Review of the clinical diagnosis of sacroiliac dysfunction in horses - Challenges and limitations.

Sacroiliac dysfunction (SID) is a condition seen in horses associated with poor performance that affects hind limb gait and impulsion. The condition comprises pain and dysfunction but there lacks clarity around the aetiopathogenesis and whether SID encompasses abnormal joint pathology, abnormal joint movement, abnormal regional biomechanical function, joint laxity and pain, or various combinations of these that may vary over time. Clinical assessment remains challenging for equine clinicians due to the deep location of the sacroiliac joint (SIJ) and surrounding structures which limits access for palpation, diagnostic imaging and joint-specific injection. There is no recognised single reference standard diagnostic test for SID. Clinical diagnosis has been based on ruling out other causes of hind limb lameness, along with combinations of ultrasonography, scintigraphy and periarticular anaesthesia of the SIJ. Recent studies have highlighted the lack of specificity of injections targeting the SIJ, with significant dispersal of injectate into surrounding structures including around the lumbosacral joint (LSJ). Advanced imaging modalities such as computed tomography offers promise for assessment of the structure and pathology of the SIJ and surrounding bony structures. However, there is a need to improve the understanding of the significance of anatomic variation of the sacroiliac region structures, with recent studies reporting detailed anatomic variation in groups of horses with and without SID. There are also limitations around functional assessment of the joint which is still largely reliant on a thorough clinical examination. This review aims to present an update on clinical approaches to the diagnosis of horses with SID, and to consider the challenges and limitations.

Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants.

Medium-chain fatty acids (FAs), found in storage lipids of certain plants, are an important renewable resource. Seeds of undomesticated California bay accumulate laurate (12:0), and a 12:0-acyl-carrier protein thioesterase (BTE) has been purified from this tissue. Sequencing of BTE enabled the cloning of a complementary DNA coding for a plastid-targeted preprotein. Expression of the complementary DNA in the seeds of Arabidopsis thaliana resulted in BTE activity, and medium chains accumulated at the expense of long-chain (greater than or equal to 16) FAs. Laurate became the most abundant FA species and was deposited in the storage triacylglycerols. These results demonstrate a mechanism for medium-chain FA synthesis in plants.

Ex vivo calibration and validation of in vivo equine bone strain measures.

REASONS FOR PERFORMING STUDY: Data are required to confirm that strain gauges recording high bone strains in Thoroughbred racehorses provide an accurate record of bone strain. OBJECTIVE: To test the accuracy and reliability of very high in vivo strain recordings made during fast exercise in Thoroughbred racehorses. METHODS: Strains were recorded during exercise from rosette gauges implanted onto the mid-shaft dorsal cortex of each third metacarpal bone (MC3) in 6 yearling and 6 mature Thoroughbreds in a previous experiment. Bulk elastic modulus (E(US)) was calculated from ultrasound speed and single photon absorptiometry measures. Each cleaned MC3 with the original gauge in situ and new gauges placed in a region of strain similarity (as shown by photoelastic coating) was loaded in a materials testing system (MTS) and strains recorded during loading. Elastic moduli were calculated from strain measures from new rosette gauges on the medial, dorsal, lateral and palmar surfaces (E(m); E(d); E(El); E(p), and bulk moduli calculated from the displacement of the MTS machine heads during loading (Ebt). Peak loads were increased incrementally to failure. RESULTS: Of 14 original gauges tested against new gauges, 11 recorded strains from 80-115% of the new gauges and 3 showed reduced function (31-40%). Ebt were similar to E(m), and E(US) were similar to E(l) and not significantly different from Ed. Maximum strains at yield were recorded by the medial gauges and ranged from -7500 to -16,000 microepsilon. CONCLUSIONS AND CLINICAL RELEVANCE: Similarities between recordings from gauges used in vivo and new gauges confirmed the reliability and likely accuracy (or possible underestimate) of very high strains (exceeding -6000 microepsilon) recorded in exercising Thoroughbred racehorses. The similarity between E(bt) and E(m) confirms that the gauges measured the true distortion of the bone in the MTS. These results confirm that mammalian bone may withstand much greater compressive loads than -4000 microepsilon under some conditions at least.

Influence of different exercise regimes on the proximal hoof circumference in young Thoroughbred horses.

REASONS FOR PERFORMING STUDY: Most lameness in horses relates to foot problems and may be associated with changes in hoof shape, but there is a lack of information on the influence of normal exercise on hoof shape. OBJECTIVES: To investigate the effect of training on proximal hoof circumference in young Thoroughbred racehorses being prepared for racing. METHODS: Thirty-seven young Thoroughbred racehorses were included in this study. Front hoof circumference immediately below the coronary band was measured weekly with a measuring tape in all horses present at the stable. Most horses accomplished a minimum of 2 training periods at the stable separated by periods of rest on a paddock. One sample t tests were used to evaluate if the mean change per week differed from zero. To estimate the repeatability coefficient, the left proximal hoof circumference of 25 horses was measured 3 times in a random order on one day. RESULTS: Most horses showed a similar pattern of change. The proximal hoof circumference decreased during the training periods (P < 0.0001) and increased when the horse was rested (P < 0.0001). The decrease of the circumference during the first training period was -0.66 mm/week on the left and -0.64 mm/week on the right. During the second training period, this was -0.58 mm/week on the left and -0.57 mm/ week on the right. During the rest period, the circumference increased by 1.03 mm/week on the left and 1.12 mm/week on the right. The repeatability coefficient for the left circumference was 1.8 mm. CONCLUSIONS: Horses showed a decrease in circumference during race training that reversed when they were rested. POTENTIAL RELEVANCE: Measurement of front hoof circumference is a simple method to assess change in hoof shape. It provides an opportunity to investigate the relationships between specific training, hoof shape and soundness.

Hypertrophy and physiological death of equine chondrocytes in vitro.

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.

Iridium(iii)-bis(imidazolinyl)phenyl catalysts for enantioselective C-H functionalization with ethyl diazoacetate.

The intermolecular enantioselective C-H functionalization with acceptor-only metallocarbenes is reported using a new family of Ir(iii)-bis(imidazolinyl)phenyl catalysts, developed based on the interplay of experimental and computational insights. The reaction is tolerant of a variety of diazoacetate precursors and is found to be heavily influenced by the steric and electronic properties of the substrate. Phthalan and dihydrofuran derivatives are functionalized in good yields and excellent enantioselectivities.

The timing and distribution of strains around the surface of the midshaft of the third metacarpal bone during treadmill exercise in one Thoroughbred racehorse.

OBJECTIVE: To confirm that the midshaft dorsal cortex of the third metacarpal bone experienced higher compressive strains during fast exercise than the medial or lateral cortices, and that the strain peak occurred earlier in the hoof-down phase of the stride on the dorsal cortex than the medial or lateral cortices. DESIGN: Observations of a single horse. PROCEDURE: Strains were collected from a single, sound, 3-year-old Thoroughbred mare during treadmill exercise from rosette strain gauges implanted onto the medial, lateral and dorsal surfaces of the midshaft of the right cannon bone, simultaneously with data from a hoof switch that showed when the hoof was in the stance phase. RESULTS: Peak compressive strains on the dorsal surface of the third metacarpal bone were proportional to exercise speed and occurred at about 30% of stance. Peak compressive strains on the medial surface of the non-lead limb reached a maximum at a speed around 10 m/s and occurred at mid-stance. Peak compressive strains on the lateral surface varied in timing and size between strides at all exercise speeds, but remained less than -2000 microstrains. CONCLUSIONS: The timing of peak compressive strains on the dorsal cortex suggests a relationship to deceleration of the limb following hoof impact, so the main determinants of their size would be exercise speed and turning (as shown in previous experiments). This experiment confirms data from other laboratories that were published but not discussed, that peak compressive strains on the medial surface occur at mid-stance. This suggests that they are related to the support of body weight. The strains on the lateral cortex occurred at variable times so may be associated with the maintenance of balance as well as the support of body weight. Understanding the loading of the third metacarpal bone will help to determine causes of damage to it and ways in which the bone might be conditioned to prevent such damage.

Third metacarpal bone laterality asymmetry and midshaft dimensions in Thoroughbred racehorses.

OBJECTIVE: The aims of this study were to test whether longer third metacarpal (MC3) bones had thicker dorsal cortices in a group of racehorses that were exercising at similar maximum speeds, and to establish if horses with larger differences in length between their right and left MC3 bones showed larger differences in the dorsal cortical thickness between the two limbs. DESIGN: An observational study. PROCEDURE: Forty Thoroughbred racehorses aged between 2 and 6 years and in training at racing speed at two racing stables were used. Two sets of radiographs of each left and right metacarpus of each horse were measured for bone length and dorsal cortical width according to standardised methods. RESULTS: The dorsal cortex thickness showed a linear relationship with bone length for the range of lengths between 25 and 30 cm for both the right MC3 (R2 = 0.30, P = 0.0003) and the left (R2 = 0.23, P = 0.002). The longer bones had thicker dorsal cortices. When results from the two limbs were combined to test if the difference in length between the right and left MC3 in an individual horse was associated with a thicker dorsal cortex in the longer MC3 there was no consistent relationship (R2 = 0.008, P = 0.58). CONCLUSION: In this sample of racehorses longer MC3 bones were likely to have been exposed to a greater dorsopalmar bending moment at the mid shaft that was reflected in a thicker dorsal cortex. The lack of a relationship between midshaft thickness and bone length within individual horses suggests that direct mechanical effects of conformation and environment were less important than the individual's level of skill (or the degree of laterality in their movements) developed before their exposure to fast exercise. It is likely that racehorses with longer right MC3 bones were more able to control the loading of the right MC3 than the left during fast exercise.

Prolonged dopamine and serotonin transporter inhibition after exposure to tropanes.

Cocaine and tropane analogs are known to interact with biogenic monoamine transporters by inhibiting amine uptake. Previous in vivo studies have demonstrated that some of these tropanes produce a longer lasting behavioral effect compared with cocaine. We have previously examined several tropane analogs and found a difference in their relative affinities for dopamine (DA) and serotonin (5-HT) transporters. The purpose of this study was to determine the recovery time of transporter function in vitro and in vivo comparing cocaine with the tropane analogs WF-11 (PTT, selective for DA transporters), WF-31 (selective for 5-HT transporters) and WF-23 (highly potent at both DA and 5-HT transporters). In vitro, using primary rat brain cultures of either midbrain or raphe regions, the recovery of the ability to transport either [3H]dopamine or [3H]serotonin, respectively was evaluated at 0, 3, 24, 48, 120 and 240 h after a 1 h exposure to cocaine and tropane analogs. The tropanes exhibited clearance half-lives ranging from 12 to 69 h, while cocaine, on the other hand, exhibited a clearance half-life of approximately 6 h. In studies utilizing [125I]RTI-55 binding, intraperitoneal injections of cocaine and WF-23 into the rat resulted in striatal clearance half-lives ex vivo that were almost identical to those obtained in vitro. These data suggest that the tropanes bind to and reduce transporter function for prolonged periods of time (up to 10-fold longer than cocaine) and those compounds with the highest affinity may produce a pseudo-irreversible inhibition of transporter function.

Synthesis of 3 beta-aryl-8-azabicyclo[3.2.1]octanes with high binding affinities and selectivities for the serotonin transporter site.

A novel entry to tropane analogs of cocaine was developed based on the reaction of rhodium-stabilized vinylcarbenoids with pyrroles. These analogs were tested in binding to dopamine, serotonin (5-HT), and norepinephrine transporters in membranes from rat striatum and frontal cortex. In all the analogs, the aryl group at the 3 position was directly bound to the tropane ring and an ethyl ketone moiety was present at the 2 position. By appropriate modification of the aryl and nitrogen substituents, highly potent and 5-HT selective tropanes were prepared. The most potent and selective compound was 3 beta-[4-(1-methylethenyl)phenyl]-2 beta-propanoyl-8-azabicyclo[3.2.1]octane (13b) which had a Ki of 0.1 nM at 5-HT transporters and was 150 times more potent at 5-HT vs dopamine transporters and almost 1000 times more potent at 5-HT vs norepinephrine transporters.

Synthesis of 2 beta-acyl-3 beta-aryl-8-azabicyclo[3.2.1]octanes and their binding affinities at dopamine and serotonin transport sites in rat striatum and frontal cortex.

A novel entry to tropane analogs of cocaine was developed on the basis of the reaction of rhodium-stabilized vinylcarbenoids with pyrroles. These analogs were tested in binding to dopamine and serotonin (5-HT) transporters in membranes from rat striatum and frontal cortex. In all the analogs, the aryl group at the 3-position was directly bound to the tropane ring (as in WIN-35,428), and methyl or ethyl ketone moieties were present at the 2-position instead of the typical ester group. The series of analogs containing a 2-naphthyl group at the 3-position were most potent, with Ki values < 1 nM in binding to both dopamine and 5-HT transporters. Although the unsubstituted 2-naphthyl analog was nonselective at dopamine and 5-HT transport sites, other compounds were selective for either site. In general, compounds with relatively small substituents on the aromatic moiety (such as p-methyl or p-fluoro) were relatively selective for the dopamine transporters, while a p-isopropylphenyl derivative was selective for the 5-HT transport sites. This latter compound represents the first N-methyltropane derivative specific for 5-HT transporters. Resolution of two of the most significant analogs was achieved by HPLC on a chiral stationary phase; the active enantiomer of a 2-naphthyl analog exhibited Ki values of < 0.1 nM at both dopamine and 5-HT transporter sites.

Synthesis of 2beta-acyl-3beta-(substituted naphthyl)-8-azabicyclo[3.2.1]octanes and their binding affinities at dopamine and serotonin transport sites.

A series of 3beta-naphthyltropane derivatives were synthesized and found to show high affinity at both the dopamine and serotonin transporter sites, leading to some of the most potent inhibitors known based on the tropane structure. Comparative molecular field analysis (CoMFA) models were developed for both dopamine and serotonin transporter binding data. These models provide insights into those factors that influence binding at the two transporters.

Evaluation of sequential plasma and urinary tumor necrosis factor alpha levels in renal allograft recipients.

The macrophage cytokine tumor necrosis factor-alpha is released early in immune activation and may be detected in the peripheral circulation. This study has investigated the occurrence of plasma and urinary TNF in 30 renal allograft recipients. Although circulating TNF may be detected in 20% of pretransplant or normal control samples, levels were significantly elevated during 65% of allograft rejection episodes. Plasma TNF levels did not rise in graft failure due to acute tubular necrosis, but were always highly raised in systemic infection. In contrast, urinary TNF was only detected in association with acute rejection (49%) or tubular necrosis (14%), and no controls had detectable urinary TNF. These findings indicate that evaluation of circulating and excreted TNF may give further insight into the immunobiology of graft rejection.

Alterations in behavior and opioid gene expression induced by the novel tropane analog WF-31.

The effects of the acute administration of the serotonin-selective tropane analog, [2beta-propanoyl-3beta-(4-isopropylphenyl)-tropane, WF-31, on spontaneous locomotor activity were measured and compared to those of the highly selective serotonin uptake inhibitor, fluoxetine and cocaine, a non-selective re-uptake inhibitor of dopamine and serotonin. WF-31 (1, 10 and 30 mg/kg)-elicited increases in locomotor behaviors when compared to vehicle-treated rats. This increased activity was blocked by pre-treatment with the dopaminergic antagonist, flupenthixol, suggesting that these effects may be mediated by dopaminergic mechanisms. Cocaine, but not fluoxetine, also elicited increases in behaviors. In addition, the effects of these three compounds on opioid peptide gene expression were also assessed using in situ hybridization histochemistry in the same animals. The acute administration of both WF-31 and cocaine increased the expression of preprodynorphin mRNA in the dorsal striatum whereas fluoxetine had no effect. Expression of striatal preproenkephalin mRNA was augmented by all three compounds. Within the nucleus accumbens, PPD mRNA levels were affected only by treatment with WF-31, an effect that was blocked by pre-treatment with flupenthixol. In contrast, the acute administration of both WF-31 and fluoxetine, but not cocaine, increased the expression of preproenkephalin mRNA. These increases, however, were not reversed by pre-treatment with flupenthixol. Despite its profile in vitro as a relatively selective serotonin re-uptake inhibitor, some of the in vivo actions of WF-31 appear to be mediated by dopaminergic mechanisms. These data further suggest that the mechanisms underlying expression of the opioid peptides in the nucleus accumbens may vary from those in the dorsal striatum.

Further evaluation of the reinforcing effects of the novel cocaine analog 2beta-propanoyl-3beta-(4-tolyl)-tropane (PTT) in rhesus monkeys.

2Beta-propanoyl-3beta-(4-tolyl)-tropane (PTT) is a cocaine analog which has been shown in rhesus monkeys to have cocaine-like discriminative stimulus effects and a long duration of action (>8 h), yet does not function as a reinforcer when substituted for cocaine in monkeys responding under a fixed-interval 5-min schedule (Nader et al. 1997). The purpose of the present study was to evaluate the reinforcing effects of PTT under a fixed-ratio (FR) schedule and to determine if decreasing the inter-injection interval would influence the reinforcing effects of PTT. Male rhesus monkeys (n=3) were trained to respond under a multiple FR 30 food-drug-food schedule. When responding was stable, cocaine (0.003-0.3 mg/kg per injection) or PTT (0.001-0.03 mg/kg per injection) was available during the drug component for at least five consecutive sessions and until stable responding was observed. To investigate whether the inter-injection interval would influence PTT-maintained response rates, the time-out (TO) following PTT injections was reduced from 180 or 300 s to 10 s for at least five consecutive sessions. Cocaine-maintained response rates were characterized as an inverted-U shaped function of dose, with peak rates maintained by 0.03 mg/kg per injection cocaine. PTT (0.001-0.03 mg/kg per injection) maintained response rates significantly higher than rates maintained by the PTT vehicle, but significantly lower than cocaine-maintained response rates; PTT intake increased with a dose. A reduction of the TO following PTT injections to 10 s did not alter PTT-maintained response rates or total session intake. Self-administered PTT was more potent than cocaine at decreasing food-maintained responding. These results suggest that for long-acting compounds like PTT, reinforcing effects are more likely to be observed when the drug is available under a ratio-based schedule, compared to an interval-based schedule.

Self-administration of two long-acting monoamine transport blockers in rhesus monkeys.

RATIONALE: 2beta-propanoyl-3beta-(4-tolyl)-tropane (PTT) is a cocaine analog with high affinity at and selectivity for the dopamine transporter (DAT). 2beta-propanoyl-3beta-(2-naphthyl)-tropane (HD-23), like cocaine, binds with approximately equal affinity to the DAT, the serotonin transporter, and the norepinephrine transporter but has over a 100-fold higher affinity for these monoamine transporters than cocaine. The reinforcing effects of these drugs have not been evaluated in cocaine-na nonhuman primates. OBJECTIVE: The primary goal of the present study was to examine the reinforcing effects of PTT and HD-23 in rhesus monkeys before and after a history of intravenous cocaine self-administration. METHODS: Monkeys (n=4) were initially trained to respond under a fixed-ratio 30 schedule of food presentation. When responding was stable, saline, PTT (0.001-0.03 mg/kg per injection), and HD-23 (0.0003-0.0056 mg/kg per injection) were made available for self-administration for least five sessions per dose. Next, a cocaine dose-effect function (0.0003-0.3 mg/kg per injection) was determined and then

Kinetic resolution and double stereodifferentiation in catalytic asymmetric C-H activation of 2-substituted pyrrolidines.

Dirhodium tetrakis(S-(N-dodecylbenzenesulfonyl)prolinate) (Rh(2)(S-DOSP)(4)) catalyzed decomposition of methyl aryldiazoacetates in the presence of 2-substituted pyrrolidines results in highly diastereoselective and enantioselective C-H insertions. These reactions can proceed with impressive levels of double stereodifferentiation and kinetic resolution, which allows for three stereocenters to be controlled during the C-H insertion step.

Stereoselectivity of methyl aryldiazoacetate cyclopropanations of 1,1-diarylethylene. Asymmetric synthesis of a cyclopropyl analogue of tamoxifen.

[formula: see text] Dirhodium tetrakis(S-(N-dodecylbenzenesulfonyl)prolinate) (Rh2(S-DOSP)4)-catalyzed decomposition of methyl phenyldiazoacetate in the presence of 1,1-diarylethylenes results in intermolecular cyclopropanation with high enantioselectivity (up to 99% ee) and moderate diastereoselectivity (up to 80% de). The reaction was applied to the asymmetric synthesis of a cyclopropyl analogue of tamoxifen.

Catalytic asymmetric allylic C-H activation as a surrogate of the asymmetric Claisen rearrangement.

[reaction: see text]. Tetrakis[N-[4-dodecylphenyl)sulfonyl]-(S)-prolinato]-dirhodium [Rh2(S-DOSP)4] catalyzed decomposition of methyl aryldiazoacetates in the presence of alkenes results in allylic C-H activation by means of a rhodium-carbene induced C-H insertion. The resulting gamma,delta-unsaturated esters are equivalent to products that would be traditionally obtained from an asymmetric Claisen rearrangement. Highly regio- and enantioselective C-H insertions can be achieved, and in certain cases, good diastereocontrol is also possible.

Asymmetric intramolecular C_H insertions of aryldiazoacetates.

[reaction: see text] The enantioselectivity of Rh(2)(S-DOSP)(4) catalyzed C-H insertion of aryldiazoacetates is very dependent on the site of the C-H insertion. The highest enantioselectivity is obtained for insertion into methine C-H bonds.