Correlation of transcranial magnetic motor evoked potentials and MRI morphometry in

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4.1 Correlation of transcranial magnetic motor evoked potentials and MRI morphometry in

herniation: A follow-up study

J.S.C.G. Siedenburg^a, H.-L. Amendt^a, K. Rohn^b, A. Tipold^a,c, V.M. Stein^a*

aDepartment of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover

bDepartment of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Hannover, Germany.

cCenter for Systems Neuroscience, Hannover, Germany.

* Corresponding author: Veronika M. Stein +49 511 953 6200 E-mail address: veronika.stein@tiho-hannover.de

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38 Abstract

Functional motor recovery after decompressive surgery in paraplegic dogs with intervertebral disc herniation (IVDH) is crucial for patients and their owners. In this prospective study the hypotheses should be investigated that transcranial magnetic motor evoked potentials (TMMEPs) reliably display the course of motor function improvement in initially paraplegic dogs with thoracolumbar IVDH after decompressive surgery and determine whether there is any association between TMMEP data and severity of neurological signs or correlation with morphometric magnetic resonance imaging (MRI) findings.

At initial presentation TMMEP data including onset latencies and peak-to-peak amplitudes were obtained in 21 paraplegic, sedated dogs from the cranial tibial muscle. Morphometric measurements of MRI of the thoracolumbar spinal cord prior to surgery included L2 normalized ratios of compression length (CLR) and of T2 weighted intramedullary hyperintensities (T2WLR). First follow-up was performed within 1-2 days after first motor function reappearance including neurological examination and TMS. Second follow-up was performed 3 months after first follow-up comprising repeated neurological examination, TMS and MRI.

At initial presentation, TMMEPs could not be recorded from pelvic limbs. However, TMMEPs could be elicited from the cranial tibial muscle in 12/21 dogs at first follow-up and in 20/21 dogs at second follow-up. Comparison of values of these TMMEPs obtained at first and second follow-up showed a significant increase of peak-to-peak amplitudes and significant decrease of onset latencies. A significant association was detected of onset latencies first follow-up and severity of neurological signs at the second follow-up. At second follow-up latencies were significantly associated and correlated with severity of neurological signs. CLR and T2WLR were not significantly correlated with TMMEP data.

In conclusion, TMMEPs reflect motor function recovery after severe spinal cord injury.

Moreover, onset latencies obtained shortly after first reappearance of motor function may provide prognostic information of further motor improvement. However, correlation of TMMEPs with MRI morphometric measurements could not be confirmed.

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Keywords: Intervertebral Disc Herniation; Transcranial Magnetic Motor Evoked Potentials;

Transcranial Magnetic Stimulation, Spinal Cord Injury; Magnetic Resonance Imaging; Dog

Introduction

Spinal cord injury (SCI) in canines is commonly caused by thoracolumbar intervertebral disc herniation (IVDH) resulting in a broad range of clinical signs from paraspinal hyperaesthesia to paraplegia and loss of deep pain perception (DPP) with concomitant impairment of micturition and defecation (Duval et al., 1996; Jeffery and Blakemore, 1999;

Ferreira et al., 2002; Olby et al., 2003; Mayhew et al., 2004; Cerda-Gonzalez and Olby, 2006;

Fluehmann et al., 2006). Transcranial magnetic stimulation (TMS) generates transcranial magnetic motor evoked potentials (TMMEPs), which can be recorded from surface or muscle electrodes (Nollet et al., 2003). These TMMEPs enable a noninvasive and fast evaluation of the functional integrity of descending motor pathways in the brain and spinal cord (Barker et al., 1985). TMS is well established in human medicine, providing information about corticospinal tract damage and lesion location in cervical spinal cord injury (Maertens de Noordhout et al., 1991; Lo et al., 2004; Shields et al., 2006; Kalupahana et al., 2008).

Moreover, it can be of prognostic value in stroke and SCI patients (Clarke et al., 1994;

Pennisi et al., 1999). In veterinary medicine diagnostic applications of TMS have been described in equine and canine species with spinal cord diseases (Sylvestre et al., 1993; Nollet et al., 2002; De Decker et al., 2011). Association of TMMEP data with severity of clinical signs and magnetic resonance imaging (MRI) findings has been described in CSM in Great Danes and Dobermann Pinschers, but was not examined in thoracolumbar IVDH so far (De Decker et al., 2011; Martin-Vaquero and da Costa, 2014).

Prognostic information gained from clinical assessment is widely accepted. In particular, presence or absence of DPP is acknowledged to be the most reliable prognostic indicator for recovery after severe SCI in dogs with plegia and has become a frequently used reference evaluating new prognostic approaches (Duval et al., 1996; Scott and McKee, 1999; Coates, 2000; Davis and Brown, 2002; Jeffery et al., 2013; Jeffery et al., 2016). In the past years a shift of focus towards CSF analysis and diagnostic imaging features revealed several associations with severity of spinal cord injury and correlations with functional outcome (Olby et al., 2003; Ito et al., 2005; Penning et al., 2006; Royal et al., 2009; Srugo et al., 2011;

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Roerig et al., 2013). Among the diagnostic imaging techniques MRI is considered the most accurate identifying characteristics of extruded disc material and parenchymal spinal cord lesions (Bos et al., 2012; Cooper et al., 2014). In previously published studies, MRI morphometric measurements on T2 weighted images were associated with severity of clinical signs and long-term ambulatory outcome after IVDH (Ito et al., 2005; Levine et al., 2009;

Boekhoff et al., 2012).

In this prospective study, the hypotheses should be proven, that a) TMS is a suitable technique for therapy monitoring in initially paraplegic dogs with IVDH after decompressive surgery and b) TMMEP data can reflect different severity of neurological signs. Further objectives were to determine whether c) TMMEP data could provide prognostic estimates about the extent of regeneration of motor function and d) whether a correlation exists between TMMEP data with morphometric MRI findings.

Material and Methods Dogs and study design

Twenty-one client-owned paraplegic dogs admitted to the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Germany, were recruited between May 2013 and May 2015. Prior to study enrollment written owner consent was obtained. The study was conducted in accordance with the guidelines of the Animal Care Committee of the Government of Lower Saxony and national regulations for animal welfare (animal experiment number 33.14-42502-04-13/1277). Dogs had to meet the following inclusion criteria: <20 kg bodyweight, acute to subacute paraplegia (onset < 28 days) with present or absent DPP and spinal cord injury due to IVDH between T3-L3 confirmed by MRI and surgery.

All dogs had a general physical and neurological examination; further clinical examinations included complete blood cell count, routine serum biochemistry and radiographs of the thorax and vertebral column. According to their neurological deficits dogs were assigned to a Sharp and Wheeler (2005) scale (Tab. 1). At initial clinical presentation TMS was performed under deep sedation, as described in previously published studies (Sylvestre et al., 1993; Van Ham et al., 1996; da Costa et al., 2006; Granger et al., 2012; Amendt et al., 2016). Afterwards,

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MRI examinations were performed to definitely localize and characterize the lesion within the spinal cord and subsequently all dogs underwent decompressive surgery. When first motor function reappearance was observed, first follow-up including neurological examination and TMS was performed within 1-2 days later. Second follow-up was performed 3 months after first follow-up comprising repeated neurological examination, TMS and MRI.

TMMEPs

Dogs were sedated with acepromazine (0.02 - 0.05 mg/kg, Vetranquil, CEVA Tiergesundheit GmbH) and levomethadone/fenpipramide (0.2 - 0.4 mg/kg, L-Polamivet, Intervet Deutschland GmbH) intravenously (IV) while heartbeat and body temperature were monitored and TMS performed in lateral recumbency or sternal positioning. TMMEPs were recorded as described in previous studies with minor modifications (Sylvestre et al., 1993; da Costa et al., 2006; Martin-Vaquero and da Costa, 2014; Amendt et al., 2016). A Magstim 200² (Magstim Co Ltd) stimulator, capable of producing a maximum 4.0 Tesla magnetic field (complies with a 100 % intensity) with a 50 mm ring coil was used. The coil was held tangentially to the skull in close contact to the skin with the center of the coil lateral to the vertex to stimulate the motor cortex. The current flow within the coil ran in clockwise direction and four consecutive stimulations were delivered for generation of TMMEPs recorded from each limb. These recordings were obtained after contralateral stimulation by use of an electromyograph (Nicolet™ NicVue 2.9.1, Natus Medical Incorporated). The recording muscle-electrode was positioned in the middle of the muscle belly of the cranial tibial and the extensor carpi radialis muscle, respectively. The reference electrode was positioned subcutaneously one cm distal to the muscle electrode, while the ground electrode was placed subcutaneously on the dorsal midline of the cranial thoracic region and caudal lumbar region, respectively.

To display, adjust and save TMMEP waveforms VikingSelect-Software Version 11.0 (Viasys healthcare, CareFusion) was used. Total recording time was 200 ms following the stimulus;

sensitivity depended on amplitude values and ranged from 0.2 to 10 mV/division for all recordings in the thoracic and pelvic limbs. Onset latency and peak-to-peak amplitude were measured as described by van Ham et al. (1994) by manually directed cursors on the

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oscilloscope. Onset of latencies was measured in milliseconds [ms], being defined as interval between stimulus artifact and first subsequent deflection from the baseline. Peak-to-peak amplitudes were measured in microvolts [mV] and calculated from the peak of the negative wave to the nadir of the first positive wave. Neuronal path length was measured starting at the vertex via the estimated course of nerve fibers to the muscle needle positioned within the cranial tibial and extensor carpi radialis muscle, respectively, contralateral to the stimulation site (Martin-Vaquero and da Costa, 2014).

MRI

To confirm presumptive diagnosis of IVDH the thoracolumbar spinal cord was assessed by MRI. All patients were assessed under general anesthesia using a 3.0 Tesla (T) magnet (Philips Achieva, Philips Medical Systems) with a phased array SENSE (sensitivity encoding)-spine-coil with 15 channels. The thoracolumbar spinal cord was scanned with the following sequences: T2-weighted sequence was a TSE (turbo-spin-echo) sequence with sagittal (TR = 3100 ms, TE = 120 ms, slice thickness 1.8 mm, interslice gap 0.2 mm) and transversal (TR = 8418 ms, TE = 12 ms, slice thickness 1.8 mm, interslice gap 0.2 mm) planes. Images were complemented by transversal planes of T1-weighted (TR = 491 ms, TE = 8 ms, slice thickness 1.8 mm, interslice gap 0.2 mm) and mFFE (multi-echo fast field echo (TR = 21 ms, TE = 7 ms, slice thickness 1.8 mm, interslice gap 0.2 mm) sequences.

MRI data sets of all dogs were evaluated as DICOM formatted images by use of easyVET (IFS GmbH). Evaluation of discernible T2 weighted intramedullary hyperintensities rested upon assessment of sagittal images (Ito et al., 2005; Penning et al., 2006; Levine et al., 2009).

Corresponding transversal T2-weighted images (T2WI) were evaluated for detection of their length expansion (Griffin et al., 2015). T1 weighted sequences were assessed in transversal planes to exclude presence of T1 weighted hyperintensities (Levine et al., 2009). Detection of extruded intervertebral disc material was assessed in sagittal and transversal T2WI, complemented by transversal T1 weighted sequences and mFFE sequences (Levine et al., 2009; Griffin et al., 2015). Quantification of spinal cord compression was achieved by measurements on sagittal T2WI, transversal T1 weighted images were used to confirm longitudinal extent of spinal cord compression-spinal cord (Levine et al., 2009; Griffin et al., 2015). The length of L2 was used to calculate standardized T2 weighted hyperintensity length

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ratio (T2WLR) and spinal cord compression length ratio (CLR), as described before (Ito et al., 2005; Levine et al., 2009; Boekhoff et al., 2012).

Statistical methods

TMMEP variables (latencies and amplitudes) recorded in each dog from the right and left limbs were averaged to give a mean value for each dog and variable, then the means were calculated of all the dogs. At initial presentation in dogs with grades IV-V no TMMEPs could be recorded in pelvic limbs (Tab. 1). Thus, comparison of TMMEP data series was limited to dogs (n = 11) with recordable TMMEPs either at first and second follow-up. Latencies and amplitudes at fist follow-up were normally distributed. Thus, comparison of follow-up TMMEP data was performed by use of parametric tests. Association and correlation between latencies and amplitudes with severity of neurological signs were examined by use of t-test and Spearman correlation, respectively. For comparison of MRI features at initial presentation and at second follow-up, non-parametric methods (Wilcoxon’s signed rank test, Kruskal-Wallis test) were used, since not all data sets were normally distributed. Association between initial CLR, T2WLR and second follow-up TMMEP features with grades of neurological impairment at initial presentation and both follow-up examinations was calculated by use of Wilcoxon two sample test and t-test, respectively. Correlation of CLR and T2WLR obtained at initial presentation with TMMEPs at first follow-up and second follow-up respectively, was calculated with a Pearson and Spearman correlation. Analogously, correlation of MRI features with TMMEPs was calculated with values of second follow-up examinations. P-values of < 0.05 were considered significant. The statistical analyses were performed with a commercially available software program (SAS Enterprise Guide 7.1, SAS Institute Inc.).

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44 Results

Clinical data and neurological status

Twenty-one paraplegic dogs with an IVDH at T3-L3 spinal cord segments were enrolled in this study. Onset of clinical signs was acute to subacute (median: 3 days; range, < 24 hours-23 days). Dogs had a median age of 5.4 years (range, 2.6-13.1 years) and had a median bodyweight of 8.5 kg (range, 3.9-19.6 kg). The study population consisted of 5 sexually intact males, 7 neutered males, 4 sexually intact females and 5 spayed females. The study comprised 7 Dachshunds (33.3 %), 4 mixed breed dogs (19 %), 2 dogs of each of the following chondrodystrophic breeds: French Bulldog, Jack Russell Terrier, Lhasa Apso and Shih Tzu and 1 dog of each of the following breeds: Bolognese, Bolonka Zwetna and Havanese.

16/21 dogs were paraplegic with DPP and therefore classified as grade IV, whereas 5/21 were paraplegic with absent DPP and classified as grade V. All dogs showed reappearance of motor function after decompressive surgery (median 18 days; range, 4-37 days). Overall, 18/21 dogs (85.7 %) became ambulatory during follow-up, whereas 3/21 dogs (14.3 %) with absent DPP prior to surgery remained non-ambulatory (Tab. 1).

TMMEPs and comparison of TMMEPs and neurological status

No TMMEPs could be generated in plegic dogs with grade IV and V at initial presentation. At first follow-up, TMMEPs were recorded in 12/21 dogs. In 6 of these paraparetic dogs (n = 3 with grade II and n= 3 with grade III) TMMEP generation was limited to one pelvic limb. The 9 paraparetic dogs without measurable TMMEPs were all still non-ambulatory (grade III). At the second follow-up examination TMMEPs could be obtained in 19/21 dogs from both pelvic limbs. In one non-ambulatory dog (grade III) recorded TMMEPs were limited to one pelvic limb. TMMEPs could not be elicited in one dog that had ambulatory paraparesis (grade II).

TMMEP values generated at first- and second follow-up of eleven dogs were available. The comparison of the TMMEPs revealed a significant decrease of onset latency (p < 0.02) and a significant increase of peak-to-peak amplitudes (p < 0.02) during the course of therapy monitoring after decompressive surgery (Fig. 1; Tab. 1, 2).

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Table 1. Course of functional motor recovery and TMMEP data during follow-up study

1Grades of severity of neurological signs according to Sharp and Wheeler (2005) Grade I = spinal hyperaesthesia without neurological deficits

Grade II = ambulatory paraparesis and ataxia Grade III = non-ambulatory paraparesis

Grade IV = paraplegia with deep pain perception Grade V = paraplegia with loss of deep pain perception

2Mean of onset latencies and amplitudes

3Values in round brackets represents range of onset latencies and amplitudes

4Number in square brackets represents number of dogs Neurological grade

according to Sharp and Wheeler (2005)¹

Grade Latencies^{2, 3} [ms] Amplitudes^{2, 3} [mV] Grade Latencies^{2, 3} [ms] Amplitudes^{2, 3} [mV]

At initial presentation First follow-up examination Second follow-up examination

[number of dogs n = 21]⁴ [n = 21] [TMMEPs in n = 12] [n = 21] [TMMEPs in n = 20]

5 [5] 3 [5] 93.59 (35.98-151.20)[2] 0.19 (0.10-0.29)[2] 3 [3] 87.37 (44.15-118.4) [3] 0.26 (0.10-0.49) [3]

2 [2] 36.01 (31.11-41.05) [2] 0.36 (0.26-0.45) [2]

4 [16] 3 [12] 80.48 (34.80-118.40)[6] 0.33 (0.10-0.74)[6] 2 [8] 63.13 (33.73-99.85) [8] 0.51 (0.10-1.43) [8]

1 [4] 37.14 (28.28-61.36) [4] 1.04 (0.20-2.48) [4]

2 [4] 83.47 (48.95-89.98)[4] 0.21 (0.10-0.40)[4] 2 [3] 58.24 (39.73-76.75) [2] 0.39 (0.10-0.84) [2]

1 [1] 44.98 [1] 0.20 [1]

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Table 2. Association between TMMEP results, MRI morphometric data and the severity of neurological signs

#According to Sharp and Wheeler (2005)

* indicates statistical significance (p <0.05)

εwas performed three months after first follow-up Course of study

(ambulatory vs. non-ambulatory) <0.05* 0.36

Association between 3a), 4a) and 5a)

(grade IV vs grade V) <0.02* 0.17

Association of 3a), 4a) and 5c)

(ambulatory vs. non-ambulatory) <0.02* 0.72

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Latencies and amplitudes were not significantly different between dogs with grades II and III at first follow-up (p = 0.76; p = 0.51; Tab. 2). Interestingly, dogs with grade II in the second follow-up examination had significantly longer TMMEP latencies compared to those that were grade I. Hence, a significant association (p < 0.05) was detected between latencies obtained at first follow-up and severity of neurological sings assessed at second follow-up.

TMMEP latencies at second follow-up were significantly longer in non-ambulatory (grade III) compared to ambulatory dogs (grades I, II; p < 0.05; Tab. 2), consequently, latencies were significantly associated with severity of neurological sings obtained at second follow-up. In addition, TMMEP latencies were significantly correlated with deficits of motor function at the second follow-up (p < 0.02; r = 0.54; Tab 2).

Fig. 1. Comparison of peak-to-peak amplitudes and onset latencies of transcranial magnetic motor evoked potentials (TMMEPs) recorded at first and second follow-up in 11 dogs.

Statistical comparison of these values revealed a significant increase of peak-to-peak amplitudes (1a; p < 0.02) and a significant decrease of onset latency (1b; p < 0.02). Boxes in graphs represent values of latencies and amplitudes respectively, recorded from the cranial tibial muscle. At first follow-up, TMMEPs could be obtained from the cranial tibial muscle in 17 pelvic limbs of 11 dogs; at second follow-up TMMEPs were recorded from the cranial tibial muscle in 22 pelvic limbs of 11 dogs. Upper and lower horizontal bar indicate 95%

interval, the wide bar illustrates the median. Asterisks indicate significant differences between the two groups.

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MRI data and comparison of MRI data and neurological status

Imaging data could be obtained in all dogs at initial presentation and at the second follow-up.

At initial presentation hyperintensities in the spinal cord were detected on T2WI in 13/21 (62

%) dogs. These were all five dogs with grade V and 8/16 dogs with grade IV. At the second follow-up MRI, 19/21 (90.5 %) dogs had discernible T2W hyperintensities in the spinal cord.

The two dogs without intramedullary hyperintensities were initially presented with grade IV.

Moreover, T2WLR was associated with grade of neurological sings at initial presentation, as dogs with grade V had significantly higherT2WLR (p = 0.01; median 1.97; range, 1.78-2.99), than dogs with grade IV (median 0.80; range, 0.00-3.16). At initial presentation, dogs had a median CLR of 1.54 (range, 0.27-4.06) with no significant difference (p = 0.17) between groups with grade IV and V.

T2WLR obtained at initial presentation was significantly associated with severity of neurological signs at the second follow-up, as dogs with non-ambulatory paraparesis (grade III) had a significantly higher (p < 0.02) T2WLR at initial presentation than dogs with ambulatory paraparesis (grade II). No significant differences were found between T2WLR obtained at initial presentation compared to results of MRI examination at the second follow-up (p = 0.67). However, the CLR was significantly lower at second follow-follow-up examination compared to data obtained at initial presentation (p < 0.001).

Correlation of MRI data and TMMEPs

No significant correlation was found of MRI morphometric data obtained at initial presentation and second follow-up with TMMEP parameters recorded in paraparetic dogs at first and second follow-up.

Discussion

In paraplegic dogs with thoracolumbar intervertebral disc herniation, therapeutic surgical intervention alleviates spinal cord compression and creates preconditions for motor function recovery to varying degree depending on initial compressive and contusive injury (Scott, 1997; Ruddle et al., 2006; Aikawa et al., 2012; Jeffery et al., 2013; Jeffery et al., 2016). This

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study outlines that recording of TMMEPs is a feasible method to assess initial recovery and further improvement of motor function after severe spinal cord injury. In the current study TMMEP reflected severity of spinal cord injury. In paraplegic dogs recording of TMMEPs was not possible at initial presentation. A missing generation of TMMEPs in plegic dogs is also described by Sylvestre et al. (1993). As a result of contusion and compression in naturally occurring SCI after IVDH the primary injury consists of edema and hemorrhage due to vascular damage and blood-spinal cord barrier disruption, axonal swelling and loss of myelin sheaths (Smith and Jeffery, 2006). In guinea pig models of compression induced acute SCI, node of Ranvier disruption and concomitantly increased juxtaparanodal potassium channel activity due to structural alterations were found to reversibly inhibit axonal conductivity (Ouyang et al., 2010). Secondary injury processes leading to inflammation, marked hemorrhage, edema and apoptosis, characterize the following phase (Rowland et al., 2008; Boekhoff et al., 2012; Jeffery et al., 2013). White matter axons become degraded accompanied by spreading apoptosis of oligodendrocytes, whereas gray matter shows cystic characteristics and necrosis of neurons (Beattie, 2004; Smith and Jeffery, 2006). In severe

Im Dokument Transkranielle Magnetstimulation bei Hunden mit motorischer Regeneration nach thorakolumbalen Bandscheibenvorfällen (Seite 45-70)