Enteric dysganglionosis resembling intestinal neuronal dysplasia in a foal with bacterial colitis.

A 5-day-old quarter horse colt with a history of hypothermia, agonal breathing, and diarrhea was euthanized. At necropsy, numerous slightly raised, discrete, closely approximated submucosal nodules were observed in the colon and small intestine. Histologically, these nodules were composed of expanded submucosal mesenchyme that contained numerous neurons either individually or in ganglia. Thirty-two percent of these ganglia included 8 or more neurons, in contrast to 6% in an age-matched foal. Some nodules had necrosuppurative inflammation with vasculitis, thrombosis, and bacterial colonization. A few heterotopic neurons were randomly distributed in the mucosa and the muscularis mucosa. Histologic changes were most consistent with intestinal neuronal dysplasia, a disease of the submucosal plexus described in humans.

Differential effects of adult and perinatal lead exposure on morphine-induced locomotor activity in rats.

The effects of adult and perinatal lead treatment on the development of locomotor sensitization produced with repeated morphine administration was investigated. In Experiment 1, adult male rats received a diet containing 250 ppm lead acetate or a control diet for 43 days. Animals then received 10 mg/kg morphine sulfate or water vehicle (ip) and locomotor activity was monitored for 14 consecutive days. While both control and lead-exposed animals demonstrated a locomotor sensitization to morphine, the magnitude of the increased locomotor response was reduced in lead-treated animals. Subsequent analysis of blood-lead in the adult lead-exposed animals indicated residue levels ranging between 20 and 30 microg/dl. In Experiment 2, adult female rats were treated daily with 0, 8, or 16 mg lead via gavage for 30 days before breeding with non-exposed males. Lead exposure in dams continued through gestation and until pups were weaned at postnatal day (PND) 21. At PND 60, male offspring received morphine or vehicle challenges identical to those described in Experiment 1. Animals perinatally exposed to dams receiving 16 mg lead daily demonstrated an enhanced behavioral response to morphine relative to control animals. Analysis of offspring blood indicated lead levels below detectable limits (<1 microg/dl) for all animals. The results suggest exposure to lead at environmentally relevant levels produces long-lasting changes in drug-induced behavior, and the developmental period in which lead exposure occurs is a significant contributor to the manifestation of these effects.

Dietary cadmium exposure alters characteristics of training, substitution, and tolerance when morphine is used as a discriminative stimulus.

This study examined the possibility that cadmium, a toxicant in high concentration in all tobacco products, may alter the stimulus properties of morphine. Adult male rats were exposed to regular laboratory chow (Group Control) or chow containing 100 ppm added cadmium chloride (Group Cadmium). Following an initial 30 day exposure period, control and cadmium-exposed animals were trained to discriminate between i.p. injections of 3.00 mg/kg morphine sulfate and vehicle (distilled water) in a two-choice drug discrimination task. Subsequently, the morphine dose-effect generalization function (0.75-6.00 mg/kg) was determined for control and cadmium-exposed animals. Additional substitution tests were conducted with increasing doses of the high efficacy mu agonist fentanyl (0.0016-0.04 mg/kg), the intermediate efficacy mu agonist (-)-metazocine (0.60-5.00 mg/kg), and the kappa agonist (+/-)-bremazocine (0.03-0.12 mg/kg). Also, increasing doses of the selective mu antagonist naloxone (0.0008-0.50 mg/kg) were presented against the training dose of morphine (3.00 mg/kg) and 0.02 mg/kg fentanyl. Finally, training was discontinued, and control and cadmium-exposed animals were injected with 8.00 mg/kg morphine in the home cage every 12 hr for 2 weeks, prior to redetermining the morphine dose effect function. Following a 1 week recovery period where morphine injections were discontinued, a final determination of the morphine dose-effect function was made. The results of the investigation indicated that cadmium exposure, without affecting the rate-changing properties of the drugs, slowed initial acquisition of the morphine discrimination, decreased the potency of selective doses of naloxone with respect to antagonizing the stimulus effects of morphine and fentanyl, and blocked the development of tolerance to morphine. Morphine, fentanyl, and (-)-metazocine generalized (substituted) equally across both groups, while (+/-)-bremazocine failed to substitute for the morphine stimulus in either group. These findings add to the growing literature on the interaction between metal poisoning and drug selection/abuse.

Differential ability of astroglia and neuronal cells to accumulate lead: dependence on cell type and on degree of differentiation.

The apparent ability of astroglia to serve as a lead (Pb) sink in the mature brain may result from either their strategic location, between the blood-brain barrier and neurons, or from intrinsic differences between the ability of astroglia and neurons to accumulate this metal. This phenomenon may be dependent on the degree of cell differentiation. In order to address the latter possibility, Pb accumulation was compared among the following cell culture models: (1) mature and immature rat astroglia, (2) undifferentiated SY5Y human neuroblastoma cells and SY5Y cells differentiated with nerve growth factor, (3) immature rat astroglia grown in differently conditioned media, some of which induce partial differentiation, and (4) rat astroglia and SY5Y cells in co-culture. Astroglial cultures, prepared from 1-day-old rat cerebral hemispheres, were exposed to 1 microM Pb after either 14 (immature) or 21 (mature) days in culture. Pb content of the cells was measured by atomic absorption spectroscopy. Immature astroglia took up less Pb when glutathione (GSH) was added to the medium, suggesting that GSH may regulate Pb uptake in these cells. Undifferentiated neuroblastoma cells accumulated more Pb than did the differentiated ones. Astroglia accumulated up to 24 times more Pb than did neuronal cells. This ability was enhanced by exposure to conditioned medium from a neuroblastoma cell line, but not by endothelial cell-conditioned medium, although this medium induced the expression of a glutamate-activated Ca2+ response. Our findings are in agreement with in vivo studies, and thus validate the use of these cell-culture models for future studies on differential mechanisms of Pb uptake.

Effect of lead exposure and accumulation on copper homeostasis in cultured C6 rat glioma cells.

C6 rat glioma cells resemble rat astroglia in culture in that both cell types accumulate lead (Pb) intracellularly from the medium. As such, C6 cells are a model for Pb accumulation by the brain. In this study, an increase in intracellular Pb accumulation induced by p-chloromercuribenzoate (PCMB) after exposure to 10 microM Pb acetate suggests a role for sulfhydryl groups in Pb retention. Stimulation of Pb accumulation by nifedipine suggests the entry of Pb into these cells by a novel path. Most of the intracellular Pb from exposure for 7 days to 1 microM Pb was associated with high-molecular weight components in cytosol. Pb exposure increased the abundance of three proteins with the following characteristics on two-dimensional gels: 81 kDa with pI of 5.6, 81 kDa with pI of 4. 9, and 71 kDa with pI of 5.6. The levels of five other proteins, ranging in size from 37-41 kDa with pIs of 6.0-6.8 declined. Exposed C6 cells accumulated copper (Cu) intracellularly, and Cu accumulation after Pb exposure was shown by kinetic analysis with 67Cu to result from an increased uptake and a decreased efflux for Cu. Pb-exposed cells also showed increased Cu binding to membranes, which is consistent with the increase of Cu uptake. These data indicate that intracellular Pb interacts with high molecular weight proteins in C6 cells, and exposure also alters membrane transport properties for copper.

Brain and plasma levels of cocaine and benzoylecgonine in lead-expose and cadmium-exposed rats following acute or chronic intraperitoneal administration of cocaine.

Previous investigations of metal/cocaine interactions have shown that chronic oral exposure to inorganic lead or cadmium attenuates the psychoactive effects of acute or repeated administration of cocaine. The purpose of this investigation was to assess the possibility that such interactive effects may derive from metal-induced disturbances in cocaine pharmacokinetics, i.e., delivery of cocaine to critical biologic sites may be disrupted by metal contamination. In this study, adult male rats were exposed to purified diets containing 250 ppm lead acetate (Group Lead), 100 ppm cadmium chloride (Group Cadmium), or unadulterated laboratory chow (Group Control); n = 48/exposure condition. Following ad libitum access to their respective diets in the home cage for 45 days, half the animals from each exposure regimen received single daily IP injections of 5, 10, or 20 mg/kg cocaine HCl for a period of 7 days (n = 8/group). The remaining half the animals received repeated daily injections of saline during this pretreatment phase. On the day following pretreatment, animals previously receiving cocaine injections were administered a single cocaine test challenge at a dose equal to that received in pretreatment. Similarly, saline pretreatment animals received either 5, 10, or 20 mg/kg cocaine. The results of this investigation did not reveal reliable evidence of metal-related differences in brain levels of cocaine. Plasma cocaine and benzoylecgonine (BE) levels also were essentially the same for control and metal-exposed animals. The failure to show that lead or cadmium alters the disposition of cocaine in brain or plasma underscores the need to pursue alternative accounts of metal/cocaine interactions.

Cadmium exposure attenuates the initiation of behavioral sensitization to cocaine.

Employing a paired-watering procedure to control for differential fluid intake confounds, adult male rats were exposed in the home cage to water containing 100 ppm cadmium chloride, or a control solution containing no added cadmium chloride. On Day 61 of exposure to their respective watering regimens, half the animals from each condition received 12 repeated daily i.p. injections of 10 mg/kg cocaine-HCl, or saline. Locomotor activity (total distance traveled) was recorded in Digiscan Activity Monitors for a 20-min baseline period prior to each injection, and for a 40-min period post-injection. On Day 13 of testing, all animals received saline injections only in the test chambers, in an effort to evaluate the role of conditioned cues in the expression of cocaine sensitization. On Day 14-16 of testing, all animals received successive daily challenges of 10, 20, and 40 mg/kg cocaine in the test chamber. The results indicated that the initiation (development) of behavioral sensitization to 10 mg/kg cocaine was attenuated in cadmium-exposed rats. Moreover, the supersensitivity to higher doses of cocaine during dose-effect testing that was registered by control animals pretreated with cocaine, was not evident in cadmium-exposed pretreatment animals. These data suggests that environmental contaminants may alter drug responsiveness, and thereby may influence patterns of drug selection and use.

The effects of cadmium exposure on ethanol pharmacokinetics.

Twenty-four adult male rats were exposed in the home cage to water containing 100 ppm added cadmium chloride. An additional 24 animals were pair-watered with water containing no added cadmium. Following 60 days of exposure to their respective watering regimens, one third of the animals in each exposure group (N = 8/condition) received IP injections of 1.0, 2.0, or 3.0 g/kg ethanol (20% v/v). Serum alcohol concentrations were measured at 15, 30, 60, 120, 180, 240, and 360 min postinjection. Although serum alcohol concentrations increased with dose for both cadmium-exposed and control animals, there was no indication at any dose of group differences. The lack of differences in ethanol pharmacokinetics reported here is instructive with respect to improving our understanding of the mechanisms underlying cadmium/ethanol interactions.

Lead (Pb) alters the norepinephrine-induced secretion of luteinizing hormone releasing hormone from the median eminence of adult male rats in vitro.

In the present study, we evaluated the in vitro effects of lead (Pb) on basal and stimulated luteinizing hormone releasing hormone (LHRH) and Prostaglandin E2 (PGE2) secretion. Median eminences (ME) were removed from brains of adult male rats and preincubated for 15 minutes in Krebs-Ringer bicarbonate glucose buffer in an atmosphere of 95% O2-5% CO2. These media were discarded and all MEs were subjected to one of the following experiments. In Experiment 1, all MEs were incubated for 30 minutes in medium only. These media were collected and replaced with medium only (controls) or with medium containing Pb doses ranging from 5 to 20 microM. After this 60-minute incubation, media were collected, then replaced with new medium containing 60 microM norepinephrine (NE), or NE plus each dose of Pb, then incubated for a final 30-minute period. Experiment 2 was conducted as above, except PGE2 (2.8 microM) replaced the NE. In both experiments, the amounts of LHRH released was measured by RIA. In experiment 3, NE was again used for the challenge; however, this time, the amount of PGE2 released was measured by RIA. Results indicate that Pb did not alter basal LHRH release, but compared with controls, significantly blocked NE-induced LHRH release in a dose-related manner. Conversely, Pb had no effect on the PGE2-induced release of LHRH. Additionally, Pb did not alter basal PGE2 release; however, it significantly blocked the NE-induced release of PGE2. Since NE-induced LHRH release is mediated by PGE2, these results support the hypothesis that Pb is capable of altering the hypothalamus and suggest that this effect is due, at least in part, to the diminished PGE2 synthesis/release within the ME, resulting in diminished LHRH secretion.

The effects of cadmium on ethanol self-administration using a sucrose-fading procedure.

Rats were exposed for 70 days to either a diet containing 100 ppm cadmium (Group Cadmium) or a control diet with no additives (Group Control). Subsequently, all animals were trained to lever press for a 20% sucrose solution. Across several phases, sucrose was faded out as the reinforcer and gradually replaced with a 10% ethanol solution. A subsequent operant choice (concurrent) test, during which pressing one lever resulted in a dipper presentation of ethanol and the other lever provided water, was followed by a single-lever test where sucrose was reinstated as the reinforcer. The results showed that although cadmium-treated rats lever pressed more than controls during the early phases of the sucrose-fading procedure, animals exposed to cadmium lever pressed at lower rats than controls for ethanol during the concurrent test. There were no group differences on the final sucrose test. The possibility that cadmium may alter sensitivity to ethanol is discussed.

Behavioral antagonism between lead and cadmium.

Adult male rats were exposed to one of four dietary conditions for a period of 60 days. Group Control-Diet received a diet containing no added lead or cadmium, group Lead-Diet received a diet that contained 500 ppm added lead, group Cadmium-Diet received a diet that contained 100 ppm added cadmium, and group Lead-Cadmium-Diet received a diet that contained both 500 ppm added lead and 100 ppm added cadmium. Subsequent to exposure, animals were tested in a Digiscan activity monitor. Animals were then sacrificed and metal concentrations were determined in blood and brain. The results from this experiment showed that lead alone increased movement and vertical activity. Cadmium alone decreased movement and increased rest time. Cotreatment with lead and cadmium failed to produce behavioral differences relative to controls; thus, it seems that the changes in activity caused by one metal are antagonized by the other. Results from the analyses of residues in tissues revealed that blood lead concentrations were lower in the cotreatment condition than the lead along condition. However, brain residue accumulations were not different for these two exposure conditions. There was no evidence that the presence of lead attenuated increases in cadmium residues in blood or brain. Overall, the residue data agree with a central, as contrasted with a peripheral, account of lead/cadmium interaction effects, at least as relates to behavior. Because lead and cadmium were additive with regard to producing decreased body weights, it seems that the toxic effect of these metals is antagonized by cotreatment in some instances, and augmented in others.

The effects of naltrexone on cadmium-induced increases in oral ethanol self-administration.

Adult male rats were exposed to a standard laboratory diet (N = 20), or an adulterated diet containing 100 ppm added cadmium (N = 20), for 60 days. On Day 61, half the animals from each dietary condition received subcutaneous implants of two 30 mg naltrexone pellets, and the remaining half the animals received identical implants of 30 mg placebo pellets. One week later, animals from groups created by this interaction (Groups Control-Placebo, Control-Naltrexone, Cadmium-Placebo, Cadmium-Naltrexone) were tested in an ethanol self-administration paradigm that presented a 10% ethanol solution (v/v) in both a choice and nonchoice format. The results indicated that cadmium exposure increased the oral self-administration of ethanol in the choice setting where water was offered as an alternative, and the opiate antagonist naltrexone failed to attenuate this effect.

Effects of combined lead and cadmium exposure: changes in schedule-controlled responding and in dopamine, serotonin, and their metabolites.

Adult male rats were maintained on 1 of 4 ad-lib diets: Group Control-Diet received a normal laboratory diet that contained no added chemicals: Group Lead-Diet received a diet containing 500 ppm (parts per million) lead: Group Cadmium-Diet received a diet containing 100 ppm cadmium: and Group Lead-Cadmium-Diet received a diet containing both 500 ppm lead and 100 ppm cadmium. After 60 days of exposure to their respective diets, animals were placed on restricted diets (15 g/day) of the identical food received during the exposure period. Each animal was trained to lever press on a fixed-interval 1-min schedule for 21 sessions (1 session day). The results of schedule training showed that lead alone or cadmium alone was associated with increased lever pressing relative to control diet. However, when lead and cadmium were exposed jointly, performance was not significantly different from control performance. Similar attenuation of effects were observed for central neurotransmitter functions. Specifically disturbances in dopamine and serotonin turnover that were produced by lead alone were attenuated by the cotreatment of cadmium and lead. Possible accounts of the apparent antagonism between cadmium and lead are discussed.

The effects of cadmium on the self-administration of ethanol and an isocaloric/isohedonic equivalent.

During a period of baseline fluid intake recording, adult male rats were presented with a three-bottle, two-fluid choice test that offered either a 10% ethanol solution (v/v) and tap water as alternatives, or a sucrose/quinine solution and tap water as alternatives. The sucrose/quinine solution was equivalent to the ethanol solution both in terms of calories and palatability. After intakes stabilized, half of the animals from each test condition were placed on a diet containing 100 ppm cadmium and the remaining half of the animals were placed on a standard laboratory diet. After 60 days of exposure to their respective diets, all animals were presented their earlier test solutions, both in a nonchoice and choice format. The results from the choice test indicated that although cadmium treatment did not produce a clear preference for ethanol over water, cadmium exposure was associated with a significant increase in ethanol consumption. Moreover, the self-administration of the isocaloric/isohedonic equivalent (sucrose/quinine solution) was unaffected by cadmium contamination. These data are discussed in terms of their implications for both nutritional and sensory-impairment accounts of metal-related changes in the volitional intake of ethanol.

Cadmium exposure results in decreased responsiveness to ethanol.

Adult male Sprague-Dawley rats were maintained on an ad lib diet containing 100 ppm cadmium (Group Cadmium-Diet) or a control diet with no added cadmium. On Day 61, all animals (N = 10/group) were challenged with a single hypnotic dose of ethanol (3.5 g/kg IP), prepared from a 20% v/v solution of tap water and a stock solution of 95% ethanol. The latency from the time of the injection until the loss of the righting reflex was recorded, as well as the latency for recovery of the reflex. The results showed a nonsignificant trend for animals exposed to cadmium to lose the righting reflex less rapidly than controls, and Cadmium-Diet animals regained the righting reflex significantly more rapidly than controls. These findings suggest that the pharmacologic effectiveness of ethanol is altered by chronic exposure to dietary cadmium. The implications of these data for other studies of cadmium/ethanol interactions are discussed.

Effects of lead on viability and intracellular metal content of C6 rat glioma cells.

Cultured C6 rat glioma cells were exposed to lead (Pb) acetate (0, 1, 10, or 100 microM) for 3-4 d. Cells were analyzed for changes in viability and intracellular lead, iron, and copper concentrations after Pb treatment was discontinued. The results were compared with previous findings on astroglia and oligodendroglia in culture in order to evaluate C6 cultures as a model for Pb toxicity in glia. Viability was measured by three methods on the day Pb was removed from the cells (designated d 0), and 2 and 9 d after Pb treatment was discontinued (designated d 2 and 9). The methods used were trypan blue dye exclusion, total cell counts, and incorporation of [3H]-L-leucine into proteins. Small, dose-dependent reductions were observed on d 2 in the percentages of cells excluding dye. Decreased cell numbers were seen at all three Pb doses only on d 0. With respect to these two viability measurements, C6 cells responded like astroglia in culture to Pb, but not like oligodendroglia, which are more Pb-sensitive. We expected decreased amino acid incorporation to accompany the decreased viability of the cultures. Instead, increased amino acid incorporation, which indicates increased protein synthesis, was seen on d 0 and 2 at all three Pb doses, though total cellular protein did not increase. A similar response has been reported previously in oligodendroglial cultures. C6 cells treated for 3 with 1 or 100 microM Pb acetate were analyzed for intracellular metal content by atomic absorption aspectroscopy on d 4 and 11 after exposure to Pb was discontinued. The cells were found to take up large amounts of Pb intracellularly and store it for at least 11 d. Cells treated with FeCl2 instead of Pb took up Fe, but required a higher extracellular Fe concentration to achieve an intracellular Fe level comparable to that of Pb in Pb-treated cells. Pb uptake did not affect intracellular Fe or Cu concentrations. With respect to Pb and Fe uptake, C6 cells closely resembled immature astroglia in culture. Unlike C6 cells, however, astroglia showed elevations of intracellular Fe and Cu after Pb treatment. Thus, Pb effects on C6 cells resembled those on cultured oligodendroglia and astroglia in some respects but not in others. C6 cells appear to be an adequate model for selected events in glial toxicosis, such as Pb-stimulated protein synthesis in oligodendroglia and Pb uptake in astroglia, but not Pb-induced alterations of intracellular Cu and Fe in astroglia. Their use as a model for glial progenitor cells in Pb toxicity studies remains to be determined.

Ethanol self-administration in rats following exposure to dietary cadmium.

Rats were maintained on an ad lib diet containing 100 ppm cadmium (Group Cadmium-Diet) or a control diet with no added cadmium (Group Control-Diet). After 55 days of exposure to their respective diets, animals were tested for fluid intake using a nonchoice procedure that presented a 15% ethanol solution in the home cage for 5 days. Subsequently, all animals were offered a 10% ethanol solution or tap water in a 3-bottle, 2-fluid choice test in the home cage. This fluid intake test was conducted for a 5 day baseline period, and then again concurrently with avoidance acquisition (14 days) and extinction (4 days) training on a free operant (Sidman) avoidance task that required animals to lever press to avoid electric footshock. After training was terminated the choice test was continued further in the home cage for a 15 day post-avoidance period. Ethanol intake was greater for animals exposed to cadmium on all tests of fluid consumption, and all animals consumed more ethanol during the periods following termination of the stressor (avoidance extinction, post-avoidance) than during the actual period of stress (avoidance acquisition). Interpretive comments focus on the effects of cadmium on stress reactivity, sensory processing, and metabolism.

Effects of lead treatment on intracellular iron and copper concentrations in cultured astroglia.

Astroglia are implicated in the pathogenesis of lead (Pb) neurotoxicity in two capacities: as a lead sink that sequesters lead and as a target for direct cellular damage. A proposed cellular mechanism of Pb neurotoxicity is the alteration of metal concentrations, particularly the intracellular accumulation of Cu2+. We measured Pb uptake and the effects of Pb acetate on intracellular trace metal concentrations in astroglial cultures prepared from 0- to 4-day-old rat cerebral hemispheres. Mature Sprague Dawley and immature Wistar rat astroglia in culture took up lead from the medium. This finding replicates in vitro the finding reported by others that astroglia in the brain take up Pb. Intracellular Cu and Fe concentrations (micrograms per 2 x 10(6) cells) were increased fourfold or more by treatment with 100 microM Pb for 3 days in the cultures of immature astroglia. Cu levels were also increased twofold or more in mature astroglia treated for 1-3 days with 100 microM Pb. The significance of this finding is that Cu is a potent inhibitor of Na+, K+-ATPase, an enzyme by which astroglia are thought to remove K+ from the extracellular fluid in the brain. Thus, this finding supports the hypothesis that elevated [Cu], and perhaps [Fe], is a subcellular mechanism of neurotoxicity.

Cellular targets of lead neurotoxicity: in vitro models.

Four types of cells in culture were exposed to lead (Pb) acetate (0.1-1000 microM): astroglia-enriched, oligodendroglia-enriched, and meningeal fibroblast cultures prepared from neonatal rat brains; and human neuroblastoma cultures prepared from the SK-N-SH-SY5Y cell line. The viability (trypan blue dye exclusion and proliferation) of these cell types after Pb exposure was compared in order to identify cellular targets in the central nervous system that were directly susceptible to cytotoxicity. Of the 4 cell types tested, only oligodendroglia showed marked sensitivity to Pb treatment. However, proliferation of the SY5Y cells was temporarily inhibited if the cells were treated 1 day (but not 3 days) after seeding. The potential for thiamin, which is used to treat Pb intoxication in cattle, to prevent this effect was tested. Rather than preventing this toxic effect, however, thiamin (1 mM) exacerbated that inhibition of proliferation. Astroglia and meningeal fibroblasts, which were resistant to Pb toxicity, were shown by atomic absorption analysis to take up Pb from the culture medium and concentrate it intracellularly to at least 55X the extracellular concentration, thus supporting hypotheses that these cells act as Pb sinks in the brain.

Pudendal and caudal rectal nerve blocks in the horse - An anesthetic procedure for reproductive surgery.

The pudendal and caudal rectal nerves in four male and five female adult crossbred horses were anesthetized with a local solution. The injection site was located at the foramen for the caudal gluteal artery and vein in the sacrosciatic ligament. Twenty milliliters of local anesthetic solution were injected via a 15-cm, 18-gauge needle. Quantitative data on anesthesia were determined from these injections. Dye was injected with the anesthetic in four additional horses so that accurate placement of the solution could be determined at postmortem examination. Satisfactory anesthesia of the anus, perineum, and vulva in the mare and of the anus, perineum, glans penis and penile layer of the prepuce in the male was achieved by placement of anesthetic near the foramen for the caudal gluteal vessels in the sacrosciatic ligament. Penile extrusion also occurred.