Analgesic efficacy of gabapentin as add-on medication in the postoperative period after hemilaminectomy in dogs

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Analgesic efficacy of gabapentin as add-on medication in the postoperative period after hemilaminectomy in dogs

Thesis

Submitted in partial fulfilment of the requirements for the degree -Doctor of Veterinary Medicine-

Doctor medicinae veterinariae ( Dr. med. vet. )

by

SONJA ANAHITA AGHIGHI

Hannover

Hannover 2010

Univ.-Prof. Dr. Sabine Kästner Univ.-Prof. Dr. Andrea Tipold

Small Animal Clinic

1. Referee: Univ.-Prof. Dr. Sabine Kästner Small Animal Clinic

2. Referee: Univ.-Prof. Dr. Wolfgang Löscher

Institute for Pharmacology, Toxicology and Pharmacy

Day of the oral examination: 07/09/2010

Contents

I. Abbreviations

II. Introduction

1. Manuscript I

Abstract 18

1.1 Introduction 20

1.2 Materials and methods 21

1.2.1 Animals ... 21

1.2.2 Study design and drugs... 22

1.2.3 Evaluation of patients ... 23

1.2.4 Rescue analgesia protocol ... 24

1.2.5 Concomitant medication... 24

1.2.6 Blood sampling, analysis of serum samples ... 24

1.3 Statistics 25 1.4 Results 26 1.5 Discussion 33 1.6 References 41

2. Manuscript II

2.2.2 Influence of general anaesthesia on pharmacokinetics of orally administered gabapentin in beagle dogs ... 56

2.2.2.1 Animals... 56

2.2.2.2 Study design ... 56

2.2.2.3 Analysis of gabapentin serum concentrations ... 58

2.2.2.4 Pharmacokinetic calculations... 58

2.2 Statistics 59 2.4 Results 59 2.4.1 Resistance of gabapentin capsules to gastric pH ... 59

2.2.2 Influence of general anaesthesia on pharmacokinetics of orally administered gabapentin in beagle dogs ... 61

2.5 Discussion 66

2.6 Literature 70

3. General discussion

3.2 Study results 82

4. Zusammenfassung

5. Summary

6. Literature

7. Appendix

8. Acknowledgements

AGHIGHI, S.A., A. TIPOLD, S.B.R. KÄSTNER (2009): Effects of gabapentin as add-on medication on pain after intervertebral disc surgery in dogs – preliminary results

10^thWorld Congress of Veterinary Anaesthesia

31^stAugust - 4^thSeptember 2009, Glasgow, United Kingdom Congress Proceedings, p. 133.

AGHIGHI, S.A., A. TIPOLD, S.B.R. KÄSTNER (2010): Perioperative analgetische Effektivität von Gabapentin als add-on Medikation bei Hunden mit thorakolumbalem Discusprolaps

18. Jahrestagung der FG Innere Medizin und klinische Labordiagnostik der DVG, InnLab 2010 6^th- 7^thFebruary 2010, Hannover, Germany

Congress Proceedings, p. A21.

AGHIGHI, S.A., A. TIPOLD, S.B.R. KÄSTNER (2010): Perioperative analgesic efficacy of gabapentin as add-on medication in the postoperative period after hemilaminectomy in dogs 23^rdAnnual Symposium of the European College of Veterinary Neurology

16^th- 18^thSeptember 2010, Cambridge, United Kingdom Congress Proceedings, p. 96, SP6.

AGHIGHI, S.A., A. TIPOLD, S.B.R. KÄSTNER (2010): Perioperative analgetische Effektivität von Gabapentin als add-on Medikation bei Hunden mit thorakolumbalem Discusprolaps

56. Jahreskongress der Deutschen Gesellschaft für Kleintiermedizin 21^st- 24^thOctober 2010, Düsseldorf, Germany

Congress Proceedings, Saturday, p. 335.

In this thesis following abbreviations were used:

A. Arteria

AG Arbeitsgemeinschaft

a. m. ante meridiem

ANOVA Analysis of variances

ASA American Association of Anaesthetists

AUC Area under the serum concentration time curve

BID bis in die

°C Celsius

Cmax maximum serum concentration

Cl total body clearance

CMPS-SF Short Form of Glasgow Composite Measure Pain Scale

Da Dalton

DAB Deutsches Arzneibuch

DAP Diastolic arterial pressure

Dipl. Diplomate

Dr. Doctor

DVG Deutsche Veterinärmedizinische Gesellschaft

ECVN European College of Veterinary Neurology

ECVAA European College of Veterinary Anaesthesia and Analgesia

EDTA ethylenediaminetetraacetate

e.g. exempli gratia

eG europäische Gemeinschaft

et al. et alii, et aliae., et alia

FG Fachgruppe

GABA γ-Aminobutyric acid

GBP gabapentin

GmbH Gesellschaft mit beschränkter Haftung

HDO High Definition Oscillometry

HPLC High Pressure Liquid Chromatography

h hour (s)

IM intramuscular

I. U. international unit

IV intravenous

IVDD intervertebral disc disease

k01 absorption half life

k10 elimination half life

kg kilogram

LAVES Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit

M1 muscarinic receptor subtype

MAP mean arterial pressure

mg milligram

Mg²⁺ Magnesium ion

min minute

ml milliliter

mM millimol

MRI Magnetic Resonance Imaging

n number of dogs included

NaCl saline solution

ng nanogram

nm nanometer

mm Hg^-1 millimeter of mercury

NSAID non steroidal anti-inflammatory drug

NO nitric oxide

OPA o-phthaldialdehyde

P placebo

p < 0.05 level of significance 5 %

pH negative decade logarithm of the molar concentration of dissolved hydrogen ions

p. m. post meridiem

Prof. Professor

R coefficient of correlation

SAID steroidal anti-inflammatory drug

SAP systolic arterial pressure

SD standard deviation

sign two tailed statistical significance

SPSS statistic software: Statistical Package for the Social Sciences

SQ subcutaneous

TID ter in die

Tmax maximum serum comcentration

µg microgram

µl microliter

VAS Visual Analogue Scale

vs. versus

QOL quality of life

II. Introduction

Intervertebral disc disease (IVDD) is the most common neurological disease in dogs with an incidence of 20 % of dogs with neurologic disease (FLÜHMANN et al. 2006). In many cases surgical treatment is needed. This results in the occurrence of a mixed type of pain. Neuropathic pain results from damage of nerve fibres of the nociceptive system through the degenerative compression of the spinal cord by the discusprolaps as well as through excitation of afferent nerve fibres through manipulation during surgery. In addition, nociceptive pain evolves by manipulation as well as damage of skin and muscles during surgery.

Therapy of neuropathic pain is challenging in human- as well as veterinary medicine. In the past opioids or non steroidal anti-inflammatory drugs (NSAIDs) were used after surgery known to be followed by neuropathic pain. Opioids offer the advantage of strong potency against nociceptive pain but do not include sufficient efficacy against neuropathic pain (ARNÉR &

MEYERSON 1988) or afford high dosages. This is accompanied by side effects like obstipation or urine retention (JENKINS 1987; SAGER 1993) and can provoke opioid tolerance (LAW et al. 1982; CHRISTIE et al. 1986; STEVENS & YAKSH 1989a, 1989b; ARDEN et al. 1995;

KEITH et al. 1996; CHAKRABARTI et al. 1997) which implies dose escalation. Also NSAIDs often do not show sufficient effect (MAX et al. 1988). In addition, their utilisation is limited by potential gastrointestinal and renal side effects, for example bleeding or ulceration of the gastrointestinal tract (REIMER et al. 1999; FIORUCCI et al. 2001) or reduction of renal perfusion (CLIVE & STOFF 1984).

Gabapentin (1-(aminomethyl)cyclohexaneacetic acid, GBP, C9H17NO2), which is structurally related to Gammaaminobutyric acid (GABA), was originally developed as an antiepileptic drug.

For this indication it is used in human medicine as add-on for partial seizures with partially secondary generalization and primary generalized seizures (CRAWFORD et al. 1987;

ANONYMOUS 1990; CHADWICK et al. 1996; KHURANA et al. 1996; BERGEY et al. 1997;

THOMAS 1997) as well as in veterinary medicine. In veterinary medicine GBP is mainly used to treat refractory epilepsy in dogs as add-on medication (GOVENDIR et al. 2005; PLATT et al. 2006) because of its short half life of 3.1 resp. 3.4 hours (RHEE et al. 2008; KUKANICH &

COHEN 2009). In addition, GBP showed efficacy in the treatment of neuropathic pain and inflammation associated pain in mice and rat models (FIELD et al. 1997; HUNTER et al. 1997;

PAN et al. 1999; PATEL et al. 2000) as well as clinical human studies. In human medicine

GBP showed improvement in diabetic neuropathy, post-herpetic neuralgia, posttraumatic neuralgia and other neuropathic pain syndromes (BACKONJA et al. 1998; TAKASAKI et al.

2001; BONE et al. 2002; TO et al. 2002; LINDBERGER et al. 2003; GORDH et al. 2008). An anxiolytic effect is also described (CHOUINARD et al. 1998; POLLACK et al. 1998;

MENIGAUX et al. 2005). Despite its use in veterinary medicine and recommendations for use in various text books only a few case reports exist which document effectiveness of GBP as add-on medication in different pain states (DAVIS et al. 2007; CASHMORE et al. 2009;

WOLFE & POMA 2010). Effects of GBP in nociceptive pain states consist of a decrease in opioid request if given additionally in human studies with opioids on demand, further in reduction of pain level in the postoperative period (DIRKS et al. 2002; PANDEY et al. 2004;

PANDEY et al. 2005; AL-MUJADI et al. 2006) and less opioid induced nausea if combined with morphine (TURAN et al. 2004a). Additionally GBP reduces other side effects of opioids like preventing opioid tolerance (GILRON et al. 2003).

The mechanism of GBP’s action is not yet completely understood, but a few hypotheses are proven. Gabapentin binds to different supraspinal and spinal targets. Best known is GBP’s effect on calcium channels: GBP binds presynaptically to the2δsubunit of high-threshold voltage dependent calcium channels in the dorsal root ganglion of the spinal cord (GEE et al.

1996; SUTTON et al. 2002). This results in a reduced release of excitatory neurotransmitters like glutamate and substance P at synapses (KUGLER et al. 2002; BEN-MENACHEM &

KUGLER 2005) and therefore reduces excitatory action potentials at the postsynaptic cell membrane of projection neurons. The subtypes of affected calcium channels are N-Type, L-type and P/Q-type channels whereby N-type channels are most susceptible (STEFANI et al. 1998;

FINK et al. 2000; SUTTON et al. 2002). Gabapentin’s affinity to2δ1subunit is superior to

2δ2and no binding occurs with2δ3(MARAIS et al. 2001). Brown and Gee (1998) identified a region inδ1which contains a zinc-like finger that is important for binding of GBP (TAYLOR et al. 1998). Gabapentin decreases the influx of calcium into presynaptic cell membranes by indirectly modulating the pore forming1subunit. Furthermore GBP possesses postsynaptic action: in inflammatory tissue the intracellular protein kinase c is elevated (MARTIN et al.

1999). Under this condition GBP becomes capable to bind to N-Methyl-D-Aspartat (NMDA) receptors of dorsal horn neurones and increases NMDA currents (GU & HUANG 2001). This action is confined to GABAergic inhibitory neurons but the derivation of this selectivity is still unknown (GU & HUANG 2002). Additionally, GBP also potentiates glycine affinity (co- activator of NMDA receptors) for NMDA receptors if glycine is not saturated (GU & HUANG 2001). This leads to changes of electrical membrane potential and releases Mg²⁺ions which

normally block NMDA receptors (MAYER et al. 1984; YAKSH 1999). Although designed to be GABA mimetic there was no evidence for GBP to influence GABA receptors. Ng and co- workers (2001) demonstrated an interaction of GBP with the postsynaptic GABAB(1a,2)receptor heterodimer complex where it activates inwardly rectifying potassium conductance which lead to hyperpolarisation and hence inhibition of neuronal excitability (NG et al. 2001). In contrast, other groups were unable to demonstrate any agonist-like activity of GBP on GABABreceptors (LANNEAU et al. 2001; JENSEN et al. 2002).

Gabapentin also exhibits supraspinal action (STEFANI et al. 1998; HAYASHIDA et al. 2007b).

It activates supraspinally the descending noradrenergic system (TANABE et al. 2005;

TAKASU et al. 2006). This leads to a release of noradrenalin which interacts with spinal2

receptors. These further recruit spinal muscarinic (M1) receptors and NO cascade and hence reduce mechanical hypersensitivity (TAKASU et al. 2006). This release of noradrenalin is mediated by inhibition of P/Q type calcium channels when stimulated by potassium and electrical pulses (DOOLEY et al. 2000). It remains unclear whether there are still other sites of its action.

It is believed that the analgesic action of GBP in dogs is comparable with that in humans (GAYNOR & MUIR 2002; DAVIS et al. 2007; CASHMORE et al. 2009; WOLFE & POMA 2010) hence the therapy in coherence with neuropathic pain in dogs could be improved by establishing the usage of GBP. Therefore clinical patients which underwent hemilaminectomy because of IVDD were medicated with GBP in comparison to a positive control group and evaluated over 5 days following surgery. Since GBP is only available as oral formulation in Germany, another aspect of this work was to estimate the effect of pre-anaesthetic GBP applied orally. It is recommended to use pre-emptive analgesia in order to possibly prevent wind up and central sensitization and hence reduce pain after surgery (DICKENSON & SULLIVAN 1987;

MCQUAY 1992; WOOLF & CHONG 1993). Although dogs in the main study experienced pain before surgery, pre-anaesthetic GBP treatment was intended (WOOLF & CHONG 1993;

KUNDRA et al. 1997; TIIPPANA et al. 2007). For further investigation of possible effects of pre-anaesthetic oral GBP, an experimental study was carried out. Beagle dogs received GBP orally at the same dose as the client owned dogs (10 mg kg^-1) and were immediately anaesthetized by the same technique and same duration to test the influence of pre anaesthetic medication on GBP’s absorption.

1. Manuscript I

Effects of gabapentin as add-on medication on pain after intervertebral disc surgery in dogs

S.A. Aghighi

, A. Tipold

, M. Piechotta

, P. Lewczuk

, S.B.R. Kästner

*Division Anaesthesia, Small Animal Clinic, University of Veterinary Medicine Hannover, Foundation, Germany

☨Division Neurology, Small Animal Clinic, University of Veterinary Medicine Hannover, Foundation, Germany

‡ Clinic for Bovine Medicine, University of Veterinary Medicine Hannover, Foundation, Germany

^Department of Psychiatry and Psychotherapy, Universtätsklinikum Erlangen, Germany

Correspondence: Sonja Aghighi, Small Animal Clinic, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, 30559 Hannover, Germany Email: sonja.aghighi@hotmail.de

Abstract

Objective To assess the effect of gabapentin (GBP) on the treatment of pain after thoracolumbar intervertebral disc surgery (hemilaminectomy) in client owned dogs as add-on therapy to µ-agonist based background analgesia.

Study designProspective randomised clinical controlled trial with blinded observer.

AnimalsSixty-three client owned dogs (aged 6.6 ± 2.4 years and weighing 9.6 ± 3.4 kg) with thoracolumbar disc disease which underwent hemilaminectomy with or without fenestration (ASA II-III) were included. Dogs that were pre-treated with antiepileptics or pain medication except non steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids were excluded. In addition dogs which underwent a re-operation during the study were excluded retrospectively.

MethodsDogs were randomly assigned to two different treatment protocols. The GBP group received gabapentin 10 mg kg^-1orally every 12 hours, the placebo (P) group received empty gelatinous capsules at the same dose interval. Both treatments started before induction of anaesthesia. Baseline analgesia was initiated with intravenous (IV) levomethadone 0.6 mg kg^-1 for premedication of anaesthesia, followed by application of a fentanyl-patch (Durogesic) directly after extubation and levomethadone 0.2 mg kg^-1subcutaneously (SQ) for 24 h (until effect of the fentanyl patch). Examinations were performed at day 0.5, 1, 2, 3, 4 and 5 after extubation. For the evaluation of pain the shortform of the Glasgow Composite Measure Pain Scale (CMPS-SF) without the category gait, Visual Analogue Scale (VAS) and blood pressure measurement were employed. Furthermore a neurological follow up examination was performed. Side effects were recorded. Gabapentin serum concentrations and cortisol concentrations were controlled at 24 and 72 h after first oral application and cortisol additionally at 48 h. Power analysis (power of 90 %) indicated a minimum of 67 dogs would be needed to detect a statistically significant difference of 10 % in the CMPS-SF (CMPS-SF difference of 2) between groups.

StatisticsStatistical analysis was performed with SPSS 16.0 and Graph Pad Prism 5.02 and included descriptive statistics, chi square test, test of normality with Kolmogorov-Smirnov test and additionally for parametric data two-way analysis of variances (ANOVA) for repeated

measurements with Dunnets and Bonferoni corrected t-tests. For non parametric data Wilcoxon test and Friedmann test were used. Correlations were tested using Spearman’s and Pearson’s correlation coefficient depending on distribution of data. For all comparisons p < 0.05 was considered significant. For analysis of agreement between VAS of trained observer and unexperienced observer (students) Bland Altman analysis was performed.

ResultsIn the GBP group median CMPS-SF was lower on day 0.5, 1, 4 and 5 than in group P.

However, the CMPS-SF and VAS showed no significant differences between groups. Both pain scales decreased significantly over time. Cortisol levels were not significantly different between groups but medians were lower on all three time points in the GBP group. Blood pressure, respiratory rate and heart rate neither showed differences over time nor between groups.

Minimal serum concentrations of GBP were above detection limit of 1 µg ml^-1in the magnitude of dogs.

Conclusions and clinical relevanceAdd-on GBP 10 mg kg^-1orally twice a day did not result in detectable less pain behaviour in dogs compared to opioid background analgesia alone.

However, a tendency towards lower pain levels was apparent and further studies are needed to determine if this is due to the potent background analgesia, too low dose of GBP employed or whether the scales used are not sensitive enough to discriminate minor differences.

Key words: neuropathic pain, intervertebral disc disease, hemilaminectomy, gabapentin, antiepileptic drug

1.1 Introduction

Intervertebral disc disease (IVDD) is the most common neurological disease in dogs, occuring with an incidence of 20 % (FLÜHMANN et al. 2006) of dogs with neurologic disease. In many cases surgical treatment is needed. The resulting pain is considered a mixed type of nociceptive and neuropathic pain. Neuropathic pain originates from the degenerative compression of the spinal cord as well as from manipulation during surgery. Nociceptive pain develops during surgery as a result of surgical manipulation as well as skin and muscle damage. There are a lot of agents available sufficient for treatment of nociceptive pain. In contrast, the treatment of neuropathic pain is more difficult. It is less susceptible to opioid (ARNÉR & MEYERSON 1988) or non steroidal pain medication (MAX et al. 1988) than nociceptive pain. However, there is the possibility to target various steps on this pain pathway with unusual medications e.g.

antiepileptics (RULL et al. 1969; SWERDLOW & CUNDILL 1981; FIELD et al. 1997;

ROGAWSKI & LÖSCHER 2004) or antidepressants (MITAS et al. 1983; WATSON 1984;

MAX 1994; MCQUAY et al. 1996).

Gabapentin (GBP), which is structurally related to Gammaaminobutyric acid (GABA), was originally developed as an antiepileptic drug. For this indication it is used in human medicine as well as veterinary medicine. In veterinary medicine GBP is used to treat refractory epilepsy in dogs (GOVENDIR et al. 2005; PLATT et al. 2006). Gabapentin is used successfully in human medicine for the treatment of different neuropathic pain syndromes (BACKONJA et al. 1998;

BONE et al. 2002; TO et al. 2002; BERGER et al. 2003; GORDH et al. 2008) as well as perioperatively (FASSOULAKI et al. 2002; TURAN et al. 2004b; HO et al. 2006; GROVER et al. 2009; SEN et al. 2009; MENDA et al. 2010; KAZAK et al. 2010). Despite its use and recommendations for use in veterinary medicine just a few case reports exist which document positive effects of GBP as add-on medication in different pain states (DAVIS et al. 2007;

CASHMORE et al. 2009; WOLFE & POMA 2010).

The mechanism of GBP’s action is not yet completely understood. Gabapentin binds presynaptically to the2δsubunit of high-threshold voltage dependent calcium channels in the dorsal root ganglion of the spinal cord (GEE et al. 1996; SUTTON et al. 2002). This results in a reduced release of excitatory neurotransmitters at synapses (KUGLER et al. 2002; BEN- MENACHEM & KUGLER 2005) and therefore reduces excitatory action potentials at the postsynaptic cell membrane of projection neurons. Furthermore GBP possesses postsynaptic

action: in inflammatory tissue the intracellular protein kinase c is elevated (MARTIN et al.

1999). Under this condition GBP becomes capable to bind to N-Methyl-D-Aspartat (NMDA) receptors of dorsal horn neurones and increases NMDA currents (GU & HUANG 2001). This action is limited to GABAergic inhibitory neurons but the derivation of this selectivity is still unknown (GU & HUANG 2002). Additionally GBP also potentiates glycine affinity (co- activator of NMDA receptors) for NMDA receptors if glycine is not saturated (GU & HUANG 2001). It exhibits also supraspinal actions (STEFANI et al. 1998; HAYASHIDA et al. 2007a).

Gabapentin activates the descending noradrenergic system supraspinally (TANABE et al.

2005). This leads to a release of noradrenaline which interacts with spinal2receptors. These further recruit spinal muscarinic (M1) receptors and NO cascades and hence reduce mechanical hypersensitivity (TAKASU et al. 2006).

In humans GBP is used for the treatment of neuropathic pain (BACKONJA et al. 1998; BONE et al. 2002; TO et al. 2002; BERGER et al. 2003; GORDH et al. 2008). Additionally it is able to reduce the opioid request in nociceptive pain states when given as add-on medication with opioids on demand. Furthermore a reduction of pain level in the postoperative period was observed (DIRKS et al. 2002; PANDEY et al. 2004; PANDEY et al. 2005; AL-MUJADI et al.

2006). Additionally GBP reduces side effects of opioids like preventing opioid tolerance (GILRON et al. 2003).

The aim of this study was to estimate the effectiveness of GBP as add-on therapy to µ- opioid based background analgesia in dogs after hemilaminectomy.

1.2 Materials and methods

1.2.1 Animals

The study was approved by the Ethics Commitee of Lower Saxony LAVES (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) trial number 33.9-42502-05- 10A037. Client owned dogs of any breed presented to the Small Animal Clinic, University of Veterinary Medicine Hannover Foundation, with thoracolumbar intervertebral disc disease which had to undergo hemilaminectomy with or without fenestration between May 2008 and December 2009 were included into this study. Dogs that were pre-treated with antiepileptics or

pain medications except NSAIDs or corticosteroids were excluded. In addition, dogs which underwent a re-operation during the first five days after the initial surgery were excluded retrospectively. Seven dogs were excluded because of re-operation. Sixty-three dogs met the inclusion criteria. They had a mean age of 6.6 ± 2.4 years and weighed 9.6 ± 3.4 kg.

1.2.2 Study design and drugs

The study was carried out as a randomized, controlled clinical trial with a blinded observer.

Dogs were randomly assigned to two different treatment protocols by a computer generated random number table: Gabapentin (group GBP) (Gabapentin 100 mg, ratiopharm GmbH, 89070 Ulm, Germany) (Gabapentin 50 mg, Löwenapotheke, 30159 Hannover, Germany) or placebo (group P) consisting of empty gelatinous capsules (Hartgelatine - leer -Kapseln, Bestellnr.

35759, WEPA Apothekenbedarf, 56204 Hillscheid, Germany) and examined over five days following surgery. The GBP group received GBP 10 mg kg^-1 orally twice a day (every 12 hours) because of suggestions in textbooks about veterinary analgesia (GAYNOR & MUIR 2002, FOX 2010), although half life is known to be short with 3.1 - 3.4 h (RHEE et al. 2008;

KUKANICH & COHEN 2009), which started within 2 minutes to 2 hours before anaesthesia whereas the group P received empty gelatinous capsules at the same time interval. Both treatments where administered over 5 days following surgery. Afterwards the GBP medication was tapered over one day by bisecting the dosage two times. Dogs were anesthetized using a standardized anaesthesia regime for hemilaminectomy. This consisted of premedication with levomethadone (L-Polamivet,levomethadonhydrochloride, fenipipramidhydrochloride, Intervet Deutschland GmbH, 85716 Unterschleißheim, Germany) 0.6 mg kg^-1and diazepam (ratiopharm GmbH, 89070 Ulm, Germany) 0.5 mg kg^-1intravenously followed by induction of anaesthesia with propofol (Narcofol, 10 mg ml^-1, cp-pharma, 31303 Burgdorf, Germany) to effect. The dogs’ tracheas were intubated and connected to a breathing circuit. Anaesthesia was maintained with isoflurane in oxygen. Background opioid analgesia was initiated with levomethadone 0.6 mg kg^-1IV as premedication for anaesthesia, followed by a fentanyl-patch (Durogesic SMAT, 12, 25, 50 µg h^-1, Janssen-Cilag GmbH, 41457 Neuss, Germany) 12.5 µg h^-1for dogs up to 5 kg, 25 µg h^-1for dogs up to 10 kg and 50 µg h^-1for dogs up to 20 kg body weight applied directly after extubation and levomethadone 0.2 mg kg^-1SQ three times with a dose interval of eight hours until effect of the fentanyl patch (after 24 h). If the fentanyl-patch did not adhere to the dry shaved dogs’ skin and dogs were painful with a CMPS-SF above 8, another fentanyl- patch was applied. During anaesthesia dogs underwent magnetic resonance imaging (MRI)

(Magnetom Impact plus, Siemens AG Medical Solutions Magnetic Resonance Imaging, Forchheim, Germany) and were operated on using hemilaminectomy with or without fenestration dependent on the surgeons’ choice.

1.2.3 Evaluation of patients

Examinations were performed at 0.5, 1, 2, 3, 4 and 5 days after extubation. For the evaluation of pain the shortform of the Glasgow Composite Measure Pain Scale (CMPS-SF) (REID et al.

2007) was used, without the category “gait” because of neurological deficits in all patients. This resulted in maximal possible score of 20. This assessment was performed by a single observer blinded to the treatment groups. Furthermore a Visual Analogue Scale (VAS) was employed by the blinded observer as well as by undergraduate students who were familiar with treating patients with neurological abnormalities. The evaluation was performed by marking the actual assumed pain on a 100 mm scale which ranged from 0 as no pain to 100 for worst pain imaginable. The dogs were visually assessed without interaction. Systolic-, (SAP) mean- (MAP) and diastolic arterial pressure (DAP) were measured by High Definition Oscillometry (VET HDO MD_15/90 Pro, Memo Diagnostic, S+B medVet GmbH, Babenhausen, Germany).

The system was used in conjunction with its analysis software (MDSwin - HDO Analyse Version 1.7) to assure linear pressure release and artefact free readings. Cuff size was chosen to obtain a cuff diameter of 40 % of tail circumference. The cuff was placed predominantly on the base of the tail (A. caudalis mediana) or alternatively (if dogs had a too short tail) on one front leg (A. mediana). On every occasion the same size and position of the cuff was chosen. Five to ten separate measures with linear deflation of cuff and minimal artefacts were recorded. Mean was calculated out of five similar measures, whereby extremes were excluded. Pulse rate was determined by means of the HDO device. Respiratory rate was counted from thoracic excursions. In addition a neurological follow up examination was performed which consisted of testing the flexor reflex, voluntary movements, deep pain sensation, muscle tone and urination.

Criteria were scored separately for both rear legs from -2 for absent reaction, -1 for decreased response, over 0 for normal reactions, +1 for exaggerated responses, to +2 for tonic responses.

Urination was also evaluated allowing a score from 2 for no bladder tone at all, unable to void urine voluntarily, affording manual help for urination, a score of 1 for decreased ability to urinate with presence of bladder tone and minimal help needed for urination and a score of 0 for normal urination. All side effects occurring during the treatment period were recorded.

1.2.4 Rescue analgesia protocol

Rescue analgesia was used if there was a marked increase in pain over a CMPS-SF level of 8 and consisted of additional levomethadone 0.2 mg kg^-1SQ. Metamizole-sodium (Novalgin 500, Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt am Main, Germany) 50 mg kg^-1 was administered to patients which were hypersensitive to opioids or showed other contraindications for increased opioid dose.

1.2.5 Concomitant medication

The use of other medication was left to the discretion of the clinician in charge of the dog. Main therapy consisted of balanced electrolyte solution (Sterofundin ISO or Sterofundin HEG-5, B.

Braun Melsungen AG, 34209 Melsungen, Germany), ceftiofur-sodium (Excenel 1 g, Pharmacia GmbH, 76139 Karlsruhe, Germany), ranitidine (Ranitidin 75, 1 A Pharma GmbH, 82041 Oberhaching, Germany) or omeprazol (Omeprazol 10; 20, ratiopharm GmbH, 89070 Ulm, Germany), and if ability to urinate was limited phenoxybenzamine hydrochloride (Dibenzyran 5, 10, esparma GmbH, 39171 Osterweddingen, Germany) and bethanechol hydrochloride (Myocholin 10, 25, Glenwood GmbH, 82302 Starnberg, Germany). Acepromazine maleate (Vetranquil 1 %, CEVA Tiergesundheit GmbH, 40472 Düsseldorf, Germany) was given to excited, hyperactive dogs which developed hyperthermia.

1.2.6 Blood sampling, analysis of serum samples

Serial blood samples for measurement of GBP and cortisol serum concentrations were collected by peripheral venipuncture at predetermined time points. Serum samples were drawn into serum tubes, centrifuged at 4000 g for 5 minutes and stored frozen at -75 ºC until further analysis.

Blood samples for minimum GBP serum concentration were collected 12 hours after last oral administration of GBP capsules which implied directly before next oral administration of GBP on 24 h and 72 h after first GBP administration. Blood samples for cortisol concentrations were drawn at 24 h, 48 h and 72 h.

Gabapetin concentration in the serum samples was analyzed with the reverse phase high- performance liquid chromatography (HPLC) method (VERMEIJ & EDELBROEK 2004) with slight modifications. One hundred µl of a serum sample, a standard (Chromsystems, 81243 Munich, Germany), or a control sample (Chromsystems, 81243 Munich, Germany) was mixed

with 50 µl of 20 % trichloroacetic acid, and following centrifugation, 15 µl of the deproteinized supernatant was collected. The derivatization was performed with o-phthaldialdehyde (OPA) and 2-mercaptoethanol under alkaline conditions (borate buffer, pH 10). Twenty µl of the reaction product was injected into the HPLC system (Knauer, 14163 Berlin, Germany), and the gradient elution was performed at the rate of 0.7 ml min^-1with acetonitrile (buffer A), and acetic buffer 50 mM, pH 6.8 (buffer B) on the Supersphere 60-4 RP column (Knauer, 14163 Berlin, Germany). The fluorescence was monitored with a fluorescence detector set at the excitation wavelength of 330 nm and the emission wavelength of 450 nm. Detection limit was > 1 µg ml^-1. Intra-assay imprecision was 3.5 % at 5.3 µg ml^-1 and 6.3 % at 13.2 µg ml^-1. Inter-assay imprecision was 10.1 % at 1.9 µg ml^-1and 9.1 % at 15.2 µg ml^-1.

Detection of cortisol concentrations was performed analysing serum samples with solid-phase, competitive chemiluminescent enzyme immunoassay (IMMULITE 1000 systems, Siemens Healthcare Diagnostics GmbH, 65760 Eschborn, Germany).

1.3 Statistics

The statistical analysis was performed with SPSS 16.0, Win Episcope 2.0 and Graph Pad Prism 5.02 and included descriptive statistics, chi square test, test of normality with Kolmogorov- Smirnov test and additionally for parametric data two-way analysis of variances (ANOVA) for repeated measurements with Dunnets and Bonferoni corrected t-tests. For non parametric data (CMPS-SF, Cortisol, neurologic follow up) Wilcoxon test and Friedmann test were used.

Correlations were tested using Spearman’s and Pearson’s correlation coefficient depending on distribution of data. For all comparisons p < 0.05 was considered significant, p < 0.1 was considered as a tendency towards statistically significance. Data are presented as mean and standard deviation or median and range. For analysis of agreement between VAS of trained observer and unexperienced observer (students) Bland Altman analysis was performed.

1.4 Results

In this study 63 dogs were included. Seven dogs were excluded because of a second operation within the first five days after first hemilaminectomy surgery. Resulting in 32 dogs in the P group and 31 in the GBP group (Table 1).

Main breed in this study was the dachshund with 65 % followed by different other breeds.

There were no significant differences between groups for demographic data (Table 1).

The CMPS-SF decreased over time which was significantly different to day 0.5 on day 4 and day 5 in group GBP (Figure 1). In group P there was a significant decrease from day 0.5 to day 3, 4 and 5. Median CMPS-SF was lower in the GBP group on days 0.5, 1, 4 and 5. There were no statistically significant differences (p < 0.05) between groups. Dogs did not show a marked sensitization to touch independent of group (Figure 2).

The VAS of the blinded observer showed a significant decrease over time from day 0.5 to day 3, 4 and 5 and to day 2, 3, 4, and 5 in group GBP and group P, respectively (Figure 3). No statistically significant differences were detected between groups. The VAS for undergraduate students points to similar results with significant decrease from day 0.5 to day 4 and 5 in both groups and no significant differences between groups (data not shown). Correlation between VAS and CMPS-SF was weak (two tailed significance with R = 0.395, p < 0.01). There was also only a weak correlation between VAS of different observers (two tailed significance with R

= 0.284, p < 0.01). Bland–Altman analysis showing the difference between VAS values of postoperative pain assessed by 2 different groups revealed wide limits of agreement. The difference between the two observer groups increased as pain increased (Figure 8).

Systolic arterial pressure, MAP and DAP did not show any statistically significant changes over time and between groups (Figure 4). Pulse rate and respiration rate were not different between groups and over time (data not shown).

Neurologic follow up examination was not significantly different between groups for deep pain sensation, voluntary movements, muscle tone, flexor reflex, proprioception and urination. A significant improvement over time was seen for muscle tone from baseline value (day 0.5) to day 3, 4 and 5 in both groups for both rear legs and additionally in group P for the right rear leg on day 2. Voluntary movements improved significantly on day 3, 4 and 5 in both groups. Flexor

reflex improved significantly on day 4 and 5 in both groups and additionally on day 3 in groups GBP left and right rear leg and P left rear leg. Proprioception increased significantly in group P on day 4 and 5 and in group GBP for the right rear leg on day 5. Urination was significantly improved on day 5 (Figure 7).

Cortisol levels were not statistically significant different between groups and did not significantly change over time but medians were lower on all three days in group GBP (Figure 5) and there was a tendency towards lower cortisol levels on 24 and 72 hours in group GBP.

Minimum serum levels of GBP were above detection limit in the magnitude of dogs at both time points: they were very variable and ranged from > 1.0 (detection limit) to 9.08 at 24 h and from 1.0 to 11.00 µg ml^-1 at 72 h measurement with median levels of 2.45 and 1.36, respectively. There was only weak correlation between serum levels in general or serum levels above 3.0 µg ml^-1and CMPS-SF. There was no statistically significant difference in CMPS-SF between dogs with serum levels < 3 and dogs with > 3 µg ml^-1(Figure 6).

The application of a second fentanyl patch was necessary in 6 cases, because the first patch did not stick well. Three dogs in each group received this additional treatment. Three dogs received metamizole. Vomitus was present in 3 dogs in group GBP and 1 dog in group P. Two dogs, one of each group, developed pyometra post surgery.

Table 1Demographic data of dogs after hemilaminectomy included in this study. Gender and breed distribution between group gabapentin (GBP) and placebo (P), weight and age are differentiated.

GBP P

examined (n) 34 36

included (n) 31 32

Female 11 18

(intact/ spayed) (3 / 8) (13 / 5)

Male 20 14

(intact / neutered) (17 / 3) (10 / 4) body-weight [kg] 9.4 ± 3.4 9.8 ± 3.5 age [years] 6.5 ± 2.1 6.7 ± 2.6

Dachshund (20 / 31) (22 / 32)

Terrier (2 / 31) (2 / 32)

Mixed breed (3 / 31) (1 / 32)

Beagle (1 / 31) (2 / 32)

Poodle (2 / 31) (0 / 32)

Shi-tzu (0 / 31) (2 / 32)

Hound (1 / 31) (1 / 32)

Cocker Spaniel (1 / 31) (1 / 32)

Bulldog (0 / 31) (1 / 32)

Schnauzer (1 / 31) (0 / 32)

Figure 1Postoperative pain determined by means of Glasgow Composite Measure Pain Scale (CMPS-SF) over time. Diagram contains data of 63 dogs in the postoperative period after hemilaminectomy. Dogs are divided into two different groups: Gabapentin and Placebo. Data are presented as box plots over time. The box contains the interquartile range (mean 50 % of data). Whiskers show data from minimum to maximum. The horizontal line inside the box marks the median. Statistically significant (p < 0.05) difference to baseline value 0.5 is depicted as asterisk.

Figure 2Postoperative sensitivity to touch taken from the shortform of Glasgow Composite Measure Pain Scale (CMPS-SF) over time. Diagram contains data of 63 dogs in the postoperative period after hemilaminectomy. Dogs are divided into two different groups: Gabapentin and Placebo. Data are presented as boxplots. The box contains interquartile range (mean 50 % of data). Whiskers show data from minimum to maximum. (p < 0.05).

Figure 3Postoperative pain determined by means of Visual Analogue Scale (VAS) of the blinded observer over time. Diagram contains data of 63 dogs in the postoperative period after hemilaminectomy. Dogs are divided into two different groups: Gabapentin and Placebo. Data are presented as means + SD. Statistically significant (p < 0.05) difference over time to baseline value 0.5 is depicted as asterisk.

Figure 4Mean arterial blood pressure (MAP) of dogs after hemilaminectomy over time. Dogs are divided into two groups: gabapentin and placebo. Data are presented as means + SD (p < 0.05).

Figure 5Postoperative cortisol serum concentrations over time of dogs after hemilaminectomy. Dogs are divided into two groups: gabapentin and placebo. Data are presented as boxplots. The box contains interquartile range (mean 50 % of data). The horizontal line inside the box marks the median. Whiskers show data from minimum to maximum (p < 0.05).

Figure 6Gabapentin serum concentrations on 24 h and 72 h after first oral administration versus the shortform of Glasgow Composite Measure Pain Scale (CMPS-SF) over time. Dogs are divided into two groups: serum concentrations above or beneath 3 µg ml^-1. Data are presented as boxplots. The box contains interquartile range (mean 50 % of data). The horizontal line inside the box marks the median. Whiskers show data from minimum to maximum (p < 0.05).

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

GBP0.5 P0.5

GBP1 P1

GBP2 P2

GBP3 P3

GBP4 P4

GBP5 P5

0 = unassisted urination 1 = assisted urination 2 = no bladder tone, manual bladder emptying

Figure 7Postoperative ability of urination of dogs after hemilaminectomy over time of 63 dogs. Dogs are divided into two different groups: first row for each time point is group gabapentin (n = 31) and second row is placebo (n = 32). Data are presented as percentage. Statistically significant (p < 0.05) difference to baseline value 0.5 is depicted as asterisk.

Figure 8Bland-Altman plot of 44 dogs after hemilaminectomy illustrating the difference between Visual Analogue Scale (VAS) values of postoperative pain assessed by 2 different groups: students and observer against the average of these 2 values independent of time. The bias is indicated as horizontal continuous black line, limits of agreement (95 % Confidence level) are indicated as horizontal dashed lines.

* *

1.5 Discussion

In this study CMPS-SF and VAS were not able to detect clear clinically relevant analgesic effects of GBP added to µ-opioid analgesia in dogs after hemilaminectomy. This might be related to several factors.

The presence of IVDD is associated with a mixed type of nociceptive and neuropathic pain. The nociceptive pain elements are sensitive to opioid analgesia (IRESON 1970; PLEUVRY &

TOBIAS 1971; HILL & PEPPER 1978) which provides a very strong background analgesia, whereas the neuropathic pain component can be refractory to this treatment.

Under the given background treatment the remaining pain was not very high. A maximum CMPS-SF of 20 was possible. Dogs after hemilaminectomy achieved median CMPS-SF levels between 3 and 1 in group GBP and between 5 and 1 in group P. Additionally extreme values were only noticed in individual animals and did not exceed a score of 11. Mean VAS scores also remained within the lower third of the VAS from 0 to 100.

This condition of potent nociceptive background analgesia requires an additional analgesic with high potency as a combination of receptor binding capability and intrinsic activity or which targets a different receptor on an alternative pathway to show its additional analgesic action and to further decrease pain scores (GAYNOR & MUIR 2002).

Inter-individual variability of the scores of CMPS-SF in dogs in both groups was high and contributed to the fact that observed differences between treatment groups did not reach significance. Power analysis aiming at a power of 90 % with a confidence interval of 95%

indicated that a minimum of 67 dogs would be needed to achieve a statistically significant difference of 10 % between groups (CMPS-SF difference of 2), when using data from the first 20 patients. However, variability of data became higher than initially expected. Data from 70 dogs were originally collected for this study. From these dogs, 7 had to be excluded retrospectively because of a second surgery needed during the first 5 days after surgery.

However, the heterogeneity of clinical patients with IVDD could be considered advantageous because the investigation has been able to test the clinical reality of this analgesic strategy.

In humans an opioid sparing effect was seen when giving opioids and GBP together in patients with opioid therapy on demand (CHENG & CHIOU 2006; MATHIESEN et al. 2007; TURAN et al. 2007). In dogs that kind of study design is not possible, particularly if dogs are client owned and experience a natural injury versus laboratory animals which can be trained to different circumstances and are held under standardized conditions. Thus it is not possible to discriminate whether less opioids would have been sufficient to treat perioperative pain when combined with GBP.

There are no sufficient experience based prescriptions for GBP dosage in dogs and particularly no references for analgesia. Thus knowledge is based exclusively on recommendations of few published trials. The available recommendations are very variable and do not provide effective serum levels. They either report administered doses and their clinical efficacy (CASHMORE et al. 2009; WOLFE & POMA 2010) or pharmacokinetics after defined oral intake as concentration - time curves without correlation to pharmacodynamic effects (STEVENSON et al. 1997; RHEE et al. 2008; KUKANICH & COHEN 2009).

In human medicine recommended doses for antiepileptic treatment are 900 - 3600 (6000) mg d^-1 (which translates to approximately 11 - 45 mg kg^-1 d^-1). (BRODIE & TREIMAN 1996;

BACKONJA & GLANZMAN 2003). Gabapentin’s absorption is dependent on the L-amino acid transporter in the small intestine and therefore serum concentrations increase linearly up to about 1800 mg d^-1and then dose escalations flatten the increase of plasma concentration in most individuals because of assumed saturable kinetics. Dosage is commonly titrated in dependence of effectiveness and side effects. The total dose is divided into three applications because GBP shows a short half life of 5 - 9 hours (BRODIE & TREIMAN 1996; BACKONJA &

GLANZMAN 2003; PATSALOS et al. 2008). Effective plasma concentrations of GBP are not dose proportional and range from 4.1 to 17.2 µg ml^-1(dose applied 1200 - 6000 mg) (BRODIE

& TREIMAN 1996). These plasma levels show a marked interindividual variation and are therefore not used routinely as monitoring parameter. Patsalos and co- workers (2008) advise a reference range of 2 - 20 µg ml^-1for adults. Mirza et al. (1999) recommend little lower values of 12 - 60 µmol l^-1which translates to 2.1 - 10.3 µg ml^-1. Furthermore an increase of adverse effects is reported when serum levels exceed 146 µmol l^-1(25 µg ml^-1) (JOHANNESSEN et al.

2003).

Gabapentin is mainly excreted by the kidneys. In contrast to other species in dogs GBP is metabolized in the liver by 34 % (RADULOVIC et al. 1995). In dogs mean elimination half-

life is 3.1 to 3.4 hours (VOLLMER et al. 1986; RHEE et al. 2008; KUKANICH & COHEN 2009) which implies it is even shorter than in humans which have a half life of 5 to 6 hours (VOLLMER et al. 1986).

In veterinary medicine there are two studies available on antiepileptic treatment with GBP as add - on therapy in dogs. Platt and co-workers (2006) administered 30 mg kg^-1d^-1(10 mg kg^-1 three times a day (TID)). They also measured minimum serum concentrations of GBP which ranged from 2.2 to 20.7 µg ml^-1with a mean of 8.4 µg ml^-1. Govendir et al. (2005) chose doses of 17 - 50 mg kg^-1d^-1GBP orally to dogs whereby a dose interval of twice a day (BID) and only in one case an interval of TID was used. In both studies a decrease of seizure frequency occurred. Govendir and co-workers (2005) additionally achieved a decrease of seizure intensity.

For the treatment of neuropathic pain in humans the same doses as for the antiepileptic treatment (900 - 3600 mg d^-1) are used as well (BACKONJA & GLANZMAN 2003). Early studies used lower levels from 300 - 1200 mg d^-1. It is recommended to use incremental doses from 300 mg once a day over twice a day to three times a day increasing to a daily dose with side effects remaining still tolerable. This treatment is then continued as long term therapy.

Recommendations are made without consideration to minimum serum levels needed as effective analgesic therapy (BACKONJA et al. 1998; TAKASAKI et al. 2001; BONE et al.

2002; TO et al. 2002; LINDBERGER et al. 2003; GORDH et al. 2008).

In the current study a dose of 20 mg kg^-1d^-1was chosen, based on textbooks and empiric suggestions (GAYNOR & MUIR 2002, FOX 2010), for dogs since analgesic efficacy seems to afford lower doses than antiepileptic treatment. Further a dose interval of BID was chosen to achieve better compliance of the owners since a number of patients with IVDD can be released from the clinic shortly after surgery and still require analgesic treatment at home. It has to be considered that the current study’s aim was to measure minimum serum concentrations to estimate the interdose decrease of a dosing schedule of twice daily not maximum or mean serum concentrations as they are listed in other studies. In 21.5 % of the dogs (24 h) and 23.1 % (72 h) respectively GBP serum concentrations fell below detection limit. Because there are no plasma concentration recommendations for analgesic effect in dogs a minimum effective concentration of 3 µg ml^-1was chosen arbitrarily based on human data to compare pain scores of patients with levels beneath and above this limit. There were no differences in pain scores between dogs which had a serum concentration above or below 3 µg ml^-1. Further studies are

warranted to investigate if further dose titration to a higher daily dose can decrease pain behaviour.

New studies which were published recently also evaluate the effect of adjunctive analgesia of GBP in dogs with assumed neuropathic pain. Wolfe & Poma (2010) administered GBP orally 10 mg kg^-1BID or TID respectively in addition to prednisone to dogs with syringomyelia. This treatment resulted in a reduction in cervical pain and stop of scratching episodes. Wagner et al.

(2010) administered GBP orally, initally 10 mg kg^-1as pre-operative loading dose, followed by 5 mg kg^-1BID postoperatively to dogs undergoing amputation of a forelimb in addition to local anaesthetics and fentanyl continuous rate infusion. Wagner and co-workers had a very small group of patients (29 dogs) and they did not define a rescue analgesia. Lower concomitant analgesic treatment in group GBP was administered while dogs were housed in the clinic. The dogs had lower mean scores in CMPS-SF, VAS and University of Melbourne Pain Scale at most time points, but these results were not statistically significant different. Wagner et al. drew the conclusion that GBP had no effect at all, even though their results also indicate a weak tendency towards beneficial effects of GBP. The dose applied in this study (with exception of the loading dose) was half of the dose used in the current study. This is presumably the reason for an even weaker effect seen in this study compared to the current data. These two studies also tested GBP in diseases with presumable contribution of neuropathic pain or combined nociceptive and neuropathic pain elements. The employed dosage was similar to the dosage used in the current study. Though different diseases are evaluated and the pain scale values differ between the different studies, all show a positive tendency toward beneficial effects of GBP in analgesic treatment, without achieving statistically significance. In all studies including our data GBP did not cause major adverse effects.

Interindividual differences in pain perception develop in dependence of the amount and nature of former experienced pain (pain memory) because of underlying plasticity of neuronal fibres (SHERRINGTON 1893; WEDDELL et al. 1941; BRADLEY & HORN 1979; BLUMBERG &

JÄNIG 1985; MCQUAY & DICKENSON 1990; JÄNIG & KOLTZENBURG 1991; WOOLF et al. 1992). Furthermore the emotional component of pain/ disability seems to play an important role. For example one dog is affected very strongly when its mobility is hampered and whimpers whereas another dog starts running around trying to interact with its surrounding by just using the front legs and ignoring the loss of the function of the rear legs. In addition, transdermal fentanyl can result in very variable plasma fentanyl concentrations and variations in the degree of the analgesic effect (KYLES et al. 1996; EGGER et al. 1998; PETTIFER &

HOSGOOD 2003; DAVIDSON et al. 2004; HOFMEISTER & EGGER 2004; PETTIFER &

HOSGOOD 2004; SOLSSOL et al. 2005). However, this type of background analgesia was chosen because we did not want to deviate from the standard clinical routine.

Additionally GBP’s effectiveness could be questioned because of discovery of paid and manipulated studies as well as retention of studies with undesirable results by the company Parke-Davis which was exposed in the year 2009 (VEDULA et al. 2009).

An important question is if acute IVDD is an appropriate disease pattern for neuropathic pain in dogs to prove GBP’s effectiveness or whether it is dominated by the nociceptive pain component even if nerve damage is present.

From laboratory experimental studies it is known that GBP alone does not have an antinociceptive effect in naïve rats, a minor effect in sham-operated rats (which have nociceptive pain due to surgery but no neuropathic pain) but a significant dose dependent effect on the inhibition of neuronal responses in the dorsal root ganglion of nerve injured rats (MATTHEWS & DICKENSON 2002). A combination of GBP and morphine indicates inhibitory effects in spinal nerve ligated and sham-operated rats, whereby the greatest effects are seen in nerve injured rats (MATTHEWS & DICKENSON 2002). IVDD should be a good neuropathic pain disease pattern because of temporarily existing nerve injury. In rat studies a nerve ligation is used as the neuropathic pain model which is caused by operative nerve ligation 14 days before the trial and confirmed by comparing the response to von Frey filaments or cooling acetone on the ipsilateral paw prior to surgery with 14 days after surgery (MATTHEWS

& DICKENSON 2002). Thus in rats a central sensitization was assured.

In the current study central sensitization was assumed but not all animals showed considerable symptoms like signs of hypersensitivity to touch when tested. This could be due to short duration of spinal cord compression or incomplete spinal cord injury. In this context a minor analgesic effect of GBP could be explained as comparable with sham-operated rats where the invasive surgery without major nerve damage seemed to be sufficient to induce a distinct degree of inflammation and enabled GBP to act. This could suggest that the chosen spontaneous disease IVDD is dominated by inflammatory and nociceptive pain but not preferentially by neuropathic pain components. In contrast, the lack of overt allodynia in both groups could also be related to mild inhibitory effects of methadone, which acts as NMDA receptor antagonist, on central sensitisation (GORMAN et al. 1997).

Mean systolic-, median- and diastolic blood pressure were not different between groups and over time. Mean systolic blood pressure ranged from 145 ± 11 to 156 ± 21 (group GBP) versus 150 ± 15 to 158 ± 17 mmHg (group P). These values are close to the reference range for dachshunds (142 ± 10 mmHg) which constituted the main breed in this study with 65 % of dogs (and have the highest blood pressure of dogs included in this study)(EGNER 2002). A rise of blood pressure is not necessarily a sign of nociceptive sympathetic stimulation since blood pressure in conscious animals is also influenced by anxiety or stress, so its informative value is limited.

Cortisol levels in the GBP treated group tended to be lower than in the control group. Although blood sampling times among dogs varied in dependence of time of surgery, cortisol levels were analysed exclusively by group because the circadian excretion rhythm which is known in humans (ADER & FRIEDMAN 1968), monkeys and rats (DALLMAN et al. 1978) is not existing in dogs. Already Kemppainen and Sartin (1984) considered the cortisol activity of dogs to be episodic not circadian. Further Castillo (2009) confirmed two peak levels a day, the first one at 8 a.m., the second between 2 and 6 p.m.. High cortisol levels can also be due to stress because of handling or venipuncture (STANFORD et al. 1967). It can be assumed that these stress artefacts were present equally in both groups and were therefore ignored for analysis.

The VAS of the different examiners (student vs. observer) showed only moderate agreement.

This is attributed to the lack of experience in evaluating animals in pain and subjectivity of the scale, because pain evaluation - especially when using a VAS without defined criteria - needs experience to capture special behaviours completely which are presumably related to pain or discomfort (FREYD 1923; WEWERS & LOWE 1990). In addition, agreement between examiners was better in dogs which were less painful than in dogs with a higher degree of pain.

In the current study the VAS was limited to visual evaluation without interaction with the dog.

Thus CMPS-SF and VAS led to regard of different aspects of pain. This explains that the correlation between VAS and CMPS-SF was weak.

GBP showed minor side effects in dogs. Vomitus was present in 3 dogs in group GBP and one dog in group P but could be explained by additional pyometra in two cases, one of which not having been treated with GBP. In two cases the vomitus is unexplained in the GBP group and could be either attributable to GBP medication (ANONYMOUS 2009) or also due to opioids whereby concurrence with GBP treatment is accidentally (STANLEY et al. 1996;

WOODHOUSE et al. 1999; CEPEDA et al. 2003).

The study design of this study has some limitations. The shortform of the Glasgow Composite Measure Pain Scale was very low leading to minor discrimination between groups GBP and P.

Furthermore pre-treatment as well as concomitant treatments were not standardized and could have influenced results since interaction of GBP with other medications is not known for all drug combinations (ANONYMOUS 2009). Patients referred to the Small Animal Clinic are often treated conservatively before with corticoids by the referring veterinarian. Therefore opioids were chosen as background analgesia instead of NSAIDs to decrease the risk of gastrointestinal side effects (TOOMBS et al. 1980; MOORE & WITHROW 1982; DAVIES 1985).

Out of 70 examined dogs 7 were excluded retrospectively due to re-operation. Second surgery was performed if neurological outcome did not improve or worsened or if the degree of pain was constantly very strong. Partially this was also dependent on owners’ wish or financial limitations. Results in the treatment of these dogs after the first surgery were excluded to assure homogeneity in the observed group. Results after second operation were excluded due to previously received analgesic treatment possibly affecting later pain perception. Also the possibility of stronger sensitization led to exclusion of these data.

In conclusion,GBPwas well tolerated without major side effects. A tendency in the GBP group towards lower pain scores and lower cortisol levels was detected but without reaching statistical significance. Further investigations are needed to test if higher doses and more frequent dosing will result in clinically relevant improvement of analgesia and reduction of pain behaviour in dogs.

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

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