Daniel L. Ward

EFFECT OF ADIPOSE-DERIVED NUCLEATED CELL FRACTIONS ON TENDON REPAIR IN HORSES WITH COLLAGENASE-INDUCED TENDINITIS

OBJECTIVE To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis. ANIMALS 8 horses. PROCEDURES Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed. RESULTS Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons. CONCLUSIONS AND CLINICAL RELEVANCE ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.

Endoscopic Balloon Dilation of Benign Esophageal Strictures in Dogs and Cats

Endoscopic balloon dilation of benign esophageal strictures was performed in 18 dogs and 10 cats with a median age of 4 years. Stricture formation was associated with a recent anesthetic episode in 18 patients. Regurgitation was the most common clinical sign and was present a median of 4 weeks before dilation. Most animals had a single stricture; median diameter was 5 mm, and median length was 1 cm. Esophagitis and mucosal fibrosis were detected in 9 patients each. Dilation was performed with progressively increasing diameter balloons, from 6 to 20 mm. After dilation, mucosal hemorrhage was mild to moderate in most patients. Esophageal perforation was the only serious complication and occurred in 1 patient. Postdilation therapy consisted of administration of cimetidine, metoclopramide, sucralfate, and prednisone in most animals. The median number of dilation procedures performed in each animal was 2, with a range of 1-5. The median interval between dilations was 13 days. Stricture diameter markedly increased with subsequent dilations. Median duration of follow-up was 131 weeks. A successful outcome occurred in 88% of patients, with most animals able to eat canned, mashed, or dry food without regurgitation. Mucosal fibrosis was associated with a better clinical response score, while increasing age was weakly associated with fewer dilations. The dilation protocol used in this group of animals was safe and efficacious.

Treatments to promote colonic hydration: enteral fluid therapy versus intravenous fluid therapy and magnesium sulphate

Although large intestine impactions are commonly treated with i.v. fluids combined with the osmotic laxative MgSO4, enteral fluids are less expensive and also appear to be efficacious for impactions. Therefore, this study was conducted to compare the systemic and gastrointestinal effects of enteral fluids with the changes produced by i.v. fluids combined with MgSO4. Four horses with a fistula in the right dorsal colon alternately received both treatments in 2 periods one week apart. Sixty litres of fluids were administered continuously (10 l/h) through a venous catheter or a nasogastric tube. Magnesium sulphate (1 g/kg bwt) was administered via nasogastric tube before i.v. fluid therapy. Two horses had mild abdominal discomfort at the end of enteral fluid therapy. Pollakiuria, hypostenuria, increased bodyweight, increased faecal and ingesta hydration, and decreased PCV, plasma protein and plasma magnesium were produced by both treatments. Abdominal distention and more pronounced changes in bodyweight and ingesta hydration were seen with enteral fluids. Intravenous fluids plus MgSO4 produced hypocalcaemia and more pronounced changes in plasma protein. These results indicate that enteral fluid therapy is more effective in promoting ingesta hydration and produces less pronounced systemic effects than i.v. fluid therapy plus MgSO4.

Nonspecific Stimulation of the Maternal Immune System. II. Effects on Gene Expression in the Fetus

BACKGROUND Maternal immune stimulation reduces malformations caused by chemical teratogens. Mechanisms for this effect are not known. Altered expression of regulatory molecules (e.g., transforming growth factor [TGF-beta], tumor necrosis factor-alpha [TNF-alpha]) has been reported in fetuses from immunostimulated mice, which may affect gene expression. Expression of selected genes that function to control proliferation, differentiation, or apoptosis was evaluated in chemical-exposed fetuses, with or without maternal immunostimulation. METHODS Ethyl carbamate (urethane) was given to pregnant ICR mice on day 10 of gestation to induce cleft palate. Before teratogen administration, the immune system of the female mice was stimulated by footpad injection with Freunds complete adjuvant (FCA) or by intraperitoneal injection with interferon-gamma (IFN-gamma). RESULTS Maternal immunostimulation with interferon-gamma (IFN-gamma) decreased severity of the cleft palate lesion caused by urethane, while FCA decreased both incidence and severity of cleft palate. Gestation day 14 fetuses from urethane-exposed mothers displayed decreased expression of cell cycle/apoptotic genes bcl2alpha, bcl2beta, pkCalpha, and p53 in fetal heads. Immune stimulation with IFN-gamma-normalized expression of bcl2alpha, bcl2beta, and pkCalpha to control levels. Urethane also decreased the ratio of expression of bclalpha/p53, bclbeta/p53, and pkCalpha/p53, while maternal injection with IFN-gamma restored these expression ratios to control levels. Maternal immunization with FCA also significantly increased bcl2alpha/p53, bcl2beta/p53, and pkCalpha/p53 gene expression ratios. CONCLUSIONS These results suggest that (1) the maternal immune system may possess heretofore unrecognized regulatory activity in fetal development, and (2) protection against urethane-induced cleft palate may be mediated through maternal immune regulation of fetal gene expression.

Basal Ganglia Accumulation and Motor Assessment Following Manganese Chloride Exposure in the C57BL/6 Mouse

Equivocal clinical evidence for involvement of manganese in development of Parkinson’s disease necessitates experimental studies on this issue. The aged, 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine–treated C57BL/6 mouse is one of the most common models for Parkinson’s disease. However, there is little information on brain bioaccumulation of manganese, and little or no information on clinical/behavioral manifestations of manganese neurotoxicity, in this strain. Male C57BL/6 retired breeder mice were given a single subcutaneous injection of either 0, 50, or 100 mg/kg of MnCl2 (single-dose regimen) or three injections of either of these doses over 7 days (multiple-dose regimen). Behavioral assessment was performed 24 h after final injection, followed by sacrifice, and body weight was recorded each day. There was a 105% increase in striatal manganese concentration 1 day after a single 100 mg/kg injection, and 421% and 647% increases, respectively, 1 day after multiple doses of 50 or 100 mg/kg of MnCl2. One day after a single injection, there were respective 30.9% and 38.9% decreases in horizontal movement (grid crossing) for the 50 and 100 mg/kg doses and a 43.2% decrease for the multiple dose of 100 mg/kg. There was no significant main effect of dose level on rearing, swimming, grip strength, or grip fatigue. Unlike previous work with the C57BL/6 strain using smaller intraperitoneal doses, this study established dosing regimens that produced significant increases in basal ganglia manganese concentration reminiscent of brain increases in the CD-1 mouse following subcutaneous doses close to our lowest. A decrease in locomotor behavior, significant but not severe in this study, has been reported following manganese exposure in other mouse strains. These data, particularly the significant increase in basal ganglia manganese concentration, provide guidance for designing studies of the potential role of manganese in Parkinson’s disease using the most common animal model for the disorder.

Nonspecific stimulation of the maternal immune system. I. Effects on teratogen-induced fetal malformations

BACKGROUND Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freunds complete adjuvant (FCA). RESULTS Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Induction and recovery characteristics and cardiopulmonary effects of sevoflurane and isoflurane in bald eagles

OBJECTIVE To compare induction and recovery characteristics and cardiopulmonary effects of isoflurane and sevoflurane in bald eagles. Animals-17 healthy adult bald eagles. PROCEDURES Anesthesia was induced with isoflurane or sevoflurane delivered in oxygen via a facemask in a crossover design with 4 weeks between treatments. Eagles were intubated, allowed to breathe spontaneously, and instrumented for cardiopulmonary measurements. Time to induction, extubation, and recovery, as well as smoothness of recovery, were recorded. RESULTS Administration of sevoflurane resulted in a significantly quicker recovery, compared with isoflurane. Temperature, heart rate, and respiratory rate significantly decreased over time, whereas systolic (SAP), diastolic (DAP), and mean arterial blood pressure (MAP) significantly increased over time with each treatment. Temperature, heart rate, SAP, DAP, and MAP were significantly higher with isoflurane. Blood pH significantly decreased, whereas PaCO(2) significantly increased over time with each treatment. Bicarbonate and total carbon dioxide concentrations significantly increased over time with each treatment; however, there was a significant time-treatment interaction. The PaO(2) and arterial oxygen saturation increased over time with isoflurane and decreased over time with sevoflurane with a significant time-treatment interaction. Six eagles developed cardiac arrhythmias with isoflurane, as did 4 with sevoflurane anesthesia. CONCLUSIONS AND CLINICAL RELEVANCE Isoflurane and sevoflurane administration resulted in smooth, rapid induction of and recovery from anesthesia similar to other species. Isoflurane administration resulted in tachycardia, hypertension, and more arrhythmias, compared with sevoflurane. Sevoflurane was associated with fewer adverse effects and may be particularly beneficial in compromised bald eagles.

Triple Antimicrobial Therapy and Acid Suppression in Dogs with Chronic Vomiting and Gastric Helicobacter spp

BACKGROUND Helicobacter pylori is a common cause of gastritis and peptic ulcers in humans. Many dogs, including those with gastritis and chronic vomiting, are infected with Helicobacter spp. HYPOTHESIS Triple antimicrobial therapy will eradicate Helicobacter infection, improve gastritis, and reduce clinical signs. The addition of acid suppression medication will not improve results. ANIMALS Twenty-four pet dogs with chronic vomiting and gastric Helicobacter spp. METHODS Dogs were randomly assigned to triple antimicrobial therapy with or without famotidine. Gastroduodenoscopy was performed 4 weeks and 6 months after therapy. Helicobacter spp status was determined by histologic assessment of gastric mucosal biopsy specimens. RESULTS Eradication rates for each treatment were not significantly different and combined were 75 and 42.9% at 4 weeks and 6 months, respectively. A greater improvement in gastritis scores occurred in dogs that became Helicobacter spp negative. Overall, the frequency of vomiting was reduced by 86.4%, but there were no differences between treatments. CONCLUSIONS AND CLINICAL IMPORTANCE Eradication rates of Helicobacter spp with both treatments were not significantly different. Eradication rates at 6 months were modest, and more effective treatments should be developed. Acid suppression is not a necessary component of treatment protocols for dogs. Eradication of gastric Helicobacter spp was associated with improvement in gastritis scores. Dramatic reduction of the vomiting frequency occurred with both treatment protocols. Gastric Helicobacter spp may cause or contribute to chronic vomiting and gastritis in some dogs.

Interpretation of the detection of Sarcocystis neurona antibodies in the serum of young horses.

Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24h of age (postsuckle), and at monthly intervals. All foals sampled before suckling were seronegative. Thirty-three foals from 33 seropositive mares became seropositive with colostrum ingestion at 24h of age, confirming that passive transfer of S. neurona maternal antibodies occurs. Thirty-one of the 33 foals became seronegative by 9 months of age, with a mean seronegative conversion time of 4.2 months. These results indicate that evaluation of exposure to S. neurona by WB analysis of serum may be misleading in young horses.

Reduced birth defects caused by maternal immune stimulation may involve increased expression of growth promoting genes and cytokine GM-CSF in the spleen of diabetic ICR mice

Maternal immune stimulation in mice decreases fetal abnormalities caused by diverse etiologies. Growth factors produced by activated immune cells were proposed to be key mediators that may exert their effects on placenta or embryo. Diabetes disrupts the secretion of cytokines, which may associate with diabetic embryopathy. Three different methods of maternal immune stimulation that result in approximately equal reduction of diabetic embryopathy were used in the present studies: footpad injection with complete Freunds adjuvant (CFA), intraperitoneal (i.p.) injection with granulocyte-macrophage colony-stimulating factor (GM-CSF), or i.p. injection with interferon-gamma (IFN-gamma). A gene microarray was then used to examine expression of a selected gene panel in splenic leukocytes. We hypothesized that maternal immune stimulation may act by overcoming altered gene expression patterns of immune cells in the diabetic mice, which partially mitigates the teratogenic effect of diabetes. It further seemed likely that a shared profile of splenic gene expression changes induced by the different immune stimulation procedures may be identified and related to reduced teratogenesis. The three procedures produced a common altered gene expression profile. Significantly affected genes included apoptotic and anti-apoptotic genes, and genes controlling cellular proliferation, and likely reflect a state of immune activation. The GM-CSF gene was up-regulated by all three immune stimulation procedures. The protein product of this gene regulates placental development, and was recently associated with reduced cleft palate in immune-stimulated pregnant mice after exposure to urethane. These data suggest that further studies of GM-CSF as mediator of reduced birth defects in teratogen-challenged, immune-stimulated mice are warranted.