Conscious Ferrets Research Articles

Differential hypoglycaemic, anorectic, autonomic and emetic effects of the glucagon-like peptide receptor agonist, exendin-4, in the conscious telemetered ferret.

BackgroundRodents are incapable of emesis and consequently the emetic potential of glucagon-like peptide-1 receptor (GLP-1R) agonists in studies designed to assess a potential blood glucose lowering action of the compound was missed. Therefore, we investigated if the ferret, a carnivore with demonstrated translation capability in emesis research, would identify the emetic potential of the GLP-1R agonist, exendin-4, and any associated effects on gastric motor function, appetite and cardiovascular homeostasis.MethodsThe biological activity of the GLP-1R ligands was investigated in vivo using a glucose tolerance test in pentobarbitone-anesthetised ferrets and in vitro using organ bath studies. Radiotelemetry was used to investigate the effect of exendin-4 on gastric myoelectric activity (GMA) and cardiovascular function in conscious ferrets; behaviour was also simultaneously assessed. Western blot was used to characterize GLP-1R distribution in the gastrointestinal and brain tissues.ResultsIn anesthetised ferrets, exendin-4 (30 nmol/kg, s.c.) reduced experimentally elevated blood glucose levels by 36.3%, whereas the GLP-1R antagonist, exendin (9–39) (300 nmol/kg, s.c.) antagonised the effect and increased AUC0–120 by 31.0% when injected alone (P < 0.05). In animals with radiotelemetry devices, exendin-4 (100 nmol/kg, s.c.) induced emesis in 1/9 ferrets, but inhibited food intake and decreased heart rate variability (HRV) in all animals (P < 0.05). In the animals not exhibiting emesis, there was no effect on GMA, mean arterial blood pressure, heart rate, or core body temperature. In the ferret exhibiting emesis, there was a shift in the GMA towards bradygastria with a decrease in power, and a concomitant decrease in HRV. Western blot revealed GLP-1R throughout the gastrointestinal tract but exendin-4 (up to 300 nM) and exendin (9–39), failed to contract or relax isolated ferret gut tissues. GLP-1R were found in all major brain regions and the levels were comparable those in the vagus nerve.ConclusionsPeripherally administered exendin-4 reduced blood glucose and inhibited feeding with a low emetic potential similar to that in humans (11% vs 12.8%). A disrupted GMA only occurred in the animal exhibiting emesis raising the possibility that disruption of the GMA may influence the probability of emesis occurring in response to treatment with GLP-1R agonists.

Quantifying Insulin Sensitivity and Entero-Insular Responsiveness to Hyper- and Hypoglycemia in Ferrets

Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5–6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes.

Evaluation of intraocular pressure in conscious ferrets (Mustela putorius furo) by means of rebound tonometry and comparison with applanation tonometry

To determine normal intraocular pressure (IOP) values by means of rebound tonometry in unanaesthetised ferrets, and to compare rebound and applanation tonometry, 55 clinically healthy ferrets were included in an...

Evaluation of drug-induced cardiac conduction disturbance using conscious ferrets—An alternative small animal telemetry model

Evaluation of drug-induced cardiac conduction disturbance using conscious ferrets—An alternative small animal telemetry model

Hemodynamic, ECG and function telemetry in conscious ferrets administered verapamil

Hemodynamic, ECG and function telemetry in conscious ferrets administered verapamil

Prolonged anti-emetic activity and 5-HT3-receptor antagonism by BRL 46470 in conscious ferrets.

The anti-emetic activity of oral and intravenously-administered BRL 46470 (endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3yl)-2,3-dihydro-3,3- dimethyl-indole-1-carboxamide HCl) has been assessed in conscious ferrets. BRL 46470 (0.05-0.5 mg kg-1, p.o.) dose-dependently prevented emesis evoked over a 2 h period by total body X-irradiation. This anti-emetic activity occurred with oral or intravenously-administered BRL 46470 even when dosed 3-4 h before radiation. In conjunction with data obtained in other species, we conclude that BRL 46470 has a potent and long-lasting ability to antagonize actions that are mediated by the 5-HT3 receptor in-vivo.

Echocardiographic and electrocardiographic examination of clinically healthy, conscious ferrets

To assess the feasibility of performing electrocardiography (ECG) and echocardiography on conscious ferrets and to establish reference values for this species under these conditions. End-systolic and end-diastolic left ventricular (LV) diameters, LV and interventricular septal thicknesses were measured by M-mode echocardiography, while left atrial, aortic and pulmonary trunk diameters were determined by two-dimensional echocardiography in 46 ferrets. In some animals the maximum velocities of blood flow in the aorta and the pulmonary trunk were also measured by spectral Doppler. ECG examinations were performed by using limb leads both in right lateral recumbency and in the so-called "hanging" position. In the course of the ECG examinations (n=43), the occurrences, directions, durations and amplitudes of the P, QRS and T waves and the duration of the PR- and QT intervals were recorded for all six leads. ECG and echocardiography were successfully performed on non-sedated ferrets in 90 and 96% of the cases, respectively. Males were significantly heavier and had larger cardiac dimensions compared to females. Performance of both echocardiography and ECG are feasible techniques in conscious ferrets, and reference values are now available for future comparisons.

Inhibition of Transient Lower Esophageal Sphincter Relaxation and Gastroesophageal Reflux by Metabotropic Glutamate Receptor Ligands

Transient lower esophageal sphincter relaxation (TLESR) is the major mechanism of gastroesophageal acid reflux. TLESR is mediated via vagal pathways, which may be modulated by metabotropic glutamate receptors (mGluRs). Group I mGluRs (mGluR1 and 5) have excitatory effects on neurons, whereas group II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) are inhibitory. This study determined the effect of mGluRs on triggering of TLESR and reflux in an established conscious ferret model. Esophageal manometric/pH studies were performed in ferrets with chronic esophagostomies. TLESR were induced by a gastric load of 25 mL glucose (pH 3.5) and 30 mL air. In control treated animals, gastric load induced 3.52 +/- 0.46 TLESRs per 47-minute study, 89.7% of which were associated with reflux episodes (n = 16). The mGluR5 antagonist MPEP inhibited TLESR dose dependently, with maximal 71% +/- 7% inhibition at 35 micromol/kg (n = 9; P < .0001). MPEP also significantly reduced reflux episodes (P < .001) and increased basal lower esophageal sphincter pressure (P < .05). MPEP inhibited swallowing dose dependently, suggesting a common action on trigger mechanisms for swallowing and TLESR. The more selective analogue, MTEP, had more potent effects (90% +/- 6% inhibition TLESR at 40 micromol/kg; n = 8; P < .0001). In contrast, the group I agonist DHPG tended to increase TLESR. The group II agonist (2R, 4R)-APDC was ineffective, whereas the group III agonist L-(AP4 slightly reduced TLESR (33% at 11 micromol/kg; P < .05). The selective mGluR8 agonist (S)-3, 4-DCPG inhibited TLESR by 54% at 15 micromol/kg (P < .01). mGluR5 antagonists potently inhibit TLESR and reflux in ferrets, implicating mGluR5 in the mechanism of TLESR. mGluR5 antagonists are therefore promising as therapy for patients with GERD.

Triggering of transient LES relaxations in ferrets: role of sympathetic pathways and effects of baclofen.

Activation of gastric vagal mechanoreceptors by distention is thought to be the trigger for transient lower esophageal sphincter relaxations (TLESR), which lead to gastroesophageal reflux. The contribution of higher-threshold gastric splanchnic mechanoreceptors is uninvestigated. GABA(B) receptor agonists, including baclofen, potently reduce triggering of TLESR by low-level gastric distention. We aimed to determine first whether this effect of baclofen is maintained at high-level distention and second the role of splanchnic pathways in triggering TLESR. Micromanometric/pH studies in conscious ferrets showed that intragastric glucose infusion (25 ml) increased triggering of TLESR and reflux. Both were significantly reduced by baclofen (7 micromol/kg ip) (P < 0.05). When 40 ml of air was added to the glucose infusion, more TLESR occurred than with glucose alone (P < 0.01). These were also reduced by baclofen (P < 0.001). TLESR after glucose/air infusion were assessed before and after splanchnectomy (2-4, 9-11, and 23-25 days), which revealed no change. Baclofen inhibits TLESR after both low- and high-level gastric distention. Splanchnic pathways do not contribute to increased triggering of TLESR by high-level gastric distention.

Inhibition of transient LES relaxations and reflux in ferrets by GABA receptor agonists.

Transient lower esophageal sphincter (LES) relaxation is the major mechanism of gastroesophageal reflux. This study uses an established ferret model to evaluate GABA(B) receptor agonists' ability to reduce triggering of transient LES relaxations. One hundred sixty manometric/pH studies were performed on 18 conscious ferrets. In untreated animals, intragastric infusion of 25 ml glucose (pH 3.5) led to 2.0 +/- 0.6 reflux episodes over the first 30 min. Twenty-nine of forty-seven reflux episodes occurred during transient LES relaxation, and 18 occurred after downward drifts (<1 mmHg/s) in basal LES pressure. The GABA(B) receptor agonists baclofen (7 micromol/kg ip), CGP-44532, and SKF-97541 (both ED(50) <0.3 micromol/kg) reduced reflux episodes and transient LES relaxations. The putative peripherally selective GABA(B) receptor agonist 3-aminopropylphosphinic acid (80-240 micromol/kg) was ineffective, as was the GABA(A) receptor agonist muscimol (5 micromol/kg). Baclofen's inhibition of transient LES relaxations and reflux was unaffected by low-affinity GABA(B) receptor antagonists CGP-35348 and CGP-36742 at 100 micromol/kg but was reversed by higher-affinity CGP-54626 and CGP-62349 (0.7 micromol/kg) or by CGP-36742 at 200 micromol/kg. Therefore, GABA(B) receptor inhibition of reflux shows complex pharmacology. Our and other data indicate the therapeutic potential for these drugs.

Mechanisms of gastro-oesophageal reflux in the ferret.

Transient lower oesophageal sphincter (LOS) relaxation is the major mechanism of gastro-oesophageal reflux in humans--an event unassociated with swallowing. Mechanisms involved in triggering transient LOS relaxation are poorly understood, and their further study requires a small animal model. In this study we aimed to establish methods for prolonged ambulant oesophageal manometry in ferrets, and to determine motor events associated with reflux episodes and their triggering by different gastric nutrient loads. Forty-two studies were performed on nine ferrets with chronic cervical oesophagostomies, through which a manometric assembly was introduced and secured to a collar, which incorporated a microphone for detection of swallows. The assembly included a gastric feeding channel, one gastric and four oesophageal manometric sideholes, a 2.5-cm-long LOS sleeve sensor, and an oesophageal pH electrode. Intragastric infusions were given over 2 min, the first after a 30-min control recording period, and in 29/42 studies, a second infusion was given 60 min later. Infusions were either 25 mL 10% dextrose solution, pH 3.5 (22 studies), 25 mL triglyceride emulsion (Intralipid) pH 3.5 (11 studies), or 25 mL air (nine studies). Episodes of oesophageal acidification were absent before gastric infusions. After infusion, 2.1 +/- 0.2 episodes occurred over the first 30 min. After glucose infusion, 15/18 acidification episodes (83%) occurred during transient LOS relaxation, and 3/18 (17%) occurred after gradual (< 1 mmHg sec-1) downward drifts in basal LOSP to < 2 mmHg. After lipid infusion two acidification episodes occurred, both during transient LOS relaxation. Mean duration of transient LOS relaxation was 8.0 +/- 0.4 sec. All infusions increased occurrence of transient LOS relaxation to a similar extent, each of which ended with primary peristalsis. We conclude that gastric infusion of glucose, lipid and gas are all effective in provoking gastro-oesophageal reflux in ferrets. Reflux occurs through similar mechanisms to those seen in humans, i.e. increased triggering of transient LOS relaxation. The conscious ferret is therefore an appropriate model for future studies of manipulation of mechanisms giving rise to gastro-oesophageal reflux.

Gastrointestinal motor activity in conscious ferrets

Gastrointestinal motility was measured with force transducers in conscious ferrets. The gastrointestinal motility pattern in both the interdigestive and digestive states was similar to that reported for humans. The activity front, phase III contractions of the migrating motor complex, occurred cyclically in the antrum and migrated to the duodenum and ileum in the interdigestive state, and relatively low-amplitude contractions were sustained in the antrum, duodenum and ileum in the digestive state. Colonic motility was characterized by basal relatively low-amplitude contractions and a single high-amplitude contraction preceding defecation. Cisparide (0.3–3 mg/kg s.c.) enhanced antral and colonic motility. This ferret model will help the investigation and evaluation of drug effects on gastrointestinal motility in humans.

Plasticity of autonomic control of emesis in irradiated ferrets.

To investigate the plasticity of the emetic response to irradiation, five groups of ferrets (with the appropriate controls) had either the vagal or the splanchnic nerves transected and either were immediately irradiated under urethan anesthesia or were irradiated in a conscious state at 0.5-4, 4-9, and 24 h and 7 days after nerve transection. All the ferrets used in this study had the left carotid artery and the right jugular vein catheterized several hours before irradiation so that blood pressures could be monitored. In ferrets that were irradiated while anesthetized, bilateral abdominal vagotomy performed immediately before 6-Gy irradiation prevented emesis. Acute bilateral abdominal splanchnicectomy markedly reduced emesis in such cases but had no statistically significant effect in conscious ferrets. The incidence of emesis in conscious controls laparotomized 0.5-4 h before irradiation was 50% after 2 Gy and 100% after 6 Gy. Intact conscious controls were tested with 10 Gy only and showed a 100% incidence of emesis after this dose. Bilateral abdominal vagotomy, performed on ferrets 4-9 h before 8-Gy irradiation of conscious animals, markedly reduced the incidence of radiation-induced emesis. When performed 24 h before irradiation, it abolished emesis. In contrast, vagotomy was ineffective when performed 7 days before irradiation. The data suggest that, after 2-8 Gy, the vagi are the major emetic pathway in conscious ferrets and the splanchnic nerves may, under certain conditions, constitute second or alternate pathways.

Pharmacological properties of dolasetron, a potent and selective antagonist at 5‐HT3 receptors

AbstractIn the rabbit isolated perfused hear, dolasetron (MDL 73,147) was found to be a potent (PA2 = 9.8) antagonist at 5‐hydroxytryptamine3 (5‐HT3) receptors present on sympathetic nerve terminals. In anesthetized rats, intravenous (iv), intraduodenal (id), or oral administration of dolasetron blocked the von Bezold‐Jarisch reflex elicited by iv injection of 5‐HT; the iv ED50 was approximately 3 μg/kg and following a dose of 140 μg/kg iv the reflex was abolished for &gt;85 min. The compound displayed no significant affinity for 5‐HT1, 5‐HT2, or a variety of other radioligand binding sites at concentration of 10 μM. In conscious ferrets, dolasetron suppressed the vomiting induced by an iv injection of the anti‐cancer drug cisplatin (10 mg/kg). Intravenous doses (0.05–0.5 mg/kg) administered 30 min before and 45 min prior to cisplatin, were clearly anti‐emetic and single oral doses of 0.5 or 2 mg/kg, given 30 min prior to cisplatin were also effective. Minimal changes in the behaviour of mice were observed at doses up to 100 mg/kg given ip or subcutaneously (sc). It is concluded that dolasetron is a potent, selective, and reversible antagonist at neuronal 5‐HT3 receptors, which is well tolerated. The compound is effective at low doses in an animal model predictive of clinical efficacy in cytotoxic drug‐induced vomiting. © 1993 Wiley‐Liss, Inc.

Are all 5-HT 3 receptor antagonists the same?

A number of 5-HT 3 receptor antagonists are currently in clinical development as antiemetics. In this paper we focus on two of these antagonists, granisetron and ondansetron, and compare their antiemetic activity against cisplatin (10 mg/kg i.v.)- or whole body X-irradiation (200 rads)-induced emesis in the conscious ferret. The results presented here have been discussed in the light of the recently published literature. Our data suggest that in comparison to ondansetron, granisetron is a more potent, longer acting and pharmacologically “cleaner” compound with a more conventional dose-response profile. The possible impact of these features upon the performance of these compounds in the clinic is discussed particularly with respect to dosing regimens and clinical efficacy. Differences appear to be emerging between granisetron and ondansetron in both these respects, although a direct head-to-head clinical comparison has yet to be carried out. This would involve studies monitoring a sufficiently high number of patients receiving severely emetogenic regimes to allow real clinical differences to be detected with the appropriate statistical power.

Changes in FSH and LH secretion in the ferret associated with the induction of ovulation by copper acetate.

The changes in FSH and LH secretion associated with the induction of ovulation by i.v. injection of 5 mg copper acetate were followed in the ferret and found to be influenced by barbiturate anesthesia. In anesthetized estrous animals, the metal ion produced a small initial increase in plasma LH concentration which was followed by a gradual but sustained rise. Anestrous animals responded with a large initial surge of LH release which declined to a plateau some 4 times higher than the basal level and was maintained for at least 6 h. Compared with the anesthetized animals, treatment of conscious estrous ferrets with copper acetate caused an abrupt and much greater initial increase in plasma LH concentration, while in conscious anestrous ferrets the initial surge in plasma LH content was significantly greater than seen under anesthesia, but was followed by a steady decline toward control values. The changes in plasma FSH concentration produced by copper acetate were somewhat similar to those for LH, but were less pronounced.

Vagal afferent discharge from mechanoreceptors in different regions of the ferret stomach.

1. The rate and volume of gastric filling was estimated in conscious ferrets by measuring the amount of milk they would drink after an overnight fast. The mean volume was 94.5 +/- 7.5 ml. at a rate of 13.0 +/- 0.74 ml./min. An intragastric infusion rate of 10 ml./min to a total of 50 ml. was selected as a standard distension stimulus. 2. Action potentials were recorded from single gastric afferent fibres in the cervical vagus. All but two of thirty-six afferent units were tonically active when the stomach was deflated. 3. Afferent fibres arising from receptors in the antrum showed modulation in phase with spontaneously occurring antral contractions. Afferent fibres from the corpus and fundus, however, discharged at irregular rates between 0.35 and 7.5 Hz with no correlation with the intragastric pressure rises associated with the antral contractions. 4. Inflation of the stomach with 50 ml. 0.9% NaCl at a rate of 10 ml./min stimulated antral motility and the rhythmic afferent discharge from the antrum was enhanced Receptors in the corpus and fundus increased their rate of discharge with increasing gastric volumes. Receptors in the region between the antrum and the corpus had the property of both types so that they responded to both distension and contractions. 5. On distension with 0.9% NaCl, fluid is distributed unevenly in the stomach. 80% was accommodated in the corpus and fundus, the remainder of the fluid entering the antrum. 6. The tension was measured in strips of stomach wall taken from corpus and antrum. For equal increments of stretch the development of tension was greater in the antral than in corpus strips. This physical property together with neurally mediated receptive relaxation of the corpus is the reason for the fluid distribution described above. 7. It is concluded that the properties of the tension receptor are determined by their site in the stomach. Those in the body and fundus signal the degree of distension and those in the antrum signal information concerning the amplitude, rate and duration of antral contractions.

Simple technique for bleeding ferrets (Mustela putorius furo).

3-5 ml of blood can be obtained routinely from the tail of a conscious ferret without affecting the wellbeing of the animal. The development of this technique makes the ferret more readily available as an alternative species for multidose toxicity studies.

Gastric secretion after pentagastrin and histamine in the basal secreting ferret

Gastric secretory experiments were conducted with pentagastrin and histamine on the chronic fistula ferret in order to evaluate the physiologic similarity of this preparation to man. Results indicated that the experimental model is suitable for gastric studies, and that the conscious ferret has a consistent basal secretion, uniquely similar to man. In addition, the ferret is highly sensitive to pentagastrin, like man and the cat and dog. Peak acid output in the chronic fistula ferret was observed following a single intraperitoneal dose of 10 μg. pentagastrin/kg. Protease output was also significantly increased following 2 μg. pentagastrin/kg. The secretory outputs following maximal pentagastrin stimulation in this preparation exceeded the maximal outputs following maximal histamine stimulation, although histamine was also a potent stimulant. Due to the consistent basal secretion observed in this species, it was possible to compare maximal stimulated responses to basal secretion in individual animals. The maximal response of individual animals correlated well with the level of basal secretion of acid. Because of the foregoing characteristics, and because of the gastric anatomic similarity to man and surgical tolerance, the ferret appears to be a suitable model for studies of gastric physiology.