Diagnosing and Characterizing Neuropathic Pain in Dogs with Spinal Cord Injury

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the

Graduate School of The Ohio State University

By

Austin Kerns, DVM

Graduate Program in Comparative and Veterinary Medicine

The Ohio State University

2018

Thesis Committee Members:

Dr. Sarah Moore, Advisor

Dr. Ronaldo da Costa

Dr. Laurie Cook

Dr. Nina Kieves

Copyright by Austin Kerns

2018

i

Abstract

Electronic von Frey anesthesiometry (VFA) has been previously reported by our

laboratory and others as a useful method of mechanical quantitative sensory testing (QST) for

evaluating neuropathic pain in dogs. Intraobserver agreement has been previously shown to be

good to excellent; however, interobserver agreement has not been evaluated and is vital to the

use of this technique in multicenter veterinary clinical trials in neuropathic pain. The goal of this

study was to evaluate the interobserver agreement of sensory thresholds obtained using

electronic VFA in a group of normal small breed dogs.

Twenty healthy dogs (< 20kg) were recruited from the general practice population at the

Ohio State University Veterinary Medical Center. Three novice evaluators used an electronic

von Frey device (IITC Life Science; Woodlands, CA) to measure mechanical sensory threshold

(ST) after a brief standardized training session conducted by an expert evaluator. Each dog was

evaluated by all three investigators on the same day with both evaluator and limb test order

randomized and testing sessions separated by five minutes.

Mean ST values were averaged for all four limbs to produce a single value per dog for

comparison between evaluators. Agreement between evaluators was determined using the intra-

class correlation coefficient (ICC; two-way model for consistency, single measures). ICC across

all three evaluators was 0.48, indicating moderate agreement. Moderate interobserver agreement

is not sufficient to support the use of this technique in multi-center clinical trials, and our results

underscore the importance of using a single evaluator for this QST technique in canine

neuropathic pain studies.

ii

VFA was then used to objectively measure mechanical ST in 21 dogs with acute spinal

cord injury (SCI) caused by spontaneous thoracolumbar intervertebral disc extrusion (IVDE) at

10 days, 30 days, and 90 days after decompressive hemilaminectomy. ST values were compared

over time and correlated with locomotor scores.

There was no significant change in ST in the thoracic limbs over the 90 day recovery

period. Pelvic limb ST were highest at 10 days consistent with hypoalgesia and declined over

time, though the decline from 30 to 90 days was not statistically significant (means ± standard

error of the mean ST for pelvic limbs at 10 days = 209.2 ± 14.1 g, at 30 days = 173.8 ± 11.1 g,

and at 90 days = 162.6 ± 12.3 g). A significant difference was identified in the pelvic limbs when

comparing ST at 10 and 30 days (mean difference 35.4 g; P=0.01) and at 10 and 90 days (mean

difference 46.7 g; P=0.001), but not at 30 and 90 days (mean difference 11.2 g; P=0.93). There

was an inverse correlation between locomotor score and pelvic limb ST over time (r = -0.5; P =

0.03). These findings are suggestive of recovery of sensorimotor spinal cord function, and

potentially the development of hyperesthesia indicative of neuropathic pain. This is the first

study to evaluate ST over an extended period of time to evaluate for chronic neuropathic pain in

dogs and results suggest the need for further longitudinal studies related to neuropathic pain in

dogs after SCI.

iii

Acknowledgements

I would like to thank my advisor and mentor, Dr. Sarah Moore for her guidance and

support throughout this process. I would also like to thank my examination committee for their

input and advice. I would like to thank Amanda Disher, Heather Anderson, Dr. Daniella

Vansteenkiste, and Dr. Ashley Hechler for their assistance with data collection. I would like to

thank Dr. Laurie Cook and Dr. Nina Kieves for their participation. Additionally, I would like to

thank the entire Neurology and Surgery service for their help in case recruitment.

iv

Vita

May 2005....................................................... El Camino High School

May 2009....................................................... B.S. Neurobiology, Physiology, & Behavior, UC

Davis

June 2013....................................................... D.V.M., UC Davis

2013 to 2014.................................................. Intern, University of Georgia

2014-2015...................................................... Neurology Intern, Veterinary Specialty Hospital of

San Diego

2015 to present .............................................. Residency in Neurology and Neurosurgery,

Veterinary Medical Center, The Ohio State

University

Publications

1. Kerns A, Brakel K, Premanandan C, Saffire A, Moore A. Extranodal non-B, non-T-cell

lymphoma with bilateral tympanic bulla involvement in a cat. J Fel Med Surg Open Rep.

2018: 1-5.

2. Kerns A, Kieves N, Cook L, Moore S. Interobserver Agreement of Mechanical Sensory

Thresholds in Normal Dogs. Intended for submission to The Veterinary Journal.

3. Kerns A, Cook L, da Costa R, Moore S. von Frey anesthesiometry to diagnose chronic

neuropathic pain after acute spinal cord injury caused by thoracolumbar intervertebral disc

extrusion in dogs. Intended for submission to The Veterinary Journal.

v

Fields of Study

Major Field: Comparative and Veterinary Medicine

vi

Table of Contents

Abstract……………………………………………………………………… …………………...i

Acknowledgements………………………………………………………………………....……iii

Vita……………………………...……………………………………………….………….…….iv

Publications………………………………………………………………………….………....…iv

Fields of Study……………………………………………………………………..………....…...v

Table of Contents………………………………………………………………………....………vi

List of Tables……………………………………………………………………..…………..…viii

List of Figures…………………………………………………………………...……………..…ix

Chapter 1: Introduction……………….………………………………………….………………..1

Chapter 2: Literature review…………………………………….…………………….………..…4

2.1 Pain syndromes after spinal cord injury…………………....………………………..……..4

2.1.1 Pain taxonomy…………………..…………………...………………………..…..……4

2.1.2 Neuropathic pain after spinal cord injury…………………….………..…………….....5

2.1.3 Pathophysiology of neuropathic pain after spinal cord injury…….…………….…......6

2.2 Von Frey anesthesiometry………………………...……………………………….……...12

2.3 Treatment of neuropathic pain after spinal cord injury…………………....……………...14

Chapter 3: Interobserver agreement of mechanical sensory thresholds in normal dogs ………...21

3.1 Abstract……………………………………...………………………………………........20

3.2 Introduction………………...………………………………………………………….….21

3.3 Materials and methods…………………...……………………………………….……….22

3.4 Results……………………………………………………………...………….………….24

3.5 Discussion………………………………………………………………..……………….25

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3.6 Conclusions…………………………………………………………………..…………...28

3.7 Conflict of interest statement…………………………………………..…………………28

3.8 Acknowledgements………………………………………………..………………….…..28

3.9 References…………………………………………………………..………….…………28

Chapter 4: von Frey anesthesiometry to diagnose chronic neuropathic pain after acute spinal cord

injury caused by thoracolumbar intervertebral disc extrusion in dog…………...….……………35

4.1 Abstract……………………………………………...…………………………………....35

4.2 Introduction……………………………………………………………...……….……….35

4.3 Materials and methods…………………...……………………………...………….…….37

4.4 Results………………………………………………………………….………...……….38

4.5 Discussion…………………………………………………………………………..…….41

4.6 Conclusions………………………………………………………………………..……...44

4.7 Conflict of interest statement……………………………………………………..………45

4.8 Acknowledgements………………………………………………………………...……..45

4.9 References………………………………………………………………………...………45

Chapter 5: Conclusions and future directions……………....……..……………………....…..…52

References………………………………………………………………………………………..55

viii

List of Tables

Table 1: List of drugs commonly used for treatment of neuropathic pain……….....……………15

Table 2: Comparison of mean sensory threshold for each observer for each dog. Sensory

threshold values from all four limbs in each dog are averaged to provide a mean sensory

threshold for each observer………………….…………………………………………………...32

Table 3: Intraclass correlation coefficient for each pair of observers and average for all observers

combined………………………………………………………………………….……………...33

Table 4: Summary of oral medications at day 10. No dogs were receiving medication at 30 or 90

days……………………………………………………………………………………………....48

Table 5: Mean sensory threshold for each limb across three testing sessions over 90 days…….49

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List of Figures

Figure 1: The electronic von Frey device and patient positioning for mechanical quantitative

sensory testing in dogs. The device consists of a load cell (A), handle (B), recording device (C),

and tip (D). The system measures and displays the amount of pressure applied to a subject, from

0.1 to 1000 g……………………………....………………………………………………..……33

Figure 2: Total mean sensory threshold and standard deviation for each evaluator. Sensory

threshold values for all 20 dogs are averaged for each evaluator...…………………....……...…34

Figure 3: Mean ± SEM for combined thoracic limb and combined pelvic limb ST values at each

time point ……………………………………………………....………………………………..51

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Chapter 1

Introduction

Spinal cord injury (SCI) is a common condition in both domestic animals and people. An

estimated 276,000 people in the United States currently live with disabilities from SCI, with

approximately 17,000 new cases of SCI reported each year (National Spinal Cord Injury

Statistical Center [NSCISC], 2016). Causes of SCI in dogs include intervertebral disc extrusion,

spinal fracture, and fibrocartilaginous embolism. Intervertebral disc extrusion (IVDE) is by far

the most common cause of acute SCI in dogs, affecting approximately 3.5% of dogs under the

age of 12 (Bergknut et al., 2012), and comprising 2% of all veterinary hospital visits (Brisson et

al., 2010; Webb et al., 2010; Levine et al., 2011). SCI results in permanent changes in sensory

and motor function, and the life-altering and life-limiting consequences after injury are

appreciated across species. The most common and most debilitating of these are impaired

mobility and development of neuropathic pain (Sjolund et al., 2002; Kloos et al., 2005; NSCISC

2016).

Neuropathic pain is a type of pain caused by damage to or dysfunction of nervous tissue

and is generated by the subsequent changes in formation and modulation of nociceptive signaling

(Treede et al., 2008). This pain persists long after resolution of the initial injury and is often

associated with burning, stabbing, and tingling sensations (Merskey et al., 2002). Neuropathic

pain syndromes are characterized by allodynia (pain associated with a non-painful stimulus) and

hyperesthesia (heightened sensitivity to a painful stimulus). This is distinguished from

nociceptive pain which is caused by damage to non-nervous tissue and inflammation resulting in

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stimulation of nociceptors. Nociceptive pain is often described as a sharp or throbbing pain that

decreases with resolution of the primary insult.

Up to 90% of people report chronic neuropathic pain after SCI (Boldt et al., 2014), which

is severe and disabling in up to 63% of cases (Turner et al., 2001). Estimates of the prevalence of

neuropathic pain have increased dramatically over the past decade as better methods for

detecting such pain have been developed (Freynhagen et al., 2006). Despite the fact that

neuropathic pain syndromes are well documented in humans with SCI, there is limited

information regarding its existence in dogs (Moore et al., 2013). This is likely because the

presence of neuropathic pain in people is typically self-reported and this is not possible in

veterinary patients. Further, sensory testing in dogs with SCI has been historically limited to

testing for the presence or absence of a behavioral response to pinching the toes, which is

interpreted as presence or absence of nociception, also referred to in the literature as deep pain

perception (Lascelles et al., 2013). While this relatively crude method is useful and important in

the clinical setting for determination of prognosis for recovery after SCI, it does not allow for

identification and quantification of neuropathic pain.

Given the prevalence of neuropathic pain in patients with SCI and its negative impact on

quality of life, further investigation into objective methods for sensory testing in veterinary

patients is of immense importance. Methods for quantitative sensory testing (QST) have been

developed for use in rodent models of SCI to test neuropathic pain and have also been employed

in the human clinical setting. Mechanical methods of QST typically test an objective value called

sensory threshold (ST), defined as the minimum amount of a particular stimulus (e.g. pin-prick,

3

light touch) that can be detected or is considered painful. QST allows objective evaluation of

allodynia and hyperesthesia, manifest as a decrease in ST (Felix et al., 2009; Walk et al., 2009).

Von Frey anesthesiometry (VFA) is a technique used for QST that tests ST for a

mechanical stimulus (a plastic pressure probe applied to the skin). Variations of this technique

have been applied in rodent models, dogs, and human patients (Detloff et al., 2012; Tena et al.,

2012; Lascelles et al., 2013). Patients with diminished sensation (hypoesthesia) have a higher

than normal ST, whereas patients with hyperesthesia and allodynia have a lower than normal ST

(Hoschouer et al., 2010). VFA has recently been shown to be a valuable technique to evaluate ST

in both normal dogs and dogs with SCI caused by IVDE (Moore et al., 2013; Song et al., 2016).

Not only does this offer an objective and reliable way to identify neuropathic pain in veterinary

patients, but also allows the use of dogs with SCI due to IVDE as a naturally-occurring model of

SCI and neuropathic pain. This is important for outcome assessment where therapeutic

interventions are intended for human clinical translation (Felix et al., 2009; Lascelles et al.,

2013).

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Chapter 2

Literature Review

2.1 Pain syndromes after SCI

2.1.1 Pain Taxonomy

Specific types of pain syndromes are associated with SCI. A three-tier pain taxonomy has

been developed by the SCI Pain Task Force of the International Association for the Study of Pain

(IASP), which has assisted with standardization in description of pain in the human clinical

setting (Bryce et al., 2012). The first tier divides pain into the broad categories of nociceptive

and neuropathic pain. The second tier sub-divides these categories into organ systems:

Nociceptive pain can be visceral or musculoskeletal, while neuropathic pain is described in

reference to the level of SCI (at-level, above-level, or below-level). The third tier identifies

specific anatomical structures or etiologies as the source of pain (e.g. myocardial infarction for

visceral nociceptive pain, cauda equina compression for at-level neuropathic pain). An additional

pain category includes those syndromes that do not fit well into either nociceptive or neuropathic

pain, including fibromyalgia and complex regional pain syndrome. While these conditions have

characteristics of neuropathic, nociceptive, and mixed pain, unlike these causes of pain there is

no original tissue damage, and the source of pain appears to be functional in origin (Arnold et al.,

2016). Depending on the cause of SCI, several different nervous and non-nervous tissues can be

affected resulting in multiple concurrent pain syndromes.

The International Spinal Cord Injury Basic Pain Data Set was developed to aid in the

consistent collection and reporting of pain in the human SCI population (Widerstrom-Noga et al.,

2008, 2014). The data set contains clinically relevant core questions concerning SCI-related pain.

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Questions cover the type, location, intensity, frequency, and duration of pain. The data set also

addresses the impact of pain on physical, emotional, and social function, and sleep. The basic

pain data set is intended to be used by health-care professionals in daily clinical practice and

provides a framework for standardized evaluation of pain in clinical studies.

2.1.2 Neuropathic pain after SCI

Neuropathic pain after SCI can be spontaneous without a peripheral stimulus, or evoked

due to an innocuous (allodynia) or noxious (hyperalgesia) stimulus (Baron et al., 2006).

Development of neuropathic pain is typically delayed weeks to months after SCI, and in rare

instances longer than a year (Tasker et al., 1992). Pain syndromes after SCI are classified as

central neuropathic pain because lesions affect the central nervous system. In the human clinical

setting, central neuropathic pain is diagnosed based on patient history and clinical sensory

examination. Clinical criteria include the presence of hyperalgesia, allodynia, or loss of

sensation, distribution of affected dermatomes, and an identified underlying cause (Treede et al.,

2008). There are three major distributions of chronic neuropathic pain with SCI. Above-level

pain occurs in dermatomes cranial to the site of injury, at-level pain occurs in dermatomes at the

site of injury, and below-level neuropathic pain occurs in dermatomes caudal to the neurological

lesion (Hulsebosch et al., 2009). These are described based on somatosensory mapping using

mechanical and thermal sensory testing (Vogel et al., 2016).

At-level pain can be present at the time of injury, or can develop directly afterward. This

pain is described as shooting, tingling, burning, pins and needles, and electric, and is associated

with allodynia and hyperesthesia (Widerstrom-Noga et al., 2008). At-level pain requires a lesion

in the spinal cord or nerve roots, and in many cases nerve root injury results in spontaneous pain

6

(Yezierski et al., 2000). A potential complication of SCI is syringomyelia, where a syrinx forms

at the site of SCI and expands with time resulting in further spinal cord damage. Syringomyelia

is implicated when new symptoms develop, particularly at-level neuropathic pain, months to

years after SCI (Tasker et al., 1992; Brodbelt et al., 2003).

Below-level pain is the most common central pain syndrome after SCI and is the most

difficult to treat (Turner et al., 2001). This pain is typically associated with a burning sensation

and can be spontaneous or evoked by different auditory stimuli or movements (Widerstrom-

Noga et al., 2008). Various regions of the limbs, hands/feet, and digits are commonly reported

sites of below-level pain (Soler et al., 2010). At-level hypersensitivity, determined by QST, after

complete SCI has been associated with below-level pain (Vogel et al., 2016). Injuries that affect

spinothalamic tracts are associated with below-level pain, where changes in neuronal excitability

of residual spinothalamic afferents are suspected to play a role (Finnerup et al., 2014).

Above-level pain develops reliably in rodents with a variety of SCI, typically manifested

as forelimb hyperalgesia after thoracic SCI (Mills et al., 2001; Wang et al., 2008; Carlton et al.,

2009; Densmore et al., 2010). However, the limited QST data in humans and primates suggests

that above-level pain is uncommon (Defrin et al., 1999; Finnerup et al., 2003; Masri et al., 2012),

which may implicate important species differences in neuropathic mechanisms for above-level

pain.

2.1.3 Pathophysiology of neuropathic pain after SCI

In its functional state, pain serves a protective role to prevent or minimize tissue damage.

It provides an important defense mechanism by alerting the body to noxious stimuli including

mechanical, thermal, and chemical insults. When pain persists without potential or actual tissue

7

damage it is maladaptive, and becomes the disease itself. The generation of pain in response to a

noxious stimulus involves four basic steps: 1. Transduction of the noxious stimulus into a

nociceptive signal, 2. Transmission of the nociceptive signal from the site of injury to the central

nervous system via Aẟ and C fibers, 3. Modulation of the signal by ascending, descending, and

local facilitation and inhibition, and 4. Perception of pain manifested as cognitive and emotional

responses that are a key component of clinical pain (Cohen et al., 2014). In the physiologic state

these steps are well regulated, but in the pathophysiologic state after neurotrauma (e.g. after SCI)

there is dysregulation resulting in neuropathic pain.

The four steps for generation of pain described previously allow for accurate coding of

external stimuli as noxious or non-noxious. Specifically, dorsal horn neurons along the spinal

cord are a key component of the nociceptive apparatus to correctly characterize external stimuli

(Thalhammer et al., 1994). Alteration of dorsal horn neuronal function results in loss of accurate

sensory perception and abnormal sensory recognition, and may ultimately lead to the perception

of non-noxious input as noxious (allodynia) or enhance noxious input (hyperesthesia) (Drew et

al., 2004; Hains et al., 2005; Gwak et al., 2008). For this reason, a primary cause of neuropathic

pain after SCI is hyperexcitability of dorsal horn neurons for which a variety of underlying

mechanisms have been identified including peripheral sensitization, reorganization of synaptic

circuits, altered intracellular pathways, disinhibition, and activation of glial cells (Hulsebosch et

al., 2009; Gwak et al., 2011; Cohen et al., 2014). These mechanisms are further discussed here.

Peripheral sensitization occurs when there is a decreased threshold for firing of

nociceptors and/or increased frequency of firing in response to stimulation. Electrophysiological

study of peripheral nociceptors after SCI has revealed peripheral sensitization with both

8

enhanced activity to evoked stimuli (mechanical and thermal) and increased spontaneous activity

in the absence of external stimuli (Carlton et al., 2009). This chronic spontaneous activity in

primary peripheral nociceptors after SCI has been shown to result in pain-related behavior (Bedi

et al., 2010). Dorsal root ganglion neurons also play an important role in peripheral sensitization.

There are a variety of isoforms of voltage-gated sodium channels (the Nav family) in dorsal root

ganglion neurons, classified as either tetrodotoxin sensitive (TTX-S) or tetrodotoxin resistant

(TTX-R) (Goldin et al., 2000; Yin et al., 2016). After SCI, there are dramatic changes in

expression of some of these sodium channels, with an increase in expression of TTX-S isoforms

and decrease in expression of TTX-R isoforms in affected dorsal root ganglion neurons (Yin et

al., 2016). This altered transcription of Nav genes may result in abnormal action potential firing

and contribute to spontaneous pain.

Further support for the role of sodium channels in the development and maintenance of

neuropathic pain is that a particular TTX-S isoform, Nav1.3, which is normally not expressed in

dorsal root ganglion neurons, is expressed after peripheral nerve injury (Black et al., 1999).

Recent evidence has shown that inhibition of Nav1.3 in injured dorsal root ganglia diminishes

allodynia (Samad et al., 2013; Tan et al., 2015). Dorsal root ganglion neurons appear to

contribute to below-level pain after SCI as demonstrated by systemic, but not intrathecal,

administration of sodium-channel blockers resulting in diminished allodynia and hyperalgesia

(Weston et al., 2009; Hama et al., 2010).

In addition to peripheral sensitization, primary changes in the function and distribution of

dorsal horn neurons after SCI contribute to neuropathic pain. Dorsal horn neurons are

categorized as low threshold (which respond primarily to non-noxious stimuli), high threshold

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(which respond primarily to noxious stimuli), and wide dynamic range neurons (which respond

to several stimuli but maximally to noxious stimuli) (Maixner et al., 1986; Chung et al., 1986;

Leem et al., 2010). Normally, low threshold neurons are found in the deep dorsal horn where

non-noxious information is received from the periphery. High threshold neurons are found in the

superficial dorsal horn where noxious information is received predominantly from Aẟ and C

fibers. Wide dynamic range neurons are found throughout the dorsal horn (Menetrey et al.,

1977). In vivo electrophysiological data has shown that there is increased excitability in all three

types of dorsal horn neurons after SCI over the course of a month compared to controls (Gwak et

al., 2011). Not only do dorsal horn neurons become more excitable, but their proportions change

as well, with the proportion of low threshold neurons decreasing in the deep dorsal horn and

proportion of wide dynamic range neurons increasing in all areas of the dorsal horn after injury

(Hao et al., 2004; Gwak et al., 2011).

The mechanism underlying this altered distribution is unknown, but one possible

explanation is a dorsal horn neuronal phenotypic switch: After nerve injury there are significant

changes in gene expression that can result in changes in excitability, transduction, and

transmission. Ultimately, this can result in a change in the phenotype of neurons, where certain

genes are now upregulated or downregulated allowing a neuron to take on different functional

characteristics. This has been demonstrated in injured nerves where non-nociceptive fibers begin

expressing neuromodulators normally expressed by C fibers such as calcitonin gene related

peptide or substance P (Ueda et al., 2006). In the case of dorsal horn neurons this may, for

example, result in low threshold neurons that normally receive and transmit non-nociceptive

signals taking on the characteristics of wide dynamic range neurons, which could result in

allodynia.

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Once dorsal horn neuronal hyperexcitability develops it continues for months, and

persists after injury. One possible explanation for continued hyperexcitability is reorganization of

synaptic circuits in the dorsal horn after SCI. SCI results in damage to ascending and descending

pathways in the spinal cord. After the primary injury, secondary injury occurs that results in

further disruption of synaptic circuits with continued neuronal death (Wasner et al., 2008).

Following injury, surviving neurons and microglia produce neurotrophic factors such as nerve

growth factor, which promotes regeneration and axon sprouting of primary afferent fibers and

subsequent newly formed synaptic circuits in the dorsal horn (Christensen et al., 1997).

Normally, primary afferents carrying non-noxious information would terminate in the deep

dorsal horn and those carrying noxious information would terminate in the superficial dorsal

horn. However, expression of nerve growth factor by primary afferent fibers after SCI results in

atypical axonal sprouting and inappropriate synapse formation. As a result, deep dorsal horn

neurons receive nociceptive signals and superficial dorsal horn neurons receive non-nociceptive

signals. This results in neuronal hyperexcitability and abnormal neuronal response properties

(Braz et al., 2009). When treated with anti-nerve growth factor after SCI, there is reduced

primary afferent sprouting, decreased dorsal horn excitability, and decreased neuropathic pain

(Christensen et al., 1997; Romero et al., 2000). Together, changes in the dorsal horn neuronal

populations and synaptic circuits offer an explanation for the permanent changes seen in dorsal

horn neuronal hyperexcitability and subsequent chronic central neuropathic pain.

Another mechanism for post-SCI dorsal horn neuron hyperexcitability and neuropathic

pain is altered intracellular pathways. SCI results in large increases in extracellular glutamate

concentrations (McAdoo et al., 1999), which then leads to receptor activation and increased

11

intracellular calcium levels (Tator et al., 1991). These changes lead to activation of various

protein kinases and downstream transcriptional factor pathways that result in central sensitization

(Crown et al., 2006). For example, it has been shown that a number of mitogen-activated protein

kinases (MAPKs) are activated after SCI, which then phosphorylate and activate transcription

factors (Crown et al., 2006). One of these transcription factors, cyclic AMP response element

binding protein (CREB), has been highlighted for its role in nociception (Ji et al., 2003). In a rat

model of SCI, both MAPKs and CREB had increased activity in microglia, astrocytes, and dorsal

horn neurons in rats that went on to develop at-level allodynia, compared to normal activity in

rats that did not develop neuropathic pain (Crown et al., 2006, 2008). Further, use of a MAPK

inhibitor after SCI resulted in dose-dependent attenuation of allodynia and decreased dorsal horn

neuronal hyperexcitability (Crown et al., 2008).

In addition to activation of certain intracellular pathways, disinhibition also plays an

important role in the development of neuropathic pain. Gamma-amino butyric acid (GABA) acts

to inhibit dorsal horn cells via GABA receptors distributed in presynaptic and postsynaptic

neurons and glial cells (Malcangio et al., 1996). After SCI there is altered ratios of apoptotic and

antiapoptotic factors resulting in death of GABAergic neurons (Rafati et al., 2008). Furthermore,

there is proinflammatory cytokine activation of caspases that result in apoptosis of GABA

interneurons in the dorsal horn (Meisner et al., 2010). There is increasing evidence that this loss

of GABAergic tone after SCI contributes to the development and maintenance of central

neuropathic pain. Several SCI models have shown good correlation between loss of GABA

function, dorsal horn hyperexcitability, and neuropathic pain (Hao et al., 1991; Gwak et al.,

2008). When GABAergic tone is returned by a variety of mechanisms, there is decreased

12

neuronal hyperexcitability, allodynia, and hyperesthesia (Liu et al., 2004; Eaton et al., 2007; Kim

et al., 2010).

2.2 Von Frey anesthesiometry

Quantitative sensory testing (QST) analyzes the response to a quantified external

stimulus, typically applied to the skin. A variety of external stimuli can be used, the most

common being touch, vibration, pin-prick and thermal (cold, warmth, burning) stimuli.

Assessment of mechanical stimuli can be used to assess large fiber and dorsal column function,

while thermal and pain thresholds can be used to assess small fiber (Aẟ and C fibers) and

spinothalamic tract function (Nathan et al., 1986). Methods for QST have long been developed

for use in rodent models of SCI and in the human clinical setting. QST has been promoted for

more accurate evaluation of somatosensory dysfunction in neuropathic pain populations

(Greenspan et al., 2001), and standardized protocols and normal values have been published

(Rolke 2006). Many of these methods test an objective value called sensory threshold (ST),

defined as the minimum amount of a particular stimulus that can be detected or is considered

painful. However, when performing QST in animals, they cannot communicate verbally and thus

ST is by convention defined as the strength of stimulus required to produce a conscious

behavioral response to that stimulus (Mills et al., 2001; Detloff et al., 2012; Moore et al., 2013).

Use of QST and ST allows for evaluation of somatosensory hypofunction and hyperfunction,

both of which are common after SCI.

Von Frey Anesthesiometry (VFA) is one method of QST that has primarily been used in

rodent models and human clinical setting. This technique, first described by Max von Frey in

1895, tests ST by applying a mechanical stimulus to the skin, allowing for assessment of

13

mechanical hyperesthesia. Von Frey monofilaments have been used historically and are still used

today, but the more recent electronic VFA has simplified the procedure. Electronic VFA uses a

plastic probe to apply a punctate mechanical stimulus, with the pressure at which ST is achieved

being automatically recorded. Variations of this technique have been applied in rodent models,

dogs, and human patients (Detloff et al., 2012; Tena et al., 2012; Lascelles et al., 2013). Patients

with diminished sensation have a higher than normal ST because they require a larger stimulus

for detection, while patients with hyperesthesia or allodynia have a lower than normal ST

(Hoschouer et al., 2010). The use of electronic VFA has allowed for more rapid and reproducible

results in the human clinical setting (Tena et al., 2012).

VFA has shown promise in veterinary medicine as a useful clinical and research

tool. VFA has been used in dogs to assess the pharmacodynamics and anti-nociceptive effects of

a variety of medications (KuKanich et al., 2005, 2011, 2016; Abimussi et al., 2014), identify

sensory abnormalities in dogs with orthopedic disease (Brydges et al., 2012; Tomas et al., 2014),

and quantify postoperative pain with different surgery techniques for ovariohysterectomy (Case

et al., 2011). Recently, a modified dorsal technique for VFA in dogs has been shown to be a

reliable method for QST in normal dogs and in dogs with SCI due to IVDE, and has shown a

difference in ST between normal and SCI dogs (Moore et al., 2013; Song et al., 2016). Chronic

neuropathic pain after SCI has yet to be investigated in dogs. Spontaneous SCI is common in

dogs (Levine et al., 2011), and results in similar histologic and structural changes in the spinal

cord as humans (Smith et al., 2006; Bock et al., 2013). Not only does VFA offer an objective and

reliable way to identify neuropathic pain in veterinary patients, but also allows the use of dogs

with SCI due to IVDE as a naturally-occurring model of SCI and neuropathic pain. This is

14

important for outcome assessment where therapeutic interventions are intended for human

clinical translation (Felix et al., 2009; Lascelles et al., 2013).

2.3 Treatment of neuropathic pain after spinal cord injury

SCI is common in people and domestic animal species. A large majority of people report

chronic neuropathic pain after SCI (Boldt et al., 2014), which is severe and disabling in many

cases (Turner et al., 2001). Neuropathic pain is also a common and reliable outcome in SCI

rodent models, but has not yet been thoroughly explored in veterinary medicine. While rodent

models have allowed elucidation of several potential mechanisms and treatment targets for

neuropathic pain, translation to effective treatment in people has not yet been borne out. Despite

significant improvement in managing other comorbidities after SCI (e.g. cardiorespiratory, skin,

bladder, and bowel complications), neuropathic pain has remained largely refractory to treatment

and continues to be the most debilitating problem that people face (Finnerup et al., 2003;

Cardenas et al., 2009). Clinical resolution of pain is often unrealistic, and modulation of pain

may be a more reasonable and achievable goal.

A variety of pharmacological interventions have been used to treat neuropathic pain after

SCI. These include anticonvulsants (gabapentin, pregabalin), antidepressants (amitriptyline,

trazodone), analgesics (lidocaine, ketamine, opioids), cannabinoids (tetrahydrocannabinol) and

antispasticity medications (baclofen, botulinum toxin) (Table 1). Treatment is generally long-

lasting and so the expected benefits and potential side effects should be carefully considered.

Two recent systematic reviews evaluated the evidence for more than twenty pharmacologic

agents after SCI. Both concluded that only gabapentin and pregabalin have strong evidence for

their ability to modulate neuropathic pain, and are now considered first-line treatment (Teasell et

15

al., 2010; Hagen et al., 2015). Amitriptyline, a tricyclic antidepressant, may have some effect on

certain types of neuropathic pain including pain associated with diabetic neuropathy and

postherpetic neuralgia (Moore et al., 2012). Injectable analgesics in general can help to decrease

neuropathic pain, but these only provide short-term relief and require the patient to be in the

hospital. In particular, opioids are commonly prescribed for both neuropathic and nociceptive

pain. After nerve injury, dorsal root ganglia have decreased expression of μ opioid receptors and

dorsal horn neurons become less responsive to opioids (Kohno et al., 2005). In contrast,

inflammation can increase expression and affinity of opioid receptors that enhances the efficacy

of opioids (Przewlocki et al., 2001). This may explain why patients with chronic neuropathic

pain require higher doses of opioids compared to those with nociceptive pain (Benedetti et al.,

1998). Unique to opioid exposure in people is opioid-induced hyperalgesia, characterized by a

paradoxical response where a patient receiving opioids to treat pain may actually become more

sensitive to that pain. While several mechanisms for opioid-induced hyperalgesia have been

proposed, none have been confirmed (Lee et al., 2011). The remainder of treatments show no

effect or inconclusive results (Teasell et al., 2010; Hagen et al., 2015).

Table 1 Medications used for neuropathic pain.

Analgesics Major Mechanism of Action Selected References (Human; Veterinary)

Anesthetics/Antiarrhythmics

Ketamine NMDA receptor antagonist Eide 1995; Kaka 2016

Lidocaine voltage-gated Na+ channel blocker Finnerup 2005; Kaka 2016

Mexiletine voltage-gated Na+ channel blocker Chiou-Tan 1996; Not evaluated

Opioids Alfentanil μ-opioid receptor agonist Eide 1995; Arndt

1986 Morphine μ-opioid receptor agonist Attal 2002; KuKanich

16

2005 Tramadol μ-opioid receptor agonist; SNRI Norbrink 2009;

Schutter 2017 Antidepressants

Amitriptyline SNRI Moore 2012; Cashmore 2009

Duloxetine SNRI; Na+ channel blocker Vranken 2011; Not evaluated

Lithium Unknown Yang 2012; Not evaluated

Trazodone serotonin receptor antagonist Davidoff 1987; Not evaluated

Antiepileptics Carbamazepine voltage-gated Na+ channel blocker Salinas 2012; Not

evaluated Gabapentin voltage-gated Ca++ channel blocker Levendoglu 2004;

Plessas 2015 Lamotrigine voltage-gated Na+ channel blocker Finnerup 2002; Not

evaluated Levetiracetam binds synaptic vesicle glycoprotein 2A Finnerup 2009; Not

evaluated Pregabalin voltage-gated Ca++ channel blocker Siddall 2006; Salazar

2009 Valproate voltage-gated Na+ channel blocker;

increases brain GABA levels Drewes 1994; Not evaluated

Cannabinoids Cannabis cannabinoid receptor agonist Wade 2003; Not

evaluated Dronabinol cannabinoid receptor agonist Rintala 2010; Not

evaluated Tetrahydrocannabinol cannabinoid receptor agonist Phillips 2010; Not

evaluated NMDA, N-methyl-D-aspartate ; SNRI, serotonin-norepinephrine reuptake inhibitor ; GABA, gamma-aminobutyric acid

Gabapentin was initially designed to mimic the structure and function of the inhibitory

neurotransmitter GABA, and thereby act as an anticonvulsant. While it is structurally similar to

GABA, it was subsequently discovered to be useful in the treatment of pain. Gabapentin appears

to act via binding the α2ẟ subunit of voltage gated calcium channels resulting in decreased

17

calcium influx and diminishing excitatory neurotransmitter release (Offord et al., 2015). One to 2

weeks after nerve injury, there is increased expression of calcium channels containing the α2ẟ

subunit in and around the dorsal root ganglia, which results in increased excitability (Luo et al.,

2001). Administration of gabapentin has been shown to decrease nociceptive signaling (Hendrich

et al., 2008), and provides adequate relief of neuropathic pain in about one third of patients

(Moore et al., 2014). While side effects are common, they are generally mild and well-tolerated.

The most common side effects seen are dizziness and somnolence, and severe effects were found

to be no more common than with placebo (Moore et al., 2014). Gabapentin is also commonly

prescribed for veterinary patients to alleviate pain, though its efficacy has not been established

for chronic neuropathic pain in animals. While gabapentin’s side effects have not been

thoroughly investigated in veterinary medicine, it appears to have a similar safety profile as in

people, perhaps with even more mild sedative effects at higher dosages.

There are limited clinical studies evaluating the analgesic effects of gabapentin in dogs.

In a study evaluating gabapentin as an adjunctive analgesic in dogs after mastectomy, dogs

receiving gabapentin required 44% less rescue analgesia with morphine compared to placebo,

despite no differences in pain scores (Crociolli et al., 2015). A similar study evaluated

gabapentin in dogs undergoing hemilaminectomy for IVDE, where gabapentin was started 12

hours prior to surgery, and no differences in pain score were found compared to placebo

(Aghighi et al., 2012). A study evaluating gabapentin in dogs undergoing forelimb amputation

also found no difference in pain scores postoperatively compared to placebo (Wagner et al.,

2010). Of note is that a gabapentin dose of 5-10 mg/kg given every 12 hours was used in these

studies. Based on available pharmacokinetic data in dogs, a dose of 10-20 mg/kg given every 8

hours may be necessary to maintain minimum effective plasma concentrations, and in some dogs

18

an even higher dose or frequency may be necessary (Kukanich et al., 2011). Further, as

previously discussed the primary molecular target for gabapentin is the α2ẟ subunit of voltage

gated calcium channels in the dorsal root ganglion and dorsal horn of the spinal cord, and

upgregulation of these calcium channels does not occur in the immediate postoperative period

(Luo et al., 2001). These studies are therefore likely evaluating the immediate postoperative

analgesic effects of gabapentin on nociceptive pain rather than a chronic pain state, which may

explain why no change in pain score was identified in dogs undergoing these various surgical

procedures. For this reason, the results of these studies are difficult to translate to dogs with

chronic neuropathic pain.

One study has evaluated the use of gabapentin at approximately 10 mg/kg given every 8

hours in dogs with Chiari-like malformation and syringomyelia, with clinical signs suggestive of

chronic neuropathic pain. No difference was found in the quality of life of these dogs when

gabapentin and carprofen were used compared to carprofen alone (Plessas et al., 2015). Even less

information about pregabalin exists in veterinary medicine. A pharmacokinetic study using a

single dose of 4 mg/kg in normal dogs resulted in plasma levels remaining above the reported

therapeutic minimum in people for slightly over 11 hours, suggesting that dosing pregabalin

every 12 hours may be adequate (Salazar et al., 2009). No clinical studies have evaluated the

efficacy of pregabalin in dogs, and the significantly higher cost of pregabalin compared to

gabapentin often precludes its use in the veterinary clinical setting, particularly for chronic

administration.

Given the significant impact of neuropathic pain on daily function and quality of life in

people, and the difficulty in treating neuropathic pain, further research is necessary to improve

19

our ability to diagnose and treat neuropathic pain in veterinary patients. This would not only

allow for better care and improved quality of life in veterinary patients, but also open an entire

field of inquiry to translate this information to the human clinical setting. One of the primary

methods for evaluating neuropathic pain in people is via quantitative sensory testing, which has

only just begun to be explored in the veterinary clinical setting. Further validation of mechanical

sensory testing in veterinary patients, including interobserver reliability, must be performed prior

to implementing this technique in multi-center trials allowing for large-scale pain studies in dogs.

Once validated, the prevalence and character of neuropathic pain in the veterinary population can

be detailed, and subsequently the efficacy of various treatments, including gabapentin, can be

explored. The aims of this project were to 1. Determine the interobserver reliability of VFA in

normal dogs, and 2. Use VFA to evaluate the existence and time course for development of

neuropathic pain in dogs with acute SCI due to IVDE.

20

Chapter 3

Interobserver Agreement of Mechanical Sensory Thresholds in Normal Dogs

3.1 Abstract

Electronic von Frey anesthesiometry (VFA) has been previously reported as a useful

method of mechanical quantitative sensory testing (QST) for evaluating neuropathic pain in

dogs. Intraobserver agreement has been previously shown to be good to excellent; however,

interobserver agreement has not been evaluated and is vital to the use of this technique in

multicenter veterinary clinical trials in neuropathic pain. The goal of this study was to evaluate

the interobserver agreement of sensory thresholds obtained using electronic VFA in a group of

normal small breed dogs.

Twenty healthy dogs (< 20kg) were recruited from the general practice population at the

Ohio State University Veterinary Medical Center. Three novice evaluators used an electronic

von Frey device (IITC Life Science; Woodlands, CA) to measure mechanical sensory threshold

(ST) after a standardized training session conducted by an expert evaluator. Each dog was

evaluated by all three investigators on the same day with both evaluator and limb test order

randomized and testing sessions separated by five minutes.

Mean ST values were averaged for all four limbs to produce a single value per dog for

comparison between evaluators. Agreement between evaluators was determined using the intra-

class correlation coefficient (ICC; two-way model for consistency, single measures). ICC across

all three evaluators was 0.48, indicating moderate agreement. Moderate interobserver agreement

is not sufficient to support the use of this technique in multi-center clinical trials, and our results

21

underscore the importance of using a single evaluator for this QST technique in canine

neuropathic pain studies.

3.2 Introduction

Neuropathic pain is generated by the changes in formation and modulation of nociceptive

signals in response to injury due to damage or dysfunction of nervous tissue (Treede et al.,

2008). Up to 90% of people report chronic neuropathic pain after spinal cord injury (SCI) (Boldt

et al., 2014), which is severe and disabling in up to 63% of cases (Turner et al., 2001). Despite

the fact that neuropathic pain syndromes are well documented in people with SCI, there is

limited information regarding its diagnosis and treatment in dogs with SCI (Moore et al., 2013).

Given the prevalence of neuropathic pain in people with SCI and its negative impact on

quality of life, further investigation into quantitative methods for assessing neuropathic pain in

veterinary patients is of immense importance. Methods for quantitative sensory testing (QST)

have been developed for use in rodent models of SCI and in the human clinical setting. These

methods test an objective value called sensory threshold (ST), defined as the minimum amount

of a particular stimulus (e.g. pinprick, light touch, temperature) that can be detected or is

considered painful. In veterinary patients specifically, mechanical ST has been defined as the

strength of mechanical stimulus required to produce a conscious behavioral response to that

stimulus (Moore et al., 2013). QST allows quantitative evaluation of hyperesthesia, which

manifests as a decrease in ST (Felix et al., 2009; Walk et al., 2009).

Von Frey anesthesiometry (VFA) is a technique used for QST that tests ST for a

mechanical stimulus (a plastic pressure probe applied to the skin). Variations of this technique

have been applied in rodent models, dogs, and people (Detloff et al., 2012; Tena et al., 2012;

22

Lascelles et al., 2013,). VFA has recently been shown to be a feasible technique to evaluate ST

in both normal dogs and dogs with SCI caused by intervertebral disc extrusion (IVDE) (Moore

2013 et al.; Song et al., 2016). While intraobserver reliability of the technique has been

established to be strong (Moore 2013 et al.; Knazovicky et al., 2017); interobserver agreement

using the technique has not been previously reported. Recently, there has been a surge of interest

in the use of dogs with IVDE as a clinical model of SCI (Smith et al., 2006; Levine et al., 2011;

Moore et al., 2017), therefore veterinary clinical trials assessing treatments to improve

neuropathic pain after SCI may provide important advances in translational pain management

strategies. An understanding of interobserver agreement related to measurements of QST is

necessary before multi-institutional veterinary pain studies can be undertaken and their findings

interpreted with confidence. The goal of this study was to evaluate the interobserver reliability of

one method of mechanical QST, electronic VFA, in normal dogs.

3.3 Materials and methods

Animals

This study was approved by the Institutional Animal Care and Use Committee (protocol

# 2015A00000054) and the Clinical Research Advisory Committee of The Ohio State

University Veterinary Medical Center (OSU VMC). Written informed consent was obtained

from owners of all dogs prior to enrollment. Healthy dogs were recruited from the general patient

population at the OSU VMC. All dogs enrolled were ≤20 kg, were reported by their owners to be

systemically healthy, had a normal physical and neurologic examination performed by a single

investigator (AK), had not received any medications during the month prior to study enrollment,

and had no owner-reported history of neurologic or orthopedic disease.

23

Evaluator training

To mirror the expected landscape of a multicenter veterinary clinical trial scenario, three

novice QST evaluators (one veterinary neurology resident, one board certified veterinary surgeon

specializing in orthopedics, and board certified veterinary neurologist) were recruited to

participate in the study. Each novice evaluator underwent a standardized training session prior to

initiation of the study which was led by an expert evaluator (SAM). The training session

consisted of a demonstration of proper VFA technique as described below using a normal,

cooperative dog and focused on method and location of probe application and evaluating

behavioral response to the stimulus. After demonstration, each evaluator performed the VFA

technique on the same normal dog with feedback from the expert evaluator until both the trainer

and the trainee felt comfortable with the technique being performed. Each evaluator had a single

training session which lasted 10-20 minutes depending on speed with which the evaluator

became comfortable with the technique.

Electronic von Frey device and technique

The device used and patient positioning are shown in Figure 1. An electronic von Frey

device (IITC Life Science; Woodlands, CA) with a 1000 g probe and rigid 0.8 mm diameter tip

was used for all measurements. Mechanical ST was measured in every dog by each of three

investigators using a technique previously published by our laboratory (Moore et al., 2013; Song

et al., 2016). After an acclimation period of 5 minutes, dogs were placed in lateral recumbency

and maintained there with minimal restraint. A testing session consisted of a single investigator

performing QST on each of the dog’s four limbs in a randomized order. Five measurements were

obtained in a blinded fashion from each limb, with the highest and lowest values excluded and

the middle three values averaged for a given limb to produce a mean ST for each limb. Dogs

24

were provided a five minute rest period between sessions, during which time they were allowed

to freely roam about the testing room. Each dog underwent a total of three testing sessions, one

with each investigator. Investigator test order was determined via a random number generator.

The total session time (in minutes) required for testing to be completed by each observer was

recorded.

Statistical analysis

For ease in statistical comparisons, mean ST for each of four limbs as determined by a

single investigator were averaged to produce a mean overall ST for each individual dog and

investigator. Agreement was compared between investigators by calculating an intra-class

correlation coefficient (ICC) using a two-way model for consistency of single measures and

where <0.2 was considered slight agreement, 0.21-0.40 was considered fair agreement, 0.41-0.60

was considered moderate agreement, 0.61-0.80 was considered substantial agreement, and 0.81-

1.00 was considered almost perfect agreement (Landis and Koch, 1977; Tena et al., 2012;

Andersen et al., 2015). Statistical analysis was performed using MedCalc software.

3.4 Results

A total of 20 normal dogs were enrolled. Dogs ranged in age from 1 to 11 years (median

5 years), and weighed between 3.6 and 15.3 kg (median 7.7 kg). There were 13 castrated males

and 7 spayed females. Breeds were as follows: 7 mixed breed dogs, 3 Border Terriers, 2

Dachshunds, 2 Terrier mixes, 1 Boston Terrier, 1 Pomeranian, 1 Silky Terrier, 1 Sealyham

Terrier, 1 Pug, and 1 Pembroke Welsh Corgi. Mean overall ST for all dogs, as determined by

each of three investigators, is shown in Figure 2.

25

The ICC across each of the 3 investigators based on ST values for all dogs is summarized

in Table 2. ICC between investigators A and B was 0.62 (95% CI 0.26-0.83), between A and C

was 0.48 (95% CI 0.07-0.76), and between B and C was 0.35 (95% CI 0-0.68). ICC for all 3

investigators was 0.48 (95% CI 0.21-0.73). Mean total time required to complete a testing

session in a single dog for an individual investigator was 23.8 minutes (range 20.0-30.2 minutes).

3.5 Discussion

Electronic VFA is ideal for sensory testing in veterinary patients as it is relatively quick,

well-tolerated, and can automatically record ST allowing the evaluator to be blinded to the

pressure applied. Previous reports of good intraobserver and intersession reliability (Moore et al.,

2013; Knazovicky et al., 2017) established the technique as a potentially promising modality

through which to diagnose and monitor neuropathic pain in the veterinary clinical setting.

However, because veterinary clinical trials are becoming more frequently multi-center in nature,

a firm understanding of the degree of interobserver agreement with the technique was needed to

support its clinical use. The results of our study suggest that interobserver agreement is only

moderate for novice, but clinically experienced evaluators who are provided a brief standardized

training session. Moderate agreement between evaluators is likely not adequate for useful

compilation and analysis of sensory data from multiple institutions for the purpose of large-scale

studies and our results underscore the need for either use of a consistent evaluator or refinement

in training techniques for multi-center studies.

The interobserver agreement of electronic VFA in both healthy and post-operative people

has been reported to have better reliability than what we found in our current study, typically

showing substantial or almost perfect agreement (Tena et al., 2012). This is similarly true using

26

other mechanical sensory tests including monofilaments and pin-prick (Andersen et al, 2015,

Marcuzzi et al., 2017). This discrepancy may reflect the inherent challenge in interpreting the

variety of behavioral responses dogs may exhibit toward a stimulus, with different investigators

being more or less observant of certain types of responses during testing. In addition to

differences in investigator interpretation of patient responses, there may have also been

differences in patient responses to each investigator. Different investigators may have given

different behavioral cues causing dogs to anticipate or react differently to the stimulus, and dogs

may have developed learned behaviors over the course of the testing sessions as they were

exposed to a repeated stimulus (Jones et al., 2007). To help control the latter we randomized the

order of the investigators and the testing order of the limbs for each dog.

Another potential contributor to our lower ICC values may be related to training of our

investigators, who spent only a brief training session becoming familiar with the von Frey device

and sensory testing procedure prior to acquiring patient data. In people, more extensive QST

training is recommended prior to collecting and reporting sensory testing data. For example, the

German Research Network on Neuropathic Pain offers an eight hour QST training program that

includes information about equipment, technique, quality control, and analysis and interpretation

of results using established standardized protocols and normal QST values (Rolke et al., 2006).

Further, many investigators have significantly more experience in QST in addition to this

recommended training (Marcuzzi et al., 2017). Laboratories can become QST certified through

this training and the use of validated QST protocols, resulting in enhanced reliability of QST data

(Geber et al., 2011). Indeed, early multi-center QST studies in people prior to the implementation

of standardized protocols and training showed consistently poor interobserver reliability

(Armstrong et al., 1991; Valensi et al., 1993; Geber et al., 2008). This was initially attributed to

27

the effect of multiple observers, but improved substantially once more extensive training and

standardized protocols were implemented (Delaney et al., 1993; Geber et al., 2011). Rigorous

QST training programs do not currently exist for veterinarians, and in the present study we

designed our training protocol to mimic what would be feasible for novice evaluators in the

clinical setting; however, our results suggest that the development of standardized training and

QST protocols will likely be needed before undertaking multi-center veterinary pain studies

using QST as an outcome measure.

Several other factors have been identified that influence QST results in people. Circadian

rhythm may affect the stability of QST measures (Geber et al., 2011). Our testing was performed

on the same day and at the same time of day with just 5 minutes between testing sessions by all 3

investigators, which controlled for this in the present study. In people it has also been shown that

age and sex affect ST (Chesterton et al., 2007). Each patient acted as its own control in our study

with respect to age and sex, eliminating impact of signalment on sensory threshold as obtained

by any single investigator. The effect of time of day, age, and sex on QST in dogs has not been

previously reported and when implementing a multicenter approach it may be important to

standardize these aspects of study design. Further evaluation of the effects of environmental and

dog-specific factors on the results of QST are needed.

Our results suggest that interobserver reliability is not currently adequate to support the

use of electronic VFA to collect QST data in multi-institutional studies. However, QST as a

means to evaluate ST and document neuropathic pain has only recently been investigated in

veterinary medicine, and this study highlights the need for further research to better establish

standardized QST protocols, define normal reference values for sensory threshold, and develop

28

effective standardized training protocols for investigators using QST. This may allow for

improved interobserver reliability and ultimately large-scale multi-center veterinary pain studies,

as has occurred over the last few decades in human medicine.

3.6 Conclusions

The results of the present study suggest that a brief training session provided to

investigators with advanced clinical training, but no prior experience with VFA does not result in

sufficient interobserver reliability to support the use of the technique in multi-center veterinary

clinical studies. This, along with previous reports of good intraobserver reliability, suggests that

a single investigator should be used to collect data for mechanical QST studies in dogs,

especially when evaluating changes over time. However, refinement of investigator training may

enhance reliability of the technique and will be vital before multi-center pain studies can be

effectively undertaken.

3.7 Conflict of interest statement

None of the authors has any financial or personal relationships that could inappropriately

influence or bias the content of the paper.

3.8 Acknowledgements

The study was funded by the Gray Lady Foundation [grant number]. In addition to the

funding source, the authors gratefully acknowledge Mrs. Amanda Disher, Mrs. Heather

Anderson, and Dr. Ashley Hechler for their assistance with the study.

3.9 References

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Turner, J.A., Cardenas, D.D., Warms, C.A., McClellan, C.B., 2001. Chronic pain associated with spinal cord injuries: A community survey. Archives of Physical Medicine and Rehabilitation 82, 501-508. Valensi, P., Attali, J.R., Gagant, S., 1993, Reproducibility of parameters for assessment of diabetic neuropathy. The French Group for Research and Study of Diabetic Neuropathy. Diabetes Medicine 10, 933–939. Walk, D., Sehgal, N., Moeller-Bertram, T., Edwards, R.R., Wasan, A., Wallace, M., Irving, G., Argoff, C., Backonja, M.M., 2009. Quantitative sensory testing and mapping – A review of nonautomated quantitative methods for examination of the patient with neuropathic pain. The Clinical Journal of Pain 25, 632–640.

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Tables Table 2 Comparison of mean sensory threshold (ST) of clinically normal dogs for each observer for each dog. To aid in statistical comparison, sensory threshold values from all four limbs in each dog are averaged to provide a mean sensory threshold for each observer.

Mean Sensory Threshold (g)

Dog Observer 1 Observer 2 Observer 3

1 131.3 61.7 67.2

2 73.9 187.5 82.1

3 184.8 241.9 81.9

4 163.1 123.3 80.4

5 165.2 133.8 187.4

6 168.0 153.3 284.8

7 207.4 175.4 151.6

8 311.7 316.3 278.9

9 166.0 153.9 167.3

10 126.4 105.1 156.0

11 182.3 79.5 139.9

12 122.3 56.8 153.7

13 101.0 67.5 220.1

14 150.0 115.1 156.4

15 52.7 47.7 90.1

16 188.7 76.3 97.5

17 111.1 180.8 129.3

18 146.7 114.4 126.1

19 92.2 96.7 92.2

20 127.6 103.2 92.9

Total Mean ST 148.6 129.5 141.8

Std Deviation 55.7 67.0 62.9

33

Table 3 Intraclass correlation coefficient for each pair of observers and average for all observers

combined.

Observers Intraclass Correlation Coefficient (95% CI)a

Observers 1 & 2 0.62 (0.26-0.83)

Observers 1 & 3 0.49 (0.07-0.76)

Observers 2 & 3 0.35 (0.0-0.68)

Mean All Observers 0.48 (0.21-0.73)

a95% CI, 95% Confidence interval.

Figures

Figure 1 The electronic von Frey device and patient positioning for mechanical quantitative

sensory testing in dogs. The device consisted of a load cell (A), handle (B), recording device (C),

and tip (D). The system measures and displays the amount of pressure applied to a subject, from

0.1 to 1000 g. Figure originally published by Moore et al., 2013.

34

Figure 2 Total mean sensory threshold and standard deviation for each evaluator. Sensory

threshold values for all 20 dogs are averaged for each evaluator.

35

Chapter 4

von Frey anesthesiometry to diagnose chronic neuropathic pain after acute spinal cord

injury caused by thoracolumbar intervertebral disc extrusion in dogs

4.1 Abstract

Von Frey anesthesiometry (VFA) was used to objectively measure mechanical sensory

threshold (ST) in 21 dogs with acute SCI caused by spontaneous thoracolumbar intervertebral

disc extrusion (IVDE) at approximately 10 days, 30 days, and 90 days after decompressive

hemilaminectomy. ST values were compared over time and correlated with locomotor scores.

There was no significant change in ST in the thoracic limbs over the 90 day post-

operative period. Pelvic limb ST were highest at 10 days consistent with hypoalgesia and

declined over time, though the decline from 30 to 90 days was not statistically significant (means

± standard error of the mean ST for pelvic limbs at 10 days = 209.2 ± 14.1 g, at 30 days = 173.8

± 11.1 g, and at 90 days = 162.6 ± 12.3 g). A significant difference was identified in the pelvic

limbs when comparing ST at 10 and 30 days (mean difference 35.4 g; P=0.01) and at 10 and 90

days (mean difference 46.7 g; P=0.001), but not at 30 and 90 days (mean difference 11.2 g;

P=0.93). There was an inverse correlation between locomotor score and pelvic limb ST over

time (r = -0.5; P = 0.03). These findings are suggestive of recovery of sensorimotor spinal cord

function, and potentially the development of hyperesthesia indicative of neuropathic pain. This is

the first study to evaluate ST over an extended period of time to evaluate for chronic neuropathic

pain in dogs and results suggest the need for further longitudinal studies related to neuropathic

pain in dogs after SCI.

4.2 Introduction

36

Damage to or dysfunction of nervous tissue can result in neuropathic pain, which is

generated by changes in formation and modulation of nociceptive signals (Treede 2008). This

pain persists long after resolution of the initial injury and is often associated with burning,

stabbing, and tingling sensations (IASP 2002). Neuropathic pain syndromes are characterized by

allodynia (pain associated with a non-painful stimulus) and hyperesthesia (heightened sensitivity

to a painful stimulus). This is distinguished from nociceptive pain which is caused by

damage to non-nervous tissue and inflammation resulting in stimulation of nociceptors.

Von Frey anesthesiometry (VFA) is a technique used for quantitative sensory testing

(QST) that tests ST for a mechanical stimulus (a plastic pressure probe applied to the skin).

Variations of this technique have been applied in people, dogs, and rodent models (Detloff 2012,

Tena 2012, Lascelles 2013). Patients with diminished sensation have a higher than normal ST,

whereas patients with hyperesthesia and allodynia have a lower than normal ST (Hoschouer et

al., 2010). Up to 90% of people report chronic neuropathic pain after spinal cord injury (SCI)

(Boldt 2014), which in the majority of individuals is severe and debilitating (Turner 2001).

Despite the fact that neuropathic pain syndromes are well documented in people with SCI, the

presence of this phenomenon has been explored only in a limited fashion in dogs with SCI. Our

group and others have recently reported short-term results of mechanical and thermal

quantitative sensory testing (QST) in this veterinary patient population (Gorney et al. 2016; Song

et al., 2016), suggesting that sensory thresholds and thermal latencies are altered after SCI;

however, long term studies documenting the existence and time course for developing

neuropathic pain in dogs with SCI are lacking. Studies focusing on this phenomenon in dogs are

important, both for effective management of our veterinary patients and to facilitate translational

studies of neuropathic pain. The goal of the present study was to provide a longitudinal, long-

37

term assessment of mechanical QST in dogs with intervertebral disc extrusion (IVDE)-associated

SCI. Based on previous work, we hypothesized that ST would decline over time in SCI-affected

dogs, consistent with development of a neuropathic pain phenotype as dogs progressed to the

chronic injury state.

4.3 Materials and methods

Animals

This study was approved by the IACUC (protocol # 2015A00000054) and the Clinical

Research Advisory Committee of The Ohio State University Veterinary Medical Center (OSU

VMC). Written consent was obtained from the owner for all dogs prior to enrollment. Dogs

weighed ≤20 kg, had SCI due to IVDE in the T3-L3 region of the vertebral column, and were

determined to have intact pain sensation below the level of injury based on subjective response

to noxious stimuli in the pelvic limbs and tail at the time of initial evaluation by their attending

clinician. All dogs underwent routine decompressive hemilaminectomy after CT or MRI

confirmed the site of IVDE, and post-operative management was at the discretion of the

attending clinician.

Modified dorsal von Frey technique

An electronic von Frey device (IITC Life Science) with 1000 gram probe and rigid 0.8 m

diameter tip were used for evaluation of mechanical ST via a technique that has been recently

described by our laboratory. (Moore 2013, Song 2016). Briefly, dogs were introduced to a small

quiet room where the testing was performed and allowed to investigate the room for

approximately five minutes prior to the start of testing. Dogs were then placed in lateral

recumbency which was maintained with minimal restraint. Limb test order was randomized. The

38

probe tip was applied to the dorsal surface of the metacarpus in the thoracic limbs and metatarsus

in the pelvic limbs, seating the probe between the fourth and fifth digits. The pressure was then

gradually increased manually until a behavioral response was elicited, and this pressure was

recorded as the ST. The evaluator was blinded to the pressure recording until the patient elicited

a behavioral response. This method was repeated for a total of five pressure recordings for each

limb, waiting one minute between measurements to prevent windup and hypersensitization

(KuKanich 2005, Detloff 2010).

Testing schedule

Dogs were evaluated at approximately 10, 30, and 90 days after hemilaminectomy. At

each time point a neurologic exam was performed by a single evaluator (AK), and a locomotor

score was assigned using the Olby Spinal Cord Injury Scale (OSCIS) (Olby et al., 2001).

Statistical analysis

The highest and lowest ST values were excluded for each limb, and the remaining three

values were averaged to determine a ST for each limb. Sensory values are reported as means ±

standard error of the mean (SEM) for each limb. To ease statistical comparison, right and left

thoracic limb ST were combined to give a single thoracic limb value, and right and left pelvic

limb ST were combined to give a single pelvic limb value. A repeated measures ANOVA and

trend analysis were used to compare ST values over time. The relationship between ST and

OSCIS score was determined using a Pearson correlation. P values were corrected for multiple

comparisons using the Bonferroni method. For all statistical analyses, P ≤0.05 was considered

significant.

4.4 Results

39

Twenty-five dogs with IVDE-associated SCI were prospectively enrolled; however, four

dogs did not complete the study due to suspected (three dogs) or confirmed (one dog) recurrent

IVDE during the 90 day study period. One dog had a confirmed L4-L5 IVDE two months after

its L1-L2 hemilaminectomy, one dog had recurrent right pelvic limb paresis and lameness

similar to its original clinical signs caused by a L3-L4 IVDE 1.5 months earlier, one dog

presented with cervical pain 1.5 months after its T12-T13 hemilaminectomy, and one dog was

presented paraplegic with lack of nociception two months after its L3-L4 hemilaminectomy with

the suspected recurrent IVDE further cranial at the thoracolumbar junction based on discomfort

during palpation and the level of a cutaneous trunci cut-off. These four dogs were not included in

statistical analysis of ST at any time point. A total of 21 dogs completed the study. Ages ranged

from three to 11 years (median six years), and dogs weighed between 5.0 and 10.4 kg (median

6.4 kg). There were 10 spayed females, 10 neutered males, and one sexually intact male. With

respect to breed: there were 19 Dachshunds, one Shih Tzu, and one mixed breed.

Single or multi-site decompressive hemilaminectomy was performed between T10-T11

and L3-L4 intervertebral disc spaces in all dogs, with lateral disc fenestrations performed at one

or more sites based on imaging findings and the discretion of the surgeon. Post-operative pain

management was provided in all cases and drug selection was based on surgeon’s preference but

included combinations of a fentanyl constant rate infusion for 12 to 24 h, a fentanyl patch placed

immediately post-operatively and removed three to four days later, tramadol, and gabapentin. All

dogs were placed on a single post-operative anti-inflammatory medication based on surgeon

preference which included prednisone, carprofen, firocoxib, or deracoxib. Medication doses and

frequency were recorded at each testing session. Oral anti-inflammatory and analgesic

medications were continued for a minimum of seven days post-operatively in all dogs, with 16

40

dogs still receiving at least one of these oral medications at their initial 10 day recheck. A single

dog was also receiving trazodone to help control anxiety at the 10 day recheck. Medication doses

at the 10 day testing time are summarized in Table 4. No dogs were receiving any medication at

the 30 and 90 day testing times.

Mean ST values for each individual limb as well as combined thoracic limbs and

combined pelvic limbs at each of the three time points are listed in Table 2, and summary

statistics for combined thoracic limb and combined pelvic limb ST values at each time point are

compared in Figure 3. Mean ± SEM sensory threshold for the thoracic limbs at 10 days was

156.5 ± 15.8 g, at 30 days was 128.3 ± 12.1 g, and at 90 days was 136.3 ± 14.6 g. A significant

difference was not detected in the thoracic limbs when comparing ST at different time points

(P=0.22 between 10 and 30 days; P=0.39 between 10 and 90 days; P=1.0 between 30 and 90

days).

Mean ± SEM sensory threshold for pelvic limbs at 10 days was 209.2 ± 14.1 g, at 30 days

was 173.8 ± 11.1 g, and at 90 days was 162.6 ± 12.3 g. A significant difference was identified in

the pelvic limbs when comparing ST at 10 and 30 days (mean difference 35.4 g; P=0.01) and at

10 and 90 days (mean difference 46.7 g; P=0.001), but not at 30 and 90 days (mean difference

11.2 g; P=0.93).

Mean ± SEM OSCIS score at 10 days was 8.0 ± 0.5, at 30 days was 9.6 ± 0.4, and at 90

days was 11.4 ± 0.3. There were significant differences in OSCIS score when comparing all time

points, with scores increasing over time (mean difference in score at 10 and 30 days 1.6,

P<0.0001; 10 and 90 days 3.4, P<0.0001; 30 and 90 days 1.8, P<0.0001). There was a

significant inverse correlation between pelvic limb ST and OSCIS at 10 days (P=0.03) and 30

41

days (P=0.003), but not at 90 days (P=0.49). OSCIS was not significantly correlated with

thoracic limb ST at any time point.

4.5 Discussion

While a previous study assessed short-term alterations in ST in dogs with IVDE-

associated SCI (Song et al., 2016), the current study is the first to assess long-term alterations in

ST in this population. Another recent study assessed ST using thermal and mechanical sensory

testing in normal dogs and dogs with SCI caused by IVDE over the course of about two months,

though only a few dogs were tested beyond 30 days (Gorney et al., 2016). While thoracic limb

ST values did not change over the course of our study, pelvic limb ST values were significantly

lower at 30 and 90 days after surgery compared to 10 day post-operative values. This pattern is

consistent with initial hypoalgesia likely due to a combination of SCI and post-operative pain

medications, followed by gradual recovery of injured spinal cord sensory pathways, cessation of

pain medication, and potentially the development of neuropathic pain as ST continued to

decrease (Hoschouer et al., 2010). While ST values were decreased at both 30 and 90 days after

surgery, this finding achieved statistical significance only for the 30 day values. There was no

difference in thoracic limb ST over time, indicating that the decline in pelvic limb ST over time

is likely related to true alterations in sensory function and not simply discontinuation of post-

operative analgesics.

We chose a 90 day time frame for observation of ST based on literature from both the

human clinical setting as well as in animal models (Nakae et al., 2011). Development of

neuropathic pain is typically delayed weeks to months after SCI, and in rare instances longer

than a year (Tasker 1992). In a human study, approximately 34%, 55%, and 62% of people with

42

SCI developed neuropathic pain at 1 month, 6 months, and 12 months respectively (Finnerup et

al., 2014). Allodynia was reported by 78% of people at 2 weeks post-SCI (Siddall et al., 1999).

Based on the prevalence of neuropathic pain reported by people and detected in people and rats

over the first few months after SCI via sensory testing, we expected to capture evidence of the

development of neuropathic pain during the 3 month testing period. However, given that the

number of patients that develop neuropathic pain may continue to increase beyond 3 months,

future studies may need to extend the testing time frame.

There are three major distributions of chronic neuropathic pain with SCI. Above-level

pain occurs in dermatomes cranial to the site of injury, at-level pain occurs in dermatomes at the

site of injury, and below-level neuropathic pain occurs in dermatomes caudal to the neurological

lesion (Hulsebosch 2009). These are described based on somatosensory mapping using

mechanical and thermal sensory testing (Vogel 2017). We tested the metacarpal and metatarsal

region of the limbs in dogs with caudal thoracic and cranial lumbar SCI, and therefore tested for

above-level and below-level (but not at-level) pain.

Below-level pain is the most common central pain syndrome after SCI in people and

rodents, and is the most difficult to treat (Turner 2001). This pain is typically associated with a

burning sensation, and can be spontaneous or evoked by different auditory stimuli or movements

(Widerstrom-Noga 2008). Various aspects of the limbs, hands/feet, and digits are commonly

reported sites of below-level pain (Soler 2010). Injuries that affect spinothalamic tracts are

associated with below-level pain, where changes in neuronal excitability of residual

spinothalamic afferents are suspected to play a role (Finnerup 2014). In our study, the gradual

43

decrease in ST in the pelvic limbs over the course of three months may be consistent with the

development of below-level neuropathic pain (Hoschouer et al., 2010).

Above-level pain develops reliably in rodents with a variety of SCI, typically manifested

as forelimb hyperalgesia after thoracic SCI (Mills 2001, Wang 2008, Carlton 2009, Densmore

2010). However, the limited QST data in humans and other primates suggests that above-level

pain is uncommon (Defrin 1999, Finnerup 2003, Masri 2012), which may implicate important

species differences in neuropathic mechanisms for above-level pain. We did not find any change

in thoracic limb ST over time, suggesting a lack of development of above-level neuropathic pain.

Most of the dogs in our study were receiving an oral anti-inflammatory and/or analgesic

medication at the 10 day testing time, and one dog was receiving trazodone. The increased ST at

day 10, indicative of hypoalgesia, was likely due to a combination of disruption of sensory

pathways in the spinal cord as well as the effect of analgesic medications. Several of these drugs

have been shown to alter sensory testing results in animals (Lascelles et al., 1998; Wegner et al.,

2008; KuKanich and Papich, 2011; Kögel et al., 2014). While the magnitude and duration of

impact with each of these medications on ST has not been evaluated in dogs, the anticipated

result is that they increase ST while they are being administered and their effect on ST is

removed shortly after the drug is stopped and cleared. Ideally, a placebo-controlled study would

be performed to help determine the magnitude of impact of various medications on ST in dogs

with SCI, but withholding post-operative analgesia would be unethical for obvious reasons.

Alternatively, a placebo-controlled trial could be performed in normal dogs using these various

common analgesic and anti-inflammatory medications to help determine their impact on ST

using VFA. Given the lack of change in thoracic limb ST when the majority of dogs were

44

receiving medications at 10 days compared to later time points may suggest that these

medications likely had minimal effect on the 10 day ST, and that the change in ST over time in

the pelvic limbs primarily reflects alteration of sensory CNS pathways.

As expected for dogs with incomplete SCI caused by IVDE, there was a significant

improvement in pelvic limb locomotor scores over the course of the 3 month testing period,

indicating partial recovery of locomotor function. Locomotor score had a strong and significant

inverse correlation with ST at 10 days and 30 days. This relationship was also present at day 90

but was not statistically significant. This inverse correlation is consistent with improving

sensorimotor function over time, and is in agreement with recent reports showing similar

correlations (Gorney et al. 2016; Song et al., 2016).

A limitation of this study was the higher than expected dropout rate due to presumed or

confirmed recurrence of IVDE, which limited our statistical power. Four dogs out of the original

25 did not complete the study due to confirmed or suspected recurrent IVDE (of which three

were likely at a different site). These dogs were not included in data reporting or statistical

analysis, as repetitive SCI would be expected to alter sensory processing to an ever larger degree,

further increasing the chances of an individual developing neuropathic pain, and potentially

increasing the magnitude of pain beyond what would occur in our more routinely managed

canine SCI population. To our knowledge, no studies exist examining the occurrence and

character of neuropathic pain caused by recurrent SCI.

4.6 Conclusions

This is the first study to evaluate changes in ST over an extended period of time in dogs

with IVDE-associated SCI. Pelvic limb ST decreased over the course of three months after

45

injury, though the decline from 30 to 90 days was not statistically significant. There was an

inverse relationship between locomotor score and pelvic limb ST over time. These findings are

suggestive of recovery of sensorimotor spinal cord function, and potentially the development of

hyperesthesia indicative of neuropathic pain. The main limitation of this study was the relatively

small number of dogs which limited statistical power. However, our results suggest the need for

further studies with larger numbers of dogs and potentially over a longer period of time to help

confirm the existence of chronic neuropathic pain in dogs. Ultimately the goal of future

neuropathic pain studies in dogs would be to better identify and treat chronic pain in our patients

and to validate the use of a spontaneous acute SCI dog model of neuropathic pain for

translational therapeutic research.

4.7 Acknowledgements

The study was funded by The Ohio State University Canine Grant [No. 2015-22]. In

addition to the funding source, the authors gratefully acknowledge Mrs. Amanda Disher, Mrs.

Heather Anderson, Dr. Nina Kieves, Dr. Daniella Vansteenkiste, and Dr. Ashley Hechler for

their assistance with the study.

4.8 References

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Defrin, R., Ohry, A., Blumen, N., Urca, G., 1999. Acute pain threshold in subjects with chronic pain following spinal cord injury. Pain 83, 275–282.

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Densmore, V.S., Kalous, A., Keast, J.R., Osborne, P.B., 2010. Above-level mechanical hyperalgesia in rats develops after incomplete spinal cord injury but not after cord transection, and is reversed by amitriptyline, morphine and gabapentin. Pain 151, 184–193.

Detloff, M.R., Fisher, L.C., Deibert, R.J., Basso, D.M., 2012. Acute and chronic tactile sensory testing after spinal cord injury in rats. Journal of Visualized Experiments 62, e3247. Finnerup, N.B., Johannesen, I.L., Bach, F.W., Jensen, T.S., 2003. Sensory function above lesion level in spinal cord injury patients with and without pain. Somatosensory & Motor Research 20, 71-76. Finnerup, N.B., Norrbrink, C., Trok, K., Piehl, F., Johannesen, I., Sørensen, J., Jensen, T.S., Werhageny, L., 2014. Phenotypes and predictors of pain following traumatic spinal cord injury: a prospective study. The Journal of Pain 15, 40-48 Gorney, A.M, Blau, S.R., Dohse, C.S., Griffith, E.H, Williams, K.D., Lim, J.H., Knazovicky, D., Lascelles, B.D.X., Olby, N.J., 2016. Mechanical and Thermal Sensory Testing in Normal Chondrodystrophoid Dogs and Dogs with Spinal Cord Injury caused by Thoracolumbar Intervertebral Disc Herniations. Journal of Veterinary Internal Medicine 30, 627-635.

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Kögel, B., Terlinden, R., Schneider, J., 2014. Characterisation of tramadol, morphine and tapentadol in an acute pain model in Beagle dogs. Veterinary Anaesthesia and Analgesia 41, 297–304.

KuKanich, B., Papich, M.G., 2011. Pharmacokinetics and antinociceptive effects of oral tramadol hydrochloride administration in Greyhounds. American Journal of Veterinary Research 72, 256–262.

Lascelles, B.D., Cripps, P.J., Jones, A., Waterman-Pearson, A.E., 1998. Efficacy and kinetics of carprofen, administered preoperatively or postoperatively, for the prevention of pain in dogs undergoing ovariohysterectomy. Veterinary Surgery 27, 568–582.

Lascelles, B.D., 2013. Getting a sense of sensations. The Veterinary Journal 197, 115–117. Levine, G.J., Levine, J.M., Budke, C.M., Kerwin, S.C., Au, J., Vinayak, A., Hettlich, B.F., Slater, M.R., 2009. Description and repeatability of a newly developed spinal cord injury scale for dog. Preventive Veterinary Medicine 89, 121–127. Masri, R., Keller, A., 2012. Chronic pain following spinal cord injury. Advances in Experimental Medicine and Biology 760, 74-88.

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Merskey, H., Bogduk, N. Classification of chronic pain: Descriptions of chronic pain syndromes and definitions of pain terms. 2nd ed. Seattle: IASP Press, 2002.

Mills, C.D., Hains, B.C., Johnson, K.M., Hulsebosch, C.E., 2001. Strain and model differences in behavioral outcomes after spinal cord injury in rat. Journal of Neurotrauma 18, 743-756. Nakae, A., Nakai, K., Yano, K., Hosokawa, K., Shibata, M., Mashimo, T., 2011. The Animal Model of Spinal Cord Injury as an Experimental Pain Model. Journal of Biomedicine and Biotechnology 2011, 1-11. Olby, N.J., De Risio, L., Muñana, K., Wosar, M.A., Skeen, T.M., Sharp, N.J., Keene, B.W., 2001. Development of a functional scoring system in dogs with acute spinal cord injuries. American Journal of Veterinary Research 62, 1624–1628. Siddall, P., Taylor, D., McClelland, J., Rutkowski, S., Cousins, M., 1999. Pain report and the relationship of pain to physical factors in the first 6 months following spinal cord injury. Pain 81, 187-197. Soler, M.D., Kumru, H., Vidal, J., Pelayo, R., Tormos, J.M., Fregni, F., Navarro, X., Pascual-Leone, A., 2010. Referred sensations and neuropathic pain following spinal cord injury. Pain 150, 192-198. Song, R.B., Basso, D.M., da Costa, R.C., Fisher, L.C., Mo, X, Moore, S.A., 2016. von Frey anesthesiometry to assess sensory impairment after acute spinal cord injury caused by thoracolumbar intervertebral disc extrusion in dogs. The Veterinary Journal 209, 144-149. Tasker, R.R., DeCarvalho, G., Dolan, E.J., 1992. Intractable pain of spinal cord origin: clinical features and implications for surgery. Journal of Neurosurgery 77, 373-378. Tena, B., Escobar, B., Jose Arguis, M., Cantero, C., Rios, J., Gomar, C., 2012. Reproducibility of electronic von Frey and von Frey monofilaments testing. Clinical Journal of Pain 28, 318-323. Treede, R.D., Jensen, T.S., Campbell, J.N., Cruccu, G., Dostrovosky, J.O., Griffin, J.W., Hansson, H., Hughes, R., Nurmikko, T., Serra, J., 2008. Neuropathic pain: Redefinition and a grading system for clinical research purposes. Neurology 70, 1630-1635. Turner, J.A., Cardenas, D.D., Warms, C.A., McClellan, C.B., 2001. Chronic pain associated with spinal cord injuries: A community survey. Archives of Physical Medicine and Rehabilitation 82, 501-508. Vogel, C., Rukwied, R., Stockinger, L., Schley, M., Schmelz, M., Schleinzer, W., Konrad, C., 2017. Functional characterization of at-level hypersensitivity in patients with spinal cord injury. Journal of Pain 18, 66-78.

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Wang, G., Thompson, S.M., 2008. Maladaptive homeostatic plasticity in a rodent model of central pain syndrome: thalamic hyperexcitability after spinothalamic tract lesions. The Journal of Neuroscience 28, 11959-11969. Wegner, K., Horais, K.A., Tozier, N.A., Rathbun, M.L., Shtaerman, Y., Yaksh, T.L., 2008. Development of a canine nociceptive thermal escape model. Journal of Neuroscience Methods 168, 88–97.

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Tables

Table 4 Summary of oral medications at day 10. No dogs were receiving medication at 30 or 90 days.

Medication Mean Dose (Range) (mg/kg) Frequency (h) No. Dogs

Prednisone 0.4 (0.3-0.7) 48 6

Carprofen 2.2 (1.6-2.5) 12 5

Firocoxib 4.3 24 1

Deracoxib 1.5 24 1

Gabapentin 10.9 (9.6-17.9) 8 12

Tramadol 4.1 (2.5-6.5) 8 7

Trazodone 2.9 12 1

49

Table 5 Mean sensory threshold for each limb in 21 dogs with acute spinal cord injury caused byintervertebral disc extrusion as measured at 10, 30, and 90 days after decompressive hemilaminectomy.

Mean Sensory Threshold (g)

Day 10

Day 30

Day 90

Dog LTL RTL LPL RPL LTL RTL LPL RPL LTL RTL LPL RPL

1 105.7 210.8 177.5 227.5 190.3 136.5 185.6 255.6 166.6 155.1 200.7 173.7

2 110.5 141.1 165.4 212.7 79.9 77.5 148.1 127.2 103.3 132.0 168.1 107.7

3 275.0 149.2 293.6 236.8 98.0 236.3 208.2 140.8 130.0 188.4 164.7 228.6

4 223.9 243.8 215.2 171.2 146.7 259.1 205.3 157.2 306.4 358.0 275.6 213.7

5 266.5 227.8 304.7 225.7 77.1 138.2 125.5 149.6 176.4 191.1 261.8 200.8

6 110.8 141.0 198.2 176.7 180.6 165.7 195.9 173.4 134.7 156.2 193.2 137.9

7 134.5 201.7 208.7 226.9 113.6 122.3 191.9 175.4 107.2 96.5 177.8 153.9

8 347.8 332.4 263.6 245.8 151.3 217.1 204.4 219.0 275.8 216.9 196.0 188.5

9 107.5 124.8 241.3 231.4 110.9 204.7 261.3 123.0 134.3 141.5 196.8 176.4

10 239.7 210.8 387.8 285.9 215.1 193.8 227.1 218.3 117.2 123.0 212.6 200.8

11 155.5 133.3 162.4 118.0 155.5 81.7 110.1 57.4 106.4 48.4 118.3 64.6

12 238.7 124.1 126.9 98.8 89.2 89.2 261.3 124.3 89.0 67.9 146.9 108.7

13 101.8 32.0 212.6 245.3 73.0 118.4 207.5 154.1 95.1 43.7 73.0 120.6

14 106.0 121.7 288.0 223.3 178.0 189.0 294.3 217.4 85.0 107.4 133.3 122.9

15 76.9 88.4 81.2 141.6 80.0 52.6 70.1 106.0 63.6 53.6 77.9 101.9

16 160.1 201.7 161.8 178.7 63.3 31.5 180.6 118.9 144.4 103.6 86.9 90.7

17 62.8 71.2 301.6 300.3 88.0 46.6 249.3 211.0 69.7 51.9 267.1 187.3

50

18 120.3 296.4 247.5 133.6 182.0 233.8 170.8 153.1 96.7 222.9 142.0 89.0

19 56.5 70.4 56.8 84.5 66.1 59.8 85.9 53.1 153.3 89.5 59.9 100.0

20 67.3 83.8 157.9 289.8 64.9 63.5 209.0 270.6 74.8 72.3 212.4 315.8

21 109.8 109.2 278.0 202.5 207.1 247.5 143.4 158.3 144.8 282.2 170.7 208.6

Total Mean ± SEM 156.5 ± 15.8 209.2 ± 14.1 128.3 ± 12.1 173.8 ± 11.1 136.3 ± 14.6 162.6 ± 12.3

LTL, left thoracic limb; LPL, left pelvic limb; RTL, right thoracic limb; RPL, right pelvic limb;

SEM, standard error of the mean.

51

Figures

Figure 3 Mean ± SEM for combined thoracic limb and combined pelvic limb ST values at each

time point in 21 dogs with acute spinal cord injury secondary to intervertebral disc extrusion.

52

Chapter 5

Conclusions and Future Directions

The primary goals of this study were to evaluate the interobserver reliability of VFA in

normal dogs and to use VFA to assess for evidence of chronic neuropathic pain in dogs after

acute SCI. VFA is ideal for sensory testing in veterinary patients as it is quick, well-tolerated,

and automatically records ST while allowing the evaluator to be blinded to the pressure applied.

In dogs with SCI, VFA was used to objectively measure ST as a means to follow sensory

function in all limbs throughout their recovery. The pattern of ST values over time could then be

used to determine if dogs with SCI develop neuropathic pain.

This was the first study to evaluate interobserver reliability of VFA in dogs. The results

of this study showed that while the average ST for each observer was similar across all dogs,

there was moderate agreement for any one individual dog. This is likely not enough agreement to

justify the use of VFA is multi-center pain studies in dogs, and highlights the shortcomings of

the brief training that was provided for the observers in this study. Interobserver reliability of

QST in people also was inadequate during its early stages in human medicine, but the

implementation of standardized testing protocols and more intensive training programs resulted

in strong interobserver reliability, allowing sensory testing data to be compiled and analyzed

from people with neuropathic pain from across the globe. For example, the German Research

Network on Neuropathic Pain, a leading organization in sensory testing and neuropathic pain,

offers in-depth QST training which allows researchers and clinicians to become comfortable and

familiar with equipment, technique, quality control, and analysis and interpretation of results

using established standardized protocols and normal QST values (Rolke et al., 2006).

53

Laboratories can become QST certified through this training and the use of validated QST

protocols, resulting in enhanced reliability of QST data (Geber et al., 2011). This process took

many years, but the limitations of multi-center trials were eventually overcome. Similar

standardized QST protocols and training programs will need to be further developed and

implemented in veterinary medicine to improve interobserver reliability and allow for large-scale

multi-center pain studies.

The next part of the study was the first to evaluate ST over an extended period of time to

evaluate for chronic neuropathic pain in dogs. Pelvic limb ST decreased over the course of three

months, though the decline from 30 to 90 days was not statistically significant. There was an

inverse relationship between locomotor score and pelvic limb ST over time. These findings are

suggestive of recovery of sensorimotor spinal cord function, and potentially the development of

allodynia indicative of neuropathic pain. The main limitation in this study was the relatively

small number of dogs which limited statistical power. However, the promising results in this

study indicate the need for further studies with larger numbers of dogs and potentially over a

longer period of time to help confirm the existence of chronic neuropathic pain in dogs.

Ultimately the goal of future neuropathic pain studies in dogs would be to better identify and

treat chronic pain in our patients and to validate the use of a spontaneous acute SCI dog model of

neuropathic pain for translational therapeutic research.

In addition to mechanical sensory testing such as VFA, the other primary form of QST

employed in the research and clinical setting is thermal sensory testing, which was not used in

this study. A recent study evaluated thermal sensory testing in normal dogs and dogs with SCI

caused by IVDE (Gorney et al., 2017). Thermal thresholds were tested using a handheld probe

54

set at 49°C (for heat) and 5°C (for cold) applied to the dorsal metatarsal region until a conscious

response was elicited. The thermal latency (time from applying the probe to the time of patient

response) was recorded. This study showed that thermal testing is feasible in dogs, that there was

moderate repeatability, and that there was a significant difference in latencies between normal

dogs and dogs with SCI. Thermal sensory testing is another testing modality that can be used

along with mechanical sensory testing to help evaluate for recovery and neuropathic pain in

veterinary patients, and similarly will require further research to help establish appropriate

veterinary protocols and training.

Future studies in dogs can evaluate the efficacy of various medications for the treatment

of neuropathic pain. Given that gabapentin has been shown to be one of the most effective drugs

for the treatment of neuropathic pain in people, is commonly prescribed in veterinary medicine,

and is very safe in dogs, this would be an ideal candidate for such a trial. Initially a placebo-

controlled cross-over study design could be implemented in normal dogs to evaluate how

gabapentin modulates ST. Normal dogs would be divided to receive either placebo or

gabapentin, VFA would then be performed, and after a wash-out period the treatment would be

switched and VFA repeated. If gabapentin was found to alter ST, this would lay the foundation

for further studies using gabapentin in dogs with SCI or other neuropathic conditions where there

is evidence of neuropathic pain. Once the QST data pool becomes robust in veterinary medicine,

this can also be translated to human clinical trials.

55

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