Influence of digital hypothermia on lamellar events related to IL-6/gp130 signalling in equine sepsis-related laminitis.

BACKGROUND: Interleukin-6 (IL-6) is consistently increased in the digital lamellae in different studies of sepsis-related laminitis (SRL). IL-6 signalling through the gp130 receptor activates similar signalling (i.e. mTORC1-related signalling) previously reported to be activated in models of endocrinopathic laminitis. OBJECTIVES: To assess the activation state of signalling proteins downstream of IL-6/gp130 receptor complex activation in an experimental model of SRL. STUDY DESIGN: Randomised experimental study. METHODS: Lamellar phospho-(P) protein concentrations downstream of the IL-6/gp130 receptors were assessed in the oligofructose (OF) model of SRL. Fifteen Standardbred horses were administered water (CON, n = 8) or oligofructose (OF, n = 7) via a nasogastric tube. At 12 h post-OF/water administration, one randomly assigned forelimb was exposed to continuous digital hypothermia (CDH) by placement in ice water (ICE, maintained at <7°C); the other forelimb was maintained at ambient temperature (AMB). Lamellar tissue samples were collected after 24 h of CDH from both ICE and AMB forelimbs and immediately snap-frozen. Lamellar proteins of interest were assessed by immunoblotting and immunofluorescence. RESULTS: Immunoblotting revealed increase (P<0.05) in the phosphorylation states of Akt (Ser 473), RPS6 (Ser235/236), RPS6 (Ser240/244), STAT3 (Ser727) and STAT3 (Tyr705) in lamellar tissue from OF-treated animals (AMB OF vs. AMB CON limbs); CDH resulted in decreased (P<0.05) lamellar concentrations of phosphorylated Akt, p70S6K, RPS6 (235/236), RPS6 (240/244) and STAT3 (S727) in OF-treated animals (AMB OF vs. ICE OF). Immunofluorescence showed that activated/phosphorylated forms of RPS6 and STAT3 were primarily localised to lamellar epithelial cells. MAIN LIMITATIONS: The nature, sequence and timing of sub-cellular events in this experimental model may differ from those that accompany naturally occurring sepsis. CONCLUSIONS: There were increased lamellar concentrations of activated signalling proteins downstream of the IL-6/Gp130 receptor complex in OF-treated horses; CDH inhibited this activation for the majority of the proteins assessed. These results demonstrate similar lamellar signalling (e.g. mTORC1-related signalling) and, therefore, possible therapeutic targets occurring in sepsis-related laminitis as previously reported in models of endocrinopathic laminitis.

Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis.

REASONS FOR PERFORMING STUDY: Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs. OBJECTIVES: To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis. STUDY DESIGN: Experimental study. METHODS: Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05. RESULTS: Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover. CONCLUSIONS: Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.

Laminar inflammatory events in lean and obese ponies subjected to high carbohydrate feeding: Implications for pasture-associated laminitis.

REASONS FOR PERFORMING STUDY: Acute, massive enteral carbohydrate overload is associated with laminar inflammation in equids; it is unclear if the same is true for a more prolonged period of moderate dietary carbohydrate intake. OBJECTIVES: To characterise laminar inflammation in ponies exposed to a dietary carbohydrate challenge meant to mimic acute pasture exposure. STUDY DESIGN: In vivo experiment. METHODS: Mixed-breed ponies (n = 22) received a diet of hay chop (nonstructural carbohydrate [NSC] ∼7% on a dry matter [DM] basis) for 4 weeks prior to initiation of the experimental feeding protocol. Following dietary acclimation, ponies were stratified into either Lean (n = 11, body condition score [BCS] ≤4) or Obese (n = 11, BCS ≥7) groups and each group further stratified to either remain on the control, low NSC diet (n = 5 each for Obese and Lean) or receive a high NSC diet (hay chop supplemented with sweet feed and oligofructose, total diet ∼42% NSC; n = 6 each for Obese and Lean) for a period of 7 days. Laminar samples were collected following euthanasia and sections stained immunohistochemically for CD163, MAC387/calprotectin and cyclo-oxygenase-2 (COX-2) using commercially available antibodies. The number of CD163 (+) and MAC387(+) cells was quantified for each section; the distribution of COX-2 expression was qualitatively assessed. Laminar mRNA concentrations of several proinflammatory molecules (interleukin-1ß [IL-1ß], IL-6, tumour necrosis factor-α [TNFα], IL-8, IL-10, monocyte chemoattractant protein-1 [MCP-1], MCP-2), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), E-selectin, plasminogen activator inhibitor-1 (PAI-1) and COX-2 were evaluated using real-time quantitative polymerase chain reaction (qPCR). RESULTS: High carbohydrate feeding resulted in no increase in laminar proinflammatory cytokine expression; laminar COX-2 expression was increased by high carbohydrate feeding. No laminar leucocyte infiltration was observed in response to high carbohydrate feeding. CONCLUSIONS: These results suggest that the marked laminar inflammation observed in models of sepsis-associated laminitis may not play a central role in the pathophysiology of pasture-associated laminitis.

Effect of dietary nonstructural carbohydrate content on activation of 5'-adenosine monophosphate-activated protein kinase in liver, skeletal muscle, and digital laminae of lean and obese ponies.

BACKGROUND: In EMS-associated laminitis, laminar failure may occur in response to energy failure related to insulin resistance (IR) or to the effect of hyperinsulinemia on laminar tissue. 5'-Adenosine-monophosphate-activated protein kinase (AMPK) is a marker of tissue energy deprivation, which may occur in IR. HYPOTHESIS/OBJECTIVES: To characterize tissue AMPK regulation in ponies subjected to a dietary carbohydrate (CHO) challenge. ANIMALS: Twenty-two mixed-breed ponies. METHODS: Immunohistochemistry and immunoblotting for total AMPK and phospho(P)-AMPK and RT-qPCR for AMPK-responsive genes were performed on laminar, liver, and skeletal muscle samples collected after a 7-day feeding protocol in which ponies stratified on body condition score (BCS; obese or lean) were fed either a low-CHO diet (ESC + starch, approximately 7% DM; n = 5 obese, 5 lean) or a high-CHO diet (ESC + starch, approximately 42% DM; n = 6 obese, 6 lean). RESULTS: 5'-Adenosine-monophosphate-activated protein kinase was immunolocalized to laminar keratinocytes, dermal constituents, and hepatocytes. A high-CHO diet resulted in significantly decreased laminar [P-AMPK] in lean ponies (P = .03), but no changes in skeletal muscle (lean, P = .33; obese, P = .43) or liver (lean, P = .84; obese, P = .13) [P-AMPK]. An inverse correlation existed between [blood glucose] and laminar [P-AMPK] in obese ponies on a high-CHO diet. CONCLUSIONS AND CLINICAL IMPORTANCE: Laminar tissue exhibited a normal response to a high-CHO diet (decreased [P-AMPK]), whereas this response was not observed in liver and skeletal muscle in both lean (skeletal muscle, P = .33; liver, P = .84) and obese (skeletal muscle, P = .43; liver, P = .13) ponies.

Proinflammatory cytokine and chemokine gene expression profiles in subcutaneous and visceral adipose tissue depots of insulin-resistant and insulin-sensitive light breed horses.

BACKGROUND: Insulin resistance has been associated with risk of laminitis in horses. Genes coding for proinflammatory cytokines and chemokines are expressed more in visceral adipose tissue than in subcutaneous adipose tissue of insulin-resistant (IR) humans and rodents. HYPOTHESIS/OBJECTIVES: To investigate adipose depot-specific cytokine and chemokine gene expression in horses and its relationship to insulin sensitivity (SI). ANIMALS: Eleven light breed mares. METHODS: Animals were classified as IR (SI=0.58+/-0.31x10(-4) L/min/mU; n=5) or insulin sensitive (IS; SI=2.59+/-1.21x10(-4) L/min/mU; n=6) based on results of a frequently sampled intravenous glucose tolerance test. Omental, retroperitoneal, and mesocolonic fat was collected by ventral midline celiotomy; incisional nuchal ligament and tail head adipose tissue biopsy specimens were collected concurrently. The expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, plasminogen activator inhibitor-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) in each depot was measured by real-time quantitative polymerase chain reaction. Data were analyzed by 2-way analysis of variance for repeated measures (P<.05). RESULTS: No differences in TNF-alpha, IL-1beta, IL-6, PAI-1, or MCP-1 mRNA concentrations were noted between IR and IS groups for each depot. Concentrations of mRNA coding for IL-1beta (P=.0005) and IL-6 (P=.004) were significantly higher in nuchal ligament adipose tissue than in other depots. CONCLUSIONS AND CLINICAL IMPORTANCE: These data suggest that the nuchal ligament depot has unique biological behavior in the horse and is more likely to adopt an inflammatory phenotype than other depots examined. Visceral fat may not contribute to the pathogenesis of obesity-related disorders in the horse as in other species.

In vivo and in vitro evidence of the involvement of CXCL1, a keratinocyte-derived chemokine, in equine laminitis.

BACKGROUND: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans. OBJECTIVES: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis. ANIMALS: Twenty adult horses. METHODS: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression. RESULTS: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [P= .0001]) and DTP groups (21-fold [P= .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases (P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H(2)O(2) (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA (P= .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes. CONCLUSION AND CLINICAL IMPORTANCE: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.

Calprotectin in myeloid and epithelial cells of laminae from horses with black walnut extract-induced laminitis.

BACKGROUND: Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin. HYPOTHESIS: CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis. ANIMALS: Twenty adult horses. METHODS: Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed. RESULTS: Laminar epidermal CP signal was significantly increased (P= .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups (P= .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CP-positive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.

Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response.

REASONS FOR PERFORMING STUDY: Recent research has indicated that inflammation plays a role in the early stages of laminitis and that, similar to organ failure in human sepsis, early inflammatory mechanisms may lead to downstream events resulting in lamellar failure. Characterisation of the type of immune response (i.e. innate vs. adaptive) is essential in order to develop therapeutic strategies to counteract these deleterious events. OBJECTIVES: To quantitate gene expression of pro-inflammatory cytokines known to be important in the innate and adaptive immune response during the early stages of laminitis, using both the black walnut extract (BWE) and oligofructose (OF) models of laminitis. METHODS: Real-time qPCR was used to assess lamellar mRNA expression of interleukins-1beta, 2, 4, 6, 8, 10, 12 and 18, and tumour necrosis factor alpha and interferon gamma at the developmental stage and at the onset of lameness. RESULTS: Significantly increased lamellar mRNA expression of cytokines important in the innate immune response were present at the developmental stage of the BWE model, and at the onset of acute lameness in both the BWE model and OF model. Of the cytokines characteristic of the Th1 and Th2 arms of the adaptive immune response, a mixed response was noted at the onset of acute lameness in the BWE model, whereas the response was skewed towards a Th1 response at the onset of lameness in the OF model. CONCLUSIONS: Lamellar inflammation is characterised by strong innate immune response in the developmental stages of laminitis; and a mixture of innate and adaptive immune responses at the onset of lameness. POTENTIAL RELEVANCE: These results indicate that anti-inflammatory treatment of early stage laminitis (and the horse at risk of laminitis) should include not only therapeutic drugs that address prostanoid activity, but should also address the marked increases in lamellar cytokine expression.

Increased expression of MAIL, a cytokine-associated nuclear protein, in the prodromal stage of black walnut-induced laminitis.

REASONS FOR PERFORMING STUDY: The mediators and signalling cascades important in the initiation of laminitis remain unclear. We therefore wanted to explore the genes and overall signalling mechanisms that play an important role in the developmental stage of laminitis. OBJECTIVE: To use a broad genomic screening technique to identify novel genes that are differentially regulated in the equine lamellae during the developmental period of laminitis. METHODS: Differential mRNA display (DRD) was performed to discover regulated genes, and real-time quantitative polymerase chain reaction (RT-qPCR) was then used to evaluate lamellar mRNA levels of a regulated gene (MAIL) and mediators related to that gene (IL-1beta and IL-6) in control horses (n = 5) and horses administered black walnut extract (BWE; n = 5). RESULTS: Using DRD, MAIL was identified as a regulated gene. RT-qPCR indicated a 4-fold increase in expression of the MAIL mRNA in BWE lamellae compared to controls. A 30-fold increase in IL-1beta, and a 160-fold difference in IL-6 mRNA expression was present in BWE lamellae. Differences in MAIL, IL-1beta and IL-6 mRNA expression were statistically significant between groups (P < 0.05). CONCLUSIONS AND POTENTIAL RELEVANCE: The data strongly support a role for inflammatory cytokines in the developmental stages of laminitis, possibly inducing the vascular and metabolic alterations reported to occur in the affected digit. These results potentially support the use of anti-inflammatory drugs in horses at risk of laminitis, and warrant further investigation of the link between systemic disease processes associated with laminitis and the reported digital inflammation.

Laparoscopic ovariectomy using sequential electrocoagulation and sharp transection of the equine mesovarium.

OBJECTIVE: To describe in horses and ponies a laparoscopic ovariectomy technique facilitated by electrosurgical instrumentation. STUDY DESIGN: Elective ovariectomy was performed in 23 mares using laparoscopic electrosurgical instrumentation. ANIMALS OR SAMPLE POPULATION: Twenty-three mares (13 horses, 10 ponies), aged from 2 to 21 years and weighing 90 to 545 kg. METHODS: Food was withheld for a minimum of 12 hours. Mares were sedated with detomidine hydrochloride (0.02 to 0.03 mg/kg) or xylazine hydrochloride (0.5 to 1.0 mg/kg). Excluding the pony mares, all other mares were restrained in stocks. Portal sites in the paralumbar fossa region were desensitized with 2% mepivacaine. Abdominal insufflation was achieved through a teat cannula positioned in the ventral abdomen or a Verres-type needle placed through the paralumbar fossa. After trocar and laparoscope insertion, the ipsilateral ovary and mesovarium were identified, and the mesovarium, tubal membrane, and proper ligament were infiltrated with 2% mepivacaine. The mesovarium was coagulated using bipolar or monopolar electrosurgical forceps and transected sequentially from cranial to caudal until the ovary was completely freed and then removed. The contralateral ovary was removed in a similar fashion through the opposite paralumbar fossa. RESULTS: Bipolar and monopolar electrosurgical forceps were easy to use and provided adequate coagulation of vessels within the mesovarium. Two mares were euthanatized after the procedure for unrelated reasons. One mare had mild signs of colic 24 hours after ovariectomy. In 1 pony mare, the incision used to remove one ovary dehisced on the 5th postoperative day and was allowed to heal by second-intention. No long-term complications had occurred in 11 horses and 10 ponies, 6 to 24 months after surgery. CONCLUSION: Laparoscopic ovariectomy and hemostasis of the mesovarium can be easily accomplished using electrosurgical instrumentation. CLINICAL RELEVANCE: Standing laparoscopic ovariectomy, using electrosurgical instrumentation, is an effective and safe technique to provide hemostasis of the mesovarium in mares.

Investigation of mRNA expression of tumor necrosis factor-alpha, interleukin-1beta, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin.

OBJECTIVE: To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin- (IL)-1beta from cultured equine smooth muscle cells (SMC). SAMPLE POPULATION: Segments of palmar digital artery harvested from 6 clinically normal adult horses. PROCEDURE: Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 microg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-alpha, and IL-1beta was performed, using isolated total cellular RNA. RESULTS: Although no message was detected for IL-1beta or TNF-alpha in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1beta and TNF-alpha mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis.

Identification of a sex-specific quantitative trait locus mediating nonopioid stress-induced analgesia in female mice.

It is increasingly appreciated that the sexes differ in their perception of noxious stimuli and in their responsivity to exogenous and endogenous analgesic manipulations. We previously reported the existence of qualitative sex differences in the neurochemical mediation of nonopioid (i.e., naloxone-insensitive) stress-induced analgesia (SIA) produced by forced swims and suggested that female mice possess a sex-specific SIA mechanism. This female-specific system is now known to be estrogen-dependent, to be ontogenetically organized, and to vary with reproductive status; however, its neurochemical identity remains obscure. In an attempt to identify candidate genes underlying SIA in both sexes, we performed a two-phase quantitative trait locus (QTL) mapping experiment using the BXD/Ty recombinant inbred (RI) set derived from DBA/2J (D2) and C57BL/6J (B6) inbred mouse strains and (B6xD2)F2 hybrid mice derived from these same progenitors. All mice were subjected to 3 min forced swims in 15 degrees C water; nociceptive sensitivity on the 54 degrees C hot-plate assay was assessed immediately before and 2 min after cessation of the swim. We report the localization of a QTL statistically associated with SIA magnitude [p = 0.00000012; logarithm of the odds (LOD) = 6.1] in female mice only. This female-specific QTL, which we name Fsia1, is located on chromosome 8 at 52-84 cM from the centromere and accounts for 17-26% of the overall trait variance in this sex. The present data provide further evidence of the existence of a female-specific SIA mechanism and highlight the important role of both genetic background and gender in the inhibition of pain.

Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies.

Choosing the best genetic strains of mice for developing a new knockout or transgenic mouse requires extensive knowledge of the endogenous traits of inbred strains. Background genes from the parental strains may interact with the mutated gene, in a manner which could severely compromise the interpretation of the mutant phenotype. The present overview summarizes the literature on a wide variety of behavioral traits for the 129, C57BL/6, DBA/2, and many other inbred strains of mice. Strain distributions are described for open field activity, learning and memory tasks, aggression, sexual and parental behaviors, acoustic startle and prepulse inhibition, and the behavioral actions of ethanol, nicotine, cocaine, opiates, antipsychotics, and anxiolytics. Using the referenced information, molecular geneticists can choose optimal parental strains of mice, and perhaps develop new embryonic stem cell progenitors, for new knockouts and transgenics to investigate gene function, and to serve as animal models in the development of novel therapeutics for human genetic diseases.

Hypoxia selectively induces proliferation in a specific subpopulation of smooth muscle cells in the bovine neonatal pulmonary arterial media.

Medial thickening of the pulmonary arterial wall, secondary to smooth muscle cell (SMC) hyperplasia, is commonly observed in neonatal hypoxic pulmonary hypertension. Because recent studies have demonstrated the existence of multiple phenotypically distinct SMC populations within the arterial media, we hypothesized that these SMC subpopulations would differ in their proliferative responses to hypoxic pulmonary hypertension and thus contribute in selective ways to the vascular remodeling process. Expression of meta-vinculin, a muscle-specific cytoskeletal protein, has been shown to reliably distinguish two unique SMC subpopulations within the bovine pulmonary arterial media. Therefore, to assess the proliferative responses of phenotypically distinct SMC subpopulations in the setting of neonatal pulmonary hypertension, we performed double immunofluorescence staining on pulmonary artery cryosections from control and hypertensive calves with antibodies against meta-vinculin and the proliferation-associated nuclear antigen, Ki-67. We found that, although neonatal pulmonary hypertension caused significant increases in overall cell replication, proliferation occurred almost exclusively in one, the meta-vinculin-negative SMC population, but not the other SMC population expressing meta-vinculin. We also examined fetal pulmonary arteries, where proliferative rates were high and meta-vinculin expression again reliably distinguished two SMC subpopulations. In contrast to the hypertensive neonate, we found in the fetus that the relative proliferative rates of both SMC subpopulations were equal, thus suggesting the existence of different mechanisms controlling proliferation and expression of cytoskeletal proteins in the fetus and neonate. We conclude that phenotypically distinct SMC populations in the bovine arterial media exhibit specific and selective proliferative responses to neonatal pulmonary hypertension. Distinct SMC subpopulations may, thus, contribute in unique ways to vascular homeostasis under both normal and pathologic conditions.

A one-stage repair of third-degree perineal lacerations and rectovestibular fistulae in 17 mares.

Third-degree perineal lacerations or rectovestibular fistulae in 17 mares were repaired surgically by a one-stage method. Primary healing occurred in 14 mares; there were one complete dehiscence and two partial dehiscences with fistula formation. Twelve of 13 mares that were bred became pregnant; nine carried foals to term and two are still pregnant. Two mares have each produced one unthrifty foal. One mare repeatedly aborts in the first trimester. Four mares have produced several healthy foals with no further problems. One mare suffered further perineal trauma while foaling.

Commonalities between gas anesthetics (nitrous oxide, nitrogen and/or argon) and ethanol intoxication in hot and cold selection line mice.

Argon, nitrogen, nitrous oxide were administered hyperbarically in doses (atmosphere) that caused loss of righting reflex (LORR). Nitrous oxide requires pressure somewhat less than two atmospheres, eighteen atmospheres were required for argon and thirty-six atmospheres roughly for nitrogen all in 0.5 atmospheres oxygen. Loss of righting reflex was assessed by using a rolling cage method of Wilson and Miller. Since nitrogen is the least liposoluble and nitrous oxide the most liposoluble of these three gases, greater pressures were needed for nitrogen to attain sufficient concentration in the membrane for anesthesia. Due to the low lipid solubility (1.4), nitrous oxide was administered hyperbarically at a compression rate of less than 0.5 atm/min at chamber temperature of 86 degrees plus or minus 2 degrees. Body temperatures were measured by minimitter transmitters. Two types of transmitters: an AM frequency and an FM frequency were used; a comparison of the two systems were made. The ED50 (atmospheres) required to produce a given score on the LORR were determined for each strain or line of mice. This ED50 value was determined for the Hot and Cold selection lines which have been specifically bred to differ as much as possible in a hypothermic response to acute doses of ethanol. These experiments demonstrate quite clearly a degree of commonality exists among CNS depressants with regard to anesthesia, loss of righting reflex and hypothermia.

Ganglioneuroma as a cause of small intestinal obstruction in the horse: a case report.

The clinical signs, medical and surgical management, and pathological findings are described for a ganglioneuroma, an atypical intestinal tumor, that caused colic because of small intestinal obturation.