Evaluation of the diagnostic value of transcranial electrical stimulation (TES) to assess neuronal functional integrity in horses.

Medical imaging allows for the visualization of spinal cord compression sites; however, it is impossible to assess the impact of visible stenotic sites on neuronal functioning, which is crucial information to formulate a correct prognosis and install targeted therapy. It is hypothesized that with the transcranial electrical stimulation (TES) technique, neurological impairment can be reliably diagnosed. Objective: To evaluate the ability of the TES technique to assess neuronal functional integrity in ataxic horses by recording TES-induced muscular evoked potentials (MEPs) in three different muscles and to structurally involve multiple ancillary diagnostic techniques, such as clinical neurological examination, plain radiography (RX) with ratio assessment, contrast myelography, and post-mortem gross and histopathological examination. Methods: Nine ataxic horses, showing combined fore and hindlimb ataxia (grades 2-4), were involved, together with 12 healthy horses. TES-induced MEPs were recorded bilaterally at the level of the trapezius (TR), the extensor carpi radialis (ECR), and tibialis cranialis (TC) muscles. Two Board-certified radiologists evaluated intra- and inter-sagittal diameter ratios on RX, reductions of dorsal contrast columns, and dural diameters (range skull-T1). Post-mortem gross pathological and segmental histopathological examination was also performed by a Board-certified pathologist. Results: TES-MEP latencies were significantly prolonged in both ECR and TC in all ataxic horses as opposed to the healthy horses. The TR showed a mixed pattern of normal and prolonged latency times. TES-MEP amplitudes were the least discriminative between healthy and ataxic horses. Youden's cutoff latencies for ataxic horses were 24.6 ms for the ECR and 45.5 ms for the TC (sensitivity and specificity of 100%). For healthy horses, maximum latency values were 22 and 37 ms, respectively. RX revealed spinal cord compression in 8 out of 9 involved ataxic horses with positive predictive values of 0-100%. All ataxic horses showed multi-segmental Wallerian degeneration. All pathological changes recorded in the white matter of the spinal cord were widely dispersed across all cervical segments, whereas gray matter damage was more localized at the specific segmental level. Conclusion: TES-MEP latencies are highly sensitive to detect impairment of spinal cord motor functions for mild-to-severe ataxia (grades 2-4).

Trapezius Motor Evoked Potentials From Transcranial Electrical Stimulation and Transcranial Magnetic Stimulation: Reference Data, Characteristic Differences and Intradural Motor Velocities in Horses.

Reason for Performing Study: So far, only transcranial motor evoked potentials (MEP) of the extensor carpi radialis and tibialis cranialis have been documented for diagnostic evaluation in horses. These allow for differentiating whether lesions are located in either the thoraco-lumbar region or in the cervical myelum and/or brain. Transcranial trapezius MEPs further enable to distinguish between spinal and supraspinal located lesions. No normative data are available. It is unclear whether transcranial electrical stimulation (TES) and transcranial magnetic stimulation (TMS) are interchangeable modalities. Objectives: To provide normative data for trapezius MEP parameters in horses for TES and TMS and to discern direct and indirect conduction routes by neurophysiological models that use anatomical geometric characteristics to relate latency times with peripheral (PCV) and central conduction velocities (CCV). Methods: Transcranial electrical stimulation-induced trapezius MEPs were obtained from twelve horses. TES and TMS-MEPs (subgroup 5 horses) were compared intra-individually. Trapezius MEPs were measured bilaterally twice at 5 intensity steps. Motoneurons were localized using nerve conduction models of the cervical and spinal accessory nerves (SAN). Predicted CCVs were verified by multifidus MEP data from two horses referred for neurophysiological assessment. Results: Mean MEP latencies revealed for TES: 13.5 (11.1-16.0)ms and TMS: 19.7 (12-29.5)ms, comprising ∼100% direct routes and for TMS mixed direct/indirect routes of L:23/50; R:14/50. Left/right latency decreases over 10 > 50 V for TES were: -1.4/-1.8 ms and over 10 > 50% for TMS: -1.7/-3.5 ms. Direct route TMS-TES latency differences were 1.88-4.30 ms. 95% MEP amplitudes ranges for TES were: L:0.26-22 mV; R:0.5-15 mV and TMS: L:0.9 - 9.1 mV; R:1.1-7.9 mV. Conclusion: This is the first study to report normative data characterizing TES and TMS induced- trapezius MEPs in horses. The complex trapezius innervation leaves TES as the only reliable stimulation modality. Differences in latency times along the SAN route permit for estimation of the location of active motoneurons, which is of importance for clinical diagnostic purpose. SAN route lengths and latency times are governed by anatomical locations of motoneurons across C2-C5 segments. TES intensity-dependent reductions of trapezius MEP latencies are similar to limb muscles while MEP amplitudes between sides and between TES and TMS are not different. CCVs may reach 180 m/s.

Flexibility of equine bioenergetics and muscle plasticity in response to different types of training: An integrative approach, questioning existing paradigms.

Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian horses were aquatrained (n = 5) or dry treadmill trained (n = 7) (8 weeks) and monitored for: evolution of muscle diameter in response to aquatraining and dry treadmill training, fiber type composition and fiber cross-sectional area of the M. pectoralis, M. vastus lateralis and M. semitendinosus and untargeted metabolomics of the M. pectoralis and M. vastus lateralis in response to dry treadmill training. Aquatraining was superior to dry treadmill training to increase muscle diameter in the hindquarters, with maximum effect after 4 weeks. After dry treadmill training, the M. pectoralis showed increased muscle diameter, more type I fibers, decreased fiber mean cross sectional area, and an upregulated oxidative metabolic profile: increased ß-oxidation (key metabolites: decreased long chain fatty acids and increased long chain acylcarnitines), TCA activity (intermediates including succinyl-carnitine and 2-methylcitrate), amino acid metabolism (glutamine, aromatic amino acids, serine, urea cycle metabolites such as proline, arginine and ornithine) and xenobiotic metabolism (especially p-cresol glucuronide). The M. vastus lateralis expanded its fast twitch profile, with decreased muscle diameter, type I fibers and an upregulation of glycolytic and pentose phosphate pathway activity, and increased branched-chain and aromatic amino acid metabolism (cis-urocanate, carnosine, homocarnosine, tyrosine, tryptophan, p-cresol-glucuronide, serine, methionine, cysteine, proline and ornithine). Trained Friesians showed increased collagen and elastin turn-over. Results show that branched-chain amino acids, aromatic amino acids and microbiome-derived xenobiotics need further study in horses. They feed the TCA cycle at steps further downstream from acetyl CoA and most likely, they are oxidized in type IIA fibers, the predominant fiber type of the horse. These study results underline the importance of reviewing existing paradigms on equine bioenergetics.

Comparison of Muscle MEPs From Transcranial Magnetic and Electrical Stimulation and Appearance of Reflexes in Horses.

INTRODUCTION: Transcranial electrical (TES) and magnetic stimulation (TMS) are both used for assessment of the motor function of the spinal cord in horses. Muscular motor evoked potentials (mMEP) were compared intra-individually for both techniques in five healthy horses. mMEPs were measured twice at increasing stimulation intensity steps over the extensor carpi radialis (ECR), tibialis cranialis (TC), and caninus muscles. Significance was set at p < 0.05. To support the hypothesis that both techniques induce extracranially elicited mMEPs, literature was also reviewed. RESULTS: Both techniques show the presence of late mMEPs below the transcranial threshold appearing as extracranially elicited startle responses. The occurrence of these late mMEPs is especially important for interpretation of TMS tracings when coil misalignment can have an additional influence. Mean transcranial motor latency times (MLT; synaptic delays included) and conduction velocities (CV) of the ECR and TC were significantly different between both techniques: respectively, 4.2 and 5.5 ms (MLT TMS --MLT TES ), and -7.7 and -9.9 m/s (CV TMS -CV TES ). TMS and TES show intensity-dependent latency decreases of, respectively, -2.6 (ECR) and -2.7 ms (TC)/30% magnetic intensity and -2.6 (ECR) and -3.2 (TC) ms/30V. When compared to TMS, TES shows the lowest coefficients of variation and highest reproducibility and accuracy for MLTs. This is ascribed to the fact that TES activates a lower number of cascaded interneurons, allows for multipulse stimulation, has an absence of coil repositioning errors, and has less sensitivity for varying degrees of background muscle tonus. Real axonal conduction times and conduction velocities are most closely approximated by TES. CONCLUSION: Both intracranial and extracranial mMEPs inevitably carry characteristics of brainstem reflexes. To avoid false interpretations, transcranial mMEPs can be identified by a stepwise latency shortening of 15-20 ms when exceeding the transcranial motor threshold at increasing stimulation intensities. A ring block around the vertex is advised to reduce interference by extracranial mMEPs. mMEPs reflect the functional integrity of the route along the brainstem nuclei, extrapyramidal motor tracts, propriospinal neurons, and motoneurons. The corticospinal tract appears subordinate in horses. TMS and TES are interchangeable for assessing the functional integrity of motor functions of the spinal cord. However, TES reveals significantly shorter MLTs, higher conduction velocities, and better reproducibility.

Extramuscular Recording of Spontaneous EMG Activity and Transcranial Electrical Elicited Motor Potentials in Horses: Characteristics of Different Subcutaneous and Surface Electrode Types and Practical Guidelines.

INTRODUCTION: Adhesive surface electrodes are worthwhile to explore in detail as alternative to subcutaneous needle electrodes to assess myogenic evoked potentials (MEP) in human and horses. Extramuscular characteristics of both electrode types and different brands are compared in simultaneous recordings by also considering electrode impedances and background noise under not mechanically secured (not taped) and taped conditions. METHODS: In five ataxic and one non-ataxic horses, transcranial electrical MEPs, myographic activity, and noise were simultaneously recorded from subcutaneous needle (three brands) together with pre-gelled surface electrodes (five brands) on four extremities. In three horses, the impedances of four adjacent-placed surface-electrode pairs of different brands were measured and compared. The similarity between needle and surface EMGs was assessed by cross-correlation functions, pairwise comparison of motor latency times (MLT), and amplitudes. The influence of electrode noise and impedance on the signal quality was assessed by a failure rate (FR) function. Geometric means and impedance ranges under not taped and taped conditions were derived for each brand. RESULTS: High coherencies between EMGs of needle-surface pairs degraded to 0.7 at moderate and disappeared at strong noise. MLTs showed sub-millisecond simultaneous differences while sequential variations were several milliseconds. Subcutaneous MEP amplitudes were somewhat lower than epidermal. The impedances of subcutaneous needle electrodes were below 900 Ω and FR = 0. For four brands, the FR for surface electrodes was between 0 and 80% and declined to below 25% after taping. A remaining brand (27G DSN2260 Medtronic) revealed impedances over 100 kΩ and FR = 100% under not taped and taped conditions. CONCLUSION: Subcutaneous needle and surface electrodes yield highly coherent EMGs and TES-MEP signals. When taped and allowing sufficient settling time, adhesive surface-electrode signals may approach the signal quality of subcutaneous needle electrodes but still depend on unpredictable conditions of the skin. The study provides a new valuable practical guidance for selection of extramuscular EMG electrodes. This study on horses shares common principles for the choice of adhesive surface or sc needle electrodes in human applications such as in intraoperative neurophysiological monitoring of motor functions of the brain and spinal cord.

Multipulse transcranial electrical stimulation (TES): normative data for motor evoked potentials in healthy horses.

BACKGROUND: There are indications that transcranial electrical stimulation (TES) assesses the motor function of the spinal cord in horses in a more sensitive and reproducible fashion than transcranial magnetic stimulation (TMS). However, no normative data of TES evoked motor potentials (MEP) is available. RESULTS: In this prospective study normative data of TES induced MEP wave characteristics (motor latency times (MLT); amplitude and waveform) was obtained from the extensor carpi radialis (ECR) and tibial cranialis (TC) muscles in a group of healthy horses to create a reference frame for functional diagnostic purposes. For the 12 horses involved in the study 95% confidence intervals for MLTs were 16.1-22.6 ms and 31.9-41.1 ms for ECR and TC muscles respectively. Intra-individual coefficients of variation (CV) and mean of MLTs were: ECR: 2.2-8,2% and 4.5% and TC: 1.4-6.3% and 3.5% respectively. Inter-individual CVs for MLTs were higher, though below 10% on all occasions. The mean ± sd of MEP amplitudes was respectively 3.61 ± 2.55 mV (ECR muscle left) and 4.53 ± 3.1 mV (right) and 2.66 ± 2.22 mV (TC muscle left) and 2.55 ± 1.85 mV (right). MLTs showed no significant left versus right differences. All MLTs showed significant (p < 0.05) voltage dependent decreases with slope coefficients of linear regression for ECR: - 0.049; - 0.061 ms/V and TC: - 0.082; - 0.089 ms/V (left; right). There was a positive correlation found between height at withers and MLTs in all 4 muscle groups. Finally, reliable assessment of MEP characteristics was for all muscle groups restricted to a transcranial time window of approximately 15-19 ms. CONCLUSIONS: TES is a novel and sensitive technique to assess spinal motor function in horses. It is easy applicable and highly reproducible. This study provides normative data in healthy horses on TES induced MEPs in the extensor carpi radialis and tibialis cranialis muscles bilaterally. No significant differences between MLTs of the left and right side could be demonstrated. A significant effect of stimulation voltage on MLTs was found. No significant effect of height at the withers could be found based upon the results of the current study. A study in which both TMS and TES are applied on the same group of horses is needed.

The 5-HT4 receptor agonist prucalopride does not facilitate cholinergic neurotransmission in circular and longitudinal smooth muscle preparations of equine mid-jejunum.

BACKGROUND: Postoperative ileus (POI) remains an important cause of death in horses. The recently developed selective 5-HT4 receptor agonists such as prucalopride target 5-HT4 receptors on myenteric cholinergic neurons to enhance acetylcholine release and GI motility. No clearcut in vitro evaluation whether highly selective 5-HT4 receptor agonists enhance submaximal cholinergic neurotransmission towards the muscle layer has been performed in horses. OBJECTIVES: To identify functional 5-HT4 receptors in equine jejunum. STUDY DESIGN: In vitro experimental study. METHODS: Circular and longitudinal smooth muscle strips (mid-jejunum) were mounted in organ baths between 2 platinum electrodes allowing electrical field stimulation (EFS). To delineate the conditions to obtain purely cholinergic responses, voltage-response curves were studied. To investigate the influence of prucalopride and 5-HT, submaximal cholinergic contractions at a single voltage were induced. RESULTS: In circular and longitudinal strips, EFS induced voltage-dependent neurogenic on-contractions when the bathing medium contained a NO-synthesis inhibitor and apamin to prevent inhibitory responses to NO and ATP. Contractions at a voltage inducing 50% of maximal amplitude were cholinergic, as they were blocked by atropine. These contractions were not influenced by prucalopride (up to 3µM), even in the presence of the phosphodiesterase inhibitor isobutyl-methyl-xanthine to inhibit breakdown of the second messenger of 5-HT4 receptors, cAMP. Also the full 5-HT4 receptor agonist 5-HT did not influence the EFS-induced submaximal cholinergic contractions. Moreover, prucalopride did not influence muscle tone continuously enhanced with KCl. CONCLUSIONS: There are no functional 5-HT4 receptors on myenteric cholinergic neurons nor muscular 5-HT4 receptors in equine jejunum.