Attenuation of ischaemic injury in the equine jejunum by administration of systemic lidocaine

REASONS FOR PERFORMING STUDY: Absorption of endotoxin across ischaemic-injured mucosa is a major cause of mortality after colic surgery. Recent studies have shown that flunixin meglumine retards mucosal repair. Systemic lidocaine has been used to treat post operative ileus, but it also has novel anti-inflammatory effects that could improve mucosal recovery after ischaemic injury. HYPOTHESIS: Systemic lidocaine ameliorates the deleterious negative effects of flunixin meglumine on recovery of mucosal barrier function. METHODS: Horses were treated i.v. immediately before anaesthesia with either 0.9% saline 1 ml/50 kg bwt, flunixin meglumine 1 mg/kg bwt every 12 h or lidocaine 1.3 mg/kg bwt loading dose followed by 0.05 mg/kg bwt/min constant rate infusion, or both flunixin meglumine and lidocaine, with 6 horses allocated randomly to each group. Two sections of jejunum were subjected to 2 h of ischaemia by temporary occlusion of the local blood supply, via a midline celiotomy. Horses were monitored with a behavioural pain score and were subjected to euthanasia 18 h after reversal of ischaemia. Ischaemic-injured and control jejunum was mounted in Ussing chambers for measurement of transepithelial electrical resistance (TER) and permeability to lipopolysaccharide (LPS). RESULTS: In ischaemic-injured jejunum TER was significantly higher in horses treated with saline, lidocaine or lidocaine and flunixin meglumine combined, compared to horses treated with flunixin meglumine. In ischaemic-injured jejunum LPS permeability was significantly increased in horses treated with flunixin meglumine alone. Behavioural pain scores did not increase significantly after surgery in horses treated with flunixin meglumine. CONCLUSIONS: Treatment with systemic lidocaine ameliorated the inhibitory effects of flunixin meglumine on recovery of the mucosal barrier from ischaemic injury, when the 2 treatments were combined. The mechanism of lidocaine in improving mucosal repair has not yet been elucidated.     

Comparison of the cytotoxic effects of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes

Objective To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. Study design Prospective, experimental study. Study material Equine articular chondrocytes. Methods Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double staining of Hoechst 33258 and propidium iodide using fluorescence microscopy, and the results were confirmed using flow cytometry. Results After 30‐minute exposure, trypan blue exclusion assay revealed that cell viability of 0.5% bupivacaine group was 28.73 ± 8.44%, and those of 2% lidocaine and 2% mepivacaine were 66.85 ± 6.03% and 86.27 ± 2.00%, respectively. The viability of chondrocytes after saline treatment was 95.95 ± 2.75%. The results of MTT assay and fluorescence microscopy had similar tendency with trypan blue assay. Each result showed that bupivacaine was the most toxic of the three local anaesthetics. Mepivacaine was less toxic than lidocaine. The results of the viability test suggest that bupivacaine and lidocaine exhibit a marked chondrotoxicity, and that this is mainly due to necrosis rather than apoptosis. Conclusions and clinical relevance Bupivacaine may induce detrimental chondrotoxicity when administered intra‐articularly, especially in patients with joint disease, and we suggest that it should be used cautiously in equine practice. Mepivacaine may be an alternative to both bupivacaine and lidocaine.     

In vitro effects of lipopolysaccharides on bovine uterine contractility

Metritis is an important disorder in dairy cows during the early postpartum period. Myometrial contractility is a prerequisite for uterine involution; however, very scanty literature is available about the effect of metritis on this process and endocrine responsiveness. This study was aimed to evaluate the effect of inflammation on uterine contractility in vitro, and the inflammation was induced by incubating myometrial strips with lipopolysaccharides (LPS). Myometrial samples were collected from 17 healthy Holstein Friesian cows during caesarean section. Eight longitudinal strips from each cow were incubated in organ baths with LPS concentrations of 0 (LPS₀), 0.1 (LPS_0.1), 1 (LPS₁) and 10 µg/ml (LPS₁₀). Spontaneous contractility and contractility induced by increasing concentrations of oxytocin (10^–10 – 10^–7 mol/L) were recorded during nine 30-min intervals (T1 to T9). The minimum amplitude (minA), maximum amplitude (maxA), mean amplitude (meanA) and area under the curve (AUC) were calculated for each time interval. LPS had an effect (p ≤ .05) on maxA, meanA and AUC. In T1, myometrial strips incubated with LPS_0.1 and LPS₁ had higher (p ≤ .05) maxA, meanA and AUC than the strips incubated with LPS₀. In T9 without oxytocin, LPS₀ led to higher (p ≤ .05) maxA, meanA and AUC than LPS_0.1 and LPS₁. In T8 and T9 with oxytocin, LPS₁ had lower (p ≤ .05) maxA, meanA and AUC than the other LPS concentrations. Interestingly, the results show that LPS has a transient positive effect on myometrial contractility in vitro and that this effect is dependent on LPS concentration and duration of incubation.     

Distribution of the neurokinin-1 receptor in equine intestinal smooth muscle

REASON FOR PERFORMING STUDY: Tachykinins have profound effects on equine intestinal motility, but the distribution of the neurokinin receptors (NKRs) through which they act is unknown. This study reports the distribution of one of these receptors, the neurokinin-1 receptor (NK1R), in smooth muscle throughout the equine intestinal tract. OBJECTIVES: To quantify the distribution of the NK1R, based upon mRNA expression, in smooth muscle of different regions of the equine intestinal tract. METHODS: Nine regions of the intestinal tract were sampled in 5 mature horses. Total RNA was isolated from smooth muscle and reverse transcribed; NK1R mRNA was then quantified using real-time PCR. RESULTS: NK1R mRNA was found at all levels of the sampled intestinal tract. The smooth muscle of the proximal small intestine and the ventral colon exhibited the highest level of NK1R mRNA expression in the equine intestinal tract. CONCLUSIONS: Tachykinins probably affect intestinal contractility and propulsion in the proximal small intestine and in the ventral colon.     

Comparison of the effects of an intravenous lidocaine infusion combined with 1% isoflurane versus 2% isoflurane alone on selected cardiovascular variables and recovery characteristics during equine general anaesthesia

Objectives

To compare the effects of a lidocaine constant rate infusion (CRI) combined with 1% isoflurane versus those of 2% isoflurane alone on cardiovascular variables in anaesthetized horses, and to estimate the sample size required to detect a difference in recovery quality.

Real‐time RT‐PCR for the detection and quantitative analysis of equine rhinitis viruses

Reasons for performing study: Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. Objectives: To develop a sensitive, rapid, real‐time RT‐PCR (rRT‐PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. Methods: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5′ UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT‐PCR were verified by virus isolation and ERBV positive samples were verified by rRT‐PCR using a different set of primers. Results: The detection limit of the rRT‐PCR for both viruses was 10–100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co‐circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT‐PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp). Conclusions: The rRT‐PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV. Potential relevance: These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.     

Risk of intra‐articular injection with longitudinal ultrasound‐guided injection of collateral ligaments of the equine distal interphalangeal joint

Desmopathy of the collateral ligaments of the distal interphalangeal joint is a common cause of equine foot lameness and carries a poor prognosis with conservative management. Intralesional injections may improve healing, although accuracy of radiographically guided injections is significantly less than when guided by MRI, which requires special needles. Longitudinal ultrasound‐guided injection of the distal collateral ligament has not been evaluated objectively. In this prospective, anatomic study, seven equine cadaver limbs (14 collateral ligaments) were injected with methylene blue dye and radiographic contrast medium using ultrasound to guide the needle longitudinally into the collateral ligaments until contacting bone. The insertion site of the needle proximal to the coronary band was measured on the limb and the needles left in place for radiography and CT to evaluate the needle angulation, location of the contrast medium, and whether the contrast entered the distal interphalangeal joint. The limbs were frozen and sectioned with a band saw to identify the location of the dye. Fifty percentage of injections were in or around the collateral ligaments. However, the percentage of “successful” injections, defined as in the collateral ligament but not in the joint, was only 36%. All legs had dye and contrast in the joint after both ligaments had been injected. There were no significant differences between the needle angle and entry site for “successful” and “unsuccessful” injections. Findings from this study indicates that the success rate is low for injecting the distal portions of the distal interphalangeal joint collateral ligaments using ultrasound guidance alone.     

The vasomotor effects of 5-hydroxytryptamine on equine basilar arteries In vitro

The vasomotor effects of 5-hydroxytryptamine (5-HT) on isolated equine basilar arteries were studied. 5-HT induced contractions of equine basilar arteries in a concentration-dependent manner, with a pEC₅₀ value (with 95% confidence limits) of 7.35 (7.08–7.62). Similar results were obtained with endothelium-denuded basilar arteries. Contractions were not competitively inhibited by the 5-HT₂ receptor antagonist ketanserin at low concentrations of 5-HT. Conversely, at high concentrations of 5-HT, contractions were inhibited by ketanserin in a concentration-dependent manner, with a pA ₂ value of 8.91 (8.62–9.20). The 5-HT₁ and 5-HT₂ receptor antagonist methiothepin shifted the concentration-response curve of 5-HT downwards and to the right in a concentration-dependent manner. In the presence of 10^-6 mol/L ketanserin, however, methiothepin antagonized 5-HT-induced contractions competitively with a pA ₂ value of 7.95 (7.59–8.31). The 5-HT₃ receptor antagonist MDL 72222 had no effect on 5-HT-induced contractions. The findings of this study indicate that 5-HT₁ and 5-HT₂ receptors are located in equine basilar arterial smooth muscle cells, and that stimulation of these receptors results in contraction.Abbreviations CR concentration ratio - EC₅₀ concentration producing 50% of the maximal response - 5-HT 5-hydroxytryptamine - MDL 72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - pA ₂ negative logarithm of the molar concentration of antagonist that produces a 2-fold rightward shift of the concentration-response curve - pEC₅₀ negative logarithm of EC₅₀ - PGF₂ prostaglandin F₂     

In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa

REASONS FOR PERFORMING STUDY: Volatile fatty acids, byproducts of carbohydrate fermentation by resident bacteria, have been implicated in causing nonglandular (NG) gastric ulcers. Lactic acid (LA), also produced by stomach bacteria, may cause gastric ulcers when exposed to the equine NG mucosa. OBJECTIVES: To investigate the in vitro effects of LA on equine NG mucosa bioelectric properties, sodium transport and tissue resistance. METHODS: Gastric tissues obtained from 13 mature horses were studied in Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured, and electrical resistance (R) and conductance (G) calculated for tissues after addition of HCl and LA (5, 10, 20 and 40 mmol/l) in normal Ringer's solution (NRS). RESULTS: Mucosa exposed to HCl or LA (5, 10 and 20 mmol/l) in NRS (pH 1.5 and to a lesser extent pH 4.0) had a significant decrease in Isc and PD. Mucosa exposed to a high concentration of LA (40 mmol/l) in NRS (LRS) at pH 1.5 showed an increased G, but this increase was not significant. Values returned to baseline after solutions were returned to pH 7.0. Histological changes were consistent with HCl-induced (pH <4.0) acid damage. CONCLUSIONS: HCl induced alteration in bioelectric properties of equine NG mucosa whereas addition of LRS did not, other than those changes seen with HCl alone.     

Preliminary investigations on the effects of a Strongylus vulgaris larval extract,mononuclear factors and platelet factors on equine smooth muscle cells in vitro

Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smooth muscle cells exposed to other factors. Equine mononuclear leukocytes exposed to LAE developed prominent morphological changes, including enlargement, clumping and increased numbers of mitotic figures.     

In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis

REASON FOR PERFORMING STUDY: While immune modulators are used routinely in equine medicine, their mechanism of action is not always known. OBJECTIVES: To determine the effect of a commercial preparation of inactivated parapoxvirus ovis (Orf virus; PPVO) on cytokine gene expression by equine peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. METHODS: PBMC were prepared from 6 mixed-breed yearlings and cultured in vitro with PPVO with or without Concanavalin A (Con A) for 24 h. Effects on the expression of IFNalpha, IFNbeta IFNgamma, TNFalpha and IL-18 were analysed by real time quantitative PCR (RT-PCR). In addition, 12 yearling horses were treated with PPVO and whole blood RNA samples were prepared at regular intervals to assess effects on in vivo cytokine gene expression. Six of those yearlings were later treated with saline and served as treatment controls. Nine additional yearlings were injected intradermally with a single dose and their injection sites biopsied at 24 and 48 h for cytokine expression. RESULTS: In vitro culture of PBMC with PPVO led to a significant increase in IFNalpha and IFNbeta gene expression compared to mock-stimulated cultures. In addition, expression of IFNgamma and TNFalpha was significantly higher in PBMC stimulated with PPVO and Con A, than those stimulated with Con A alone. No changes were observed in IL-18 gene expression in vitro. Treatment of horses with a 3-dose regimen of PPVO resulted in elevation of IFNgamma gene expression, which was detected 24 h after the first dose and declined thereafter. Intradermal inoculation led to increased expression of IFNgamma along with IFNbeta, IL-15 and IL-18. CONCLUSIONS: Together these results indicate that PPVO stimulated IFNgamma production both in vitro and in vivo. Increased cytokine expression could account for its immunomodulatory activity. POTENTIAL RELEVANCE: The absence of adverse reactions and clear indications of increased expression of cytokine gene expression supports previous clinical uses for this immune modulator in those situations when increased expression of IFNgamma is warranted.     

The effects of furosemide and pentoxifylline on the flow properties of equine erythrocytes:In vitro studies

The effects of various concentrations of furosemide and pentoxifylline on equine RBCin vitro were evaluated to facilitate better understanding of the potential effects of these drugs on blood flow properties. Furosemide induced increased mean cell volume (MCV), increased RBC potassium concentration, increased whole blood viscosity, and decreased the RBC filtrability. These data indicate that furosemide may block the RBC membrane transport pathways resulting in potassium and water retention. The increase in size and the resultant decrease in the surface-area-to-volume ratio may have caused the impaired RBC filtrability and increased blood viscosity. Pentoxifylline improved RBC filtrability without changing the RBC size or the potassium or chloride concentrations, suggesting that pentoxifylline may increase the deformability of the RBC membrane. The study indicated that pentoxifylline has potential therapeutic applications for improving microvascular blood flow but that furosemide may have adverse effects on blood flow.Abbreviations cP centipoise - EIPH exercise-induced pulmonary haemorrhage - MCV mean cell volume - Na-K-2Cl sodium potassium 2-chloride - PCV packed cell volume - RBC red blood cell