that suggested Cushing`s disease (such as bulging supraorbital fossa, abnormal fat distribution, hirsutism) so the actual number of laminitic horses with Cushing`s disease may have been even higher. Although Cushing`s disease is usually considered to affect older horses, Donaldson found horses as young as 3 years of age with the condition. The median age of affected animals was 15.5 years. September was the most common month for horses with CD to develop laminitis. On the other hand horses that did not have CD most commonly developed laminitis in May. (However, these differences were not statistically significant.) Not all horses that were identified as having CD showed typical clinical signs of the condition. Only a third of the horses with CD had a long hair coat. Five horses showed no sign of CD apart from the laminitis. The most common clinical sign of CD was the abnormal distribution of body fat (along the crest, and over the back and rump.) Unlike the situation in those animals that are simply obese, the outlines of the ribs could be seen. Excessive consumption of lush pasture was considered to be a factor in the onset of laminitis in 7/28 horses with CD. This demonstrates that CD should not be discounted just because the horse is known to have eaten too much. Pergolide appeared to be useful in treating affected animals. Seventeen horses with CD were treated with pergolide and all survived. In contrast, only three of five animals that did not receive pergolide survived. Evaluation of suspected pituitary pars intermedia dysfunction in horses with laminitis. MT Donaldson, AJR Jorgensen, J Beech. JAMA (2004) 224, Research from North America sheds light on the cause of Cushing's Disease in horses. Equine Cushing's disease, (ECD) is a complex condition, in which dysfunction of the intermediate lobe (pars intermedia) of the pituitary gland leads to excessive secretion of cortisol from the adrenals. It is common in older horses. Often the first sign that a horse has Cushing's disease is hirsutism, the development of an excessively long and curly coat. Other signs include weight loss, loss of performance, excessive sweating ( hyperhidrosis ) and increased drinking and urinating. Many affected animals eventually develop laminitis. The intermediate lobe of the pituitary gland contains cells of a single type, known as melanotropes. They produce a pre-cursor molecule called POMC (Proopiomelanocortin), which they process to make a number of active substances including MSH (melanocyte stimulating hormone), b-endorphin (B-END) and ACTH. In Equine Cushing's disease it is these melanotrope cells that are affected. They increase in number, and release increased amounts of their active products. The ACTH stimulates the release of cortisol from the adrenal gland, contributing to the signs of Cushing's disease. In the normal horse, adrenal gland secretion is under the control of ACTH released from another part of the pituitary gland, the pars distalis. A feedback control mechanism operates, so that when cortisol levels are adequate, further release of ACTH is prevented. Unfortunately this feedback mechanism does not include the pars intermedia. Experimental work has shown (in cats and rats) that the secretion of the pars inter-media is controlled by nerves originating in a small part of the brain known as the peri-ventricular nucleus of the hypothalamus. These nerves release dopamine, a neurotransmitter that acts on specific receptors (called D2 receptors) on the melanotropes to inhibit their secretion. Cutting these nerves, or blocking their effect on the D2 receptors, leads to changes in the pars intermedia typical of Cushing's disease.

7 Equine Science Update 7 Dr Dianne McFarlane and colleagues at Canada's Atlantic Veterinary College have confirmed the role of degeneration of the dopamine secreting ( dopaminergic ) nerves in the development of ECD. They have also found evidence of oxidative stress in the pars inter-media of affected animals. Oxidative stress may be caused by factors such as disease, (allergy, infection); environmental factors, (pollution, radiation, physical injury) or diet (high concentrations of polyunsaturated oils). Reactive oxygen species (ROS, otherwise known as free radicals or oxidants) play an important role in the development of inflammation and cell damage. In excess they can cause cell damage, including damage to the DNA, and cell death. Normally they are kept in check by anti-oxidants, often described as free radical scavengers, which act to limit tissue damage by preventing an accumulation of oxidants. The horse has various antioxidative mechanisms, including enzymatic systems such as superoxide dismutase and catalase, and non-enzymic mechanisms such as ascorbic acid and glutathione. Oxidative stress occurs when the balance between oxidants and antioxidants changes and oxidants predominate. Using specialised staining techniques ( immuno-histochemistry ), the researchers examined microscopic sections of pituitary glands removed from horses at post mortem examinations. They compared samples from three groups of horses: those known to have CD; a group of older horses (at least fifteen years old) with no sign of CD; and young horses (less than 15 years old). The average age of the horses with Cushing's disease was similar to that of the old horse group. Firstly they used a stain to highlight tyrosine hydroxylase, an enzyme present in dopamin-ergic nerves. They found a marked difference between the horses with CD and the other two groups. Horses with CD had five times fewer dopaminergic nerves in the pars intermedia than did unaffected horses. They found no difference in the number of dopaminergic nerves present in the young and old groups of horses. This was, perhaps, surprising, as degeneration of the dopaminergic nerves increases with age in man and other species. Secondly they stained the sections for 3-nitrotyrosine. The presence of this substance in the cells is an indication of oxidative stress. They found that in horses with ECD a much higher proportion of the nerves cells in the pars inter-media showed evidence of oxidative stress. There was a slight but significant increase in cells showing oxidative stress in the older age group compared with the young horses. This was not unexpected, as one of the consequences of aging is the accumulation of damage due to oxidative stress. This work confirmed that ECD is associated with degeneration of dopamine-secreting nerves in the pars intermedia of the pituitary. The role of oxidative stress in the development of the disease had not been identified previously. Dr McFarlane suggested that further research should be carried out to identify the process that leads to the development of oxidative stress. Reference. The role of dopaminergic neurodegeneration in equine pituitary pars intermedia dysfunction (equine Cushing's disease) Dianne McFarlane, Mark T Donaldson, Tarek M Saleh, Alastair E Cribb. Proc AAEP (2003) 49, Treating respiratory infections. A new approach to treating respiratory tract infection has been supported by recent research In treating any infection it is important to expose the bacteria to an adequate concentration of an effective antibacterial agent. One antibiotic that may be effective against the bacteria involved in the more serious respiratory tract infections is gentamicin. However, it can cause kidney damage when high concentrations are present in the blood. One way of reducing the risk of such side effects is to deliver the drug directly into the airways. Researchers at the Marion dupont Scott Equine Medical Center, part of the Virginia-Maryland Regional College of Veterinary Medicine, reported recently on an investigation into the use of gentamicin by once daily aerosol administration directly into the respiratory tract. Harold C McKenzie III and Michael J Murray described their findings in a report in the American Journal of Veterinary Research. Their work followed on from research they had done previously, which showed that a single dose of gentamicin, administered by aerosol gave a concentration twelve times greater in the airways than that produced by intravenous injection. They wanted to check that repeated doses did not lead to accumulation of the drug in the respiratory tract or in the blood. If that were the case it might be necessary to reduce the dosage for repeated administration. They also wanted to check that repeated doses did not cause respiratory tract inflammation. Thirteen healthy horses, with no apparent respiratory disease, were used in the study. Eight were treated daily; five were left untreated for comparison. Each horse in the treated group breathed an aerosol spray containing gentamicin (50mg/ml in 0.45% saline solution) for ten minutes every day for a week. The aerosol spray was produced by an ultrasonic nebulizer and was administered to the horses using a tight-fitting facemask. To assess the effect on the airway of the inhaled gentamicin, McKenzie and Murray collected samples of fluid from the smaller airways ( bronchial lavage ). Thirty milliliters of sterile saline were introduced into the airway through an endoscope. The fluid was then withdrawn and examined for cell content. The same parts of the lung were sampled each time. Samples were collected at 0.5, 4, 8, and 24 hours after final treatment They found no sign of respiratory inflammation as a result of the treatment. Although the injectable solution used to make up the aerosol contained preservatives that have been associated with bronchoconstriction and coughing the researchers noticed no coughing in the treated horses. Blood samples taken immediately before each bronchial lavage gave no evidence of gentamicin accumulating in the blood. Administering antimicrobials by aerosol can produce high concentrations of the medications in the airways whilst minimising the systemic effects. A limitation of the technique is that aerosol administration is not effective if the lungs are collapsed or poorly ventilated, in which case the antibacterial aerosol may be unable to spread throughout the lungs. The researchers concluded that repeated daily aerosol treatment with gentamicin appeared to be safe and might be a useful procedure for treating bacterial infections of the airways.

8 Equine Science Update 8 Removing cheek teeth. Thyroid function and fertility. Cheek teeth may be successfully removed through the mouth in the sedated horse according to a recent report. Equine dentistry has evolved over the years. For a long time the only way to remove cheek teeth was through the mouth. Then, as general anaesthetics became more reliable it became popular to remove the teeth by repulsion, - that is by making a window in the bone overlying the tooth, and using a dental punch and mallet used to drive the tooth from the socket. Now that effective drugs are available to keep a horse sedated for long periods, removal of cheek teeth through the mouth has once again become popular. Oral extraction in the sedated horse has advantages over repulsion under general anaesthetic. For a start it is much less traumatic. Secondly, general anaesthesia is still a relatively high-risk procedure in horses despite improvements in recent years. Indications for cheek tooth removal in the horse include tooth root infections, severe periodontal disease with the accumulation of pockets of food material between or around the teeth, and displaced teeth. Teeth are firmly anchored to the alveolar bone of the tooth socket by Sharpey`s fibres, strong bundles of collagen fibres that cross the periodontal space. It is necessary to break down these attachments before the tooth can be removed. Disrupting these fibres occupies the major part of the tooth extraction procedure. Veterinary surgeon Graham Duncanson, based in the UK in Norfolk, has a special interest in equine dentistry. He reports a series of 125 cases that required removal of cheek teeth in the June issue of Equine Veterinary Education. Some were cases that he encountered in his own practice, and others were referred for treatment from other practitioners or dental technicians. The most common reason for removing the teeth was the tooth being loose. This applied in ninety-one of the 125 horses. Other reasons included apical (tooth root) infections and teeth that had been fractured during previous dental work. In most cases, inspection of the teeth was all that was needed to make a diagnosis. In a few cases radiography was used. Generally, the horses were sedated, and a Haussman`s gag used to provide access to the mouth. The first step after identifying the affected tooth was to loosen its attachments to the alveolar bone. A dental pick was used to separate the gum from around the base of the tooth. Then an instrument called a molar spreader was used to separate the tooth from its neighbours. Once this preliminary loosening has been done, molar extractors were fixed tightly to the tooth and the handle rocked to and fro in a horizontal plane to loosen the tooth. This was the time-consuming part of the procedure. Duncanson worked in 20-minute sessions. After each session he released the gag and allowed the horses a couple of minutes rest. Only when the tooth was loose in its socket did he try to extract it. In the ninety-one horses with loose teeth to start with the procedure was relatively quick, taking less than 20 minutes. In eight horses the teeth were so loose that they could be removed without using instruments and even without sedating the horse. Teeth with apical infections took longer to remove, as the tooth was still firmly anchored in its socket. Fourteen horses (11%) fell into this category. In all of these cases it took over an hour to remove the teeth, sometimes as long as two hours. In three horses the tooth root fractured and the fragments had to be removed separately. Typically apical infections occurred in younger horses. Most other reports stress the need to pack the empty tooth socket, especially after removing the teeth by repulsion, to prevent it filling with food material, or allowing food into the paranasal sinuses. Duncanson explains that he does not feel it is necessary when removing the teeth through the mouth. Certainly, in this series there were no adverse effects as a result. Refernece: A case study of 125 horses presented to a general practitioner in the UK for cheek teeth removal. GR Duncanson Equine Veterinary Education (2004) Thyroid dysfunction is uncommon in broodmares and thyroid supplements should not be given according to recent research from the USA. The thyroid hormones have widespread functions throughout the body. There is hardly any metabolic process that they do not affect. A deficiency of biologically active thyroid hormone ( hypothyroidism ) has been suggested as a cause of poor growth, failure of milk production, muscle problems, prolonged gestation, poor performance and laminitis. The thyroid gland secretes two main hormones, thyroxine (T4) and triiodothyronine (T3). T4 is produced in greater quantities; but T3 is the more biologically active hormone. T3 is also produced by metabolism from T4 in tissues such as the liver and kidneys. Much of the circulating thyroid hormone is bound to proteins in the blood. Only the free, un-bound, hormone is biologically active. Young horses in which thyroid deficiency is induced experimentally stop growing and their body temperature falls. They become lethargic and anaemic. They develop swelling of the hind legs, and a dull hair coat. However, mares may still become pregnant and carry a foal to term after their thyroid gland has been removed. Hypothyroidism is associated with infertility in women. But it is unclear whether it plays an important role in reproductive function in mares. Nevertheless, thyroid supplements are frequently given to brood mares in the belief that the practice will improve fertility. However, giving thyroid hormones to horses that do not need them can cause disuse atrophy of the thyroid gland. So researchers at the University of Pennsylvania's New Bolton Center have been investigating the role of thyroid function in fertility in mares. A single measurement of thyroid hormone levels in the blood may be misleading. The levels may appear to be low when in fact the thyroid gland is functioning normally. Dr Travis Meredith and Dr Ina Dobrinski used a thyroid stimulation test, which they claim gives a more reliable assessment of

9 Equine Science Update 9 continued thyroid function than a single measurement. Sixty-eight standardbred and eleven Thoroughbred mares were included in the study. All were apparently healthy. The researchers assessed the thyroid function during the diestrus phase of the oestrus cycle a few days before each mare was bred. They collected a blood sample immediately before injecting 1mg of thyrotropin releasing hormone (TRH), which stimulates thyroid secretion, and then collected further blood samples two and four hours later. They measured T3 in the 0 and 2 hour samples and T4 in the 0 and 4 hour samples. They then determined whether the mares were pregnant by ultrasound examination starting 16 days after breeding. On average, T3 measured in 0 and 2-hour sample showed an increase of about 4.5, and T4 measured in 0 and 4-hour sample showed an increase of 2. Two mares failed to show an increase in T4 in response to TRH but their baseline levels were normal. The researchers found that the baseline levels of T3 and T4, and the stimulated values for T3 and T4, did not differ significantly between mares that became pregnant and those that did not. They also noticed that 6 mares had baseline levels of T3 below normal and 44 had T4 below normal. However, the stimulation test confirmed that most of these mares had normal thyroid function. In all mares the T3 increased and all but two mares showed an increase in T4 after stimulation. Only 2 of the 79 mares had reduced thyroid function in that they failed to show an increase in T4 after stimulation. One of those became pregnant and the other did not. Too few mares showed reduced thyroid function to allow meaningful statistical evaluation. However, overall there was no association between baseline and stimulated T3 and T4 levels and future pregnancy status. Meredith and Dobrinski concluded that their study gave no support to the view that thyroid dysfunction is common in broodmares. They advise that until there is good evidence of a link between hypothyroidism and subfertility brood mares should not be given thyroid hormone supplements. Racecourse fractures. Analysis of the type and number of fatal fractures that horses suffer whilst racing helps to identify risk factors, and may prompt changes to make the sport safer. Scientists based at the Liverpool Vet School recently published the results of a two-year study into fatal limb fractures in racehorses. The study only considered fractures affecting the fore or hind legs below the knee or hock. During a two-year period they examined a total of 109 distal (lower) limb fractures. A detailed post mortem examination, including radiography, was carried out on each fracture. Overall, fatal fractures occurred in less than one in a thousand starts. The research team looked at the relative risk of fractures occurring in the five types of races that are held in the UK. These are: flat racing on turf, or all weather surfaces; National Hunt (jumping) races over hurdles or steeplechase fences, and National Hunt flat (NHF) races. They found considerable differences in the risk of fracture between the different types of race. Flat racing presented the least risk of fatal lower limb fractures and National Hunt flat racing the greatest risk. Of every 1000 horses starting a flat race on turf 0.38 suffered a fracture. In contrast, 2.17 of every 1000 horses starting National Hunt flat races had a fracture. Putting it another way, a horse running in a NHF race was nearly six times more likely to have a fatal lower limb fracture than a horse racing on the turf. Thyroid function and pregnancy status in broodmares Travis B Meredith, Ina Dobrinski JAVMA (2004) 224, All weather surfaces were associated with a greater risk of fracture. Horses were about twice as likely to be injured when racing on all weather surfaces compared with turf. Factors other than the nature of the surface may be involved. Poorer performing horses tend to be selected for all weather flat races in the UK because the prize money is generally lower than for races on turf. These horses may have previous injuries that make them more likely to suffer further damage. There were marked differences in the most common fatal lower limb fractures occurring in the five different types of racing. For example, fatal fractures of the third metacarpal (cannon) bone were most likely to occur in NHF racing. Fractures of the knee were over four times more likely in hurdle racing than in any other type of race. Racing on all weather tracks carried a particular risk of a fracturing both sesamoid bones of one leg (described as a biaxial sesamoid fracture) This type of fracture was ten times more likely to occur in all weather flat races than in any other race. In flat horses racing on the turf, the bone most commonly fractured was the first phalanx (the long pastern bone). The study is continuing, and the researchers hope to be able to identify risk factors for each type of fracture. They will then be able to recommend changes to minimise the risks to racing horses. Risk of fatal distal limb fractures among thoroughbreds involved in the five types of racing in the United Kingdom. TDH Parkin, PD Clegg, NP French, CJ Proudman, CM Riggs, ER Singer, PM Webbon, KL Morgan. Veterinary Record (2004) 154,

10 Equine Science Update 10 Heat from powered dental tools damages teeth. The cheek teeth of the horse have a complex structure composed of folds of tough enamel, surrounded by softer dentine and cement. As the teeth wear, the softer dentine and cement are ground down, exposing ridges of enamel, which are used for grinding the food. The upper jaws are set further apart than the lower jaws. As a result, there tends to be less wear on the inner edge of the lower cheek teeth and outer edge of the upper (maxillary) cheek teeth. This leads to sharp enamel points forming on the outer (labial) edge of the upper cheek teeth and the inner (lingual) edge of the lower teeth. Rasping (or floating) the cheek teeth to remove the sharp points that arise due to normal wear is an important part of routine equine dental care. Significant abnormalities of tooth wear may occur in older horses. For example, a missing tooth on one jaw may lead to a marked overgrowth of the tooth on the opposite jaw, a condition known as step-mouth. Such abnormalities often require prolonged grinding with power tools to correct them. Powered grinders generate a considerable amount of heat during their use. Concern has been expressed that the heat may damage the odontoblasts that produce the dentine. Eventually, this would lead to exposure of the sensitive pulp cavity. Human dental research has found that an increase in temperature of only 5.5 o C caused irreversible damage to 15% of teeth. An increase of 16.7 o C caused necrosis of the sensitive pulp tissue in 100% of teeth. Dr Martin Allen, of Firethorn Veterinary Services, Lancaster, Ohio, has been investigating the effect of power grinding on the temperature within horses` teeth. He carried out the research in conjunction with colleagues at the University of Illinois College of Veterinary Medicine. For the study they used teeth from the lower jaws of horses that had been recently killed at an abattoir. They drilled two small holes on the lingual (inner) surface of the teeth, 15mm and 25 mm below the grinding surface. Into these holes they placed thermocouples to measure the temperature. The tips of the thermocouples lay close to the pulp cavity. In most cases the 15mm hole was at about gum level. The researchers assessed the effect of three different procedures on the temperature within the teeth. A circular carbide powered blade was used at a speed of 4000 revolutions per minute. The first group of teeth were subjected to grinding for one minute. This was to simulate the amount of work that would be done to reduce the sharps points of normal tooth wear. The second group of teeth were ground for two minutes to simulate the treatment that might be given to reduce large overgrowths. In reality, the time needed might well be longer. The third group of teeth were also ground for two minutes but were cooled with water at room temperature (a constant 20.3 o C). The researchers measured the temperatures within the teeth before. and immediately after, grinding. They had planned to record temperature changes during grinding, but found that the vibration prevented meaningful readings. They found that even the teeth ground for one minute reached temperatures that could damage the pulp cavity. The average temperature increase was 6.6 o C at the upper thermocouple. Only one tooth failed to show a temperature rise above 5 o C. The temperature at the lower thermocouple remained below the 5 o C limit throughout the procedure. There was a marked increase in temperature in teeth ground for two minutes without cooling. The average temperature increase at the upper thermocouple was 24.3 o C. The temperature recorded at the lower thermocouple rose by an average of 4.1 o C. In contrast, most of the teeth that were cooled while being ground showed a decrease in temperature. The researchers conclude that long dental procedures using power grinders are likely to cause damage to the pulp cavity through thermal injury. They point out that, as far as they know, there are no powered equine dental tools that are approved for use with water. So they warn that a circuit breaker should always be used to reduce the risk to horse and dental technician. Type of bedding affects time spent lying. Horses spend more time lying on their side when bedded on straw than when bedded on shavings according to a recent report. Sleep can be divided into different states according to the electrical activity in the brain, which can be recorded using an electroencephalogram (EEG). Generally the frequency of the brain waves decreases and the amplitude increases with lowering alertness. There are two main patterns of electrical activity in the sleeping horse- slow wave sleep (SWS) and paradoxical sleep (PS). PS is so called because although the animal is deeply asleep the brain wave pattern resembles that of wakefulness. It is also known as rapid eye movement (REM) sleep or deep sleep. Although horses can rest while standing in a state of drowsiness, they need to lie down to experience deep sleep. Paradoxical sleep occurs with the horse lying flat on its side with its head on the floor (lateral recumbency). The muscles are completely relaxed. Short bursts of REM may occur, often associated with twitching of the ears, face and lips. The horse may even whinny. PS appears to be important. Experiments have shown that people deprived of PS become irritable, anxious and have difficulty concentrating. SWS usually occurs with the horse in sternal recumbency.(ie lying more or less upright with the legs flexed). SWS is possible in the standing horse. If a horse is unable to lie down it may increase the amount of SWS to compensate for the lack of PS. Researchers at Denmark's Royal Veterinary and Agricultural University, Reference. In vitro study of heat production during power reduction of equine mandibular teeth. Martin L Allen, Gordon J Baker, David E Freeman, Kenneth R Holmes, Sandra Manfra Marretta, R Dean Scoggins, Peter Constable. JAVMA (2004) 224,

11 Equine Science Update 11 Continued Electrochemotherapy for sarcoids. and the Danish Institute of Agricultural Sciences have been studying the influence that bedding has on the time horses spend lying down. Gunhild Riemann Pedersen, Eva Sondergaard, and Jan Ladewig reported their findings in the April edition of Journal of Equine Veterinary Science. The study was performed at the Holte Rideklub, a large riding club stables, housing about 150 horses. The horses, which were mostly Danish Warmblood geldings used for dressage, were kept in individual boxes 3metres square. All were familiar with the routine in the stables, where they had been living for at least three weeks before the start of the study. The researchers observed eight horses bedded on straw and eight bedded on shavings, each for two nights. They used time-lapse video recordings to log the time spent lying in sternal recumbency and lateral recumbency. They found that there was no difference in the total time spent lying down between the two groups. But horses housed on straw spent three times longer lying on their side than did horses bedded on shavings. This may indicate that horses bedded on shavings spend less time in paradoxical sleep than those kept on straw, as horses must lie on their side to experience paradoxical sleep. If that is so, the authors question whether the use of shavings as bedding adversely affects the horses` welfare. However, horses at pasture spend less time lying down than housed horses. This is probably an evolutionary adaptation to protect them from predators. The researchers also noticed that horses often tried to roll before standing up. They suggested that this might be due to the horse's movement being restricted by the size of the stable. Maybe the horses were trying to move away from the wall to make it easier to get up. They point out that the behaviour may explain why some horses become cast with their feet up the wall. The influence of bedding on the time horses spend recumbent. GR Pedersen, E Sondergaard, Jan Ladewig J Eq Vet Sci (2004) A new technique may provide a more effective treatment for sarcoids. Sarcoids are the most common type of tumour in the horse, and account for 90% of equine skin tumours. They can occur anywhere on the body, but are most often found on the head, around the eyes, lips and ears, on the legs and underneath the body. They are locally invasive swellings involving fibroblasts, the cells responsible for producing connective tissue. Sarcoids are highly likely to return if removed. They do not metastasise (spread) and rarely regress spontaneously. Sarcoids are notoriously difficult to treat. Numerous treatments have been described, but no single treatment is effective for all types of sarcoid. Ineffective treatment may be worse than doing nothing. It may make the sarcoid more aggressive. Cisplatin is a cytotoxic drug (ie it kills cells). It has been shown to be effective for treating nodular or small fibroblastic sarcoids. One factor that limits its usefulness is that it does not penetrate the tissues well and so is best suited for treating small sarcoids. Experimental studies have shown that passing electrical pulses through tissues makes the cells more permeable to cisplatin. This procedure, known as electropermeabilization, enhances the cytotoxic effect. In one study, it increased the efficacy of cisplatin by twenty times. The use of a cytotoxic drug like cisplatin in conjunction with electropermeabilization is known as electrochemotherapy, or ECT. It has been used to treat a variety of tumours in humans, and has been used successfully in laboratory animals. It is still only at the experimental stage in horses. Dr Y Tamzali, and colleagues, working in the Ecole Nationale Veterinaire, in Toulouse, have been studying the use of ECT with cisplatin for treating sarcoids. Over a three-year period they treated 46 sarcoids on 25 horses. Only verrucose (warty), fibroblastic or nodular forms of sarcoid that were less than 5cm diameter were included in study. They treated the horses under short duration general anaesthesia. This was to avoid problems with muscle twitching in response to the electrical impulses. It took about 15 minutes to treat a single sarcoid. Up to 40 minutes were required if several sarcoids were present. So far, the technique has been restricted to smaller sarcoids. Five minutes after injecting the cisplatin into the sarcoid and surrounding skin they applied the ECT, using specially designed paired parallel electrodes, each 0.9cm long and 0.9 cm apart. The electrical impulse generator provided pulses of 0.1ms duration at 1Hz and 1.3kV. On average, each sarcoid received 160 pulses over 15 minutes. They repeated the ECT at two-week intervals. Up to four treatments were required. They monitored the response to treatment and followed cases for at least two years to check for recurrence of the sarcoid. All cases responded successfully. The researchers found that lesions less than 1cm diameter responded quickest, only needing one treatment. They suggest this may be due to fact that the electrical field was able to penetrate the whole lesion. No adverse effects were noticed in any of the cases. Reporting the findings to the annual Convention of the American Association of Equine Practitioners, Dr Tamzali concluded that the combination of cisplatin and ECT seemed to be safe and effective, and gave encouraging results. First horse sarcoid treatment by electrochemotherapy: preliminary experimental results. Y Tamzali, J Teissie, MP Rols. Proc AAEP (2004)