Diane E. Mason

Potassium Channel Antagonists Influence Porcine Granulosa Cell Proliferation, Differentiation, and Apoptosis

Abstract This investigation determined the effects of K+ channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol IKs antagonist 293B, the benzodiazepine IKs antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27kip1 and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K+ channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three IKs antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K+ channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K+ channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

Transnasal Laryngoscopy for the Diagnosis of Laryngeal Paralysis in Dogs

Four dogs with clinical signs of laryngeal paralysis and three normal dogs were evaluated with transnasal laryngoscopy. Six of these dogs subsequently underwent standard laryngoscopy. For transnasal laryngoscopy, a video endoscope was passed through the left nasal passage after intramuscular sedation and topical anesthesia. The laryngeal opening was observed during spontaneous ventilation. Laryngeal paralysis was diagnosed in four dogs and was confirmed with traditional laryngoscopy in three dogs. Normal motion of the arytenoid cartilages was present in the other three dogs; however, two required mechanical stimulation of the laryngeal mucosa for full evaluation. Transnasal laryngoscopy provided a means for diagnosing laryngeal paralysis in dogs without general anesthesia.

4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

BackgroundIon channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.MethodsGranulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size.Results4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production.Conclusion4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Effects of lidocaine administration via continuous rate infusion on the minimum alveolar concentration of isoflurane in New Zealand White rabbits (Oryctolagus cuniculus)

OBJECTIVE To evaluate the effect of a continuous rate infusion (CRI) of lidocaine on the minimum alveolar concentration (MAC) of isoflurane in rabbits. ANIMALS Five 12-month-old female New Zealand White rabbits (Oryctolagus cuniculus). PROCEDURES Rabbits were anesthetized with isoflurane. Baseline isoflurane MAC was determined by use of the tail clamp technique. A loading dose of lidocaine (2.0 mg/kg, IV) was administered followed by a CRI of lidocaine at 50 μg/kg/min. After 30 minutes, isoflurane MAC was determined. Another loading dose was administered, and the lidocaine CRI then was increased to 100 μg/kg/min. After 30 minutes, isoflurane MAC was determined again. Plasma samples were obtained for lidocaine analysis after each MAC determination. RESULTS Baseline isoflurane MAC was 2.09%, which was similar to previously reported values in this species. Lidocaine CRI at 50 and 100 μg/kg/min induced significant reductions in MAC. The 50 μg/kg/min CRI resulted in a mean plasma lidocaine concentration of 0.654 μg/mL and reduction of MAC by 10.5%. The 100 μg/kg/min CRI of lidocaine resulted in a mean plasma concentration of 1.578 μg/mL and reduction of MAC by 21.7%. Lidocaine also induced significant decreases in arterial blood pressure and heart rate. All cardiopulmonary variables were within reference ranges for rabbits anesthetized with inhalation anesthetics. No adverse effects were detected; all rabbits had an uncomplicated recovery from anesthesia. CONCLUSIONS AND CLINICAL RELEVANCE Lidocaine administered as a CRI at 50 and 100 μg/kg/min decreased isoflurane MAC in rabbits. The IV administration of lidocaine may be a useful adjunct in anesthesia of rabbits.

Venous blood gas analytes during isoflurane anesthesia in black-tailed prairie dogs (Cynomys ludovicianus)

OBJECTIVE To describe changes in venous blood gas analytes during isoflurane anesthesia in black-tailed prairie dogs (Cynomys ludovicianus). DESIGN Prospective study. ANIMALS 16 black-tailed prairie dogs. PROCEDURES Black-tailed prairie dogs were placed in an anesthesia chamber for induction of general anesthesia, which was maintained with isoflurane in oxygen delivered via mask. Immediately following anesthetic induction, a venous blood sample was obtained from the medial saphenous vein; a second venous blood sample was obtained just prior to anesthetic gas shutoff. An evaluation of venous blood gas analytes was performed on each sample. General linear mixed models with repeated measures were used for data analyses. RESULTS Median anesthetic time was 90 minutes (range, 60 to 111 minutes). A significant increase from immediately after induction to completion of anesthesia was observed in Pco2 and mean blood chloride ion, BUN, and creatinine concentrations. A decrease in Po2, mean blood pH, and anion gap was observed from induction of anesthesia to completion. No significant differences during anesthesia were observed in mean base excess or blood bicarbonate, sodium, potassium, calcium, magnesium, blood glucose, lactate, and total CO2 concentrations. No complications occurred during or after anesthesia for any animal. CONCLUSIONS AND CLINICAL RELEVANCE Examination of prairie dogs often requires general anesthesia, with isoflurane currently the inhalation agent of choice. Results suggested respiratory acidosis and relative azotemia may occur during isoflurane anesthesia of prairie dogs. Given the increased risk associated with anesthesia in small mammals and the propensity for respiratory disease in prairie dogs, insight into physiologic changes associated with isoflurane anesthesia in healthy prairie dogs can aid in perioperative evaluation and anesthetic monitoring in this rodent species.

Respiratory Emergencies in the Adult Horse

Responding to an equine respiratory emergency requires rapid localization of the problem and appropriate choices for therapy. Localizing the cause of respiratory distress is aided by history and thorough physical examination. When examining the patient, one must focus on the presenting signs as indicators of URT or LRT dysfunction. Table 3 summarizes the characteristic presenting signs based on respiratory tract location and suggests the initial treatment course indicated. Respiratory distress in the absence of signs related to the pulmonary system suggests inadequate oxygen delivery secondary to a nonpulmonary problem such as shock or severe anemia, which is just as compromising to the animal but requires an entirely different therapeutic approach (see Allen and Schertel, this issue). Thus, localization of the source of respiratory distress is always the first step in determining successful treatment.

EVALUATION OF THE EFFECTS OF STERNAL VERSUS LATERAL RECUMBENCY ON TRENDS OF SELECTED PHYSIOLOGIC PARAMETERS DURING ISOFLURANE ANESTHESIA IN ZOO-HOUSED BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS)

Abstract Isoflurane gas anesthesia is often used for immobilization of prairie dogs in field studies, laboratory research, and veterinary clinical purposes. The goals of this prospective study were to evaluate the effects of sternal versus right lateral recumbency on trends of selected physiologic parameters during isoflurane anesthesia in black-tailed prairie dogs (Cynomys ludovicianus). Fourteen adult, zoo-housed black-tailed prairie dogs were tested during the study. Animals were anesthetized using isoflurane and randomly placed in either sternal or right lateral recumbency to evaluate changes in trends of physiologic parameters, measured selectively every 30 min throughout a 60-min anesthesia period. Results were analyzed using linear mixed modeling. Right lateral recumbency resulted in a decrease in anion gap of about 4.6 mEq/L (95% confidence interval [95% CI]: 3.1–6.0, P < 0.001), whereas sternal recumbency resulted in a lower decrease of 2.1 mEq/L (95% CI: 0.7–3.6, P = 0.02). However, the absolute values at the beginning and at the end of the anesthesia time were not significantly different between the right lateral and sternal recumbency (all P > 0.57). Body position did not have any effect on any other variables, and most of the observed physiologic changes were due to the duration of anesthesia. Our results indicate no significant effect on trends of selected physiologic parameters between sternal recumbency and right lateral recumbency during 1 hr of isoflurane anesthesia in black-tailed prairie dogs.