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Thoracolaryngeal reflex latencies in Thoroughbred horses with recurrent laryngeal neuropathy
The Veterinary Journal The Veterinary Journal 170 (2005) 67–76 www.elsevier.com/locate/tvjl
Thoracolaryngeal reflex latencies in Thoroughbred horses with recurrent laryngeal neuropathy R.A. Curtis
a,*
, C.N. Hahn b, D.L. Evans
a,*
, T. Williams c, L. Begg
c
a
b
Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia Royal (Dick) School of Veterinary Studies, University of Edinburgh, EH259RG, UK c Randwick Equine Centre, 3 Jane St, Randwick, NSW 2031, Australia Accepted 30 March 2004
Abstract Electrolaryngeography was used to study the latencies of the thoracolaryngeal adductor reflex in Thoroughbred horses with and without recurrent laryngeal neuropathy (RLN). Latencies were compared in horses with grades 1 and 2 RLN, diagnosed by endoscopy in resting horses. The reliability of the measurements, effect of sedation and correlations of latencies with age of the horse were also studied. There was no effect of sedation on reflex latency periods. The latency of the reflex period measured to a convolved peak of the electromyographic response was significantly different in horses with grades 1 and 2 disease; medians and quartile ranges were 0.067 (0.065–0.073) and 0.072 (0.068–0.074) s, respectively (P < 0.05). Significant associations were found between reflex latencies and both horse age and the grade of RLN. Reflex latency measurements are reliable and sensitive, and may assist with the clinical appraisal of Thoroughbred horses with RLN. 2004 Elsevier Ltd. All rights reserved. Keywords: Horse; Electrolaryngeograph; Thoracolaryngeal reflex; Recurrent laryngeal nerve
1. Introduction Recurrent laryngeal neuropathy (RLN) is a prominent and incurable disease of domestic horses. It is characterised by a distal axonopathy of the recurrent laryngeal nerve supplying the intrinsic musculature of the larynx, and affects the left side more severely than the right side. The pathology underlying RLN was first described by Cole (1946), who described the nerve lesions as a primary axonal lesion of sensory or motor axons in which degeneration first occurs in the distal axon and which tends to preferentially affect the largest and longest fibres. This has since been corroborated by the
*
Corresponding authors. Tel.: +61 2 93512474; fax: +61 2 93513957. E-mail address: [email protected] (D.L. Evans).
1090-0233/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.03.020
work of Cahill and Goulden (1986a), who confirmed that the primary insult is axonal in nature, characterized by the presence of axonal atrophy. The resulting impaired abduction of the left arytenoid cartilage produces upper respiratory airway obstruction during exercise in 3–9% of Thoroughbreds (Lopez-Plana et al., 1993). Estimates of the number of horses with subclinical disease range from 41.6% (Gunn, 1972) to 91% (Cook, 1988). A subjective increase in severity of clinical signs over time suggests that the disease may be progressive (Dixon et al., 2002). Clinical diagnosis is based on abnormal respiratory noise, exercise intolerance, palpable atrophy of the left cricoarytenoideus dorsalis muscle and endoscopic evidence of left arytenoid dysfunction (Lane, 1993). Poor adduction of the vocal fold is demonstrated with the thoracolaryngeal adductor (ÔslapÕ) test (Cook and
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Thalhammer, 1991). The thoracolaryngeal adduction reflex (TAR) is induced by slapping the skin caudal to the withers during expiration, while the larynx is observed with an endoscope (Mayhew, 1989). The reflex is reduced or abolished in tense or frightened horses. The TAR is believed to follow a complex pathway whereby sensory information is carried from the skin over the dorsal part of the scapula to the spinal cord via segmental dorsal cutaneous nerves. Impulses are then carried to the somatic motor nucleus of the vagus nerve in the lateral funiculus of the spinal cord. Axons from this nucleus course caudally in the vagosympathetic trunk and then re-ascend as the recurrent laryngeal nerve to innervate the majority of the intrinsic laryngeal musculature. This reflex response consists of a brief adduction of the arytenoid cartilage, and relies on the integrity of neural pathways through the spinal cord and recurrent laryngeal nerve. In horses with recurrent laryngeal neuropathy the response from the left arytenoid is absent or weak. Grading systems based on the subjective assessment of laryngeal movement have been established to describe the severity of endoscopic changes (Lane, 1993; Hackett et al., 1991). A summary of results of several studies found that there was a wide range of reported prevalence, from 0.96% to 95% (Lane, 1993). The subjective nature of grading the laryngeal movement may account for this wide range. Hackett et al. (1991) found a 79% agreement between three observers as to the grade of disease. This system employed four grades of severity. The majority (79%) of the disagreements were due to differences in differentiation of grades 1 and 2. Use of only 4–6 grades for describing the severity of the disease also makes it difficult to diagnose subtle changes over time. Comparisons between studies that used different grading systems are also problematic. The limitations of qualitative assessment of the severity of the disease in resting horses is clear. An objective technique for assessing the function of the affected motor unit is therefore desirable. A quantitative method to assess nerve function by measuring the reflex latency after inducing the thoracolaryngeal reflex has been described (Cook, 1988). This technique may represent a more accurate and sensitive test for evaluating horses with RLN. The period between the stimulus of a TAR and the electromyographic response at the larynx was measured at a differential
electrode pair in a standardized location over the larynx (Cook and Thalhammer, 1991). This evoked potential test is known as electrolaryngeography. Reflex latencies were obtained on the left and right sides, and the conduction velocity of the reflex was calculated with use of an estimate of the length of the recurrent laryngeal nerves as indices of the length of the left and right reflex pathways (Cook, 1998; Cole, 1946). Data obtained on the right side was used as a reference against which the conduction velocity of the left side could be compared. This technique has not been systematically assessed in Thoroughbreds. The aims of this study were to assess the repeatability of the measurements obtained by electrolaryngeography, and investigate the effects of sedation on the measurements. It was hypothesised that latencies would be significantly different in normal and horses with low grade RLN. The study evaluated horses with grades 1 and 2 RLN, as clinicians generally have no problems differentiating normal from severely affected horses. The ELG technique would be most useful if it is sensitive enough to objectively evaluate animals with mild disease. The correlations of the reflex latency measurements with horse age, body morphology and endoscopically observed grades of RLN were also investigated.
2. Materials and methods 2.1. Laryngoscopy A flexible endoscope was placed into the pharynx through the right nares of a resting horse to enable inspection of the upper airways and movements of the arytenoid cartilage. Laryngoscopy was performed before every ELG test in this study by an experienced equine veterinary surgeon. Laryngeal function was graded according to the system suggested by Lane (1993), Table 1. All endoscopic evaluations were performed without sedation. 2.2. Electrolaryngeography The ELG test was performed according to the recommendations of Cook and Thalhammer (1991) and Cook (1993). Compound motor action potentials were re-
Table 1 Recurrent laryngeal neuropathy grading system (Lane, 1993) Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
Adductory and abductory movements at rest and after exercise fully synchronous and symmetrical Gross adductory and abductory movements symmetrical. Transient asynchrony, flutter or delayed or biphasic abduction may be seen – especially in the left arytenoid Left arytenoid capable of full abduction, but activity greatly reduced compared with right. Periods of prolonged asymmetry Left arytenoid no longer capable of full abduction and during adduction compensation by the right arytenoid crossing the midline may be evident. Asymmetry is marked. True hemiplegia – total paresis of the left arytenoid. No response to the Ôslap testÕ
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69
Fig. 1. Position of ELG recording electrodes over the larynx for recording signals generated by the thoracolaryngeal adductor test. A, active electrode; R, reference electrode; G, ground electrode.
corded after stimulating the skin by slapping the dorsal part of the cranial scapula using a glove incorporating a piezo shock sensor. This triggered the ELG instrumentation into recording the resultant evoked compound muscle action potentials between two 0.3 · 12 mm platinum trans-dermal electrodes sited over the contralateral laryngeal musculature. The non-inverting (active) electrode was placed between the sternomandibularis muscle and linguofacial vein, close to the cricothyroid fulcrum (Fig. 1). The inverting (reference) electrode was placed at the edge of the mandible, approximately 5 cm dorsal and rostral to the positive electrode. A third electrode was configured as a driven ground electrode and formed a 4–5 cm equilateral triangle with the other two. Positioning of the two signal electrodes was found to be critical to obtaining a clean and consistent laryngogram recording. Five TARs were induced by slap on the left side of the chest wall while measuring the reflex latencies with electrodes on the right side of the larynx. The electrodes were then moved to the opposite side and the test repeated. 2.3. Instrumentation The ELG equipment (Cook, 1999) consists of an electromyograph instrumentation amplifier configured with a driven common mode ground output. The resultant electromyogram signal (EMG) was digitised by an analog to digital converter sampling at 3 kHz. The data were stored and processed on a laptop computer using ELG 2000 software (Crafted Software) running under the Windows operating system. Recording began when a slap signal from the glove shock sensor, and conditioned by a monostable circuit, triggered the computer
software. Once triggered, the software recorded for 100 ms, and displayed the electrolaryngogram (ELG) signal graphically. All the electronics including the computer were powered by batteries in order to electrically isolate the horse. Traces were scaled to a normalised maximum by the software. The ELG 2000 software formats the data for export into an Excel database, for statistical analysis. 2.4. Signal processing Typical recordings of left and right TAR responses in one horse are shown in Figs. 2(a) and (b). The recordings show the time period from the stimulus to the onset of a muscle action potential; the reflex response. The latency durations were expressed as the mean of five records. Latencies measured to the peaks of the EMG records were designated as Rlat and Llat for results on the right and left sides. CooksÕ original studies (Cook and Thalhammer, 1991) were based on a definition of latency duration as the time from the stimulus to the time corresponding to the peak of the evoked electromyographic response (software ELG 3.4 for MSDOS 1998). However, the time at the peak of the electromyographic response may not be the ideal location to determine the latency of the reflex pathway. The peak latency may be affected by variable shape or duration of the compound muscle action potential. An alternative approach is to measure the time interval between the stimulus and the initial deviation from baseline (Ludin, 1980). A pilot study of TAR responses in Thoroughbred horses however showed that signals were often noisy, and the visual assessment of the point of deviation from the baseline was thought to be unacceptably subjective.
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Fig. 2. Typical ELG traces showing convolution and averaging processes in records from the left and right reflex recordings in one horse. (a and b) are raw traces; (c and d) are convolved traces; (e and f) are resultant means of the convolved traces.
In an attempt to remove this subjectivity, further signal processing was developed in Matlab (Mathworks, 2001) to establish an objective mathematical model to clearly define the start of the electromyographic response. First, each trace was convolved (Kahaner et al., 1989) with a predetermined ‘‘synthesised expected peak’’ shape, as illustrated in Fig. 3. This reference or convolutor shape was estimated from the mean of selected Ônoise freeÕ peaks pooled from several horses
and was of a Ôflat top triangleÕ vector form [1 2 3 4 5 5 4 3 2 1]. The convolution process increases the power to detect the waveform that represents the electromyographic response against background noise. After convolving each of the five records, as shown in Figs. 2(c) and (d), the mean convolved record was produced (Figs. 2(e) and (f)). The time from the stimulus to the start of the electromyographic response on the convolved EMG record was defined as a start latency.
amplitude (scalar units)
6 5 4 3 2 1 0 -2
0
2
4
6
8
time (ms) Fig. 3. Flat top triangle shape used for convolution of raw data.
10
12
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Table 2 Variables derived from electrolaryngographic recordings of the thoracolaryngeal reflex, with abbreviations and units of measurement Measured variable
Abbreviation
Units
Latency period on left side to peak of the electromyographic response in raw data (average of five records). Latency period on right side to peak of the electromyographic response in raw data (average of five records). Conduction velocity of reflex on left side representing the estimated length of the reflex pathway divided by Llat Conduction velocity of reflex on right side representing the estimated length of the reflex pathway divided by Rlat Latency to start of convolved electromyographic response on left side Latency to start of convolved electromyographic response on right side Latency to the peak of the convolved electromyographic response on left side Latency to the peak of the convolved electromyographic response on left side Ratio of latency periods (see Eq. (4))
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
s s m/s m/s s s s s
The convolved record was a signal that represented the mean of five recordings. These latencies on the left and right sides were designated Lst and Rst. In addition, the convolved peak latencies (Lpk, Rpk) were calculated as the times from the stimulus to the times of the peak of the electromyographic response in the convolved record. The definitions of all reflex latency and velocity measurements are described in Table 2. 2.5. Estimation of reflex pathway velocities An estimate of the length of the reflex pathway was obtained by measuring the distance in metres from the poll to withers, withers to saddle and the height of the horse at the top of the wither using a flexible tape (Cook, 1998; Cole, 1946). These data were entered into the ELG2000 software. The software estimated left and right recurrent laryngeal nerve lengths (Lnl, Rnl) from formulae based on the methods used previously (Cook (1998), ELG 3.4 for MSDOS): LnlðmÞ ¼ 3 ðPoll–WithersÞ þ ðWithers–SaddleÞ þ 0:15:
ð1Þ
Poll–Withers is the distance (m) from the poll in line with the cranial edge of the ears to the spine of the scapula. Withers-Saddle (m) is the distance from the spine of the scapula to the lowest point of the back. Right nerve length (Rnl) was estimated as (Lnl-0.4) m (Cook, 1998). Reflex conduction velocities on the left and right sides, Lcv and Rcv, were calculated by: Lcv ¼ ðLnl=LlatÞ m=s;
ð2Þ
Rcv ¼ ðRnl=RlatÞ m=s;
ð3Þ
where Llat and Rlat are the latency times to the peaks of the electromyographic responses. A latency ratio (Lratio) was also calculated to express a relationship between left and right convolved latencies. Lratio ¼ ðLst – RstÞ=Rst:
ð4Þ
The effect of sedation was examined in a crossover study of six race-fit thoroughbred horses with ages ranging from two to six years. On day one, the ELG
test was performed on three sedated horses and three unsedated horses. On the following day at approximately the same time these treatments were crossed over. Sedation consisted of 5 mg of the a2 agonist detomidine, administered intravenously 5–10 min before the ELG test. This resulted in 30–40 min of light sedation for ease of animal handling. A quiet room or stable was used for all tests. A group of 12 sedated horses was tested to determine if there was a relationship between age and TAR latencies. These horses had months of birth that were verifiable from official records. Horse ages ranged from 24–82 months. All horses in this group were endoscopically determined to have grade 1 RLN. The relationships between endoscopic grades and ELG measurements were investigated in 60 sedated thoroughbred horses in training. Correlations between ELG measurements and body size were also examined in horses with grade 1 RLN in two age groups, two- and three-year-old horses. Repeatability was investigated by comparing measurements on two consecutive days in six horses. 2.6. Statistics Normality was not assumed; therefore non parametric statistical tests were used. Results were expressed as medians and quartile ranges. Latencies for each horse were recorded as the mean of five tests. A nonparametric alternative to the two-sample t test (Wilcoxon signed rank test) was used to compare results in sedated and non sedated horses. A non-parametric independent two-group comparison test (Mann–Whitney) was used to compare results on days 1 and 2 in the study of the repeatability of the ELG technique. The relationships between age and ELG results were investigated with linear regression analyses and Spearman Rank correlations. The relationships between ELG measurements and endoscopic grades were described with Spearman rank correlations. The association between latencies and the distance from the poll to the withers (an index of body size) was also examined by Spearman rank correlation. Results in horses with endoscopic grades 1 and 2 were compared with a Mann–Whitney test. P values
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Table 3 Description of the Thoroughbred horses used in the study Number and gender of horses
60 (34 Male, 26 female)
Number in each RLN grade (assessed by endoscopy at rest)
Grade 1, n = 35; Grade 2, n = 19 Grade 3, n = 2; Grade 4, n = 4
Number (n) of horses in each age group (years) Age n
1 8
2 8
3 10
4 9
less than or equal to 0.05 were considered statistically significant.
3. Results Table 3 describes the RLN grades and features of the 60 horses used in the study. There was no significant effect of sedation on the ELG measurements (Table 4). There were no significant differences detected with repeated measurements in sedated horses tested on consecutive days (Table 5). The right and left latencies measured to the start and peak of the convolved EMG response (Lst, Rst, Lpk, and Rpk) were significantly associated with age (Table
5 11
6 9
7 2
8 1
11 1
20 1
6; Fig. 4). Median age was 46.5 months, and quartile range was 35.2–57.7 months. Convolved left and right start latencies (Lst, Rst) were significantly related to endoscopic grade of RLN. The unconvolved latency (Rlat) and right convolved peak latency (Rpk) were also correlated with grade of RLN (Table 7). No correlations were found between left sided latencies and the poll-wither distance in two- or three-year-old horses (Table 8). Latency ratios likewise did not depend on the poll-withers distance in two- or three-year-old horses. The latencies to the peak of the convolved EMG responses (Lpk, Rpk) were significantly different in horses with grades 1 and 2 RLN (P < 0.05, Table 9). Medians
Table 4 Latencies (s) and conduction velocities (m/s) in six sedated and unsedated Thoroughbred horses Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
Without sedation
With sedation
P
Median
Quartile range
Median
Quartile range
0.072 0.057 49.37 54.63 0.065 0.052 0.073 0.058 0.26
(0.066, 0.075) (0.055, 0.057) (47.51, 53.50) (48.10, 54.95) (0.063, 0.068) (0.049, 0.053) (0.070, 0.075) (0.055, 0.059) (0.19, 0.33)
0.069 0.059 51.15 52.82 0.064 0.052 0.070 0.061 0.29
(0.068, 0.070) (0.058, 0.061) (50.05, 52.16) (51.08, 54.18) (0.061, 0.066) (0.051, 0.055) (0.068, 0.074) (0.058, 0.062) (0.15, 0.33)
0.31 0.22 0.31 1.00 1.00 1.00 0.31 0.31 0.69
For the definitions of the variables, see Table 2. P, probability statistic.
Table 5 Latencies (s) and conduction velocities (m/s) in tests repeated on consecutive days (n = 6) Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
Day 1
Day 2
P
Median
Quartile range
Median
Quartile range
0.073 0.061 53.79 57.52 0.063 0.051 0.072 0.059 0.21
(0.071, 0.076) (0.059, 0.064) (50.67, 57.20) (54.66, 62.49) (0.059, 0.063) (0.051, 0.055) (0.071, 0.074) (0.058, 0.061) (0.16, 0.22)
0.073 0.060 55.99 57.23 0.062 0.050 0.071 0.058 0.23
(0.070, 0.075) (0.058, 0.065) (53.78, 57.59) (54.13, 63.15) (0.061, 0.064) (0.050, 0.052) (0.071, 0.074) (0.057, 0.061) (0.21, 0.24)
For the definitions of the variables, see Table 2. P, probability statistic.
0.82 0.99 0.39 0.82 0.94 0.18 0.94 0.82 0.09
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Table 6 Spearman rank correlations (R) between age (months) and the ELG measurements (n = 12) Variable
Median
Quartile range
R
P
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
0.070 0.059 56.87 61.30 0.063 0.051 0.072 0.059 0.20
(0.068, 0.072) (0.057, 0.063) (54.29, 60.67) (55.72, 63.85) (0.058, 0.066) (0.049, 0.055) (0.069, 0.074) (0.057, 0.061) (0.16, 0.27)
0.18 0.28 0.01 0.04 0.53 0.64 0.56 0.77 0.05
0.5 0.32 0.95 0.87 0.05 0.01 0.03 0.01 0.84
For the definitions of the variables, see Table 2. P, probability statistic.
75.0
Convolved Latency (ms)
70.0 65.0 60.0 55.0 50.0 45.0 40.0
Regression Equation Lst y = 0.21x + 52.00 R2 = 0.48 Rst y = 0.12x + 45.20 R2 = 0.27
35.0
Lst
Rst
30.0 0.0
10.0
20.0
30.0
40.0 50.0 Age (months)
60.0
70.0
80.0
90.0
Fig. 4. Relationships between age (months) and ELG convolved left and right start point latencies (Lst, Rst). R; Spearman rank correlation (n= 12).
Table 7 Spearman rank correlations (R) between RLN grade (1–5) and the ELG measurements (n = 52) Variable
Median
Quartile range
R
P
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
0.069 0.061 56.15 59.10 0.059 0.051 0.068 0.058 0.19
(0.066, 0.074) (0.057, 0.064) (53.70, 59.40) (54.90, 62.65) (0.057, 0.066) (0.047, 0.055) (0.065, 0.074) (0.055, 0.062) (0.13, 0.27)
0.20 0.37 0.11 0.21 0.27 0.31 0.21 0.34 0.06
0.14 0.01 0.44 0.13 0.05 0.02 0.14 0.01 0.65
For the definitions of the variables, see Table 2. P, probability statistic.
of the latencies to the peak of the convolved EMG were 7.5% greater on the left side in grade 2 horses. Latencies to the start of the convolved EMG on the right side (Rst) and the unconvolved peak on the left side (Llat) were also significantly greater in grade 2 horses (Table 9).
4. Discussion ELG measurements were highly repeatable under the conditions used in this study. Tests were done under controlled conditions, with the same operators for the
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Table 8 Spearman rank correlations (R) between distance from poll to wither and the ELG measurements in two- (n = 7) and three-year-old (n = 7) Thoroughbred horses with grade 1 RLN Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
Two-year-olds
Three-year-olds
Median
Quartile range
R
P
Median
Quartile range
R
P
0.070 0.058 54.79 62.46 62.04 0.050 0.072 0.058 0.24
(0.056, 0.086) (0.051, 0.060) (51.19, 69.36) (56.60, 68.52) (0.050, 0.072) (0.046, 0.052) (0.057, 0.078) (0.051, 0.060) (0.08, 0.39)
0.68 0.21 0.36 0.64 0.18 0.38 0.32 0.31 0.18
0.07 0.60 0.39 0.09 0.66 0.34 0.44 0.44 0.66
0.067 0.060 58.59 59.56 0.058 0.048 0.068 0.057 0.18
(0.065, 0.080) (0.054, 0.064) (47.74, 64.15) (52.10, 72.51) (0.052, 0.063) (0.044, 0.062) (0.064, 0.072) (0.051, 0.067) (0.03, 0.32)
0.41 0.70 0.63 0.79 0.56 0.13 0.44 0.05 0.36
0.30 0.05 0.09 0.02 0.15 0.72 0.30 0.84 0.39
For the definitions of the variables, see Table 2. P, probability statistic.
Table 9 Latencies and conduction velocities in horses with grades 1 (n = 35) and 2 (n = 19) laryngeal hemiplegia Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
P
0.025 0.156 0.731 0.514 0.108 0.018 0.029 0.010 0.419
Grade 1
Grade 2
Median
Quartile range
Median
Quartile range
0.068 0.059 56.65 60.52 0.059 0.049 0.067 0.058 0.20
(0.065, 0.073) (0.056, 0.062) (53.71, 59.72) (56.19, 63.09) (0.056, 0.064) (0.046, 0.053) (0.065, 0.073) (0.054, 0.059) (0.14, 0.27)
0.071 0.061 55.43 57.51 0.059 0.053 0.072 0.061 0.20
(0.068, 0.075) (0.058, 0.066) (53.78, 58.78) (53.78, 63.24) (0.058, 0.065) (0.050, 0.055) (0.068, 0.074) (0.057, 0.062) (0.10, 0.26)
repeatability trial. It was found that after minimal operator practice, consistent, artefact free results could be readily obtained. It was noted during early trials that different operators could place the electrodes in slightly different positions, and variably ÔslapÕ the withers. These factors should not change the latency, but may mask an ELG trace in myographic noise or cause direct current offsets that make the ELG peaks harder to distinguish. Results from the sedation crossover study show that it is quite appropriate to use a2 agonist sedation when performing an ELG test. There were no significant effects of sedation on the right or left latency periods. This result was expected, as one of the properties of evoked potentials is their resistance to sedative drugs. Sedation enables safer handling of the animals and more consistent ELG traces without additional muscle noise. The statistical difference in the right average velocity is unlikely to be biologically significant. It may be reasonable to expect horses with lower grades of RLN to maintain normal nerve conduction velocities, because a proportion of the large axons will still be functional. This explanation was used to discuss the results obtained by Hawe et al. (2001). In that study the slap test (TLR) latency was compared to five other diagnostic techniques in Clydesdales and ponies. There
were no correlations between nerve latency periods and laryngeal function in those breeds. In the current study, latency measurements were significantly different in Thoroughbred horses with grades 1 and 2 RLN. The use of an algorithm to determine convolved latencies in the present study may have resulted in a more accurate measurement of the latencies than was possible with the analogue filter system used by Hawe et al. (2001). The current study may therefore have been more sensitive, enabling detection of small differences in latencies. Recent evidence has suggested that there is interdependence between the axon and its myelin sheath (Trapp et al., 1999), and that in a chronic disease such as RLN there may be myelin pathology secondary to axonal pathology. Schwann cell pathology is present in RLN, as shown by BungnerÕs bands, onion bulbs and wallerianlike degeneration (Cahill and Goulden, 1986b; Duncan et al., 1978). It is therefore reasonable to expect that an accurate measurement of latency will enable detection of a decrease in nerve conduction latency, even in horses with low grades of clinical disease. The novel technical approach in this study was the extensive use of standard digital signal processing techniques to process and analyse the ELG data. Digital sig-
R.A. Curtis et al. / The Veterinary Journal 170 (2005) 67–76
nal processing allows automated analysis on time domain events (take off points, slopes, wave shapes). Further, digital filtering to remove high frequency artefacts before analysis has the advantage over analog filtering of reducing phase errors and thereby maintaining the time domain accuracy required for the ELG. The standard ELG 2000 software used a simple approach to finding latency peaks. This involves placing a window around the expected latency region and finding the first major peak. This can give incorrect results, particularly when the laryngeal signal is small with respect to large amounts of myographic and electrical noise. Convolving and averaging the ELG traces significantly improved the ability to resolve wave shapes. Conduction velocities did not differentiate grades 1 and 2 horses. The calculation of velocity was based on an estimated reflex pathway length. However, this measurement is subject to error. The accuracy of the estimation of the true length of the recurrent laryngeal nerve from measurements of body morphology (Cook and Thalhammer, 1991) has not been described. There were no significant correlations between poll to wither distance and latency in similar age and grade horses. Measurements of conduction velocities based on estimates of nerve length were not useful for testing the hypotheses in this study. There were associations between horse age and convolved start and peak latencies obtained by signal processing. These results applied for data obtained from TAR reflexes studied on the left and right sides. These results confirm a suggestion by Dixon et al. (2002) that the disease is progressive. Small changes in nerve function over time might be detectable with ELG measurements of convolved latencies, and further work could investigate the rate of change in latencies in young horses. The latencies to the start of the convolved muscle action potential on the left side were not significantly different in horses with grades 1 and 2 RLN, but the latencies to the peak of the curve were. This suggests that the latency measurement that includes the rise-time of the muscle action potential is the most sensitive, potentially because the peak measures the average of all the conducting axons while the start measures only the latency of the largest axon. Even in horses with severe RLN a few large axons may still remain, and thus the latency to the start of the muscle action potential would not be affected. The significant correlations between age and latencies could have reflected an increase in body size in the older horses. However, there was no dependency of left latencies on the poll to wither distance, suggesting that the association with age reflects a progressive increase in reflex latency in normal horses. In conclusion, the use of electrolaryngeography for measurement of TAR latencies is a repeatable method
75
that is low cost and simple to perform in a clinical setting. These results support the suggestion by Cook (1988) that electrodiagnostic measurement of the latency of the thoracolaryngeal reflex has a role in evaluation of horses with RLN. Results were not affected by sedation. ELG results were correlated with age and endoscope score, and differentiated horses with endoscope scores 1 and 2. The technique has potential in the clinical evaluation of the horse, and for further studies of the nature of the disease. It would be of interest to study differences in nerve function in younger horses, and the rates of change in latencies in longitudinal studies. Acknowledgements The authors thank Thoroughbred racecourse trainer Ron Quinton and staff, particularly Alan Putland, for access to a well-trained and cared for population of thoroughbred horses. Dr. Stuart Murray and Dr. Richard Humberstone helped in conducting the tests. We are grateful to the Australian Rural Industries Research and Development Corporation for financial support.
References Cahill, J.I., Goulden, B.E., 1986a. Equine laryngeal hemiplegia. II. An electron microscopic study of peripheral nerves. New Zealand Veterinary Journal 34, 170–175. Cahill, J.I., Goulden, B.E., 1986b. Equine laryngeal hemiplegia. I. A light microscopic study of peripheral nerves. New Zealand Veterinary Journal 34, 161–169. Cole, R.C., 1946. Changes in the equine larynx associated with laryngeal hemiplegia. American Journal of Veterinary Research 7, 69–77. Cook, W.R., 1988. Recent observations on recurrent laryngeal neuropathy in the horse: applications to practice. In: 34th Convention of American Equine Practitioners, pp. 427–478. Cook, W.R., 1998. Electrolaryngeograph Version ELG 3.4 UserÕs Guide. ELG, Inc., Chestertown, pp. 49–50. Cook, W.R., 1993. Diagnosis and grading of hereditary recurrent laryngeal hemiplegia in the horse. Journal of Equine Veterinary Science 8, 432–455. Cook, W.R., 1999. The ear, nose and the lie in the throat. In: Rossdale, P.D., Greet, T.R.C., Harris, P.A., Green, R.E., Hall, S. (Eds.), Guardians of the Horse: Past, Present and Future. British Equine Veterinary Association, London, pp. 175–182. Cook, W.R., Thalhammer, J.G., 1991. Electrodiagnosis test for the objective grading of recurrent laryngeal hemiplegia. Proceedings of the Annual Convention of the American Association of Equine Practitioners 37, 275–296. Dixon, P.M., Mcgorum, B.C., Railton, D.I., Hawe, C., Tremaine, W.H., Pickles, K., Mccann, J., 2002. Clinical and endoscopic evidence of progression in 152 cases of equine recurrent laryngeal neuropathy (RLN). Equine Veterinary Journal 34, 29–34. Duncan, I.D., Griffiths, I.R., Madrid, R.E., 1978. A light and electron microscopic study of the neuropathy of equine idiopathic laryngeal hemiplegia. Neuropathology and Applied Neurobiology 4, 483–501. Gunn, H.M., 1972. Histological observations on laryngeal skeletal muscle fibres in ‘‘normal’’ horses. Equine Veterinary Journal 4, 144–148.
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Hackett, R.P., Fubini, S.L., Erb, H.N., 1991. The reliability of endoscopic examination in the assessment of arytenoid cartilage movement in the horse. Veterinary Surgery 20, 3174–3179. Hawe, C., Dixon, P.M., Mayhew, I.G., 2001. A study of an electrodiagnostic technique for the evaluation of equine recurrent laryngeal neuropathy. Equine Veterinary Journal 33, 459–465. Kahaner, D., Moler, C., Nash, S., 1989. Numerical Methods and Software. Prentice-Hall, New Jersey, pp. 458–460. Lopez-Plana, C., Sautet, J.Y., Pons, J., Navarro, G., 1993. Morphometric study of the recurrent laryngeal nerve in young ‘‘normal’’ horses. Research in Veterinary Science 55, 333–337.
Lane, J.G., 1993. Equine recurrent laryngeal neuropathy (RLN): current attitudes to aetiology, diagnosis and treatment. In: Proceedings of the 15th Bain-Fallon Memorial Lectures Australian. Equine Veterinary Association, Canberra, pp. 173–192. Ludin, H.P., 1980. Electroneurography. In: Electromyography in Practice. Georg Thieme, New York, pp. 31–34. Mayhew, I.G., 1989. Large Animal Neurology: A Handbook for Veterinary Clinicians. Lea & Febiger, Philadelphia, PA. Trapp, B.D., Bo¨, L., Mo¨rk, S., Chang, A., 1999. Pathogenesis of tissue injury in MS lesions. Journal of Neuroimmunology 98, 49–56.
Book review D. Marlin, K. Nankervis, Equine Exercise Physiology, Blackwell Science, Oxford, 2002, ISBN 0632055529, p. 296 £29.95 (Soft) Many of us who teach equine exercise physiology or field calls from horse owners, trainers, and equine clinicians should thank the authors of this book for a text that can be used by multiple audiences to understand the basics of equine exercise physiology. Dr. David Marlin and Ms. Nankervis have penned a very readable textbook that thoroughly covers the basic concepts of equine exercise physiology. The book presents the most recent information we have on these magnificent athletes in a very easy to read and unpretentious manner. Material covered in the book is well organized into three very complete sections. Part I covers the basics of biochemistry and energetics and each of the physiological systems that plays a major role in the response to exertion. The second section of the book gives the reader a detailed understanding of how each organ system responds to acute exercise and how each system adapts to the repeated challenge of exercise training. In the third section of the book, the authors do a great job of synthesizing how the basics of equine exercise physiology can be used in an applied fashion to enhance our ability to train and care for the equine athlete. However, one should note that this is not a dry scientific tome and that in addition to making this book enjoyable to read, the authors have done a great job of including an ample number of detailed, yet easy to understand figures, charts, and photographs that allow one make a full visualisation of the inner workings of the equine athlete. This doi:10.1016/j.tvjl.2005.03.011
is especially useful to those who are visual learners. Finally, at the end of each chapter they include a summary of key points. This summary of material covered is thoughtful and an excellent review for the reader that has additionally served as an excellent resource improving my own classroom lectures. My positive opinion of this book is evident and I use it as one of two required texts for my classes in equine exercise physiology. However, I use it with an appreciation for the value of a well written book. Supporting that opinion are the comments from students on their written course evaluations where they praised the bookÕs usefulness as a key resource to bridge the gap from systems physiology, where they had to memorise a great deal of material, to exercise physiology, a subject that requires an integrative understanding of the biology of exercise. To that end the authors have created an excellent resource for undergraduate, graduate, and even veterinary students looking for a great supplement to existing weighty scientific and clinically oriented books. For the horse owner, trainer, and equine clinician, this text is a great resource to pull out when one is interested in a more clear understanding of advanced integrative and applied aspects of the field of equine exercise physiology and equine sports medicine. Kenneth Harrington McKeever Department of Animal Sciences, Rutgers The State University of New Jersey, 84 Lipman Drive New Brunswick, NJ 08901-8525 USA E-mail address: [email protected]
Thoracolaryngeal reflex latencies in Thoroughbred horses with recurrent laryngeal neuropathy R.A. Curtis
a,*
, C.N. Hahn b, D.L. Evans
a,*
, T. Williams c, L. Begg
c
a
b
Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia Royal (Dick) School of Veterinary Studies, University of Edinburgh, EH259RG, UK c Randwick Equine Centre, 3 Jane St, Randwick, NSW 2031, Australia Accepted 30 March 2004
Abstract Electrolaryngeography was used to study the latencies of the thoracolaryngeal adductor reflex in Thoroughbred horses with and without recurrent laryngeal neuropathy (RLN). Latencies were compared in horses with grades 1 and 2 RLN, diagnosed by endoscopy in resting horses. The reliability of the measurements, effect of sedation and correlations of latencies with age of the horse were also studied. There was no effect of sedation on reflex latency periods. The latency of the reflex period measured to a convolved peak of the electromyographic response was significantly different in horses with grades 1 and 2 disease; medians and quartile ranges were 0.067 (0.065–0.073) and 0.072 (0.068–0.074) s, respectively (P < 0.05). Significant associations were found between reflex latencies and both horse age and the grade of RLN. Reflex latency measurements are reliable and sensitive, and may assist with the clinical appraisal of Thoroughbred horses with RLN. 2004 Elsevier Ltd. All rights reserved. Keywords: Horse; Electrolaryngeograph; Thoracolaryngeal reflex; Recurrent laryngeal nerve
1. Introduction Recurrent laryngeal neuropathy (RLN) is a prominent and incurable disease of domestic horses. It is characterised by a distal axonopathy of the recurrent laryngeal nerve supplying the intrinsic musculature of the larynx, and affects the left side more severely than the right side. The pathology underlying RLN was first described by Cole (1946), who described the nerve lesions as a primary axonal lesion of sensory or motor axons in which degeneration first occurs in the distal axon and which tends to preferentially affect the largest and longest fibres. This has since been corroborated by the
*
Corresponding authors. Tel.: +61 2 93512474; fax: +61 2 93513957. E-mail address: [email protected] (D.L. Evans).
1090-0233/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.03.020
work of Cahill and Goulden (1986a), who confirmed that the primary insult is axonal in nature, characterized by the presence of axonal atrophy. The resulting impaired abduction of the left arytenoid cartilage produces upper respiratory airway obstruction during exercise in 3–9% of Thoroughbreds (Lopez-Plana et al., 1993). Estimates of the number of horses with subclinical disease range from 41.6% (Gunn, 1972) to 91% (Cook, 1988). A subjective increase in severity of clinical signs over time suggests that the disease may be progressive (Dixon et al., 2002). Clinical diagnosis is based on abnormal respiratory noise, exercise intolerance, palpable atrophy of the left cricoarytenoideus dorsalis muscle and endoscopic evidence of left arytenoid dysfunction (Lane, 1993). Poor adduction of the vocal fold is demonstrated with the thoracolaryngeal adductor (ÔslapÕ) test (Cook and
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Thalhammer, 1991). The thoracolaryngeal adduction reflex (TAR) is induced by slapping the skin caudal to the withers during expiration, while the larynx is observed with an endoscope (Mayhew, 1989). The reflex is reduced or abolished in tense or frightened horses. The TAR is believed to follow a complex pathway whereby sensory information is carried from the skin over the dorsal part of the scapula to the spinal cord via segmental dorsal cutaneous nerves. Impulses are then carried to the somatic motor nucleus of the vagus nerve in the lateral funiculus of the spinal cord. Axons from this nucleus course caudally in the vagosympathetic trunk and then re-ascend as the recurrent laryngeal nerve to innervate the majority of the intrinsic laryngeal musculature. This reflex response consists of a brief adduction of the arytenoid cartilage, and relies on the integrity of neural pathways through the spinal cord and recurrent laryngeal nerve. In horses with recurrent laryngeal neuropathy the response from the left arytenoid is absent or weak. Grading systems based on the subjective assessment of laryngeal movement have been established to describe the severity of endoscopic changes (Lane, 1993; Hackett et al., 1991). A summary of results of several studies found that there was a wide range of reported prevalence, from 0.96% to 95% (Lane, 1993). The subjective nature of grading the laryngeal movement may account for this wide range. Hackett et al. (1991) found a 79% agreement between three observers as to the grade of disease. This system employed four grades of severity. The majority (79%) of the disagreements were due to differences in differentiation of grades 1 and 2. Use of only 4–6 grades for describing the severity of the disease also makes it difficult to diagnose subtle changes over time. Comparisons between studies that used different grading systems are also problematic. The limitations of qualitative assessment of the severity of the disease in resting horses is clear. An objective technique for assessing the function of the affected motor unit is therefore desirable. A quantitative method to assess nerve function by measuring the reflex latency after inducing the thoracolaryngeal reflex has been described (Cook, 1988). This technique may represent a more accurate and sensitive test for evaluating horses with RLN. The period between the stimulus of a TAR and the electromyographic response at the larynx was measured at a differential
electrode pair in a standardized location over the larynx (Cook and Thalhammer, 1991). This evoked potential test is known as electrolaryngeography. Reflex latencies were obtained on the left and right sides, and the conduction velocity of the reflex was calculated with use of an estimate of the length of the recurrent laryngeal nerves as indices of the length of the left and right reflex pathways (Cook, 1998; Cole, 1946). Data obtained on the right side was used as a reference against which the conduction velocity of the left side could be compared. This technique has not been systematically assessed in Thoroughbreds. The aims of this study were to assess the repeatability of the measurements obtained by electrolaryngeography, and investigate the effects of sedation on the measurements. It was hypothesised that latencies would be significantly different in normal and horses with low grade RLN. The study evaluated horses with grades 1 and 2 RLN, as clinicians generally have no problems differentiating normal from severely affected horses. The ELG technique would be most useful if it is sensitive enough to objectively evaluate animals with mild disease. The correlations of the reflex latency measurements with horse age, body morphology and endoscopically observed grades of RLN were also investigated.
2. Materials and methods 2.1. Laryngoscopy A flexible endoscope was placed into the pharynx through the right nares of a resting horse to enable inspection of the upper airways and movements of the arytenoid cartilage. Laryngoscopy was performed before every ELG test in this study by an experienced equine veterinary surgeon. Laryngeal function was graded according to the system suggested by Lane (1993), Table 1. All endoscopic evaluations were performed without sedation. 2.2. Electrolaryngeography The ELG test was performed according to the recommendations of Cook and Thalhammer (1991) and Cook (1993). Compound motor action potentials were re-
Table 1 Recurrent laryngeal neuropathy grading system (Lane, 1993) Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
Adductory and abductory movements at rest and after exercise fully synchronous and symmetrical Gross adductory and abductory movements symmetrical. Transient asynchrony, flutter or delayed or biphasic abduction may be seen – especially in the left arytenoid Left arytenoid capable of full abduction, but activity greatly reduced compared with right. Periods of prolonged asymmetry Left arytenoid no longer capable of full abduction and during adduction compensation by the right arytenoid crossing the midline may be evident. Asymmetry is marked. True hemiplegia – total paresis of the left arytenoid. No response to the Ôslap testÕ
R.A. Curtis et al. / The Veterinary Journal 170 (2005) 67–76
69
Fig. 1. Position of ELG recording electrodes over the larynx for recording signals generated by the thoracolaryngeal adductor test. A, active electrode; R, reference electrode; G, ground electrode.
corded after stimulating the skin by slapping the dorsal part of the cranial scapula using a glove incorporating a piezo shock sensor. This triggered the ELG instrumentation into recording the resultant evoked compound muscle action potentials between two 0.3 · 12 mm platinum trans-dermal electrodes sited over the contralateral laryngeal musculature. The non-inverting (active) electrode was placed between the sternomandibularis muscle and linguofacial vein, close to the cricothyroid fulcrum (Fig. 1). The inverting (reference) electrode was placed at the edge of the mandible, approximately 5 cm dorsal and rostral to the positive electrode. A third electrode was configured as a driven ground electrode and formed a 4–5 cm equilateral triangle with the other two. Positioning of the two signal electrodes was found to be critical to obtaining a clean and consistent laryngogram recording. Five TARs were induced by slap on the left side of the chest wall while measuring the reflex latencies with electrodes on the right side of the larynx. The electrodes were then moved to the opposite side and the test repeated. 2.3. Instrumentation The ELG equipment (Cook, 1999) consists of an electromyograph instrumentation amplifier configured with a driven common mode ground output. The resultant electromyogram signal (EMG) was digitised by an analog to digital converter sampling at 3 kHz. The data were stored and processed on a laptop computer using ELG 2000 software (Crafted Software) running under the Windows operating system. Recording began when a slap signal from the glove shock sensor, and conditioned by a monostable circuit, triggered the computer
software. Once triggered, the software recorded for 100 ms, and displayed the electrolaryngogram (ELG) signal graphically. All the electronics including the computer were powered by batteries in order to electrically isolate the horse. Traces were scaled to a normalised maximum by the software. The ELG 2000 software formats the data for export into an Excel database, for statistical analysis. 2.4. Signal processing Typical recordings of left and right TAR responses in one horse are shown in Figs. 2(a) and (b). The recordings show the time period from the stimulus to the onset of a muscle action potential; the reflex response. The latency durations were expressed as the mean of five records. Latencies measured to the peaks of the EMG records were designated as Rlat and Llat for results on the right and left sides. CooksÕ original studies (Cook and Thalhammer, 1991) were based on a definition of latency duration as the time from the stimulus to the time corresponding to the peak of the evoked electromyographic response (software ELG 3.4 for MSDOS 1998). However, the time at the peak of the electromyographic response may not be the ideal location to determine the latency of the reflex pathway. The peak latency may be affected by variable shape or duration of the compound muscle action potential. An alternative approach is to measure the time interval between the stimulus and the initial deviation from baseline (Ludin, 1980). A pilot study of TAR responses in Thoroughbred horses however showed that signals were often noisy, and the visual assessment of the point of deviation from the baseline was thought to be unacceptably subjective.
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Fig. 2. Typical ELG traces showing convolution and averaging processes in records from the left and right reflex recordings in one horse. (a and b) are raw traces; (c and d) are convolved traces; (e and f) are resultant means of the convolved traces.
In an attempt to remove this subjectivity, further signal processing was developed in Matlab (Mathworks, 2001) to establish an objective mathematical model to clearly define the start of the electromyographic response. First, each trace was convolved (Kahaner et al., 1989) with a predetermined ‘‘synthesised expected peak’’ shape, as illustrated in Fig. 3. This reference or convolutor shape was estimated from the mean of selected Ônoise freeÕ peaks pooled from several horses
and was of a Ôflat top triangleÕ vector form [1 2 3 4 5 5 4 3 2 1]. The convolution process increases the power to detect the waveform that represents the electromyographic response against background noise. After convolving each of the five records, as shown in Figs. 2(c) and (d), the mean convolved record was produced (Figs. 2(e) and (f)). The time from the stimulus to the start of the electromyographic response on the convolved EMG record was defined as a start latency.
amplitude (scalar units)
6 5 4 3 2 1 0 -2
0
2
4
6
8
time (ms) Fig. 3. Flat top triangle shape used for convolution of raw data.
10
12
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71
Table 2 Variables derived from electrolaryngographic recordings of the thoracolaryngeal reflex, with abbreviations and units of measurement Measured variable
Abbreviation
Units
Latency period on left side to peak of the electromyographic response in raw data (average of five records). Latency period on right side to peak of the electromyographic response in raw data (average of five records). Conduction velocity of reflex on left side representing the estimated length of the reflex pathway divided by Llat Conduction velocity of reflex on right side representing the estimated length of the reflex pathway divided by Rlat Latency to start of convolved electromyographic response on left side Latency to start of convolved electromyographic response on right side Latency to the peak of the convolved electromyographic response on left side Latency to the peak of the convolved electromyographic response on left side Ratio of latency periods (see Eq. (4))
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
s s m/s m/s s s s s
The convolved record was a signal that represented the mean of five recordings. These latencies on the left and right sides were designated Lst and Rst. In addition, the convolved peak latencies (Lpk, Rpk) were calculated as the times from the stimulus to the times of the peak of the electromyographic response in the convolved record. The definitions of all reflex latency and velocity measurements are described in Table 2. 2.5. Estimation of reflex pathway velocities An estimate of the length of the reflex pathway was obtained by measuring the distance in metres from the poll to withers, withers to saddle and the height of the horse at the top of the wither using a flexible tape (Cook, 1998; Cole, 1946). These data were entered into the ELG2000 software. The software estimated left and right recurrent laryngeal nerve lengths (Lnl, Rnl) from formulae based on the methods used previously (Cook (1998), ELG 3.4 for MSDOS): LnlðmÞ ¼ 3 ðPoll–WithersÞ þ ðWithers–SaddleÞ þ 0:15:
ð1Þ
Poll–Withers is the distance (m) from the poll in line with the cranial edge of the ears to the spine of the scapula. Withers-Saddle (m) is the distance from the spine of the scapula to the lowest point of the back. Right nerve length (Rnl) was estimated as (Lnl-0.4) m (Cook, 1998). Reflex conduction velocities on the left and right sides, Lcv and Rcv, were calculated by: Lcv ¼ ðLnl=LlatÞ m=s;
ð2Þ
Rcv ¼ ðRnl=RlatÞ m=s;
ð3Þ
where Llat and Rlat are the latency times to the peaks of the electromyographic responses. A latency ratio (Lratio) was also calculated to express a relationship between left and right convolved latencies. Lratio ¼ ðLst – RstÞ=Rst:
ð4Þ
The effect of sedation was examined in a crossover study of six race-fit thoroughbred horses with ages ranging from two to six years. On day one, the ELG
test was performed on three sedated horses and three unsedated horses. On the following day at approximately the same time these treatments were crossed over. Sedation consisted of 5 mg of the a2 agonist detomidine, administered intravenously 5–10 min before the ELG test. This resulted in 30–40 min of light sedation for ease of animal handling. A quiet room or stable was used for all tests. A group of 12 sedated horses was tested to determine if there was a relationship between age and TAR latencies. These horses had months of birth that were verifiable from official records. Horse ages ranged from 24–82 months. All horses in this group were endoscopically determined to have grade 1 RLN. The relationships between endoscopic grades and ELG measurements were investigated in 60 sedated thoroughbred horses in training. Correlations between ELG measurements and body size were also examined in horses with grade 1 RLN in two age groups, two- and three-year-old horses. Repeatability was investigated by comparing measurements on two consecutive days in six horses. 2.6. Statistics Normality was not assumed; therefore non parametric statistical tests were used. Results were expressed as medians and quartile ranges. Latencies for each horse were recorded as the mean of five tests. A nonparametric alternative to the two-sample t test (Wilcoxon signed rank test) was used to compare results in sedated and non sedated horses. A non-parametric independent two-group comparison test (Mann–Whitney) was used to compare results on days 1 and 2 in the study of the repeatability of the ELG technique. The relationships between age and ELG results were investigated with linear regression analyses and Spearman Rank correlations. The relationships between ELG measurements and endoscopic grades were described with Spearman rank correlations. The association between latencies and the distance from the poll to the withers (an index of body size) was also examined by Spearman rank correlation. Results in horses with endoscopic grades 1 and 2 were compared with a Mann–Whitney test. P values
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Table 3 Description of the Thoroughbred horses used in the study Number and gender of horses
60 (34 Male, 26 female)
Number in each RLN grade (assessed by endoscopy at rest)
Grade 1, n = 35; Grade 2, n = 19 Grade 3, n = 2; Grade 4, n = 4
Number (n) of horses in each age group (years) Age n
1 8
2 8
3 10
4 9
less than or equal to 0.05 were considered statistically significant.
3. Results Table 3 describes the RLN grades and features of the 60 horses used in the study. There was no significant effect of sedation on the ELG measurements (Table 4). There were no significant differences detected with repeated measurements in sedated horses tested on consecutive days (Table 5). The right and left latencies measured to the start and peak of the convolved EMG response (Lst, Rst, Lpk, and Rpk) were significantly associated with age (Table
5 11
6 9
7 2
8 1
11 1
20 1
6; Fig. 4). Median age was 46.5 months, and quartile range was 35.2–57.7 months. Convolved left and right start latencies (Lst, Rst) were significantly related to endoscopic grade of RLN. The unconvolved latency (Rlat) and right convolved peak latency (Rpk) were also correlated with grade of RLN (Table 7). No correlations were found between left sided latencies and the poll-wither distance in two- or three-year-old horses (Table 8). Latency ratios likewise did not depend on the poll-withers distance in two- or three-year-old horses. The latencies to the peak of the convolved EMG responses (Lpk, Rpk) were significantly different in horses with grades 1 and 2 RLN (P < 0.05, Table 9). Medians
Table 4 Latencies (s) and conduction velocities (m/s) in six sedated and unsedated Thoroughbred horses Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
Without sedation
With sedation
P
Median
Quartile range
Median
Quartile range
0.072 0.057 49.37 54.63 0.065 0.052 0.073 0.058 0.26
(0.066, 0.075) (0.055, 0.057) (47.51, 53.50) (48.10, 54.95) (0.063, 0.068) (0.049, 0.053) (0.070, 0.075) (0.055, 0.059) (0.19, 0.33)
0.069 0.059 51.15 52.82 0.064 0.052 0.070 0.061 0.29
(0.068, 0.070) (0.058, 0.061) (50.05, 52.16) (51.08, 54.18) (0.061, 0.066) (0.051, 0.055) (0.068, 0.074) (0.058, 0.062) (0.15, 0.33)
0.31 0.22 0.31 1.00 1.00 1.00 0.31 0.31 0.69
For the definitions of the variables, see Table 2. P, probability statistic.
Table 5 Latencies (s) and conduction velocities (m/s) in tests repeated on consecutive days (n = 6) Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
Day 1
Day 2
P
Median
Quartile range
Median
Quartile range
0.073 0.061 53.79 57.52 0.063 0.051 0.072 0.059 0.21
(0.071, 0.076) (0.059, 0.064) (50.67, 57.20) (54.66, 62.49) (0.059, 0.063) (0.051, 0.055) (0.071, 0.074) (0.058, 0.061) (0.16, 0.22)
0.073 0.060 55.99 57.23 0.062 0.050 0.071 0.058 0.23
(0.070, 0.075) (0.058, 0.065) (53.78, 57.59) (54.13, 63.15) (0.061, 0.064) (0.050, 0.052) (0.071, 0.074) (0.057, 0.061) (0.21, 0.24)
For the definitions of the variables, see Table 2. P, probability statistic.
0.82 0.99 0.39 0.82 0.94 0.18 0.94 0.82 0.09
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Table 6 Spearman rank correlations (R) between age (months) and the ELG measurements (n = 12) Variable
Median
Quartile range
R
P
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
0.070 0.059 56.87 61.30 0.063 0.051 0.072 0.059 0.20
(0.068, 0.072) (0.057, 0.063) (54.29, 60.67) (55.72, 63.85) (0.058, 0.066) (0.049, 0.055) (0.069, 0.074) (0.057, 0.061) (0.16, 0.27)
0.18 0.28 0.01 0.04 0.53 0.64 0.56 0.77 0.05
0.5 0.32 0.95 0.87 0.05 0.01 0.03 0.01 0.84
For the definitions of the variables, see Table 2. P, probability statistic.
75.0
Convolved Latency (ms)
70.0 65.0 60.0 55.0 50.0 45.0 40.0
Regression Equation Lst y = 0.21x + 52.00 R2 = 0.48 Rst y = 0.12x + 45.20 R2 = 0.27
35.0
Lst
Rst
30.0 0.0
10.0
20.0
30.0
40.0 50.0 Age (months)
60.0
70.0
80.0
90.0
Fig. 4. Relationships between age (months) and ELG convolved left and right start point latencies (Lst, Rst). R; Spearman rank correlation (n= 12).
Table 7 Spearman rank correlations (R) between RLN grade (1–5) and the ELG measurements (n = 52) Variable
Median
Quartile range
R
P
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
0.069 0.061 56.15 59.10 0.059 0.051 0.068 0.058 0.19
(0.066, 0.074) (0.057, 0.064) (53.70, 59.40) (54.90, 62.65) (0.057, 0.066) (0.047, 0.055) (0.065, 0.074) (0.055, 0.062) (0.13, 0.27)
0.20 0.37 0.11 0.21 0.27 0.31 0.21 0.34 0.06
0.14 0.01 0.44 0.13 0.05 0.02 0.14 0.01 0.65
For the definitions of the variables, see Table 2. P, probability statistic.
of the latencies to the peak of the convolved EMG were 7.5% greater on the left side in grade 2 horses. Latencies to the start of the convolved EMG on the right side (Rst) and the unconvolved peak on the left side (Llat) were also significantly greater in grade 2 horses (Table 9).
4. Discussion ELG measurements were highly repeatable under the conditions used in this study. Tests were done under controlled conditions, with the same operators for the
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Table 8 Spearman rank correlations (R) between distance from poll to wither and the ELG measurements in two- (n = 7) and three-year-old (n = 7) Thoroughbred horses with grade 1 RLN Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
Two-year-olds
Three-year-olds
Median
Quartile range
R
P
Median
Quartile range
R
P
0.070 0.058 54.79 62.46 62.04 0.050 0.072 0.058 0.24
(0.056, 0.086) (0.051, 0.060) (51.19, 69.36) (56.60, 68.52) (0.050, 0.072) (0.046, 0.052) (0.057, 0.078) (0.051, 0.060) (0.08, 0.39)
0.68 0.21 0.36 0.64 0.18 0.38 0.32 0.31 0.18
0.07 0.60 0.39 0.09 0.66 0.34 0.44 0.44 0.66
0.067 0.060 58.59 59.56 0.058 0.048 0.068 0.057 0.18
(0.065, 0.080) (0.054, 0.064) (47.74, 64.15) (52.10, 72.51) (0.052, 0.063) (0.044, 0.062) (0.064, 0.072) (0.051, 0.067) (0.03, 0.32)
0.41 0.70 0.63 0.79 0.56 0.13 0.44 0.05 0.36
0.30 0.05 0.09 0.02 0.15 0.72 0.30 0.84 0.39
For the definitions of the variables, see Table 2. P, probability statistic.
Table 9 Latencies and conduction velocities in horses with grades 1 (n = 35) and 2 (n = 19) laryngeal hemiplegia Variable
Llat Rlat Lcv Rcv Lst Rst Lpk Rpk Lratio
P
0.025 0.156 0.731 0.514 0.108 0.018 0.029 0.010 0.419
Grade 1
Grade 2
Median
Quartile range
Median
Quartile range
0.068 0.059 56.65 60.52 0.059 0.049 0.067 0.058 0.20
(0.065, 0.073) (0.056, 0.062) (53.71, 59.72) (56.19, 63.09) (0.056, 0.064) (0.046, 0.053) (0.065, 0.073) (0.054, 0.059) (0.14, 0.27)
0.071 0.061 55.43 57.51 0.059 0.053 0.072 0.061 0.20
(0.068, 0.075) (0.058, 0.066) (53.78, 58.78) (53.78, 63.24) (0.058, 0.065) (0.050, 0.055) (0.068, 0.074) (0.057, 0.062) (0.10, 0.26)
repeatability trial. It was found that after minimal operator practice, consistent, artefact free results could be readily obtained. It was noted during early trials that different operators could place the electrodes in slightly different positions, and variably ÔslapÕ the withers. These factors should not change the latency, but may mask an ELG trace in myographic noise or cause direct current offsets that make the ELG peaks harder to distinguish. Results from the sedation crossover study show that it is quite appropriate to use a2 agonist sedation when performing an ELG test. There were no significant effects of sedation on the right or left latency periods. This result was expected, as one of the properties of evoked potentials is their resistance to sedative drugs. Sedation enables safer handling of the animals and more consistent ELG traces without additional muscle noise. The statistical difference in the right average velocity is unlikely to be biologically significant. It may be reasonable to expect horses with lower grades of RLN to maintain normal nerve conduction velocities, because a proportion of the large axons will still be functional. This explanation was used to discuss the results obtained by Hawe et al. (2001). In that study the slap test (TLR) latency was compared to five other diagnostic techniques in Clydesdales and ponies. There
were no correlations between nerve latency periods and laryngeal function in those breeds. In the current study, latency measurements were significantly different in Thoroughbred horses with grades 1 and 2 RLN. The use of an algorithm to determine convolved latencies in the present study may have resulted in a more accurate measurement of the latencies than was possible with the analogue filter system used by Hawe et al. (2001). The current study may therefore have been more sensitive, enabling detection of small differences in latencies. Recent evidence has suggested that there is interdependence between the axon and its myelin sheath (Trapp et al., 1999), and that in a chronic disease such as RLN there may be myelin pathology secondary to axonal pathology. Schwann cell pathology is present in RLN, as shown by BungnerÕs bands, onion bulbs and wallerianlike degeneration (Cahill and Goulden, 1986b; Duncan et al., 1978). It is therefore reasonable to expect that an accurate measurement of latency will enable detection of a decrease in nerve conduction latency, even in horses with low grades of clinical disease. The novel technical approach in this study was the extensive use of standard digital signal processing techniques to process and analyse the ELG data. Digital sig-
R.A. Curtis et al. / The Veterinary Journal 170 (2005) 67–76
nal processing allows automated analysis on time domain events (take off points, slopes, wave shapes). Further, digital filtering to remove high frequency artefacts before analysis has the advantage over analog filtering of reducing phase errors and thereby maintaining the time domain accuracy required for the ELG. The standard ELG 2000 software used a simple approach to finding latency peaks. This involves placing a window around the expected latency region and finding the first major peak. This can give incorrect results, particularly when the laryngeal signal is small with respect to large amounts of myographic and electrical noise. Convolving and averaging the ELG traces significantly improved the ability to resolve wave shapes. Conduction velocities did not differentiate grades 1 and 2 horses. The calculation of velocity was based on an estimated reflex pathway length. However, this measurement is subject to error. The accuracy of the estimation of the true length of the recurrent laryngeal nerve from measurements of body morphology (Cook and Thalhammer, 1991) has not been described. There were no significant correlations between poll to wither distance and latency in similar age and grade horses. Measurements of conduction velocities based on estimates of nerve length were not useful for testing the hypotheses in this study. There were associations between horse age and convolved start and peak latencies obtained by signal processing. These results applied for data obtained from TAR reflexes studied on the left and right sides. These results confirm a suggestion by Dixon et al. (2002) that the disease is progressive. Small changes in nerve function over time might be detectable with ELG measurements of convolved latencies, and further work could investigate the rate of change in latencies in young horses. The latencies to the start of the convolved muscle action potential on the left side were not significantly different in horses with grades 1 and 2 RLN, but the latencies to the peak of the curve were. This suggests that the latency measurement that includes the rise-time of the muscle action potential is the most sensitive, potentially because the peak measures the average of all the conducting axons while the start measures only the latency of the largest axon. Even in horses with severe RLN a few large axons may still remain, and thus the latency to the start of the muscle action potential would not be affected. The significant correlations between age and latencies could have reflected an increase in body size in the older horses. However, there was no dependency of left latencies on the poll to wither distance, suggesting that the association with age reflects a progressive increase in reflex latency in normal horses. In conclusion, the use of electrolaryngeography for measurement of TAR latencies is a repeatable method
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that is low cost and simple to perform in a clinical setting. These results support the suggestion by Cook (1988) that electrodiagnostic measurement of the latency of the thoracolaryngeal reflex has a role in evaluation of horses with RLN. Results were not affected by sedation. ELG results were correlated with age and endoscope score, and differentiated horses with endoscope scores 1 and 2. The technique has potential in the clinical evaluation of the horse, and for further studies of the nature of the disease. It would be of interest to study differences in nerve function in younger horses, and the rates of change in latencies in longitudinal studies. Acknowledgements The authors thank Thoroughbred racecourse trainer Ron Quinton and staff, particularly Alan Putland, for access to a well-trained and cared for population of thoroughbred horses. Dr. Stuart Murray and Dr. Richard Humberstone helped in conducting the tests. We are grateful to the Australian Rural Industries Research and Development Corporation for financial support.
References Cahill, J.I., Goulden, B.E., 1986a. Equine laryngeal hemiplegia. II. An electron microscopic study of peripheral nerves. New Zealand Veterinary Journal 34, 170–175. Cahill, J.I., Goulden, B.E., 1986b. Equine laryngeal hemiplegia. I. A light microscopic study of peripheral nerves. New Zealand Veterinary Journal 34, 161–169. Cole, R.C., 1946. Changes in the equine larynx associated with laryngeal hemiplegia. American Journal of Veterinary Research 7, 69–77. Cook, W.R., 1988. Recent observations on recurrent laryngeal neuropathy in the horse: applications to practice. In: 34th Convention of American Equine Practitioners, pp. 427–478. Cook, W.R., 1998. Electrolaryngeograph Version ELG 3.4 UserÕs Guide. ELG, Inc., Chestertown, pp. 49–50. Cook, W.R., 1993. Diagnosis and grading of hereditary recurrent laryngeal hemiplegia in the horse. Journal of Equine Veterinary Science 8, 432–455. Cook, W.R., 1999. The ear, nose and the lie in the throat. In: Rossdale, P.D., Greet, T.R.C., Harris, P.A., Green, R.E., Hall, S. (Eds.), Guardians of the Horse: Past, Present and Future. British Equine Veterinary Association, London, pp. 175–182. Cook, W.R., Thalhammer, J.G., 1991. Electrodiagnosis test for the objective grading of recurrent laryngeal hemiplegia. Proceedings of the Annual Convention of the American Association of Equine Practitioners 37, 275–296. Dixon, P.M., Mcgorum, B.C., Railton, D.I., Hawe, C., Tremaine, W.H., Pickles, K., Mccann, J., 2002. Clinical and endoscopic evidence of progression in 152 cases of equine recurrent laryngeal neuropathy (RLN). Equine Veterinary Journal 34, 29–34. Duncan, I.D., Griffiths, I.R., Madrid, R.E., 1978. A light and electron microscopic study of the neuropathy of equine idiopathic laryngeal hemiplegia. Neuropathology and Applied Neurobiology 4, 483–501. Gunn, H.M., 1972. Histological observations on laryngeal skeletal muscle fibres in ‘‘normal’’ horses. Equine Veterinary Journal 4, 144–148.
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Hackett, R.P., Fubini, S.L., Erb, H.N., 1991. The reliability of endoscopic examination in the assessment of arytenoid cartilage movement in the horse. Veterinary Surgery 20, 3174–3179. Hawe, C., Dixon, P.M., Mayhew, I.G., 2001. A study of an electrodiagnostic technique for the evaluation of equine recurrent laryngeal neuropathy. Equine Veterinary Journal 33, 459–465. Kahaner, D., Moler, C., Nash, S., 1989. Numerical Methods and Software. Prentice-Hall, New Jersey, pp. 458–460. Lopez-Plana, C., Sautet, J.Y., Pons, J., Navarro, G., 1993. Morphometric study of the recurrent laryngeal nerve in young ‘‘normal’’ horses. Research in Veterinary Science 55, 333–337.
Lane, J.G., 1993. Equine recurrent laryngeal neuropathy (RLN): current attitudes to aetiology, diagnosis and treatment. In: Proceedings of the 15th Bain-Fallon Memorial Lectures Australian. Equine Veterinary Association, Canberra, pp. 173–192. Ludin, H.P., 1980. Electroneurography. In: Electromyography in Practice. Georg Thieme, New York, pp. 31–34. Mayhew, I.G., 1989. Large Animal Neurology: A Handbook for Veterinary Clinicians. Lea & Febiger, Philadelphia, PA. Trapp, B.D., Bo¨, L., Mo¨rk, S., Chang, A., 1999. Pathogenesis of tissue injury in MS lesions. Journal of Neuroimmunology 98, 49–56.
Book review D. Marlin, K. Nankervis, Equine Exercise Physiology, Blackwell Science, Oxford, 2002, ISBN 0632055529, p. 296 £29.95 (Soft) Many of us who teach equine exercise physiology or field calls from horse owners, trainers, and equine clinicians should thank the authors of this book for a text that can be used by multiple audiences to understand the basics of equine exercise physiology. Dr. David Marlin and Ms. Nankervis have penned a very readable textbook that thoroughly covers the basic concepts of equine exercise physiology. The book presents the most recent information we have on these magnificent athletes in a very easy to read and unpretentious manner. Material covered in the book is well organized into three very complete sections. Part I covers the basics of biochemistry and energetics and each of the physiological systems that plays a major role in the response to exertion. The second section of the book gives the reader a detailed understanding of how each organ system responds to acute exercise and how each system adapts to the repeated challenge of exercise training. In the third section of the book, the authors do a great job of synthesizing how the basics of equine exercise physiology can be used in an applied fashion to enhance our ability to train and care for the equine athlete. However, one should note that this is not a dry scientific tome and that in addition to making this book enjoyable to read, the authors have done a great job of including an ample number of detailed, yet easy to understand figures, charts, and photographs that allow one make a full visualisation of the inner workings of the equine athlete. This doi:10.1016/j.tvjl.2005.03.011
is especially useful to those who are visual learners. Finally, at the end of each chapter they include a summary of key points. This summary of material covered is thoughtful and an excellent review for the reader that has additionally served as an excellent resource improving my own classroom lectures. My positive opinion of this book is evident and I use it as one of two required texts for my classes in equine exercise physiology. However, I use it with an appreciation for the value of a well written book. Supporting that opinion are the comments from students on their written course evaluations where they praised the bookÕs usefulness as a key resource to bridge the gap from systems physiology, where they had to memorise a great deal of material, to exercise physiology, a subject that requires an integrative understanding of the biology of exercise. To that end the authors have created an excellent resource for undergraduate, graduate, and even veterinary students looking for a great supplement to existing weighty scientific and clinically oriented books. For the horse owner, trainer, and equine clinician, this text is a great resource to pull out when one is interested in a more clear understanding of advanced integrative and applied aspects of the field of equine exercise physiology and equine sports medicine. Kenneth Harrington McKeever Department of Animal Sciences, Rutgers The State University of New Jersey, 84 Lipman Drive New Brunswick, NJ 08901-8525 USA E-mail address: [email protected]