Immunohistochemical study of the ontogeny of prochymosin--and pepsinogen-producing cells in the abomasum of sheep.

The appearance and development of prochymosin- and pepsinogen-producing cells were investigated in the ovine abomasum from fetus to adult using immunohistochemistry. Prochymosin immunoreactivity appeared first in the proper gastric glands of the 100-day-old fetus. The intensity and distribution of prochymosin-immunoreactive cells increased gradually with the progress of gestation, and their most intense immunoreactivities and widest distribution were observed in 3-day-old lambs. They were subsequently reduced throughout postnatal growth. A few prochymosin-immunoreactive cells were scattered in the glands of adult sheep. Pepsinogen immunoreactivity appeared at first in a small number of cells in the base of some proper gastric glands of 120-day-old fetuses. After 130 days, pepsinogen-immunoreactive cells increased their staining intensities and distribution. They reached a peak in area at 21 days, which is comparable to adult sheep. In the pyloric glands, prochymosin- and pepsinogen-immunoreactive cells appeared from 100 and 130 days, respectively. Numbers were reduced in comparison to gastric glands and their occurrence was capricious. The results demonstrated that the ontogeny of prochymosin- and pepsinogen-immunoreactive cells in the abomasum of sheep is more similar to that in cattle than to that in the goat. The present data will contribute to the overall understanding of the development of ruminant gastric proteases.

Galanin-like immunoreactive neural elements in domestic ruminant pancreas.

The distribution and ontogeny of the galanin-like immunoreactive (Gal-IR) neural structures in the pancreas of cattle, sheep and goat were investigated immunohistochemically. The present study confirmed the previous findings on the immunolocalization of galanin both in the neural elements and endocrine cells of cattle, and reported for the first time its exclusive localization in the neural elements of sheep and goat. The frequency of Gal-IR nerve fibers and nerve cell bodies was high in cattle and low in sheep and goat. Their first detection was at the first fetal trimester in cattle and third trimester in sheep and goat. In cattle, a marked increase in the frequency of Gal-IR nerve fibers was observed from the third trimester to early neonatal stage followed by a decrease after three months postnatal. In contrast to the non-preferential distribution pattem in sheep, the Gal-IR nerve fibers in cattle and goat pancreas were predominantly associated with the acini, excretory ducts and blood vessels, but rarely detected in the pancreatic islets. The Gal-IR nerve cell bodies were observed as isolated bodies in the intra- and interlobular connective tissues and as a group within the intrapancreatic ganglia. At the vicinity of the nerve cell bodies, Gal-IR nerve fibers were observed. The present findings may suggest that: (1) galanin regulates pancreatic function as neurotransmitter/neuromodulator in ruminants; (2) galanin plays a more important role in large than in small ruminants; and (3) particularly in cattle, it exerts its most dramatic effect during perinatal development.

Evaluation of a hemostasis model for teaching ovariohysterectomy in veterinary surgery.

OBJECTIVE: To evaluate the efficacy of a reusable plastic model mimicking the anatomy and hemodynamics of the canine female genital tract for teaching basic surgical skills and ovariohysterectomy. SAMPLE POPULATION: 40 veterinary students of the class of 1998. STUDY DESIGN: Prospective study. METHODS: Students'confidence level and experience in private practice was evaluated via questionnaire before training. Students in 2 groups performed an ovariohysterectomy on cadavers (group C, n = 20) or on the model (Group M, n = 20) for 2 hours. Students' psychomotor and basic surgical skills were objectively assessed by the following tests: ligation of a foam cylinder, passing a needle through the eyelets of an electronic suture board, and ligating latex tubing. Results were compared before and after training and within and between groups. The ability of students to perform an ovariohysterectomy in a live dog after training was compared between groups with a scoring system. RESULTS: Students in both groups had similar surgical experience and basic skills before training. The results of the psychomotor and basic surgical skills tests were better in group M after training than group C. The improvement of each student in performing these tasks also increased when students were trained with the model. Scores assigned to students performing an ovariohysterectomy in a live dog were higher in group M (31.45 +/- 1.15) than in group C (20.7 +/- 1.42). CONCLUSION: The model was more effective than cadavers in teaching basic surgical skills and ovariohysterectomy in dogs.

Peripheral demyelination and neuropathic pain behavior in periaxin-deficient mice.

The Prx gene in Schwann cells encodes L- and S-periaxin, two abundant PDZ domain proteins thought to have a role in the stabilization of myelin in the peripheral nervous system (PNS). Mice lacking a functional Prx gene assemble compact PNS myelin. However, the sheath is unstable, leading to demyelination and reflex behaviors that are associated with the painful conditions caused by peripheral nerve damage. Older Prx-/- animals display extensive peripheral demyelination and a severe clinical phenotype with mechanical allodynia and thermal hyperalgesia, which can be reversed by intrathecal administration of a selective NMDA receptor antagonist We conclude that the periaxins play an essential role in stabilizing the Schwann cell-axon unit and that the periaxin-deficient mouse will be an important model for studying neuropathic pain in late onset demyelinating disease.

The neurology and enterology of equine grass sickness: a review of basic mechanisms.

Autonomic dysfunction constitutes a prominent clinical feature of equine grass sickness (EGS). Significant injury to the nervous control of the alimentary system is life threatening, partly because of dysphagia but also because of the failure of the unique regulatory mechanisms in equine digestion involving water and electrolyte balance. The neuropathology also indicates the presence of a somatic polyneuropathy. The morphological features of EGS are similar to those of excitotoxic neuronal degeneration, which resembles neuronal apoptosis. It is difficult to ascertain from published accounts the degree of damage to central neurones: the distribution is well documented and selective but the proportion of damage is poorly quantified. If lesions involve a significant number of regulatory neurones they should produce functional deficits. Any clinical assessment of horses, especially those with chronic EGS, should include a thorough neurological examination. Although this will not necessarily improve the outcome of the case, it may enable the rational selection of animals with a reasonable prognosis for recovery which is partly determined by the extent of CNS lesions. The evidence supports the following pathogenesis. There is an initial lesion in the enteric nervous system of susceptible horses. In the acute form of EGS, massive enteric neuronal damage occurs first functionally, then structurally leading to generalized alimentary smooth muscle atony, enhanced secretions and altered fluid fluxes. Severe distension of the stomach and small intestines rapidly develops, which augments the intestinal ileus by intersegmental inhibitory reflexes and causes colic and dehydration. In subacute cases, failure of intestinal bicarbonate buffer together with alimentary stasis rapidly reduces caecal-colonic fermentation. Thus the osmolality of large intestinal digesta reduces and water travels out of the bowel along osmotic gradients. Water returns to the circulation, but is eventually lost in the gastric and small intestinal secretions. The observation that pathological lesions may not be seen in the prevertebral ganglia within the first few days of acute cases supports the view that a functional deficit precedes structural lesions which may be secondary to a retrograde degeneration. It is therefore possible to resolve the observations that less damage may be seen in prevertebral ganglia and elsewhere in peracute and acute cases with the more common finding that greater neuronal damage is present in acute than in chronic cases. These different observations are probably time dependent. Chronic EGS occurs when there is less initial enteric nerve damage which may lead to less secondary prevertebral ganglionic pathology, and more time for functional and structural compensatory mechanisms to develop. Denervation hypersensitivity develops at target sites both in the gut and in peripheral somatic nerves which may account, in part, for the clinical signs of patchy sweating and muscle tremors. Raised circulating adrenaline levels may also account for generalized sweating, may contribute to gastrointestinal atony and may affect pacemakers at the pelvic flexure. Many of the features of EGS make worthwhile the re-investigation of Clostridium botulinum Group III toxins, which are known to prevent vesicular exocytosis, stimulate neurosecretion, produce neuronal chromatolysis and inhibit neutrophil migration. Also, evidence from other species suggests that increased nitrergic neuronal activity can account for many of the clinical signs of EGS, namely dysphagia, generalized ileus, gastric dilatation, sweating, peripheral vasodilatation, tachycardia, salivary hypersecretion, muscle wastage and cachexia.

Gas handling in the caecum of the horse.

Electromanometric recordings of caecal pressures were made in chronically cannulated ponies. Endoscopy allowed direct observation of the caecal base and cupola while recording intracaecal pressures and enabled a functional interpretation of caecal pressure profiles. Using gas-tight seals, the caecal gas cap baseline pressure (tone) was usually below atmosphere, becoming less negative postprandially. Diurnal variations were observed in the amplitude and frequency of caecal pressure profiles and the direction of caecal movements, during which intra-caecal gas cap pressures briefly became positive. A paraprandial (psychic) phase of enhanced caecal motility was observed. Manipulation of the caecal gas cap volume, by insufflation of nitrogen gas, demonstrated the capacity of the caecal base to regulate gas pressures and to eliminate fermenting gases.

Three-dimensional development of bovine reticular cell (Cellula reticuli).

This paper describes the three-dimensional construction of the reticular cell, cellula reticuli, in bovine reticulum and its ontogenetic development. Reticular cells of fetal suckling calf and adult tissues were investigated both at the surface of intact mucosa and macerated samples using scanning electron microscopy. At the third month of gestation, the formation of the reticular cells on the mucosal surface started from the center of the cell and just above the reticular crest. The reticular crests were observed on the surface of the macerated sample at an earlier period of gestation (third month). In the eighth month of gestation, primary, secondary and tertiary reticular crests and papillae could be observed from the mucosal surface. Macerated samples showed that those structures were already formed completely under the epithelium by the first half of the seventh month of gestation. It is suggested that the development of the reticular papillae starts from the lower to the upper parts of the reticular cell.

Effect of supplementary dietary biotin on hoof growth and hoof growth rate in ponies: a controlled trial.

The effect of dietary biotin supplementation, at a dose rate of 0.12 mg/kg bwt, on growth and growth rate of the hooves of 8 match-paired poines was investigated in a controlled feeding trial. Treatment animals had a mean hoof growth at the midline dead centre of the hoof capsule of 35.34 mm after 5 months of biotin supplementation compared to control animals 30.69 mm (P < 0.05). Comparison of regression analysis also showed that biotin supplementation produced a significantly higher (P < 0.02) growth rate of hoof horn in this trial. Treatment animals had a 15% higher growth rate of hoof horn and 15% more hoof growth at the midline dead centre, after 5 months of biotin supplementation compared to control ponies. No differences were found between feet for growth of horn, but the older animals in the trial had significantly lower hoof growth (P < 0.05) than the remaining poines.

Light microscopy of the enteric nervous system of horses with or without equine dysautonomia (grass sickness): its correlation with the motor effects of physostigmine.

Light microscopy was undertaken on sections from the caudal flexure of the duodenum and the terminal ileum proximal to the ileocaecal fold in 5 control horses, 5 horses with acute grass sickness (AGS), and 5 horses with chronic grass sickness (CGS). With the exception of the ileal submucous plexus of the CGS group, the AGS group had the lowest number of neurons as measured using a subjective scoring scheme. The proportion of abnormal neurons in the AGS group was similar in both plexuses and both regions, whereas the values for the CGS group were much higher in the duodenal region than in the ileal region. The motility of tissue adjacent to that used for histology was measured isometrically in vitro. The increase in the rate of contractions following exposure to physostigmine was greatest for the AGS group, both from the duodenal and from the ileal region. The latency was longest for the AGS group, suggesting that the material from this group had the least number of active cholinergic neurons. The studies with physostigmine thus indicated that the most severe functional damage occurred in cases of AGS. These findings confirm that extensive damage occurs in the enteric neurons in equine grass sickness. There was good correlation between the functional cholinergic responses and the extent of neuronal degeneration.

Focal heat stimulation for the determination of the minimum alveolar concentration of halothane in the rabbit.

A focal heat stimulus of 54.37 +/- 0.07 (SD) degrees C was applied for 30 s to the inner aspect of the pinna of the ear for the determination of the minimum alveolar concentration (MAC) of halothane in New Zealand White rabbits. The latency before head movement was measured electromanometrically. The MAC value was 1.05 +/- 0.09 (SD)%. Other physiological responses occurred inconsistently and could not be used as reliable end points for the determination of the MAC in the rabbit.

In vitro responses to noradrenaline of small intestine taken from normal and grass sickness-affected horses.

Small intestine was taken from the caudal flexure of the duodenum and the terminal ileum proximal to the ileocaecal fold of 25 horses, 9 with acute grass sickness (AGS), 12 with subacute grass sickness (SAGS) and 12 with chronic grass sickness (CGS). The motility in the samples was measured isometrically either within 1 h of death or after storage for 24 h at 4 degree C. In control tissue, noradrenaline produced contractions of muscle strips which did not involve a muscarinic cholinergic mechanism and which were unaffected by the alpha 1 antagonist prazosin but were blocked by the alpha 2 antagonist yohimbine. Pretreatment with the alpha antagonist phentolamine prevented the contractile response to noradrenaline and the background contractions either continued at a reduced rate and amplitude or were abolished after a few minutes. Thus, following alpha blockade, noradrenaline reduced the background contraction rate by an effect on inhibitory beta adrenoceptors. The rate of background contractions in duodenal preparations was significantly greater than that in control ideal preparations. Although cold storage for 24 h caused a reduction in the background contraction rates of the control preparations, there was no effect on the contractile responses to noradrenaline, the associated pharmacology being similar to that of fresh tissue. This suggests that noradrenaline-evoked contraction was not dependent on enteric neural elements. The response to noradrenaline by grass sickness-affected tissue was generally similar to that of tissue from control horses, with an immediate contraction which was alpha 2 sensitive. The contractile response to noradrenaline after propranolol was significantly reduced in the CGS group and there were significant differences between the AGS, CGS and control groups. There was a significant difference between the ileal preparations from the control and SAGS groups in their response to noradrenaline following pretreatment with propranolol.

Afferent activity in the superior spermatic nerve of lambs--the effects of application of rubber castration rings.

Electrophysiological techniques were used to record afferent activity in the superior spermatic nerves of young lambs under general anaesthesia. Receptive fields were identified in the pampiniform plexus and the deep tissue of the testis in response to mechanical stimulation. Application of a standard rubber castration ring to the scrotal neck evoked vigorous afferent activity, including some from formerly silent units with receptive fields particularly in the pampiniform plexus. Some of this multi-unit discharge adapted rapidly within 10 s of the application of the ring and was followed by a discharge pattern which decayed exponentially over 90 min. The rate of decay of this discharge showed more than one exponent (time constant) with inflections at approximately 90 s and 16 min. After the application of the castration ring, quantitatively controlled scrotal compression continued to excite receptors, though a declining frequency was recorded over the period of observation. It was concluded that: (a) rubber castration rings initiated afferent activity which persisted for periods in excess of 90 min, a time course which is similar to the behavioural and humoral changes in the conscious animal; (b) both standard and small rubber castration rings were ineffective in rapidly producing neuronal pressure block of the slowly conducting afferent fibres in the superior spermatic nerve; (c) intra-testicular injection of local anaesthetic rapidly blocked afferent fibres running in the superior spermatic nerve.

Detection of oestrous-related odour in ewe urine by rams.

The ability of rams to discriminate between urine odour of oestrous ewes and urine odours from ewes at other days of the oestrous cycle was determined using operant conditioning techniques. Rams could discriminate between the odour of urine of oestrous ewes and the odours of urine from ewes at day 6 to day 1 before oestrus and from ewes at day 4 to day 10 after oestrus. Rams did not discriminate between odours of urine samples from different ewes in oestrus, or between urine odour of oestrous ewes (day 0) and urine odours from ewes at day 1 to day 3 after oestrus. These results support the hypothesis that ewes in oestrus produce an odour in urine that is detectable by rams.

Vagal reflex inhibition of motility in the abomasal body of sheep by antral and duodenal tension receptors.

Vagally-mediated regulation of motility in the abomasal body by duodenal and abomasal antral motility was demonstrated in acutely prepared anaesthetized sheep. The enteric plexuses between the abomasal body, antrum and duodenum were interrupted by transection. Antral contractions were more effective than duodenal contractions at causing inhibition of the abomasal body, and antral isometric conditions were more effective than antral isotonic conditions. Inhibition of motility in the abomasal body was reduced by unilateral cervical vagotomy, was abolished by bilateral cervical vagotomy, and was reversibly inhibited by cervical vagus cold block. The demonstration of vagal pathways in abomaso-abomasal reflexes confirms a functional homology of the mechanisms in the ruminant and animals with simple forms of stomach.

Cholinergic activity of intestinal muscle in vitro taken from horses with and without equine grass sickness.

Equine grass sickness (EGS) is a pan-dysautonomia of horses that involves central and peripheral neuronal degeneration and ultimately depletion. This is the first reported functional study on the motility of equine intestine taken immediately post mortem from horses with EGS. Strips of smooth muscle from the small intestine of healthy and EGS-affected horses were suspended in an organ bath and their motility was measured isometrically. The activity of the cholinergic system was studied. Physostigmine enhanced the motility of all muscle strips. Tissues taken from horses suffering from acute grass sickness (AGS) had the longest latency before a measurable response could be obtained (p < 0.05). The ileum appeared to be damaged by EGS to a greater extent than the duodenum. For the duodenal strips the enhanced rate of spontaneous contractions was significant (p < 0.05) for both normal tissue and that affected by grass sickness but this was not the case for the ileal strips. Muscarinic receptor sensitivity investigation using bethanecol suggested a hypersensitivity of receptors with AGS material.

Electrophysiological characteristics of tension receptors in the abomasal antrum of sheep.

Unitary impulse activity from tension receptors in the abomasal antrum in sheep anaesthetized with chloralose was studied with concurrent measurements of isometric tension in the antral wall, intraluminal pressures and electromyography (EMG). Rhythmical background activity had a close correlation with intraluminal pressure changes and usually a close correlation with local increases in EMG activity and isometric tension. Impulse frequencies increased, but were not sustained, following changes from isotonic to isometric conditions. Tension receptors were excited by transmural compression, by antral distension and by local intra-arterial injections of acetylcholine (2.5-25 micrograms), pentagastrin (10-20 micrograms), 5-hydroxytryptamine (1-20 micrograms), adrenaline (10 micrograms) cholecystokinin-8 (1-5 micrograms) or bradykinin (0.5-20 micrograms), and by systemic injections of bethanecol (5-1000 micrograms). Local intra-arterial perfusions of atropine (50 micrograms) and hexamethonium (100 micrograms) did not abolish spontaneous antral movements or affect the enhanced impulse activity evoked by acetylcholine or 5-hydroxytryptamine. Most antral tension receptors were excited by increases in antral tension or passive distension; they therefore functioned 'in series' with the muscularis externa. The discharges of three tension receptors were 'off-loaded' early during the contraction evoked by local injections of pentagastrin (10-20 micrograms).

A mechanical stimulator for the determination of the minimum alveolar concentration (MAC) of halothane in the rabbit.

The minimum alveolar concentration (MAC) of halothane was determined in New Zealand White rabbits. Tracheal anaesthetic concentrations were measured using a Siemens Servo Gas Monitor. A stimulator was used to deliver precisely controlled mechanical stimuli for the determination of MAC. Movement of the rabbit's head was recorded using a force transducer attached to the teeth. Evidence is presented that for the determination of MAC a precise nociceptive threshold is preferable to the so-called supramaximal stimulus used in clinical anaesthesia and in determination of anaesthetic potency. We conclude that techniques for the determination of MAC which disregard either sensitization of sensory mechanisms by producing tissue inflammation or the possibility of nerve compression by severe mechanical stimuli are of questionable value. The use of the mechanical stimulator described, or a similar device, would help in the standardization of the determination of MAC in all species by facilitating the application of a force of controlled amplitude, duration and velocity, thereby removing some of the variables which confound comparative studies of MAC.

An excitatory body-antral reflex in the sheep abomasum.

This paper describes an abomasal body-antral excitatory reflex in acute experiments in sheep anaesthetized with chloralose. The abomasum was either surgically intact or acutely transected to form separate compartments of the abomasal body and antrum. Inflation of a balloon situated in the abomasal body and active contraction of the abomasal body increased the amplitude of antral contraction. In the intact preparation, inflation of the abomasal body increased the amplitude of antral contraction before and after extrinsic denervation. The antral response was reduced by 75% after extrinsic denervation. In the acutely transected preparation, inflation of the abomasal body and electrically induced contraction of the abomasal body also increased antral contraction amplitude. The antral response was abolished by bilateral section of the cervical vagus nerves in five of six experiments. Selective extrinsic neurotomy of either compartment abolished all responses. It was concluded that the neural mechanisms controlling abomasal motility in the sheep resemble those found in species with simple stomachs: excitation of in-series tension receptors of the abomasal body causes an increase in antral contraction amplitude by vago-vagal reflex pathways. Antral motility is also modulated by changes of tension in the abomasal body by intrinsic reflex pathways and reflexes involving abdominal preganglia may also be present. In a restricted number of experiments the splanchnic nerve was shown to play an important role.

The nervous control of intraluminal pressure in the vomeronasal organ of the domestic ram.

In order to examine whether an aspiration mechanism exists in the vomeronasal organ (VNO) of the domestic ram, experiments were conducted in chloralose-anaesthetized animals using acute electromanometry and electrophysiological techniques. Stimulation of the ipsilateral cervical sympathetic nerve activated a mechanism producing a negative (with respect to atmospheric pressure) intraluminal pressure which drew fluid into the VNO from the nasopalatine canal. The mechanism was under alpha-adrenergic control and did not seem to involve the carotid blood supply. An ipsilateral increase in intraluminal pressure occurred following electrical excitation of the maxillary trigeminal innervation. Thus a mechanism was demonstrated in the ram for conveying odours to the putative olfactory receptors in the VNO.

Motility of the oesophagus and gastro-oesophageal junction during halothane anaesthesia in sheep.

Electromanometry and electromyography were used to study gastro-oesophageal motility in two planes of halothane anaesthesia in sheep. Gastro-oesophageal motility when present was greater in light than in deep anaesthesia. The caudal thoracic oesophagus contracted more frequently and for longer than the rostral thoracic oesophagus. In light anaesthesia oesophageal movements were peristaltic in direction with a propagation velocity of 26-29 cm sec-1. Rumen pressures increased throughout anaesthesia and the rate of increase was greatest when the plane of anaesthesia was deep at the start. Gastro-oesophageal reflux (GOR) occurred in both planes of anaesthesia and must occur by passive mechanisms during deep anaesthesia because gastro-oesophageal motility was inhibited. A high pressure zone (HPZ) was demonstrated for a length of 2.9 cm at the gastro-oesophageal junction with a balloon-tipped catheter and a 'pull through' technique. Open-tipped catheters could detect the HPZ but were less sensitive. The pressure in the HPZ was not significantly influenced by the depth of anaesthesia used. In 80% of cases of light anaesthesia an increase in HPZ pressure preceeded the contraction of the cranial sac of the rumen. In deep anaesthesia the HPZ continued to have rhythmic changes in tone. Spontaneous GOR coincided with a maximum gastro-oesophageal pressure gradient in 24% of cases. Rumen insufflation with oxygen provoked GOR at a rumen pressure above 33 mmHg compared with 7.2 mmHg during spontaneous reflux. The study demonstrates that a gastro-oesophageal pressure gradient was not primarily responsible for the initiation of GOR during anaesthesia and that the HPZ at the gastro-oesophageal junction of sheep had some of the properties of a lower oesophageal sphincter and played an important role in the initiation of GOR during anaesthesia.