The present state and future of laminitis research
Although understanding of the pathophysiology of equine laminitis is far from complete, it is generally agreed that an apparently diverse set of pathological processes (e.g. sepsis, endocrinopathies, excessive weightbearing) most likely converge at the steps that result in detachment of the lamellar basal epithelial cell (LBEC) from the basement membrane, thereby undermining the structural integrity of the digit as well as support of the entire musculoskeletal system. Importantly, the LBEC appears to remain viable even after lamellar failure, suggesting that dysregulation of adhesion is central to the development of laminitis. Studies in other species have indicated that adhesion events of the epithelial cell are dynamic processes that can be rapidly disrupted by local events affecting either cellular regulation of adhesion proteins and/or events affecting underlying matrix molecules. Looking forward, the challenge for the equine research community is to determine how a diverse set of disease entities lead to dysadhesion of the LBEC from the underlying matrix.
Progress in unravelling the equine laminitis enigma requires us to address 2 overarching questions:
- What are the pathophysiological mechanisms, both systemically and locally within the lamellae, that lead to lamellar failure in the different types of laminitis?
- What are the risk factors for the different types of laminitis?
Addressing the first question will enable the development of effective diagnostic tools and therapeutic regimens, while improved knowledge of risk factors (animal and environmental) for the different types of laminitis should allow the clinician and horse owner to identify high-risk horses and potentially minimise risk of lamellar failure.
There are few diseases in veterinary medicine that have been plagued with the degree of dogma and controversy that has characterised equine laminitis over the past 50 years. Much of the dogma has persisted because of a lack of consistently used experimental models to study the different types of laminitis and, as important, technical limitations that have precluded rigorous scientific evaluation of the different theories. Technical limitations have included both a dearth of scientific techniques available to the veterinary and human researcher for studying the cellular dynamics of the lamellar tissue and, secondly, the fact that the lamellar tissue is situated between 2 extremely dense tissues (hoof wall and distal phalanx) severely limits the application of the in vivo physiological techniques developed in other species for tissues and organs that are easily accessed (this has especially limited studies of lamellar blood flow). Modelling of the different disease entities leading to laminitis is an ongoing challenge but, on a positive note, advancements made regarding the technical challenges in the last 2 decades - the development of new scientific techniques to study cell biology - have been nothing but remarkable.
Experimental models to study laminitis have included and will continue to include in vivo, ex vivo (lamellar vessels or entire lamellar tissue maintained in organ culture media) and in vitro (e.g. cell culture) models. In vivo models will continue to play a vital role in investigating the different types of laminitis, primarily due to the fact that the condition occurs due to a complex interplay of systemic and local events that cannot, at this point in time, be replaced by other modelling systems. The primary challenge investigators need to confront is establishment of repeatable, clinically accurate models for the different disease entities that are accepted and used by multiple laminitis investigators. A prime reason that more advances have been made on the pathophysiology and treatment of sepsis-related laminitis than endocrinopathic or supporting limb laminitis is the establishment of repeatable models in the form of the black walnut extract  and, most relevant, the carbohydrate-overload models (starch and oligofructose models) [2, 3]. These models have been used by multiple teams of investigators, culminating in a fairly broad database that provides a springboard for further investigations into pathophysiology and into new therapeutic targets. Because the carbohydrate-overload models more accurately mimic the events that occur in the clinical case and the fact that the oligofructose model is more consistent overall between animals than the traditional starch model, the oligofructose model is likely to be the primary model used for future sepsis-associated laminitis investigations. Models for endocrinopathic laminitis have been more variable, ranging from insulin infusions [4, 5] in normal, nonobese, equids (included in the virtual issue) to weight gain studies in different breeds of horses. Although all of these models have provided valuable data, it is imperative that one or 2 models that best replicate the naturally occurring condition are established so that progress can be made in dissecting the effects of obesity, insulin resistance and diet on systemic and local signalling events. The limited data on supporting limb laminitis coincides with a lack of modelling for this syndrome also. Models being used in currently funded investigations include brief episodes of excessive weightbearing (from minutes up to 48 h using either flexion of the other limb by an assistant or the use of a shoe with limited stability to cause preferential weightbearing on the contralateral limb). Although these models do not mimic the time commonly involved prior to the onset of this disease in the clinical arena (up to several weeks), humane concerns will limit the time aspect of supporting limb laminitis modelling.
Both ex vivo and in vitro models have been used in laminitis studies with the primary limitation being the inability to model the complex in vivo dynamics. However, this reality can be used to the investigators' advantage as we obtain more in depth information from in vivo models to dissect events and signalling mechanisms relevant to the dysregulation of the LBEC (i.e. the effect of high concentrations of insulin or decreased glucose on lamellar tissue or cells) [6, 7]. Finally, computer modelling of the disease is also a possibility in the future but will require a great deal more data from the other models prior to this being a valuable tool in laminitis research.
Regarding the first limitation of experimental techniques available to study laminitis, veterinary research has benefited immensely from advances in human research, primarily in the realm of molecular biology (the study of genes and gene expression for this discussion) and protein biochemistry (the study of regulation of protein concentrations and protein activation state for this discussion). The first discovery that transformed laminitis research and medical research in general, was the discovery of polymerase chain reaction (PCR, for which Kary Mullis received the Nobel Prize) in the late 1980s, which rapidly evolved by the late 1990s into real time PCR (quantitative RT-PCR or qPCR), a technique that enabled the accurate quantification of mRNA concentrations for genes of interest . Although the equine genome had not been sequenced yet, it was possible to design primers (probes) by aligning the sequences of genes of interest from man and other species. Although somewhat cumbersome, investigation of a gene of interest could now be performed in a matter of several weeks vs. several months or years prior to the discovery of qPCR; it resulted in the first papers on equine lamellar gene expression in the early 2000s. The impact of qPCR is evidenced by its use in 6 of the reports in this virtual issue [6, 9-13] playing a vital role in detailing inflammatory signalling in the pathophysiology of laminitis and also used to investigate the efficacy of therapies such as cryotherapy and systemic lidocaine.
The event that will likely have the greatest impact on equine laminitis research for years to come is the sequencing of the equine genome (a collaboration of the Broad Institute at MIT), which was first released for public use in 2007 . This has had a phenomenal impact on equine research in general and certainly on laminitis research. In regards to molecular biology, knowledge of the equine genome has made qPCR and other techniques routine with results available in 1–2 weeks. It has also facilitated use of the latest important advancement in high throughput genomic screening, next generation sequencing (NGS), to determine the regulation of thousands of genes at one time in a cell or tissue sample. Until the last 2–3 years, high throughput screening relied primarily on species-specific screenings such as microarray analysis that required small fragments of equine specific nucleotide sequences to be placed on a slide and probed with extracts of the tissues of interest. Although valuable data have been obtained with equine microarrays , the expense and very rigorous methodology required by individual laboratories to ensure fidelity of the technique has somewhat limited their use. The NGS techniques, which have come to the forefront of human medical research in the last 3–5 years, have rapidly evolved into extremely repeatable, accurate methods that, most importantly, are not species-specific. Thus, as the equipment and methodology also require large investments of money and the costly maintenance of well trained staff, the equine research community can now take advantage of core facilities to perform these types of procedures. We can also utilise other advances from the human research side to take full advantage of techniques such as NGS. For example, laser capture-microdissection (LCM) enables the rapid isolation and retrieval of individual cell types from a frozen tissue section from which RNA can be obtained and used to assess the signalling occurring in that cell type. State of the art bioinformatics software is also available to process the overwhelming amount of information derived from techniques such as NGS and determine the effects of disease states or treatments on the regulation of thousands of genes. We are currently using the combination of LCM, NGS sequencing and bioinformatics software analysis to compare the cellular signalling of the lamellar basal epithelial cell layer (the layer which dysadheres leading to lamellar failure in laminitis) in equine models of sepsis-related, metabolic syndrome-related and supporting limb laminitis.
Protein biochemistry techniques, as evidenced by their use in 5 reports in this issue, are still essential for determination of the localisation of a protein of interest to specific cell types (or to an area of the extracellular milieu for matrix-related proteins) and for assessing cell signalling. In disease states, much of the regulation of signalling cascades that cause cellular dysfunction occurs at the protein level through events such as protein phosphorylation/dephosphorylation (controlling the activation state of the protein) and protein degradation (controlling effective concentrations of the protein). Thus, assessment of these events (which are commonly therapeutic targets) can only be done via protein biochemistry techniques, primarily using specific antibodies to assess concentrations either of the total protein, or of the phosphorylated protein. The sequencing of the human genome has also greatly streamlined the use of protein biochemistry techniques as knowledge of the equine sequence of proteins of interest greatly assists the investigator in determining if an antibody raised against another species (the majority of commercial antibodies available are raised against human or laboratory rodent proteins) will react with the equine homologue of that protein. Collaborative efforts such as the U.S. Veterinary Immune Reagent Network and the Equine Immunology Resource Page from the Gluck Center, University of Kentucky are facilitating this effort through cataloguing of antibodies demonstrated to be effective in equine tissue and also production of equine-specific antibodies for use in protein biochemistry techniques. Finally, laminitis researchers at University of Pennsylvania are now taking advantage of advances in the field of proteomic analysis, recently presenting work in which they used ‘quantitative mass spectral counting’ to determine lamellar proteins which are increased or decreased in concentration in models of sepsis and endocrinopathic laminitis.
As regards physiological techniques, although the physical barrier of the keratinised hoof wall has limited the number of advancements from human medicine that can be used to study lamellar events in vivo, investigators are able to apply some of these advances to laminitis research. In a current investigation at the University of Queensland, investigators are able to assess lamellar ‘bioenergetics’ in the standing horse by use of microdialysis catheters placed within the lamellae enabling metabolites such as lactate and glucose to be continuously measured. This research group is currently using this technique to test the hypothesis that supporting limb laminitis occurs due to energy failure (most likely due to decreased blood flow) in the foot sustaining excessive weightbearing. This is an example of how state of the art methodology will help answer a question that has been investigated for decades without a clear answer.
The increasingly rapid emergence of technological advances will result in a vast amount of discovery in laminitis research over the next decade. Several of the reports in EVJs virtual issue demonstrate the power of these techniques in rapidly solving long-standing controversial issues such as the presence of inflammatory processes in sepsis-related laminitis  and quickly resolving the question of whether matrix metalloproteases were playing a decisive role in early lamellar failure . With these technological advances, most theories can be tested in a short period of time if an appropriate experimental model exists. Additionally, the use of molecular biology and most protein biochemistry techniques allow the use of archived samples (stored frozen and in paraffin blocks), which allows the rapid testing of new theories without the financial or humane cost of performing new animal protocols. Again, this reality drives home the necessity for well designed, well accepted in vivo models from which investigators can store and share samples. Finally, one of the greatest challenges facing laminitis research is an increasingly limited amount of funding in the face of the high cost of using state of the art research tools in molecular biology and protein biochemistry. This reality will not only require constant vigilance of the veterinary research community and horse industry to keep the investigation of this devastating disease a priority, but also drives home the need for the careful development of experimental models which are well accepted internationally and will thus allow for the sharing of samples. This will have multiple positive results including creation of a cohesive database between laboratories and, importantly, will result in decreased use of animals thereby addressing humane concerns and sharing the not insubstantial costs of performing animal protocols between investigators.
Epidemiology and risk factors
Recent systematic reviews have highlighted the lack of quality information with respect to the epidemiology of equine laminitis, including data on disease frequency, inciting causes and risk factors [16, 17]. Although data from studies using the various experimental models of laminitis have provided insight into pathological processes, a critical question relates to the applicability of this information to spontaneous cases of laminitis. These models are extreme in that they induce laminitis in the majority of animals challenged, whereas it is apparent that only a small proportion of any at risk horse population develops clinical laminitis (e.g. hospitalised animals with sepsis or a herd of horses or ponies maintained on the same pasture). These anecdotal observations suggest that risk of laminitis might be affected by intrinsic (e.g. breed, age, sex) and extrinsic (e.g. health status, nutrition, season) risk factors. In this context, there is much current discussion and debate regarding the role of underlying endocrine and/or metabolic disturbances. Observational cohort studies have reported that horses and ponies with the insulin resistant phenotype labelled equine metabolic syndrome (EMS) are at heightened risk of laminitis, while the term endocrinopathic laminitis has been loosely adopted to describe cases of laminitis in equids with clinical evidence of EMS or pituitary pars intermedia dysfunction. In various studies, obesity, regional adiposity, insulin resistance and hyperinsulinaemia have been associated with incident laminitis in ponies maintained at pasture [18-20]. In light of reports that laminitis can be induced in healthy horses and ponies by maintaining a very high (>1000 mu/l) insulin concentration [5, 21, 22], it is tempting to conclude that hyperinsulinaemia is causative in endocrinopathic laminitis. However, most of the observational studies to date have suffered from a variety of limitations (e.g. lack of clear definition of laminitis, small study populations, selection bias between cases and controls) that preclude firm conclusions regarding putative risk factors such as hyperinsulinaemia and obesity. Accordingly, an important task for the laminitis research community is to conduct well designed epidemiological studies that identify animal-level and management-risk factors for laminitis.
As noted in a previous commentary on epidemiology of laminitis , case definition is a critical component of the design of future epidemiological studies. Many studies to date combined laminitis cases regardless of associated cause, whereas a preferred approach for obtaining the most accurate epidemiological information would be studies of specific forms of laminitis (i.e. endocrinopathic, supporting limb laminitis etc). Ultimately, information derived from well designed epidemiological studies should pave the way for the development of improved methods for the identification of high-risk animals as well as disease prevention via treatments and management practices that modify risk factors.
Fortunately, organisations such as the British Equine Veterinary Association (BEVA) and American Association of Equine Practitioners (AAEP) have given high priority to investigations related to the epidemiology of laminitis. BEVA members recently contributed to a practice-based survey of prognosis in pasture-based laminitis [23, 24] as part of their Evidence-Based Medicine initiative and currently, Wylie and colleagues are completing a study in the United Kingdom, while the AAEP Research Foundation has initiated a case–control study of acute endocrinopathic laminitis in the United States. Additionally, planning is underway for a case–control study on supporting limb laminitis. We anticipate that the results on these studies will more clearly identify risk factors for laminitis and pave the way for further studies on the pathogenesis, treatment and prevention of this crippling condition.