G. F. Schusser

Immunohistochemical characterization and quantitative analysis of neurons in the myenteric plexus of the equine intestine

The present study was performed on whole-mount preparations to investigate the chemical neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall. The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from different defined areas of the small intestine and the flexura pelvina were taken from 15 adult horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed on free floating whole-mounts. Additionally, samples used for neuropeptide staining were incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The evaluation was routinely accomplished using confocal laser-scanning microscopy. For quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse.

Triple fluorescence labelling of neuronal, glial and vascular markers revealing pathological alterations in various animal models

The simultaneous detection of glia, vessels and neurons facilitates insights into the complex chemoarchitecture of the central nervous system. Here, we present a simple, robust and versatile approach for the carbocyanine triple fluorescence labelling of neuronal, vascular and glial markers. The usefulness of this procedure is shown for rat brain tissue under physiological conditions, after traumatic brain injury caused by controlled cortical impact injury, and after stroke following middle cerebral artery occlusion. Moreover, the versatility of the method is verified by its application to sections from old triple transgenic mice with age-dependent beta-amyloidosis and tau hyperphosphorylation in the hippocampus, modelling neuropathological alterations in Alzheimers disease. To exemplify the usefulness of the approach for analysis of the enteric nervous system, it was applied to whole mounts from the horse intestine. The biotinylated lectin from potato (Solanum tuberosum) is presented as an excellent tool to detect both vessels and microglia. Furthermore, this lectin revealed macrophages after experimental insults, and senile plaques in aged triple transgenic mice. A large portion of astroglia was demonstrated by immunolabelling of glial fibrillary acidic protein. Neurons were detected by monoclonal antibodies directed against neuronal nuclei and, in horse tissues, mouse-anti-HuC/D recognizing a conserved nuclear protein. Confocal laser-scanning microscopy elucidated spatial relationships of the relevant markers and their pathological alterations after experimental insults and in transgenic mice with Alzheimer-like lesions.

Serum thyroid hormone, insulin, glucose, triglycerides and protein concentrations in normal horses: Association with topical dexamethasone usage

The aim of this study was to determine if topical application of dexamethasone affected the serum concentrations of thyroid hormones (triiodothyronine T(3) and thyroxine T(4)), glucose, triglycerides, total protein and insulin in normal horses. Ten horses were treated twice daily for 10 days with 50 g dexamethasone using an ointment formulation. Thyroid hormones and insulin were assayed using standard radioimmunoassay methods, while glucose, triglycerides and total protein were determined using a standard enzymatic method and the Biuret reaction, respectively. An increase in serum glucose and triglyceride concentrations was accompanied by 2-6-fold increases in serum insulin concentrations, but there was no change in serum total protein concentration. Insulin secretion increased with concomitant hyperglycemia and hypertriglyceridemia. A non-significant decline in T(4) secretion was noted. Serum T(3) and T(4) concentrations declined continuously below baseline values from 48 h. Glucose and insulin levels returned to baseline values 3 days after treatment withdrawal, whereas triglycerides reverted to baseline by 7 days. In contrast, baseline values of serum T(3) and T(4) were not reached by 20 days following drug withdrawal. The results indicated that topical administration of dexamethasone affected thyroid function and physiological metabolic functions, which may have implications for potential doping cases in racing horses.

Dexamethasone-Induced Increase in Lymphocyte β-Adrenergic Receptor Density and cAMP Formation in vivo

The influence of dexamethasone on the density of β2-adrenergic receptors (β2-adrenergic receptors (β2AR) and on the intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) response was studied in equine lymphocytes in vivo. Dexamethasone (0.1 mg/kg/day, 1–5 days) raised the number of β2AR – Bmax as assessed by (–)-[125I]iodocyanopindolol binding (ICYP) – to 2.5- to 3.5-fold as compared with control values. The increase in β2AR number was fast (342 ± 49 vs. 960 ± 103 binding sites/lymphocyte after 24 h), reaching a maximum between 48 and 96 h (342 ± 49 vs. 1,289 ± 150 and 1,106 ± 68 binding sites/lymphocyte, respectively). The isoprenaline-induced cAMP accumulation (measured by a [3H]-cAMP radioimmunoassay system) was concomitantly enhanced by dexamethasone (1.5- to 2.4-fold). Both parameters were reversible to a similar rate at dexamethasone withdrawal. The changes in the functional responsiveness of lymphocytes were not reflected by changes in the binding affinity for ICYP of β2AR. These results demonstrate the in vivo glucocorticoid-mediated regulation of β2AR in equine lymphocytes which has already been suggested on the basis of in vitro observations in other tissues.

Anatomy and Anaesthesia of the Equine External Ear Canal

Anaesthesia of the external ear canal (external acoustic meatus) is usually performed by blocking both the great and internal auricular nerves by regional infiltration. However, exact landmarks for blocking the internal auricular nerve to accomplish effective anaesthesia have not been described yet. In this study, detailed anatomical dissection of the equine external ear canal and its nerve supply was carried out on fifteen cadaver heads. Tissue samples of the dissected nerves were taken from two cadaver heads processed and were evaluated microscopically. Prior to the dissection, the region of interest was evaluated ultrasonographically, and injection of a local anaesthetic was simulated with an injection of methylene blue on ten cadaver heads. The tympanic membranes of three cadaver heads were obtained by microdissection and processed for microscopic evaluation. The entrance point of the internal auricular nerve, which is a branch of the facial nerve, into the ear canal is formed by the styloid process of the auricular cartilage. Using ultrasound, the styloid process presented as a thin hyperechoic line 2.17–2.97 cm deep, based on the skin surface. Landmarks for performing a complete and reliable anaesthesia of the external ear canal were established, and the simulated anaesthesia with methylene blue injection was evaluated as successful in all ten cases. Additionally, the histological composition of the equine tympanic membrane is described and illustrated.

Histological Study of the External, Middle and Inner Ear of Horses

Clinical, anatomical and histological aspects of the equine acoustic organ have been poorly investigated and illustrated in literature so far. It is understood that an intact acoustic organ and hearing function are of vital importance for the well‐being of flight animals like horses. The knowledge of the acoustic organ is usually transferred analogously from other mammals to horses. The purpose of this study was to provide a detailed and complete histological description of the healthy equine auditory organ, and to determine its congruity to other mammalians. Anatomical dissections and histological preparations were carried out on ten cadaver heads. Specimens of various parts of the equine acoustic organ were taken and evaluated histologically. The histological composition of external, middle and inner ear structures are predominantly congruent to those of other mammals, especially to human beings. Unique inwardly directed rete pegs within the osseous ear canal and the prominent tensor tympani muscle are described for the first time. Results obtained in this study can be employed as references for further research on the equine acoustic organ and improve the understanding of the clinical development of hearing loss, otitis externa/media/interna or tympanosclerosis.

RNA interference protects horse cells in vitro from infection with Equine Arteritis Virus.

Equine Arteritis Virus (EAV) belongs to the Arteriviridae and causes viral arteritis in horses. In an attempt to develop novel and save therapies against the infection it was tested whether EAV is susceptible to RNA interference (RNAi) in an equine in vitro system. Horse cells were transfected with chemically synthesized small interfering RNA oligonucleotides (siRNAs) and challenged with EAV. Application of these siRNAs led to a significant protection of the cells, and virus titers decreased drastically. siRNAs derived from DNA plasmids expressing small hairpin RNAs (shRNAs) were also effective. The protection was most pronounced with two siRNAs targeting the open reading frame 1 (coding for non-structural proteins), whereas siRNAs targeting sequences for several structural proteins had less or no effect. In addition, it was investigated whether RNAi could be used to treat cells with an already established viral infection. Only application of the siRNAs shortly after viral challenge led to significant survival rates of the cells, whereas transfection at later time points caused much less benefit for the cells. These findings are discussed in a perspective of using RNAi as a therapeutic approach to combat EAV.

Clinical evaluation of serum alcohol dehydrogenase activity in horses with acute intestinal obstruction

OBJECTIVES To measure serum alcohol dehydrogenase (ADH) activity in horses with acute intestinal obstruction and to determine the diagnostic and prognostic utility of this analyte. DESIGN Prospective observational study. SETTING University Veterinary Hospital. ANIMALS Thirty healthy horses (control group) and 77 horses with acute intestinal obstruction, including 36 horses with nonstrangulating obstruction (23 with left ventral colon impaction and 13 with left dorsal displacement [G1], 22 with small intestinal strangulation [G2], and 19 with colon torsion [G3]). INTERVENTIONS Serum ADH activity was assayed spectrophotometerically in all horses. Serum lactate concentration and hepatic enzyme (aspartate aminotransferase, gamma-glutamyl transferase, glutamate dehydrogenase) activities were measured using an automatic analyzer. MEASUREMENTS AND MAIN RESULTS The median [interquartile range] serum ADH activity in healthy horses was 10.5 [8.7-11 U/L]. ADH activity was significantly increased (P<0.05) in G1=16.5 [13.8-18 U/L], G2=40 [20-74.9 U/L], and G3=63.2 [40-78 U/L] compared with healthy controls. Aspartate aminotransferase and glutamate dehydrogenase activities were also significantly increased in G3 in comparison with controls. ADH activity was correlated with serum lactate concentration in G1 and G3, respectively (P<0.01, r=0.55 and 0.8). Other liver enzymes did not show any significant correlation with lactate. ADH activity was directly related to the probability of strangulation; odds ratio=1.11. ADH activity >20 U/L had 80.6% specificity and 80.5% sensitivity for discriminating horses with strangulating obstruction. Twelve horses euthanized before surgery were excluded from the outcome analysis. Increasing ADH activity was associated with nonsurvival; odds ratio=1.03. ADH activity <80 U/L had 94.44% specificity and 66.67% sensitivity for survival. CONCLUSION Serum ADH activity may be a useful clinical parameter in detecting intestinal strangulation in horses and may provide some prognostic value in horses with acute intestinal obstruction.

Kontinuierlich unkonjugierte Hyperbilirubinämie beim Pferd – Ähnlichkeit mit dem Gilbert-Meulengracht-Syndrom*

Gegenstand und Ziel: Bei einem vierjahrigen Vollbluthengst und einem 14-jahrigen Fjordpferd-Wallach mit Leistungsdepression uber sieben Monate bzw. mehrere Jahre und Ikterus wird eine primar kontinuierlich unkonjugierte Hyperbilirubinamie nachgewiesen. Material und Methoden: Im Blut wurden die unkonjugierte und konjugierte Bilirubinkonzentration sowie die Enzymaktivitaten bestimmt. Es erfolgte eine Uberprufung auf hamolytische Anamien (infektiose Anamie, immunbedingte Erythrozytolyse, Babesiose, Ehrlichiose und primar intravaskulare Hamolyse) und chronisch entzundliche Lebererkrankungen (Ultraschall und Biopsie). Der Wallach wurde mit 1 mg Phenobarbital/kg KM zweimal taglich p. o. uber 20 Tage behandelt, um die Glucuronosyltransferase zu stimulieren und damit das unkonjugierte Bilirubin zu reduzieren. Ergebnisse: Die Konzentration des unkonjugierten Bilirubins beider Pferde lag bei Aufnahme zwischen 114,2 (konjugiert 8,3) und 76,4 (konjugiert 8,8) μmol/l und blieb ohne Therapie im Beobachtungszeitraum von vier bzw. funf Monaten konstant zwischen 111,2 (konjugiert 10,8) und 58,1 (konjugiert 9,3) μmol/l. Der Coggins- Test (infektiose Anamie) beider Pferde war negativ und der indirekte und direkte Coombs-Test konnten keine autoagglutinierenden Antikorper (immunbedingte Erythrozytolyse) nachweisen. Weder Zoiten (Babesia equi) in Erythrozyten noch Morula (Anaplasma phagocytophila) in neutrophilen Granulozyten waren nachweisbar. Die Konzentration des freien Hamoglobins im Serum lag beim Fjordpferd im Referenzbereich (zu Beginn 1,07 μmol/l; funf Monate spater 1,36 μmol/l). Beim Wallach lies sich die Konzentration des unkonjugierten Bilirubins mit Phenobarbital von 76,4 auf 61,6 μmol/l senken. Der Hengst wies zu Beginn leicht erhohte Aktivitaten der AST und γ-GT auf. Die monatliche Kontrolle der Enzyme AST, γ-GT, AP, GLDH und LDH beider Pferde ergab jedoch keine Abweichungen von den Referenzbereichen. Das Lebersonogramm des Hengstes zeigte eine normale Echogenitat des Parenchyms und keine erweiterten Gefase. Chronisch entzundliche Lebererkrankungen beim Hengst konnten mittels histologischer Untersuchung von Leberbioptaten ausgeschlossen werden. Schlussfolgerung und klinische Relevanz: Die Absenkung der Konzentration an unkonjugiertem Bilirubin durch Phenobarbital bei einem Pferd sowie der kontinuierlich erhohte Serumspiegel des unkonjugierten Bilirubins ohne Nachweis einer hamolytischen Anamie oder chronisch entzundlicher Lebererkrankungen bei beiden Pferden zeigt eine Ahnlichkeit zum hereditaren Gilbert-Meulengracht-Syndrom des Menschen. Die Aufnahme des unkonjugierten Bilirubins in die Leberzelle ist bei diesem Syndrom durch eine geringe Aktivitat der Glucuronosyltransferase vermindert. Wie in diesen Fallen mit mildem Ikterus ist die primare von der sekundaren unkonjugierten Hyperbilirubinamie (hamolytische Anamien, Lebererkrankungen) zu unterscheiden.