Relative Potency and Duration of Analgesia Following Palmar Digital Intra-Neural Alcohol Injection for Heel Pain in Horses

Relative potency and duration of analgesia following palmar digital intra-neural alcohol injection for heel pain in horses

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Christine Pariseau Schneider

Graduate Program in Veterinary Clinical Sciences

The Ohio State University

2013

Master's Examination Committee:

Alicia Bertone, Advisor

Michael Oglesbee

Lisa Zekas

Copyrighted by

Christine Pariseau Schneider

2013

Abstract

Objective: To determine the potency (percent analgesia), duration of action (up to four months), and clinical and histological effects of surgical exposure and intra-neural injection of 98% dehydrated medical-grade ethyl alcohol compared to no treatment

(negative control), sham operation (surgical control), or formaldehyde injection (positive control) to decrease experimentally-induced palmar heel pain in horses.

Animals: Six horses

Procedures: The horses were fitted with a custom pressure-inducing shoe and had outcome measurements for each heel performed before and after nerve treatments.

Outcomes included induced lameness grade and vertical peak force with pressure applied to each heel, thermal and touch sensation for each heel, and pastern circumference.

Outcomes were followed serially for 112 days when nerves were harvested for histology.

Results: Alcohol and formaldehyde reduced all measures of heel pain which progressed toward return, but persisted over the 112 days of the study (P<0.05). Pastern circumference was not different for alcohol than sham treatment, but was greater in formaldehyde than alcohol or baseline (P<0.05). Histological evaluation showed preservation of nerve fiber alignment with an intact epineurium, loss of axons (axon drop out), axon degeneration, fibrosis and inflammation in alcohol- and formaldehyde-injected

ii nerves compared to control nerves. Formaldehyde injection induced greater fibrosis and inflammation than alcohol.

Conclusions and Clinical Relevance: Alcohol injection induced effective neural blockade for months with evidence that nerve structure and function could return. Formaldehyde injection showed no advantage over alcohol and is not recommended under the conditions of our study due to soft tissue inflammation.

iii

I dedicate this thesis to my family and husband for their unending and inspiring support.

Special thanks to my incredibly giving parents, Bob and Sheila Pariseau, for their sound advice and life-long belief in my abilities. I would not be where I am today without you!

To my sister, Beth, your strength and perseverance inspire me daily to „keep swimming‟.

To my husband, Tom, thank you for your unwavering and sustaining support. Your steady guidance allows me to be at my best. Thanks for holding down the fort.

Acknowledgments

I would like to thank my committee members for their guidance and assistance with the execution of this study and the formation of this thesis. A special thanks to Dr. Alicia

Bertone for the dedication of many hours filled with discussion, editing, proof-reading, and teaching that made this study and thesis possible. Your guidance has been invaluable. Thank you to Dr. Michael Oglesbee and Dr. Lisa Zekas for agreeing to serve on my committee. Thank you, also, to Dr. Laurie Gallatin for her guidance through my residency and the creation of this thesis.

I also wish to acknowledge and thank Akikazu Ishihara. Without his assistance, this study, the accompanying figures, and manuscript would not have been possible. I am very grateful for all of your help.

Vita

June 2001 ...... Chelmsford High School

2005 ...... B.S. Agriculture, The Ohio State University

2009 ...... D.V.M., The Ohio State University

2010 to present ...... Graduate Teaching Associate, Department

of Veterinary Medicine, The Ohio State

University

Fields of Study

Major Field: Veterinary Clinical Sciences

Table of Contents

Abstract…………………………………………………………………………………...ii

Dedication………………………………………………………………………………...iv

Acknowledgements………………………………………………………………………..v

Vita……………………………………………………………………………………...... vi

List of Figures…………………………………………………………………………...viii

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

Chapter 2: Materials and Methods………………………………………………………...4

Chapter 3: Results…………………………………………………………………………9

Chapter 4: Discussion……………………………………………………………………21

Endnotes………………………………………….………………………………………24

References………………………………………………………………………………..25

vii

List of Figures

Figure 1. Photographs of injection procedure, custom shoe, and heat lamp...... …………8

Figure 2. Graph of baseline lameness without bolt………………………………………13

Figure 3. Graph of baseline VPF without bolt…………………………………………...14

Figure 4. Graphs of lameness grade, VPF, and percent lameness analgesia with bolt in

place……………………………………………………………………………...15

Figure 5. Graphs of thermal reaction time and percent thermal analgesia…...... 16

Figure 6. Graph of skin sensitivity score………………………………………………...17

Figure 7. Graph of pastern circumference across time…………………………………..18

Figure 8. Photomicrographs of sections of palmar digital nerves harvested 16 weeks post-

treatment…………………………………………………………………………19

viii

Chapter 1: Introduction

The cost of long-term management of chronic lameness due to heel pain contributes significantly to the unwanted horse population1. Navicular syndrome2, accounts for a large percentage of chronic and incurable equine lamenesses in many breeds with an over-representation of the Quarter Horse3. Nonresponsive4 palmar heel pain has been treated by palmar digital (PD) neurectomy with reports dating back to 19625. PD neurectomy is generally reserved for the chronic, refractory cases due to the complete and long-lasting effects and risks associated with complete heel desensitization. These risks include P3 sepsis, deep digital flexor tendon rupture, luxation of the distal interphalangeal joint, hoof capsule slough, and navicular bone fracture6 as well as superficial injury due to the inability to feel the heel. Less commonly, other injuries have been managed with

PD neurectomy in the athletic horse, such as distal phalanx wing fractures, sidebone, fracture of the navicular bone, and collateral ligament injuries7,8. The complication rate of

PD neurectomy has decreased significantly over the last 50 years, and the latest reported incidence of neuroma formation is ~5%6. Alternate methods of neural analgesia that do not include nerve transection, might avoid neuroma formation and other potential complications6,7, but also may produce a partial or transient (weeks to months) effect that may be desirable in performance horses.

Medical grade ethyl alcohol is approved for perineural injection in human medicine and has been used for relief of multiple painful conditions, including trigeminal neuralgia9, ankle-foot spasticity10,11, and end-stage, intractable pain due to malignancy12,13, with reports dating back to 191214. The injections described were performed percutaneously with guidance from fluoroscopy, nerve stimulator, or computed tomography to achieve perineural injection. Relief from trigeminal neuralgia was achieved immediately post- injection in 99% of patients and mean pain-free duration was 46 months9. Reduction in ankle-foot spasticity was achieved in 90% of patients for the full 6 month follow-up period11. Relief of visceral pain caused by malignancy was achieved in 100% of patients for the first 3 months (or until death) after local infiltration of alcohol around multiple plexuses13.

Although not in published literature, the American Quarter Horse Association has banned the blocking of tail motor function. Anecdotally, one substance that has been used for tail blocking is alcohol, which indicates that it has the ability to successfully block peripheral nerves in horses. Formaldehyde has been used to treat multiple disorders in the horse, including ethmoid hematomas15 and hemorrhage16 and, like alcohol, functions as a tissue fixative. Both techniques offer the potential to block nerve transduction but also preserve, in situ, the nerve structure, such as fiber alignment and the peri- and endo- neurium. By preserving peripheral nerve structure, the potential for reinnervation by nerve regeneration rather than axon sprouting with neuroma formation (as occurs after neurectomy) should be favored.

The primary purpose of this study was to determine the potency (percent analgesia) and duration of action (up to four months) of intra-neural injection of 98% dehydrated medical-grade ethyl alcohol as a chemical analgesic to decrease experimentally-induced palmar heel pain in the horse. Comparative groups included an untreated nerve, a sham- operated control nerve, and a nerve similarly-injected with formaldehyde. Our hypothesis was that both alcohol and formaldehyde would produce measurable analgesia for weeks and that formaldehyde would induce a greater and longer duration of analgesia than alcohol.

Chapter 2: Materials and Methods

Experimental Design: All experimental procedures were approved and monitored by the

Institutional Animal Care and Use Committee (IACUC) of The Ohio State University.

Six horses purchased for research purposes and meeting inclusion criteria were fitted to a custom shoe (Figure 1A). Controlled lameness was induced at the heel of each forelimb with a bolt tightened to apply pressure to each of the heels, one at a time. Baseline parameters, including gait analysis, were obtained prior to treatment (Day 0) and at defined time points for 112 days after intra-neural injection. Nerve assignment was random within treatment (formaldehyde or alcohol) and within control (sham or untreated) and completed as a blocked design to control for limb side. On Day 0, each horse was anesthetized with total intravenous anesthesia and one medial PD nerve was surgically exposed and injected intra-neurally with 98% medical-grade ethyl alcohola using a 27 gauge needle (Figure 1B). The nerve was isolated using a hemostat and secured using a soft rubber retractor to permit isolation and control of the nerve during injection. The contralateral medial PD nerve was exposed and isolated in the same fashion and was injected with formaldehydeb. One lateral PD nerve was exposed, but no injection was performed (sham control) and the remaining lateral PD nerve served as an untreated control. Clinical outcomes for each heel included thermal reaction time, lameness to the heel bolt (lameness score and vertical peak force), heel skin sensitivity

score, and for each limb, pastern circumference. Measurements were made pre-injection

(Day 0), and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 70, 84, 98, and 112 post- injection. On Day 112, specimens of 2 sham, 5 alcohol-injected, and 2 formaldehyde- injected nerves were harvested via neurectomy (to include the injected area) under general anesthesia and examined histologically.

Lameness model: For inclusion in the study, all horses were sound with a lameness score of zero. Temporary, intermittent lameness was induced with a custom-made horseshoe. The shoes were made for each horse so that the bolt contacted the palmar horned aspect of the hoof, distal to the coronary band of the heel (Figure 1A). The bolt length and tightness were adjusted to produce a grade 2 lameness on a modified

American Association of Equine Practitioners (AAEP) grading scale on Day 0 (before injection)17. The modified AAEP lameness grading scale used was as follows: grade 0 = no lameness, grade 1= intermittent lameness at the trot, grade 2 = consistent lameness at the trot and not seen at the walk, grade 3 = head bobbing consistent lameness at the trot and lameness noted at the walk, grade 4= Obvious and consistent lameness at the walk and trot, 5= non-weight bearing lameness. Half grades were assigned when lameness severity was between two scores.

Kinetic gait analysis: An in-ground, stationary force plate and computer analysis system was used to collect valid repetitions for each forelimb before and after tightening the bolt in each heel. For each repetition, a vertical peak force (VPF) vs. time curve was calculated by the computer analysis system using previously published procedures from our laboratory17. Data was obtained by trotting the horse over the force plate at a standard

rate of speed. A trial was considered valid if the foot in question struck the force plate at, or close to, the center of the force plate. Data from valid trials were averaged for each time point. Vertical peak force (newtons, N) was expressed as percent body weight17 and as percent decrease in vertical peak force when the treated heel was compared to the control heel in the same limb (relative potency). Specifically, the formula used to obtain the relative potency of analgesia for induced lameness (percent lameness analgesia) was:

% lameness analgesia = (VPFAlcohol or Formaldehyde with bolt [same limb] – VPFUntreated or Sham with bolt

[same limb as the alcohol or formaldehyde]) ÷ (VPFWithout-bolt – VPFUntreated or Sham with bolt [same limb as the alcohol or formaldehyde]) × 100.

Clinical assessments:

Thermal reaction time – An incandescent lamp (heat lamp analgesia meter)18,c was focused on the skin of the heel bulb to measure, by digital timer, avoidance to radiant heat as an assessment of cutaneous thermal analgesia (Figure 1C). In a pilot horse, it was determined that the duration of thermal application with the lamp should be capped at 22 seconds to avoid visual thermal injury to the skin (data not shown). For our study, complete thermal analgesia was defined as 22 seconds. Thermal reaction time was measured pre-injection and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 70, 84, 98, and

112 post-injection. Avoidance of radiant heat was measured in time elapsed (seconds), recorded on the digital timer, before the horse moved the foot when the heat lamp was focused on the heel bulb skin. Data was expressed as seconds and as a percent increase in thermal reaction time calculated relative to the control heel in the same limb by the

following equation: % thermal analgesia = (Reaction TimeAlcohol or Formaldehyde – Reaction

TimeUntreated or Sham in the same limb) ÷ (22 – Reaction TimeUntreated or Sham in the same limb) × 100.

Skin sensitivity score – Pressure was applied to the skin of each forefoot‟s heel bulb with a mosquito hemostat to assess the skin‟s sensitivity to touch pre-injection and on days 3,

7, 10, 14, 21, 28, 35, 42, 49, 56, 70, 84, 98, and 112 post-injection. Skin sensitivity to touch was scored with a 0-6 scoring system ranging from complete analgesia (confirmed with needle prick) to hyperalgesia (0 – no reaction (confirmed as no reaction to needle prick), 1 – notices heavy pressure, 2 – shifts weight with heavy pressure, 3 – picks up the foot with heavy pressure, 4 – notices light pressure, 5 – shifts weight with light pressure,

6 – picks up the foot with light pressure).

Pastern circumference – The circumference of the pastern (cm) of each forelimb was measured with a tape as an estimate of local soft tissue reaction associated with the skin incisions and intra-neural injection. Pastern circumference was measured pre-injection and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 70, 84, 98, and 112 post-injection.

Histopathology: Specimens of PD nerves were fixed in neutral buffered formalin for 1 week. Fixed nerves were serially sectioned (4 microns) and stained with hematoxylin and eosin, Bielschowsky‟s stain, Masson‟s Trichrome, and Luxol Fast Blued. Sections were evaluated by 3 investigators (CPS, MO, ALB) for primary demyelination, axon degeneration and axon drop-out, perineural and neural coagulation necrosis, mineralization, inflammation, and fibroplasia.

Statistical analysis: Three-way repeated-measures analysis of variance (ANOVA) by a commercial software packagee evaluated the effects of 3 factors, heel treatment, time, and

horse, using Proc Mixed models for continuous dependent variables and Proc Genmod models for categorical dependent variables19. The post-test ANOVA for multiple comparisons was made between the treatment groups at each time point. Significance level was set at P<0.05 for all analyses.

Figure 1. A – Photograph of injection procedure on a medial PD nerve. B – Photograph of custom-made shoe on horse‟s hoof. C – Photograph of incandescent lamp directed at the skin of the heel.

Chapter 3: Results

Horses: Five horses completed the study. Shoes were reset and the horses‟ feet were trimmed every 5 weeks. One horse had a celiotomy to correct a non-strangulating colon displacement within 8 hours of recovery from the first surgery and completed the study.

One horse required removal from the study at day 19 because the horse exceeded the limits of the lameness inclusion criteria in the formaldehyde limb as per IACUC protocol.

Horse was not a significant factor in the 3-way ANOVA for all outcomes.

Surgical procedure: The surgical procedure required a mean of 47.5 minutes +/- 7.1

(SEM) to complete from anesthetic induction to cessation of anesthetic drug administration. In all horses the nerve was readily identified with minimal dissection. In most nerves, some immeasurable amount of leakage of the injected substance could be observed in the surgical field.

Baseline Lameness (without bolt):

Lameness Grade – There was no significant difference in lameness grade across time, including baseline, between alcohol and formaldehyde limbs (Figure 2). Few horses exhibited a mild, intermittent (< Grade 1) lameness prior to application of the bolt in some of the alcohol-injected limbs or some of the formaldehyde-injected limbs on various days throughout the study.

Vertical peak force – There was no significant difference in VPF between the alcohol- and formaldehyde-injected limbs, including at baseline or at any time point (Figure 3).

Induced Lameness (with bolt):

Lameness Grade - The alcohol- and formaldehyde-injected heels had a significantly lower (P < 0.04) induced lameness grade (with bolt) when compared to the sham or untreated heels (Figure 4A). Induced lameness grade in the sham and untreated heels increased with time (P<0.05). Evaluation of the shape of the lameness curves for alcohol and formaldehyde showed a marked decrease in induced lameness in the first 14 days, a moderate decrease to day 28, and a subsequent decreased plateau phase for the rest of the study (day 112) (P<0.05).

Vertical peak force (percent body weight) – The vertical peak force was significantly greater (P < 0.05) in the alcohol- and formaldehyde-injected heels with the bolt in place when compared to the sham heel or untreated heel with the bolt in place (Figure 4B).

The percent lameness analgesia peaked on day 10 for alcohol (mean 90.3%) and on day 7 for formaldehyde (mean 97.3%) (Figure 4C). The percent lameness analgesia was not significantly different between alcohol- and formaldehyde-injected heels (P<0.08; Figure

2C). After 112 days, the percent lameness analgesia was still reduced by a mean of 42.2% for alcohol and a mean of 65.3% for formaldehyde compared to controls (Figure 4C).

Clinical assessments:

Thermal reaction time – Thermal reaction time was significantly longer (P < 0.05) for the alcohol- and formaldehyde-injected heels than the sham heel overall and on most days post-injection (Figure 5A). Considering all time points, the percent thermal analgesia

(relative potency) was greater for formaldehyde than alcohol (P=0.03; Figure 5B). The percent thermal analgesia produced by alcohol injection peaked on day 3 (mean of

65.3%) then decreased, but remained between 30-50% through the remainder of the study

(Figure 5B). The percent thermal analgesia produced by formaldehyde injection reached a mean of 62.3% on day 3 and stayed elevated throughout the study (between 45-70%)

(Figure 5B).

Skin sensitivity score – There was no significant difference in skin sensitivity to touch among the alcohol-injected, the sham, and the untreated heels (Figure 6). The skin sensitivity score of the formaldehyde-injected heel was significantly decreased (P < 0.02) when compared to the sham and untreated heels (Figure 6).

Pastern circumference – The circumference of the formaldehyde-injected pastern was significantly larger (P < 0.05) and more variable when compared to the alcohol-injected pastern (Figure 7). The circumference of the alcohol-injected pastern did not differ across time whereas the formaldehyde significantly enlarged with time (P < 0.04) (Figure

7).

Histopathology: Nerves injected with alcohol demonstrated primary demyelination (loss of myelin with conservation of axons) and preservation of fiber alignment with an intact epineurium and mild fibrosis. Alcohol injection also caused loss of axons (axon drop out), axon degeneration, fibrosis, and inflammation, all of which were mild. Nerves injected with formaldehyde demonstrated less preservation of fiber alignment than alcohol-injected nerves. The formaldehyde injected nerves also showed subjectively greater loss of axons (axon drop out), moderate-severe axon degeneration, and

subjectively greater fibrosis and inflammation (Figure 8). Mineralization was only noted in nerves injected with formaldehyde.

Figure 2. Graph of baseline lameness grade without bolt.

Figure 3. Graph of baseline VPF without bolt. The force plate picked up a mild, subclinical lameness22 which peaked at Day 21.

Figure 4. A – Graph of lameness grade with bolt in place (mean ± SEM). The alcohol- and formaldehyde-injected heels had a significantly lower (P < 0.04) induced lameness grade (with bolt) when compared to the sham or untreated heels. B – Graph of VPF with bolt in place (mean ± SEM). The VPF was significantly greater (P < 0.05) in the alcohol- and formaldehyde-injected heels when compared to the sham heel or untreated heel indicating partial analgesia of the heels by alcohol and formaldehyde for the duration of the study. * - significantly different than untreated and sham, # - significantly different than untreated, ‡ - significantly different than sham. C – Graph of percent lameness analgesia (mean ± SEM) demonstrating no significant difference in potency or duration of lameness analgesia between alcohol and formaldehyde after intra-neural injection. 15

Figure 5. A – Graph of thermal reaction time (mean ± SEM). Thermal reaction time of the alcohol- and formaldehyde-injected heels was significantly longer (P < 0.05) than the sham heel. * - significantly different than untreated and sham, # - significantly different than untreated, ‡- significantly different than sham. B – Graph of percent thermal analgesia (mean ± SEM). The percent thermal analgesia (relative potency) was greater for formaldehyde than alcohol across all time points (P=0.03).

Figure 6. Graph of skin sensitivity score (mean ± SEM). Only the formaldehyde-injected heels had decreased skin sensitivity (P<0.02). * - significantly different than untreated and sham, # - significantly different than untreated, @- significantly different than all other groups. 17

Figure 7. Graph of pastern circumference across time (mean ± SEM) demonstrating larger circumference in formaldehyde-injected limbs. †– significantly different from alcohol limb. 18

Figure 8. Photomicrograph of sections of palmar digital nerves harvested 16 weeks post- treatment. H&E stain; bar = 50µm. Bielschowsky stain – a silver stain in which axons are stained black; bar = 50µm. Masson‟s trichrome stain in which myelin is stained pink and connective tissue is stained blue; bar = 50µm. Luxol fast blue stain in which myelin is stained blue; bar = 50µm. In the alcohol-injected nerves there is preservation of fiber alignment, loss of axons, axon degeneration, mild fibrosis and mild inflammation. In the formaldehyde-injected nerves, in addition, there is subjectively greater fibrosis and inflammation.

Chapter 4: Discussion

Both 98% medical-grade ethyl alcohol and formaldehyde administered by intra-neural injection produced substantial, but partial, heel analgesia compared to control nerves.

This analgesia plateaued through the 4 months of the study. This lameness analgesia persisted to an average of 53.8% and thermal analgesia persisted to an average of 43.2% through the 4 months of this study, which is likely a clinically relevant reduction in heel sensation of pain. It is unknown how long this plateau in reduced heel sensation to pain could persist, but based on studies done in the human medical field, it may be possible to have some level of pain relief from alcohol injection for as long as 46 months9.

Evaluation of the data in concert suggests that alcohol injection may produce a less complete and less prolonged loss of sensation than formaldehyde. Observations that support this include the lack of heel skin desensitization, the tendency for less lameness analgesia, and the subjectively more preserved nerve alignment and less scarring seen histologically in the alcohol-injected nerves when compared to formaldehyde-injected nerves. These histological changes suggest that conditions post-alcohol injection would be optimal for re-innervation. In contrast, the histological findings post-formaldehyde injection suggest that formaldehyde injection is more of a true “chemical neurectomy” with reinnervation less likely. Regardless, both treatments induced a similar partial analgesia to heel pain for at least 4 months. In support of our interpretation, a rat model

study20 that investigated intra-neural and perineural injection of the sciatic nerve with phenol (another type of fixative) found that function of the affected muscles returned to

74% of control at the conclusion of their 5 month study. This time-line to return to partial sensation or function can be contrasted with return to sensation (and lameness) after nerve transection, as in neurectomy, which has been reported as a mean of 14 months6.

Alcohol induced minimal inflammation based on circumference of the pastern, acceptable incisional healing, and histologic evidence of mild inflammation.

Formaldehyde induced substantially greater inflammation than alcohol which resulted in clinically excessive soft tissue swelling, delayed incisional healing in some horses, and histologically-evident perineural fibrosis and inflammation. The alcohol injection produced primary demyelination and mild axon degeneration with minimal fibrosis. The degree of fibrosis seen with the alcohol permitted a neurectomy to be performed without difficulty. The degree of difficulty in isolating the nerve and dissecting the nerve free of fibrosis was greater in the formaldehyde injected nerves, but could be performed proximal to the injection site. Formaldehyde injection did not provide any measurable benefit over alcohol injection and induced unacceptable soft tissue inflammation. Intra- neural injection of formaldehyde is not recommended without further studies to investigate techniques for soft tissue protection.

Heel skin desensitization (based on the skin sensitivity score) was not different among alcohol-injected, sham, and untreated heels, despite a substantial reduction in sensitivity of the alcohol-injected heel to other causes of heel pain. Possible explanations include a

differential loss of sensation in response to chemical injection based on the type of sensory neurons within the nerve, contributions to skin sensation from branches of the nerve proximal to the injection site, or both. Afferent sensory nerves are comprised of large myelinated A delta fibers and small unmyelinated C fibers21 which may differ in injury severity or mechanism of injury after intra-neural injection of alcohol or formaldehyde.

Heel skin desensitization to touch and pressure was a unique feature of formaldehyde- injected heels not seen in alcohol injected heels. However, both formaldehyde and alcohol injection similarly reduced other measures of heel pain. This may reflect the proposed differential loss of sensation of types of sensory neurons to this chemical.

Additionally the degree of inflammation and tissue disruption caused by the formaldehyde injection may have affected the proximal nerve branches innervating the pastern area.

Our non-euthanasia lameness model using the heel bolt permitted a consistent, temporary induction of heel pain that permitted serial comparative evaluation for each heel.

Application of the bolt could be repeated reliably. The horses did develop a subclinical, but measurable by force plate, increase in sensitivity to the bolt with time in the untreated and sham heels (Figures 2 and 3). This presumably represented soreness at the site of the bolt pressure in these unblocked heels with repeated application. This finding was not associated with formaldehyde or alcohol injection as it was seen in both limbs and had an imperceptible affect on baseline lameness measurements [all horses were grade 1 (mild intermittent) or grade 0 (not lame) at all time points and not different with treatment].

Our model supported our hypotheses that intra-neural injection of alcohol or formaldehyde could produce a clinically relevant, but partial analgesia to the heel that persisted for at least 4 months. The trajectory of our lameness measurements and the histologic integrity of the epineurium suggest that this effect may last for an estimated additional 2-3 months and would be temporary. Our data also supported our hypothesis that formaldehyde would produce some measures of greater potency than alcohol.

In summary and with coalescence of our data, injection of either alcohol or formaldehyde into the palmar digital nerve potently induced analgesia of palmar heel pain (> 67% at peak) and was temporary (~42% analgesia and ~65% analgesia at 112 days for the alcohol- and formaldehyde-injected nerves, respectively). The mechanism of action of the analgesic effect appeared to be primary demyelination (with alcohol injection), and axon degeneration and dropout. Soft tissue inflammation and fibrosis was minimal with alcohol and marked with formaldehyde. Nerve alignment and intact peri-, epi-, and endo- neurium suggest re-innervation could proceed along a regenerative pathway.

Endnotes

a. dehydrated alcohol, American Regent, Inc. Shirley, New York. b. formaldehyde (40% by volume), Fisher Scientific, Fair Lawn, New Jersey. c. Heat lamp analgesia meter, Columbus Instruments International Corp, Columbus,

Ohio. d. Histology/Immunohistochemistry Lab, Comparative Pathology and Mouse

Phenotyping Shared Resource, College of Veterinary Medicine, The Ohio State

University, 1925 Coffey Road, Columbus, Ohio. e. SAS Institute Inc., Cary, North Carolina

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