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Master of Science / Department of Clinical Sciences / Amy Rankin / Objective- The purpose was to compare the efficacy and duration of 0.5% proparacaine, 0.5% bupivacaine, 2% lidocaine, and 2% mepivacaine on corneal sensitivity in clinically normal horses. Animals- 68 clinically normal horses Procedures- In group 1, 60 horses from the Kansas State University horse unit were assigned to receive one topical anesthetic in a completely randomized design. In group 2, 8 privately owned horses were sequentially treated with each of the topical anesthetics in random order with a one week washout period between drugs. Corneal sensitivity was assessed by corneal touch threshold (CTT) measurements which were taken with a Cochet-Bonnet aesthesiometer before anesthetic application (T0), 1 minute after (T1), every 5 minutes until 60 minutes (T5-T60), and then every 10 minutes until 90 minutes (T70-T90) after application. General linear mixed models were fitted to CTT in each design in order to assess the effects of topical anesthetics over time, accounting for repeated observations within individual horses. Results- Corneal sensitivity, as determined by CTT measurements, decreased immediately following application of the topical anesthetic, with persisting effects until T35 for proparacaine and mepivacaine, T45 for lidocaine, and T60 for bupivacaine. Maximal CTT reduction was achieved following application of bupivacaine or proparacaine, while mepivacaine was least effective. Conclusions and Clinical Relevance- All topical anesthetics reduced corneal sensitivity, though maximal anesthesia and effect of duration differed between drugs. For brief corneal anesthesia, 0.5% proparacaine or 2% lidocaine appeared adequate, while 0.5% bupivacaine may be most appropriate for procedures requiring longer periods of corneal anesthesia.

Master of Science / Department of Clinical Sciences / Warren L. Beard / The flexion test is routinely used in lameness and pre-purchase examinations. There is no accepted standard for duration of flexion or evidence that interpretation of results would differ with different durations of flexion. We hypothesized there would be no difference in interpretation of full hindlimb flexion for 5 or 60 seconds. Video recordings of lameness examinations of 34 client owned horses were performed that included: baseline lameness, upper hindlimb flexion for 60 seconds, and flexion of the same leg for 5 seconds. Videos were edited to blind reviewers to the hypothesis being tested. The baseline lameness video from each horse was paired with each flexion to make 2 pairs of videos for each case. Twenty video pairs were repeated to assess intra-observer repeatability. Fifteen experienced clinicians reviewed the videos and graded the response to flexion as either positive or negative. Potential associations between the duration of flexion and the likelihood of a positive flexion test were evaluated using generalized linear mixed models. A kappa value was calculated to assess the degree of intra-observer agreement on the repeated videos. Full hindlimb flexion of 60 seconds was more likely to be called positive than flexion of 5 seconds (p<0.0001), with the likelihood of the same interpretation 74% of the time. The first flexion performed was more likely to be called positive than subsequent flexions (p=0.029). Intra-assessor agreement averaged 75% with κ=0.49. Full hindlimb flexion of a horse for 5 seconds did not yield the same result as 60 seconds.

Master of Science / Department of Clinical Sciences / Warren L. Beard / Pharmacokinetics of Intramuscular Morphine in the Horse Elizabeth Devine, DVM; Butch KuKanich, DVM, PhD, DACVCP; Warren Beard, DVM, MS, DACVS Objective - To determine the pharmacokinetics of morphine after intramuscular administration in a clinical population of horses Design – Prospective, clinical study Animals – Pilot study included 2 normal horses and the clinical study included 75 horses Procedures – Morphine was administered at 0.1mg/kg, IM and 2-3 blood samples were obtained from each horse at various times from 0-9 hours after administration. Plasma morphine concentrations were measured using liquid chromatography and mass spectrometry. Results – Data was analyzed using a naïve pooled pharmacokinetic model. The half-life for the elimination phase was approximately 1.5 hours, the volume of distribution (per bioavailability) was approximately 4.5 L/kg and the clearance (per bioavailability) was approximately 35 mL/kg/min. The peak plasma concentration was 21.6 ng/mL and occurred approximately 4 minutes after administration. Plasma concentrations of morphine were below the limit of quantification by 7 hours in 74 horses. Conclusions and Clinical Relevance – The relatively short half-life of morphine indicates the need for frequent dosing to maintain targeted plasma concentrations. Adverse effects were uncommon in this study and morphine was well tolerated at a dose of 0.1 mg/kg, IM. Morphine may be a useful adjunctive therapy in painful horses, but the variable plasma concentrations suggest the dose and dosing interval may need to be adjusted to the individual patient’s response.

A depressão do sistema cardiocirculatório é um fator associado ao uso dos agentes halogenados na anestesia inalatória eqüina. Como conseqüência direta é observada a queda na perfusão tecidual, causadora de complicações como a miopatia pós-anestésica, o íleo paralítico ou a insuficiência renal. O objetivo do presente estudo foi avaliar os efeitos hemodinâmicos de dois fármacos simpatomiméticos, efedrina e fenilefrina, em eqüinos anestesiados com isoflurano que apresentaram episódio de hipotensão durante o procedimento anestésico. Foram utilizados 10 eqüinos sadios, em decúbito lateral, distribuídos aleatoriamente em dois grupos de cinco animais cada. O grupo I recebeu a infusão de efedrina (0.02mg/kg/min), e o grupo II de fenilefrina (0,002mg/kg/min), sendo que o avaliador não conhecia a natureza dos fármacos. Foram analisados os parâmetros hemodinâmicos, de oxigenação e de ventilação. Os dados foram submetidos à comparação estatística que constou de análise de variância seguida dos testes de Tukey Kramer e T de Student. Quando comparou-se os diferentes momentos do mesmo grupo, foi observada melhora ou manutenção dos parâmetros hemodinâmicos avaliados após o uso dos agentes simpatomiméticos, nos dois grupos. Porém, quando os momentos correspondentes dos grupos foram confrontados, o índice cardíaco, o conteúdo de oxigênio venoso misto, o índice de transporte do oxigênio, a pressão de oxigênio venoso misto e a saturação de oxigênio venoso misto foram superiores no grupo I. Foi concluído que os dois agentes proporcionaram os efeitos hemodinâmicos desejáveis, entretanto, a efedrina apresentou melhor resposta cardiovascular e de oxigenação que a fenilefrina. Assim, pode-se pressupor que a infusão com efedrina provavelmente proporciona melhor perfusão sangüínea, sendo portanto mais indicada para o tratamento da hipotensão nos eqüinos submetidos à anestesia geral. / Depression of the cardiovascular system is a feature of the use of halogenated anesthetics in equine anesthesia. A decrease in tissue perfusion is the main result and can be the cause of complications such as post-anesthetic myopathy, ileus and renal insufficiency. The aim of then present study was to evaluate the hemodynamic effects of two sympathomimetic agents, ephedrine and phenylephrine in horses anesthetized with isoflurane, which presented hypotension during the anesthetic procedure. Ten healthy horses, in lateral recumbence, were randomly distributed in two groups of five animals each. Group I received an infusion of ephedrine (0.02mg/kg/min) and group II an infusion of phenylephrine (0.002mg/kg/min) and the surveyor did not know the agent administered. Hemodynamic, oxygenation and ventilation parameters were evaluated and the results submitted to statistical analysis performed by means of analysis of variance followed by the Tukey-Kramer and Student-T test. When the different moments of the same group were compared, an increment or maintenance of the hemodynamic parameters evaluated after the infusion of both agents was noticed. However, when the correspondent moments of each group were compared, the cardiac index, the mixed venous oxygen content, oxygen transport index and both mixed venous oxygen saturation and partial pressure were superior in group I. It was concluded that the two agents promoted the desired hemodynamic effects, although ephedrine caused the best cardiovascular and oxygenation responses. For this reason, we can presume that ephedrine infusion probably promotes superior tissue perfusion, and therefore is more indicated for the treatment of hypotension in horses submitted to general anesthesia.

Induced pluripotent stem cells (iPSCs) are generated by reprogramming somatic cells to an embryonic state. Therefore iPSCs represent an extremely valuable tool for modelling disease and organ toxicity, with enormous potential in veterinary medicine. Several equine diseases are currently untreatable and can result in euthanasia on medical grounds. In contrast to humans, in vitro models for cellular research in equine do not exist. Therefore it has been necessary to explore the use of stem cells in constructing cell based equine models. Pluripotent stem cell populations are of great interest in this field given their ability to form the three germ layers found in the developing embryo. While a promising notion, the isolation of equine embryonic stem cells has thus far proved elusive and therefore it has been necessary to explore other pluripotent stem cell populations. A very limited number of induced PSC lines have so far been generated from equine fibroblasts but studies in humans showed that other cell types such as keratinocytes were more amenable to reprogramming and generated iPSCs with much higher efficiency; whether this may be also the case in other species has not been investigated. Moreover, iPSC lines reported so far from domestic species, including the horse, depended on complex culture conditions for growth, including feeder layers and media supplementation with several growth factors. Although a promising alternative to fibroblast for generation of induced pluripotent stem cells there is dearth in literature on equine keratinocyte culture techniques. In this work I am reporting a novel approach to generate equine iPSCs lines from keratinocytes. Skin biopsies were used to derive keratinocyte cultures. The three dimensional culture systems were developed for robust culture of equine keratinocytes. These cells were then transduced with retroviral constructs coding for murine Oct-4, Sox-2, c-Myc and Klf-4 sequences, following the original Yamanaka protocol. Following transduction, tight cell colonies with sharp boundaries staining positive for alkaline phosphatase resembling previously reported human iPSCs were generated. The reprogrammed cells were successfully maintained in feeder free and serum free conditions with LIF supplementation. Immunochemistry and qPCR analyses revealed the equine iPSCs lines expressed pluripotency markers expressed in equine embryonic stages including, OCT4, SOX2, SSEA1, LIN 28, NANOG, REX1 and DNMT3B. Equine iPSCs were able to form embryoid bodies and differentiate into derivatives of the three germ layers in vitro. Equine iPSCs were pluripotent in vivo as demonstrated by the formation of teratoma consisting of tissue derivatives of all three lineages such as bone, cartilage, pulmonary epithelium and mature neurons in SCID mice. Importantly, equine iPSCs should not only have the ability to differentiate in a non-directed manner. Therefore, the ability for efficient and directed cellular differentiation was analysed. Equine iPSCs were successfully induced to differentiate into neurospheres forming extensive neuronal projections and synapses. Equine iPSCs were differentiated to neurons using a novel and robust approach. The neurons expressed FOXG1, TUBB3 at induction before ISL1 up regulation, a potent and specific inducer of motor neurons, during terminal differentiation. The neurons tested could fire multiple action potentials and also induce TTX –sensitive action potentials. The iPSC line that showed in vivo differentiation in bone and cartilage was tested for directed differentiation into bone and results were compared to equine mesenchymal stem cells. This study provides the first demonstration of the potential of iPSCs in equine biomedicine. The ability to derive iPSC cells capable of direct differentiation in vitro opens the way for new and exciting applications in equine regenerative medicine.

Orientador: Raimundo Souza Lopes / Resumo: A cólica, também denominada desconforto abdominal agudo, consiste em uma síndrome que acomete o trato gastrintestinal de eqüinos, provocando a diminuição do trânsito intestinal e aumento da produção de endotoxinas com o conseqüente aumento da permeabilidade do epitélio intestinal. Se esse processo não for controlado pode levar a lesões vasculares generalizadas, gerando a sintomatologia observada na cólica podendo evoluir para a morte por choque endotoxêmico. Com o objetivo de avaliar as possíveis alterações fisiopatológicas em eqüinos acometidos de cólica, utilizando os exames hematológicos, bioquímicos e do líquido peritoneal, foram colhidas amostras de sangue, soro e liquido peritoneal de nove eqüinos que apresentavam sintomas de abdômen agudo, como depressão, dor, alterações de comportamento (rolar no chão e olhar para o flanco) aumento do tempo de perfusão capilar e diminuição da motilidade intestinal, devido a distúrbios gastrintestinais (cólica), e de nove eqüinos saudáveis (grupo controle) comprovado por exames clínicos e confirmado pelos exames laboratoriais. Os valores médios observados nos eqüinos com cólica foram maiores que nos eqüinos do grupo controle no eritrograma, leucometria, uréia e creatinina (no soro e no líquido peritoneal). O exame do líquido peritoneal demonstrou aspecto turvo e cor avermelhada. Conclui-se que eqüinos acometidos com cólica apresentaram hemoconcentração, reação inflamatória e azotemia. O líquido peritoneal de eqüinos com desconforto abdominal agudo apresenta características de transudato modificado. E a elevação da concentração das enzimas séricas ALT, AST, CK, no sangue e no líquido peritoneal pode ser utilizada na abordagem clínica dos eqüinos com cólica. / Abstract: Colic also known as acute abdominal pain, is a syndrome that affect the gastrointestinal tract of equines, leading to a decrease in the intestinal flow and increase in the productions of endotoxins with consequent increase in the intestinal epithelium permeability. If not controlled, this process could lead to generalized vascular lesions, and generating the colic symptoms, leading to death by endotoxemic chock. The goal of this work was to evaluate the possible alterations in equine colic, using the hematological, biochemistry and peritoneal fluid exams, were collect samples from nine horses that present symptoms of acute abdomen such as depression, pain, behavioral abnormalities (rolling and look to the side), increased capillary perfusion time and decreasing in the intestinal movements, due to gastrointestinal disturbances (colic), and nine healthy equines (control group), demonstrated by clinical and laboratory exams. The average values observed in the horses with colic were bigger than the equines from the control group in the eritrogram, leucogram, urea and creatinin (in the serum and plasma). The peritoneal fluid was turbid and reddish. In conclusion the equines with colic presented hemoconcentration, inflammatory reaction and azotemia. The peritoneal fluid of equines with acute abdominal presented the typical characteristics of modified transudate. And the elevation of serum and peritoneal fluid enzyme concentrations, such as ALT, AST and CK could be used in the clinical approach to equine colic. / Mestre

A utilização de colóides é indicada em várias situações, mas nem sempre aplicável na clínica de eqüinos. O objetivo desse trabalho foi avaliar o uso de solução concentrada de albumina eqüina (diluída a 5%) durante fluidoterapia em eqüinos com desidratação leve a moderada, comparando-a com fluidoterapia apenas com solução fisiológica. Foram utilizados dois grupos de cinco eqüinos adultos, sem alterações clínicas. Cada animal passou pelo protocolo dos dois grupos experimentais (fluidoterapia apenas com solução fisiológica; fluidoterapia com solução de albumina eqüina e solução fisiológica). A desidratação foi induzida com duas aplicações de furosemida e jejum. Durante o experimento foram realizadas várias avaliações: pesagem; exame físico geral; hematócrito; osmolalidade plasmática; gasometria; proteína total; albumina; Na; K; débito cardíaco; pressão arterial; uréia e creatinina, e cálculo da pressão oncótica e volume plasmático. Com a aplicação da solução de albumina houve diferença em relação ao outro grupo, embora nem sempre demonstrada estatisticamente, na avaliação do turgor de pele, hematócrito, proteína total, albumina, Na plasmático, pressão arterial, débito cardíaco, pressão oncótica e volume plasmático. Concluiu-se que: a aplicação apenas de pequeno volume de solução de albumina é capaz de causar efeitos comparáveis aos da infusão sob pressão de metade do volume de solução fisiológica calculado para reidratar o mesmo animal; ao final da fluidoterapia, a solução de albumina leva a maior valor de pressão arterial e de albumina sérica e menor de proteína total, mesmo sem diferença estatística, do que apenas a aplicação de solução fisiológica; ao final da fluidoterapia, com o uso de solução de albumina o turgor de pele dos animais ainda indica presença de desidratação, ao contrário dos que recebem apenas solução fisiológica onde o mesmo indica boa hidratação em todos os animais; a solução concentrada de albumina eqüina é passível de ser usada em fluidoterapia nesta espécie, com facilidade de preparação e aplicação e não demonstrando efeitos deletérios. / The colloids utilization is indicated in several situations, but not always applicable in equine practice. The objective of this study was to evaluate the use of the equine concentrated albumin solution (diluted to 5%) during fluid therapy in horses with slight to moderate dehydration, making a comparison with fluid therapy only with physiologic saline solution. Two groups of five adult horses, without clinical alterations, were used. Each animal was submitted to the protocol of each experimental group (fluid therapy only with physiologic saline solution; fluid therapy with equine albumin solution and physiologic saline solution). The dehydration was induced with two administrations of furosemide and fasting. During the experimental period, several evaluations were made: weighing; gasometry; total protein; albumin; Na; K; cardiac output; arterial pressure; urea and creatinin, and calculation of the oncotic pressure and plasmatic volume. With the administration of albumin solution, there was difference, although not always statistically demonstrated in the evaluation of the skin turgor, packed cell volume, total protein and albumin, plasmatic Na, arterial pressure, cardiac output, oncotic pressure and plasmatic volume. It was concluded that: the administration of only a small volume of albumin solution is capable of causing effects comparable to the infusion under pressure of half of the calculated volume of physiologic saline solution necessary to rehydrated the animal; at the end of the fluid therapy, the albumin solution leads to higher values of arterial pressure and serum albumin and lower values of total protein, although without statistical difference, than the single administration of physiologic saline solution; at the end of the fluid therapy, with the use of the albumin solution, the skin turgor still indicates the presence of dehydration, the opposite that occurs with the animals when receiving only physiologic saline solution, where the test indicates good hydration in all the horses; the equine concentrated albumin solution is utilizable in fluid therapy in this species, with easy preparation and administration and with no demonstration of deleterial effects.

This research study examines factors that influence a social worker’s willingness to utilize animals in therapy, specifically equine assisted psychotherapy. Equine assisted psychotherapy is an experiential therapy involving horses for the treatment of mental and behavioral health issues. The study uses quantitative data. The sample population is Bachelors of Social Work (BSW) and Masters of Social Work (MSW) students attending a University in Southern California. The participants were provided an electronic self-administered survey through their University email account. The data collected was analyzed and the results were provided to the University. The results indicate a relationship between several variables, such as previously owning and/or caring for a pet and fondness of animals, however, there are likely other factors that predict the use of equine assisted therapy that were not explored in this study. The results of this study will help raise awareness about equine assisted psychotherapy and the benefits of utilizing this non-traditional treatment.

Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia and viremia, termed chronic EIA. Most infected horses control the virus by immune mechanisms and become inapparent carriers. To further our understanding of the equine immune response to EIAV, a multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNAs. Eleven template plasmids specific to ten equine cytokine genes and the ?-actin gene were generated, from which radiolabeled anti-sense RNA probes were produced. The RPA simultaneously quantitated mRNA levels of interleukin (IL)-1, IL-1, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, interferon (IFN)-, transforming growth factor (TGF)-1 and tumor necrosis factor (TNF)- in equine peripheral blood mononuclear cells and equine monocyte-derived macrophages (EMDM). The assay detected as few as 5105 RNA molecules and displayed coefficients of variation of 0.03-0.08 when normalized to -actin expression. Using this RPA, cytokine expression in EMDM infected with 2 molecularly cloned viruses (EIAV17 and EIAV19) was determined. EIAV17 varies from EIAV19 only in env, rev and LTR and causes fatal disease in Shetland ponies. When added to EMDM cultures, virulent EIAV17 stimulated expression of IL-1, IL-1, IL-6, IL-10 and TNF-. These cytokine mRNAs were significantly elevated by 0.5 to 1 hr post infection (hpi) and returned to basal levels by 12 to 24 hpi, indicating modulation by early event(s), such as receptor binding. In contrast to EIAV17, EIAV19 is avirulent in vivo and failed to induce any of the tested cytokines in EMDM. These data show a direct correlation between the virulence of the EIAV clone and the induction of cytokines. The cytokines stimulated by EIAV17 may contribute to EIA-associated symptoms, enhance viral replication in the host, and regulate the host immune response. To determine whether cytokine induction requires EIAV17 replication, EMDM cultures were exposed to UV-inactivated EIAV17 and cytokine induction was monitored. UV-inactivation did not block cytokine induction by EIAV17, suggesting dispensability of viral replication. Given that EIAV17 induces cytokines in a rapid and replication-independent manner, the activation of cytokine expression is likely mediated by binding of EIAV17 to equine macrophage receptor(s).