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DEXAMETHASONE: THE TREATMENT OF HEAVES AND
THE RISK OF LAMINITIS

presented by
CORNELIS JAN CORNELISSE

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PhD. degree in LARGE ANIMAL CLINICAL SCIENCES

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DEXAMETHASONE: THE TREATMENT OF HEAVES AND THE RISK OF
LAMINITIS
By

Cornelis Jan Cornelisse

A DISSERTATION

Submitted
to Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Large Animal Clinical Sciences

2005

ABSTRACT
DEXAMETHASONE: TREATMENT OF HEAVES AND THE RISK OF
LAMINITIS
By

Cornelis Jan Cornelisse

Glucocorticoids are potent anti-inflammatory drugs that are used with great success in the
treatment of recurrent airway obstruction (RAO, also known as heaves) in the horse.
However, up to date clinical studies into an optimal dose have not been performed, and
the Speed of onset of action is unknown. Additionally, data regarding the clinical efficacy
of oral application of glucocorticoids to horses during an RAO-crisis are not available
either. Oral application is easy, and would facilitate the treatment of RAO-exacerbations
by the horse owner. Unfortunately glucocorticoid treatment in the horse has been
associated with reports of laminitis, a debilitating disease of the equine hoof. A vascular
etiology has been hypothesized. Up to date in vivo studies on horses regarding the
modulation of vascular tone by glucocorticoids have not been performed.

The objectives of my Ph.D. studies were: 1) to study the efficacy of various oral
doses of dexamethasone on RAO in horses, and to compare this to the effect of a standard
intravenous dose of 0.1 mg/kg dexamethasone or an equivalent volume of saline; 2) to
compare the clinical onset of improvement in lung function in RAG-affected horses after
0.1 mg/kg intravenous dexamethasone treatment to intravenous saline and 3) to study the
in viva modulation of dexamethasone on vascular responses after intradermal injections
of vasoconstricting or vasodilating substances. The changes in dermal perfusion from the

intradermal injections were studied as changes in dermal temperature with a

thermographic imager and compared between dexamethasone and saline treated horses.

The results showed that all oral applications of dexamethasone were as
efficacious as intravenous dexamethasone in the treatment of RAO in the horse.
Dexamethasone at a dose of 0.164 mg/kg given orally prior to food and intravenous
dexamethasone both resulted in clinically relevant improvement of lung function (as
judged by the APPLMAX) from 6 to 24 hours. Intravenous dexamethasone resulted in a
statistical improvement of lung function afier 2 hours and a clinical improvement afier 4
hours. Intravenous dexamethasone treatment resulted in a Significant decrease of baseline
Skin temperature, indicating an increase in vasomotor tone. The vascular responsiveness
to intradermal injections with al-agonist phenylephrine was greater (i.e. increased
efficacy) and potentiated (i.e. left Shift of the ECSO). The effects of intradermal injections
with vasoconstrictor endothelin-l were superimposed on the lower Skin baseline
temperature, resulting in a greater decrease in skin temperature compared to the saline
treatment. The statistical analysis of the data on nitric oxide controlled vasodilation from
intradermal injections of methacholine was difficult to interpret. The reasons for this
were a rise in skin temperature over time due to anxiety of the horses, likely in
combination with other confounding experimental factors.

In conclusion, oral dexamethasone is an easy applicable and efficacious treatment
for heaves with an onset, magnitude and duration of effect that is comparable to 0.1
mg/kg intravenous dexamethasone. Dexamethasone treatment in horses results in an
altered vasomotor tone with a greater and potentiated response to catecholamines. These
studies indicate that oral and intravenous dexamethasone is of great benefit for the

treatment of RAO, a disease not associated with circulatory risk factors.

Copyfightby
CORNELIS JAN CORNELISSE

2005

Dedication

ACKNOWLEDGEMENTS

This thesis would have been impossible without the help and support of many people.
First of all my warm and Special thanks to my advisor Dr. N. Edward Robinson for his
guidance and stewardship of my PhD program, and his incredible enthusiasm for my
studies. Dear Ed I hope when I am your age I still can be that much involved in projects
as you are. Also many many thanks to Dr. Fred Derksen, Dr. Greg Fink, Dr. Ray
Nachreiner and Dr. Hal Schott who as members of my graduate committee believed in
my projects, and gave me the support and confidence to see it all through. I am in great
great debt to Cathy Berney and Sue Eberberhardt. They were instrumental to the good
ending of all my experiments, some which were done under bizarre weather conditions.
Sorry for the artic exposures and thank you so much for your friendship, support and
putting up with me. Many thanks to Dr. Jeff Bunn, practitioner in Howell, who was so
gracious to lend me his thermograph imager for pilot experiments on intradermal
injections of vasoactive substances in horses. Similarly so many thanks to Christina Kobe
and Dan Boruta for that I always could count on them during the collection of the lung
function data on the horses, even if that sometimes ended up being in weekend hours. Of
course all data collected needed statistical analysis and I owe many thanks to Dr. Joe
Hauptman and Dr. Wilfried Kannaus for their interest in, and help with statistics on my
projects. It appeared that the art of editing papers is not one of my strongest points.
Luckily I could count on the expertise of Vicki Hoelzer-Maddox who did do such a great
job to get everything seemingly easy up to the required standards. Vicki thank you so
much for that. I also like to thank Heather de Feiter-Rupp, Annerose Bemdt and Lisa

Bartner for their upbeat lab spirit and helping me out on several occasions.

vi

Funding and contributions from many sources were essential to the completion of
my Ph.D program. I like to acknowledge the Matilda Wilson Equine Respiratory
Endowment and the Office of the Associate Dean for Research and Graduate Studies of
the College of Veterinary Medicine for their annual stipend for the duration of the
program. Additionally I like to thank the Office of the Dean of the Graduate School of
Michigan State University for a writing grant in support of my thesis. My warm thanks
go to Dr. Hans Swaan, Surgeon and friend of my father, and his wife Wil for financing a
laptop computer which was a great asset to me in analyzing my data and writing this
thesis in and outside the office.

Many thanks to the horses Bright, Casey, Cindy, Dancer, Lacey, Missy, Rusty,
Sioux, Sugar, Sunny, Wendelina and White Fang for their ongoing contributions to
veterinary science.

So many thanks to my run-bike-swim and Ski buddies Amanda, Amy, Bob,
James, Jean, Justin, John, Kent, Lauri, Lyndsay, Mat, Mary, Sarah, Seth, Sue and Tom
for their friendship and the awesome time we spent on great outdoors activities that
helped to keep some sanity in me. I will miss you guys. And finally I like to thank my
mother Alice and brothers Dick and Rogier for their ongoing support during all these

years of continuing education.

vii

TABLE OF CONTENTS

LIST OF TABLES AND FIGURES ................................................................... xi
LIST OF ABBREVIATIONS ........................................................................... xiii
INTRODUCTION ................................................................................................. 1
CHAPTER 1: INTRODUCTION TO GLUCOCORTICOIDS ....................... 3
Nomenclature ........................................................................................................ 3
Structure, activity and metabolism ..................................................................... 3
Mechanism of action ............................................................................................. 7
Intrinsic glucocorticoid potencies ...................................................................... 11

CHAPTER 2: GLUCOCORTICOID THERAPY FOR RECURRENT

 

 

 

AIRWAY OBSTRUCTION IN THE HORSE ..... - - - 22
Recurrent airway obstruction .......... _- - -- - ....... 22
Inflammatory mediators in RAO ...................................................................... 23
Treatment of allergic airway disease: Bronchodilators .................................. 25
Treatment of allergic airway disease: Glucocorticoids in Asthma ................. 26
Treatment of allergic airway disease: Glucocorticoids in RAO ..................... 29
Clinical efficacy of glucocorticoids in RAO ...................................................... 32
Summary Chapter 2 ............. -- - -- ........ 33

CHAPTER 3: EFFICACY OF ORAL AND INTRAVENOUS
DEXAMETHASONE IN HORSES WITH RECURRENT AIRWAY

 

OBSTRUCTION .................................................................................................. 34
Summary .............................................................................................................. 34
Introduction..-...----.--- - .............................................. - 37
Material and Methods ........................................................................................ 38
Protocol 1 .............................................................................................................. 39
Protocol 2 .............................................................................................................. 40
Instrumentation and measurements ....................................................................... 40
Drugs ................................................................................................................... 41
Data analysis ......................................................................................................... 42
Statistical analysis ................................................................................................. 42
Results .................................................................................................................. 42
Protocol 1 .............................................................................................................. 42
Protocol 2 .............................................................................................................. 43

viii

Discussion ........................... -- - - - 45
Conclusion -- - - - - - ........ . - ......... - - 48

CHAPTER 4: GLUCOCORTICOID THERAPY AND EQUINE

 

 

 

 

 

LAMINITIS; FACT OR FICTION - - - .......................... 53
Introduction. 53
IS there clinical evidence for glucocorticoid induced laminitis? - 53
Hypothesis on glucocorticoid induced laminitis - - 55
Relevance of pharmacokinetics ........ 58
Conclusion Chapter 3 - - - ........... 59

 

CHAPTER 5: EFFECTS AND MECHANISMS OF GLUCOCORTICOID

 

 

 

ACTION ON MICROCIRCULATION - 61
The microcirculation .......................................................................................... 61
Systemic regulation ............................................................................................. 62
Local regulation ............ - - - - - - - 63
Vascular effects of glucocorticoids- ........ - - ............................ 65
Implications Chapter 4. ......... - - - -- - - - ...... 68

 

CHAPTER 6: A THERMOGRAPHIC STUDY ON THE IN VI V0
MODULATION OF VASCULAR RESPONSES TO PHENYLEPHRINE

 

 

AND ENDOTHELIN-l lBY DEXAMETHASONE IN THE HORSE ............ 70
Summary ....................................................... . ...... .......... 71
Introduction--- ...... . ........... - -- -- .74
Material and Methods ........................................................................................ 76
Intradermal solutions ................................................................................. 76
Horses ....................................................................................................... 77
Study protocol .......................................................................................... 78
Image and data analysis ............................................................................ 79
Results .................................................................................................................. 80
Baseline skin temperature ......................................................................... 80
PHE-series ................................................................................................. 80
ET-l, antagonists and ET-l plus antagonist combinations (RXS) series. 81
Relative clinical potency of PHE and ET-l .............................................. 81
Environment .............................................................................................. 82
Discussion.-.......... ..... - ....... - - -- - - ----- 82

 

CHAPTER 7: A THERMOGRAPHIC STUDY OF THE IN VIVO
MODULATION OF METACHOLINE INDUCED VASODILATION BY
DEXAMETHASONE IN THE HORSE - -- - - 104

 

ix

Summary........ ..... ....... - - -- - we. - - . -104

 

 

 

 

 

 

 

 

 

 

 

 

Introduction.- - - - -- ---------------- 107
Material and methods- - ............. 108

Horses ..................................................................................................... 109

Study protocol ......................................................................................... 1 10

Image and data analysis .......................................................................... 111
Results- - - - - - - - - - - ----------------------- 112
Intradermal injections (RX) ................................................................................. 112
Strength of association between left and right side ............................................. 113
Environment ......................................................................................................... 1 13
Discussion........................................ ----- - - - - - -- - - 113
Conclusion ......................................................................................................... 115
GENERAL CONCLUSION -- - - ...... ................ 132
APPENDIX A: FOOTNOTES- ........ - ...... - 135
BIBLIOGRAPHY ............................................................................................. 137
Chapter 1 ........................................................................................................... 137
Chapter 2 - - ----- 141
Chapter 3 - 150
Chapter4-- - - - - - - - - 152
Chapter 5 ----- - ------ - - 157
Chapter 6 - --------------------------------- 162
Chapter 7 ............................................................................................................ 167

LIST OF TABLES and FIGURES

 

 

CHAPTER 1

TABLES

Table 1 .................................................................................................................. 12
Table 2 .... - - -- ------------- . 16
Table 3- ----------------------- - - 19
Table 4 .................................................................................................................. 20
FIGURES

Figure 1 .................................................................................................................. 6
Figure 2 .................................................................................................................. 8
Figure 3 ................................................................................................................ 18
CHAPTER 2

TABLES

Table 2.1 ............................................................................................................... 24
Table 2.2 ............................................................................................................... 30
Table 2.3 ............................................................................................................... 31
CHAPTER 3

TABLES

Table 3.1 ............................................................................................................... 50
FIGURES

Figure 3.1 ............................................................................................................. 51
Figure 3.2 ............................................................................................................. 52
CHAPTER 6

TABLES

Table 6.1 ............................................................................................................... 86
Table 6.2 ............................................................................................................... 87
FIGURES

Figure 6.1a ........................................................................................................... 88
Figure 6.1b ........................................................................................................... 89
Figure 6.2a ........................................................................................................... 90
Figure 6.2b ........................................................................................................... 91
Figure 6.2c ........................................................................................................... 92
Figure 6.3a ................................................... ' ........................................................ 93

xi

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6.3b -------------------- 94
Figure 6.3e - - 95
Figure 6.3d - ------- - - 96
Figure 6.3e . - -- ------ 97
Figure 6.3f-.--- ....... . ------- 98
Figure 6.3g --------- 99
Figure 6.3b -- - - 100
Figure 6.4a ------ -- - - ----------------------------------------- 101
Figure 6.4b ......................................................................................................... 102
Figure 6.4c ......................................................................................................... 103
CHAPTER 7

TABLES

Table 7.1 ............................................................................................................. 117
Table 7.2-. ------ . ----------------------------- - - 118
Table 7.3.... -- - -- - -- - - 119
Table 7.4 ............................................................................................................. 120
FIGURES

Figure 7.1 -- - - ---------------- 121
Figure 7.2a- - - -- ------ 122
Figure 7.2b--. ....... - - - - - - -- -- -- 122
Figure 7.2c ......................................................................................................... 123
Figure 7.2d ......................................................................................................... 123
Figure 7.2e -- ------------------ 124
Figure 7.2f. - -- - - - 124
Figure 7.3a--- - -- -- -- - - - -- ..... - -- - 125
Figure 7.3b ......................................................................................................... 125
Figure 7.3c ......................................................................................................... 126
Figure 7.3d ......................................................................................................... 126
Figure 7.3e - - . ..... - -- ..... 127
Figure 7.3f- - - - - ........ 127
Figure 7.4a..-..... ---- - ...... . ------------- - - -- - -- - 128
Figure 7.4b ......................................................................................................... 129
Figure 7.4c -------------------------------------- - ------- -- - - 130
Figure 7.4d ......................................................................................................... 131

xii

ACE
ARG
AT-1
BALF
BCL
BDP
Cdyn
CONC
DEX
DMSO

E050
ECE
ET-1
ETA

ETB
PF
60
GR
HDF
HDNF

LIST OF ABBREVIATIONS

Angiotensin converting enzyme
Argine

Angiotensin type-1 receptor
Bronchoalveolar lavage fluid
Beclomethasone
Beclomethasone dipropionate
Dynamic compliance
Concentration

dexamethasone

Dimethyl sulfoxide

50% effective dose
endothelin converting enzyme
Endothelin-1

Endothelin Type A receptor

Endothelin Type 8 receptor
Fluticasone dipropionate
Glucocorticoid
Glucocorticoid receptor
High dose, food

High dose, no food
Infra-articular
lntradermally

interleukin

interferon

lntravenously

Low dose, no food
NG-nitro-L-arginine methyl ester
Molar concentration
Muscarine receptor, type 2

Muscarine receptor, type 3
Methacholine

Myosin light chain kinase
Matrix metalloproteinase
Methyl prednisolone acetate
Nuclear factor

Nitric oxide

xiii

NOS
PBS
PEF
PHE

PIF

PO
PREDNL
RAO

RL
RR

Nitric oxide synthase
Phosphate buffered saline
Peak expiratory flow
Phenylephrine

Peak inspiratory Flow

Per as (oral)

Prednisolone

Recurrent airway obstruction

Airway resistance
Respiratory rate

Relative receptor affinity
various injection solutions
Saline solution
S-nitroso-N-acetylpenicillamine
Triamcinolone acetonide
Average temperature
Tracheal epithelial lining fluid
Time

triamcinolone acetonide
Tumor necrosis factor

Tidal volume
Treatment

Minute volume

Vascular smooth muscle cell

alpha adrenergic receptor, type1

BZ-adrenergic receptor, type 2

Maximal intrapleural pressure change (cm H20)

xiv

INTRODUCTION

Recurrent airway obstruction (RAO; also known as heaves) is an allergic and
inflammatory condition of the lower airway of the horse that can be induced by exposure
to organic dusts that are associated with feeding roughage and stabling on straw bedding.
The disease has many Similarities to human asthma, including bronchospasm, mucus
production and inflammatory cell infiltrates into the lower airway.

Glucocorticoids are potent anti-inflammatory drugs that successfully are used for
the treatment of exacerbations of RAO in horses. Literature indicates that the effects by
glucocorticoids are due to genomic and non-genomic actions. Genomic effects by
glucocorticoids involve the modulation of the transcription of cellular proteins. This can
result in transactivation or transrepression of these products. Non-genomic effects
involve cytosolic post translational modifications of proteins in cellular pathways. These
effects are faster and hypothesized to be more prominent with higher doses of a
glucocorticoid. In the light of this new literature it is no surprise that glucocorticoid
therapy can result in desired as well adverse effects.

Dexamethasone is a glucocorticoid that is used with great success for the
treatment of exacerbations of RAO in the horse. However up to date no clinical dose-
response studies have been conducted regarding the systemic or oral application of this
drug. Owners and veterinarians would be very interested to know what a minimal
efficacious dose of an easy way to administer glucocorticoid would be for the treatment
of RAO.

Glucocorticoid therapy in the horse has unfortunately been overshadowed by

reports of induction of laminitis, a debilitating disease of the equine foot. The reports are

 

‘_.....

few and the exact nature of how glucocorticoid treatment can result in this complication
in horses is unknown. The broad actions by glucocorticoids suggest that it is likely that
glucocorticoid induced laminitis follows the same pathways that have been hypothesized
to be important in the etiology of clinical laminitis from severe disease. This includes a
vascular model. The in vivo vascular effects of glucocorticoids in horses have not been
studied.

From the above it is clear that there exist many questions regarding the efficacy
and safety of glucocorticoid therapy in horses. This Ph.D. thesis reports on 3 studies with
dexamethasone in horses in order to help to answer some of these questions. Study 1
(chapter 3) reports on a study with different doses of oral dexamethasone in the treatment
of heaves, and compares this to the efficacy with a standard intravenous dose. Speed of
onset is additionally studied as well. In regard to the possible etiologies of glucocorticoid
induced laminitis, Study 2 and 3 investigate the in vivo effects of dexamethasone
treatment on the peripheral vasculature. Study 2 (chapter 6) reports on how
vasoconstrictive responsiveness is modulated by glucocorticoid therapy, while study 3
(chapter 7) describes the finding on vasodilatory responsiveness. The additional chapters
function to put the results of these studies in a better perspective. They include literature
discussions on the mechanisms of actions by glucocorticoids and the relationship

between glucocorticoid use in the horse and laminitis.

 

 

CHAPTER 1
INTRODUCTION T O GLUCOCORTICOIDS

Nomenclature

Glucocorticoids belong to a drug group that iS used for their potent anti-inflammatory
actions in medicine. They are structurally related to the bodies natural “stress hormone”
cortisol. It was the publication in 1949 by Hench et al. (Hench, Kendall et al. 1949), in
which they reported dramatic improvement of rheumatoid arthritis after administration of
cortisone, which sparked the interest in the anti-inflammatory actions of these drugs.
Cortisone is an inactive precursor form of the hormone cortisol. Cortisol has a steroid
structure similar to cholesterol and is produced in the zona fasciculata of the cortex of the
adrenal gland. Hence the initial name corticosteroid or corticoid for this drug group.
Cortisol has many functions in the body. It appears however that the anti-inflammatory
effect of corticosteroids parallels their key function involving the regulation of
carbohydrate metabolism (gluconeogenic effect) (Schimmer and Parker 1995). A recent
meeting of the European League Against Rheumatism (EULAR) regarding nomenclature
and treatment regimes suggested therefore that the name glucocorticoid should be
preferred over corticosteroid in order to avoid any confusion with the other adrenal
steroid hormones (aldosterone, androgens) that are produced in the adrenal cortex

(Buttgereit, da Silva et al. 2002) but which have different functional properties.

Structure, activity, and metabolism
Glucocorticoids are synthesized in the cortex of the adrenal gland from modifications of

cholesterol. AS such, glucocorticoids consists of a Similar 4-ring, 21-carbon atom

 

 

In.“ A"
I

framework. The rings are called A, B, C and D. Modifications to this framework
determines the level of glucocorticoid and mineralocorticoid activity, relative potencies,
and pharmacokinetic features of a glucocorticoid. Several of the carbon atoms have
Shown to be key positions regarding these characteristics (Figure 1). The 4,5 double bond
and 3-keto group on ring A are an absolute requirement for glucocorticoid activity. The

hydroxyl group (OH-group) on C-21 of ring-D is an absolute requirement for

 

mineralocorticoid activity. On the other hand the presence of a hydroxyl group on the l 1-

carbon atom (C-1 1) is important for glucocorticoid activity at local tissue level. It appears

 

that the configuration of this group is determined by the local balance in activities of two
isoforms of the enzyme 11-beta—hydroxysteroid dehydrogenase (respectively abbreviated
as 11-6- HSD-1 and 11-3- HSD-2). Oxidation (also referred to as dehydrogenation) of
this C-ll hydroxyl group results in the inability of the glucocorticoid to bind to the
cytoplasmic glucocorticoid receptor (GR). The glucocorticoid will as a consequence be
less active in that tissue. The picture is complicated by the fact that the two isoforms of
11-3— HSD have bidirectional action with distributions and equilibriums that are tissue
specific. For example, ll-B- HSD-2 is more prevalent in epithelial tissues such as tubular
epithelium of the kidney, colonic mucosa, Skin, epithelium of the airways as well
vascular smooth musculature (Smith, Maguire et al. 1996) with a primarily oxidative
action (leading to inactivation). 11 43- HSD-1 is, however prevalent in most tissues,
including liver and fat tissue, and has an equilibrium to the reductive Side (leading to
activation)(Li, Obeyeserkere et al. 1997; Brereton, van Driel et al. 2001). Examples of
this prereceptor metabolism are the inactivation of prednisolone into inactive prednisone

by 11-6- HSD-2 in the kidney and conversion of inactive cortisone into active cortisol

(hydrocortisone) by 11-8— HSD-1 in the liver. Consequently, renal tubular epithelium is
therefore highly insensitive for prednisolone, while liver and fat tissue contribute to
maintenance of systemic and tissue levels of cortisol (Rask, Olsson et al. 2001).
Introduction of a double bond at the 1,2 position of the A-ring increases glucocorticoid
potency and causes an enhanced glucocorticoid to mineralocorticoid potency ratio.
Halogenation, of the C-9 group results in additional increases in glucocorticoid as well as
mineralocorticoid potency. These halogenations, usually with fluorine, tilt the
equilibrium for ll-B— HSD-1 more to the reductive Side and make glucocorticoids more
resistant to 11-6- HSD-2 (Oelkers, Buchen et al. 1994; Ferrari, Smith et al. 1996; Li,
Obeyeserkere et al. 1997; Diederich, Hanke et al. 1998). AS a result the halogenated
glucocorticoids remain mainly in the activated form in tissues, including that of the renal
tubular epithelium. (Diederich, Hanke et a1. 1996; Diederich, Hanke et al. 1998).
However, it appears that the unwanted improvement in mineralocorticoid potency can be
eliminated by adding substitutions on C-16. Consequently almost all modern
glucocorticoids are therefore halogenated (budesonide is an exception) and have
additions to C-16. Additionally substitution and esterification at C-16, but also at C-17 or
C-21 with an acetate, propionate or valate group will result in an increase in lipophility

and local tissue availability of the glucocorticoid.

 

 

 

Dexamethasone

Figure 1: Chemical structure of dexamethasone. Compared to cortisol a double
bound has been added between C1 and C2. C-9 is halogenated with fluorine and a
methyl group is present on C-16.
These substitutions, therefore, also optimize glucocorticoids for use in long acting depot
formulations (Dahlberg, Thalen et al. 1983). Final elimination of glucocorticoids is
accomplished on basis of reduction of the 4,5 double bonds in hepatic and extrahepatic
tissues followed by final reduction of the 3-keto group in the liver. In the liver and kidney
these A-ring reduced steroids are subsequently conjugated with sulfate or glucuronide to

the 3-hydoxyl group, and excreted with the urine (Liddle 1961; Dahlberg, Thalen et al.

1983; Schimmer and Parker 1995).

Mechanism of action

The biological actions of glucocorticoids, especially in relation to their anti-inflammatory
properties, have been attributed to modulation of gene transcription. This involves a
pathway that starts with the lipophilic glucocorticoid hormone acting as an intracellular
ligand by binding to a cytoplasmic glucocorticoid receptor (GR). The ligand-bound GR
translocates to the nucleus and activates a glucocorticoid response element (GRE) on the
genome. The activated GRE subsequently influences transcription of nearby genes by
either activating (transactivation) or repressing (transrepression) their transcription. This
results in respectively more or less messenger RNA (mRNA) and therefore ultimately
more or less protein products from mRNA-translation (Baxter and Tomkins 1971;
Merkulova, Merkulov et al. 1997). This classical pathway (figure 2) from GR-occupation
to alterations in transcription of the genome needs time, explaining why the first effects
with most application of glucocorticoids are not observed before 2 to 4 hours after
administration. Recent studies indicate that this view of the genomic pathway of
glucocorticoid effects is too simplistic. It appears that the GR exists as GRa or GRB
splice variants that differ in the last 50 C-terminal amino acids. As a consequence GRfi is
unable to bind with a glucocorticoid. GRB also exists mainly as a free monomer in the
cytoplasm and nucleus while GRoz is sequestered in the cytoplasm as a multi-protein
complex that includes heat-Shock chaperone molecules (hSp-90 and hSp-70) and src, a
member of the MAP-kinase family. Binding of a glucocorticoid to GRor results in
dissociation of the multi-protein complex followed by dimerization of GRs. The dimers
can be in GRa/GROL, GRor/GRB or GRfi/GRfi formats. These GR-dimers translocate in the

nucleus and bind to selective DNA sequences called glucocorticoid response elements

(GRES). However only GRa/GRa-dimer binding results in full transactivation or
repression, while dimers containing GRB have impaired activity (Bamberger, Schulte et
al. 1996). The ratio GRa to GRB appears therefore to influence the level of glucocorticoid
activity. The effect on transactivation and transrepression by the ligand bound GRu/GRa-

dimers is modulated in several ways.

 

L'l's PG:
Gcs . i it
AA
82-Adrenoceptors GR
I-KB .

     

 

..

.g'jx W\ —+ 100x.2

mRNA INOS

 

 

Nucleus +GRE "635

Figure 2. Genomic and non-genomic pathways by glucocorticoids.
See text for abbreviations

 

 

 

 

For a start, the dimers can with the help of additional coactivator complexes (e.g. CBP,
p300, P/C AF etc), switch on nearby genes (Merkulova, Merkulov et al. 1997; Pelaia,
Vatrella et al. 2003). The result is newly formed mRNA that will be translated into

protein products within the cytoplasm. These products have specific anti-inflammatory

actions (e.g. IL-10 and lipocortin inhibit cPLA2) and additional effects (e.g. a new 6-
adrenergic receptor). Transactivation by ligand bound GR-dimers can also result in the
production of a Specific inhibitor (IKB) for the nuclear factor NF-KB. NF-KB appears to
have a key role in the transcription of mediators of inflammation (Barnes, Pedersen et al.
1998). Binding of cytosolic NF-KB by IKB results consequently in strong down regulation
of transcription of inflammatory interleukins and cytokines (e.g. TNFoz, RANTES) or
mediator-synthesizing enzymes (COX-2, iNOS, cPLA2). AS an alternate variant there is
evidence that ligand-bound GR-monomers can directly bind and inhibit the nuclear
factors NF-KB and AP-l factors (Barnes, Pedersen et al. 1998; Tuckerman, Reichardt et
al. 1999; Adcock and Caramori 2001).

It has, however, been suggested that transactivation and production of anti-
inflarnmatory proteins only contributes in a small amount to anti-asthmatic effects of
glucocorticoids (Pelaia, Vatrella et a1. 2003). Direct repression of genes is considered a
more important pathway via which glucocorticoids result in anti-inflammatory effects.
Repression is thought to result from steric hindrance in cases where binding of the GR-
dimers on the GRE also partially overlaps TATA boxes of other genes. The expression of
NF-KB and AP-l is indeed repressed in this manner (Hofrnann, Hehner et al. 1998;
Jaffuel, Demoly et al. 2000). Because steric hindrance is, compared to multi-factor
transactivation, relatively easily achieved, it has been suggested that (direct) repression
might require relatively lower concentrations of synthetic glucocorticoid to have effect
(Adcock and Ito 2000).

There is evidence that glucocorticoids can have faster effects than would be

expected from the 2-hour window of the transcription/repression machinery. Examples of

this are the fast lymphopenia after glucocorticoid administration, the fast relief of status
asthmaticus with prednylidene, the fast resolution of an Addisonian crisis with
fludrocortisone and treatment of spinal Shock or cerebral edema with dexamethasone or
methylprednisolone (Schmidt, Gerdes et al. 2000). These observations suggest therefore
the existence of additional pathways besides the modulation of transcription. (Buttgereit,
Brand et al. 1999; Croxtall, Choudhury et al. 2000; Buttgereit, da Silva et al. 2002;
Buttgereit and Scheffold 2002; Croxtall, van Hal et al. 2002). It appears that these rapid
effects are more prominent in higher dose ranges, what has been postulated to be the
result of an overflow effect after all GRS have been saturated with GC (Buttgereit, Brand
et al. 1999; Buttgereit and Scheffold 2002). Recent studies have uncovered some of the
non-genomic effects of glucocorticoids. For instance src, liberated from the GR-
chaperone multi-protein complex afier GC binding, will activate cytoplasmic lipocortin-
]. This activated lipocortin-1 will bind and inactivate Grb2, a protein that plays a central
role in the cellular signal transduction via MAPK-kinase pathways after, for instance,
epidermal growth factor receptor (EGF-R) activation. Normally the activated MAPK-
kinases result in phosphorylation and activation of cPLA2, an enzyme that catalyses the
release of arachidonic acid from membrane phospholipids. These phospholipids are
subsequently converted into inflammatory leukotrienes and prostaglandin by enzymes
that include COX-2. The blocking of the MAPK-kinase pathways therefore causes
cPLA2 to remain in its inactive form (figure 2b) and results in a decrease of leukotriene
and prostaglandin production (Croxtall, Choudhury et a1. 2000; Croxtall, van Hal et al.
2002). Another non-genomic pathway of glucocorticoid action might be the influence on

calcium and sodium cycling across cell membranes and the increased proton permeability

10

across the inner mitochondrial membrane. It is thought that the basis for these effects is
the insertion of the lipophilic glucocorticoid molecules in the membranes with
consequently a change in physicochemical membrane properties. The clinical effects
likely arise from decreased cytosolic calcium availability and energy production and thus
influence on cell function and protein synthesis. A decreased ion cycling makes cells also
less prone to development of edema. (Buttgereit, Brand et al. 1999; Buttgereit and

Scheffold 2002).

Intrinsic glucocorticoid potencies

Glucocorticoids are used for their anti-inflammatory and immunosuppressive actions.
Many clinical studies have indeed reported on dosage, administration and efficacy of
glucocorticoids for treating inflammatory disease in human beings (Bolland and Liddle
1958; Liddle 1961; Derendorf, Hochhaus et al. 1993). However good comparable data on
anti-inflammatory potencies between glucocorticoids, and for the species and clinical
indications they are used for is still lacking. Some data involved studies on relative
eosinopenic potencies or adverse effects in human beings (Derendorf, Hochhaus et al.
1993). However most comparative data studied bioassays on rodents or in vitro
lymphocyte proliferation. General inferences from these investigations is now known as
the classical table for comparative anti-inflammatory effects of glucocorticoids (table 1)
(Liddle 1961; Melby 1977; Schimmer and Parker 1995).

Unfortunately there are some clinical observations that are not completely in line with
this classical classification. For instance, why is it that a less potent glucocorticoid can

have as strong side effects as one that is regarded as more potent? Or why, in case of two

11

equipotent glucocorticoids, is one more effective in treating obstructive airway disease or
Spinal injury? It is likely that some of these clinical discrepancies are based on
differences between Species and differences between the inflammatory models used. For
instance some bioassays studied the effect of topical glucocorticoid application on
granuloma inhibition or experimental induced edema in extremities of rats while other
assays investigated thymolytic activity (Lerner, Turkheimer et a1. 1964; Tonelli, Thibault

et al. 1965; Dahlberg, Thalen et al. 1983).

Table 1: Classical and updated anti-inflammatory potencies as determined
by inhibition in a lymphocyte proliferation test. Mineralocorticoid activity (MA)
and relative receptor affinities (RRA) are provided as well. Data were

a
obtained from (Schimmer et al. 1995 ) and modified from (Mager et al.

 

 

 

 

 

 

 

 

 

2003)

Glucocorticoid classic“ updatedID MA” RRA"
Fluticasone 156.2 0.0 202.3
Budesonide 32.1 0.0 94.6
Beclomethasone 3.6 0.0 5.8
Dexamethasone 20-30 9.3 0.0 11.1
Betamethasone 20-30 7.6 0.0 6.4
Triamcinolone 5.0 13.9 0.0 26.1
prednisolone 4.0 1.8 0.8 1.8
Cortisol 1.0 1.0 1.0 1.0

 

 

 

 

 

 

 

The in vitro studies on lymphocytes in turn focused on the ability of glucocorticoids to
inhibit phytohemagglutinin (PHA) induced lymphocyte proliferation (Cantrill, Waltman
et al. 1975; Langhoff and Ladefoged 1983; Spahn, Landwehr et a1. 1996; Mager,
Moledina et al. 2003).

In vivo, the response of cells to a glucocorticoid is the result of the interplay

between 1) local concentration of free active glucocorticoid, 2) the ability of the cell to

12

transduce the glucocorticoid signal and 3) the genomic effects by the glucocorticoid.
(Bamberger, Schulte et al. 1996). The concentration of free and active glucocorticoid is
dependent on the pharmacokinetic properties and metabolism of the glucocorticoid. Thus
for instance acetate esterifications will enhance lipophility and local tissue availability
(Dahlberg, Thalen et al. 1983). Similarly, fluorination of glucocorticoids causes
decreased binding to plasma proteins like corticosteroid binding globulin (CBG),
decreased metabolism by the liver and decreased inactivation at local tissue level by the
11-6- HSD-isotypes. As a consequence, halogenation results in higher free and active
glucocorticoid concentrations at the local tissue level.

The relative ability to transduce the glucocorticoid Signal in the cell depends on
the affinity of the GR for the glucocorticoid, and the subsequent ability of the activated
GR to modify transcription. Glucocorticoids need to bind to the GR in order to activate it.
The number of activated GRS is therefore an very important factor for the level of
influence on the genome (Baxter and Tomkins 1971). Although the GR number per cell
can differ between tissues (Damon, Rabier et al. 1985), it appears to be within a 1/2 log
order. Therefore, unless large numbers of modified (=unresponsive) GRS are present, GR
number per cell is a relatively minor factor for explaining differences in potency between
glucocorticoids. It is therefore possible to compare relative glucocorticoid potencies on
basis of receptor affinity for a GC between different tissues or species. Affinity is a net
result from an equilibrium between the rate of association between GC and GR (defined
as constant KASS) and the rate of dissociation of the GC-GR complex (defined as constant
K9133). The net result, is reflected in the equilibrium dissociation constant Kn

(KD=KD|ss/KA33), which is an indicator for GC—GR complex stability and half life. Thus

13

an increased rate of association or a slower dissociation will result in longer GR
occupation by glucocorticoids. Consequently relatively less glucocorticoid should be
required to accomplish the same effect. KD is, therefore inversely correlated with affinity
of the GR for the GC. The number of occupied GRS translates in genomic effects, and
thus a glucocorticoid for which the GR has a low KD will have more occupied receptors
at a lower concentration, and thus genomic effects at lower concentrations. Therefore GC
receptor affinity translates in potency of the glucocorticoid. Indeed a linear relationship
has been established between glucocorticoid affinity for the GR (as determined by
glucocorticoid-GR half life) and glucocorticoid potency (Dahlberg, Thalen et al. 1983;
Spahn, Landwehr et a1. 1996; Brattsand and Axelssson 1997; Mager, Moledina et al.
2003). It appears that the KASS is almost equal amongst all glucocorticoids and therefore
it is the K9155 that mainly determines the stability of the activated glucocorticoid receptor
complex, and thus the K0 (Yeakley, Balasubramarrian et al. 1980; Rohdewald, Mollrnann
et al. 1985). Despite these differences in KD between the glucocorticoids, the binding
studies also Show a maximal receptor occupation (saturation) for fluoroglucocorticoids
between 10'7 to 10'6 M (Chen, Kohli et al. 1994) while that for cortisol and prednisolone
is approximately an order higher at 10"5 M. Acetylations and modifications to the D-ring
that cause enhanced lipophility is also one of the factors that cause differences in GR
affinity and consequently explain some of the increased potencies of drugs like
triamcinolone and dexamethasone (Manz, Grill et al. 1982). The result is also the
existence of a correlation between lipophility and potency.

Recently it has become possible to compare glucocorticoids on basis of their

ability to modify transcription with the help of gene expression assays. Although not

14

completely independent of GR—affinities these genomic expression assays allow
distinction between either agonist or antagonist activated GRS and integrate this effect
with cell line specific metabolism (Jaffuel, Roumestan et al. 2001). The genomic effects
can be plotted as effective dose-response curves (Jaffuel, Roumestan et al. 2001) from
which a more cell type specific trans-activating and trans-repressive effect of
glucocorticoids can be compared (table 2). Comparative potencies can be inferred from
these curves as the ECso which is the effective dose that results in a 50% change of
transcription. For instance in the A549 human lung carcinoma cell line and the HeLa
human epithelioid cervix cell line it appears that triamcinolone iS actually a more potent
transactivator of a GRE-dependent luciferase gene than dexamethasone and
betamethasone. In the classical order triamcinolone is however 5 fold weaker compared
to dexamethasone and betamethasone. Similarly, in a HTC rat hepatoma cell line,
triamcinolone is at least as equipotent as dexamethasone and betamethasone in the
induction of the gluconeogenic enzyme tyrosine aminotransferase (TAT) (Jaffuel,
Demoly et al. 2000; Jaffuel, Roumestan et al. 2001). On the other hand beclomethasone-
diproprionate (BDP), a popular anti—asthmatic glucocorticoid used for inhalation

treatment, has one of the weakest intrinsic potencies (table 2).

15

Table 2: Genomic potencies of glucocorticoids on transactivation and transrepression.

The data are calculated from E050 — values that were reported in gene expression
assays (Jaffuel D. et al, Am J Respir Crit Care Med 2000; 57-63)

 

 

 

 

 

 

 

 

 

 

Trans-activation 3:5er 36:11:?)
Glucocorticoid _ _

huciferase PIF;- act’iiiaation ac'iliSaIIchIn meIEtEiin

eLa cells cells

PF 66 12.5 50 10 12.5
Budesonide 27 1 8.3 2.2 3.3
TM 1 9 1 1 1 1
Dexamethasone 1 1 1
Betamethasone 9 1
BDP 6 NM 1 .3 0.5 0.5
Prednisolone 3
Cortisol 1

 

 

 

 

 

 

 

 

Also marked differences between transrepression potencies of glucocorticoids
have been reported with these gene expression assays. So in a HeLa and A549 cell lines,
fluticasone propionate (FP) is the most potent repressor for AP-l and NF-KB, and this
correlates well with the ability of this drug to suppress RANTES transcription.
Beclomethasone-diproprionate on the other hand is surprisingly weaker than
triamcinolone or budesonide (table 2) (Jaffuel, Demoly et al. 2000). RANTES is an
important cytokine in the pathogenesis of human asthma and therefore fluticasone seems
an excellent choice as a therapeutic agent against asthma. Unfortunately fluticasone also
has the strongest potential to induce gluconeogenic adverse effects (table 2). The gene
expression studies indicate that different glucocorticoids have different EC50 for different
cellular effects whether it is transactivation or transrepression. However at least for the
repression of AP-l, it appears that the relative potencies between glucocorticoids is
independent even of cell type (Jaffuel, Demoly et al. 2000). It also appears that the

approximate maximal difference in an EC50 for an effect between the modem

16

glucocorticoids is Slightly more than 1 log (===10-15x). For most effects this will translate
into glucocorticoid concentrations between 10'9 and 10'7 M. The reported concentration
necessary for maximal genomic effects is 10'7 to 10'6 M, which is similar to the earlier
discussed values obtained from glucocorticoid receptor affinity (KD) studies. Overall no
transactivation/repression activity is seen at glucocorticoid concentrations below 10'10 to
10"2 M (Jaffuel, Roumestan et al. 2001).

The genomic data are a helpful aid in better understanding certain clinical
phenomena. For instance, inhalation therapy of glucocorticoids results in local tissue
levels that are in the nanomolar range while systemic concentrations are around 10'10 M.
Comparing the EC 50’s for the repression of RANTES, AP-l and NF-KB suggests that FP
is likely to be a far more effective glucocorticoid than BDP to treat airway inflammation
with such low local concentrations. However, unlike BDP it is also likely that FP will
cause unwanted systemic gluconeogenic effects. (Roumestan, Henriquet et al. 2003). The
KD and EC50 values for glucocorticoids Show that genomic effects of glucocorticoids
manifest themselves for concentrations between 10'10 and 10'6 M. Additionally at high
doses with complete receptor saturation a trans—activating or trans-repressing effect
willbe of Similar magnitude between different glucocorticoids. However duration of
actioncan still differ and non-genomic effects could become an important factor as well.
Indeed, and as discussed before, non-genomic mechanisms of a glucocorticoid can
explain some of its specific individual clinical effects. For instance betamethasone, which
is equipotent to dexamethasone in the classical order, appears to have far less non-
genomic potency, aS measured by its ability to inhibit convalin-A stimulated respiration.

The result is therefore a Slower effect. Similarly prednilydene, a weak glucocorticoid in

17

the classical order, has stronger non-genomic potency than dexamethasone (figure 3)

(Buttgereit, Brand et al. 1999).

serouerod engmea

 

Genomic Non-Genomic

Figure 3: Comparison of relative genomic and non-genomic effects by the
glucocorticoids prednislone (PRED), betamethasone (BETA), dexamethasone
(DEX) and prednylidene (PREDNYL).

The non-genomic effects can also differbetween glucocorticoids. For instance even in

L 1 LL J' ' A
l' r

high doses, potent glucocorticoids like , budesonide,
fluticasone propionate dexamethasone and triamcinolone (table 3) (Croxtall, Choudhury
et al. 2000; Croxtall, van Hal et al. 2002). Therefore drugs like fluticasone propionate that
have strong genomic down regulation of cell growth and COX-2 production can still be
relatively ineffective in blocking the production of specific mediators like LTD4, a potent

bronchoconstrictor. It is thus interesting to note that fluticasone propionate recently has

become available as a compounded combination (Advair Diskus®) with the
bronchodilator salmeterol for the treatment of human asthma.

Table 3: Relative glucocorticoid potencies-on PGE2 production \tia C0302
repression and non-genomic cPLA2 inhibition (NS=-’no’ Based on E050

Glucocorticoid cPLA2 COX—2 PGE2

         

The new insights into genomic and non-genomic potencies have lead to a review of the
standardized nomenclature and dosage regimens for glucocorticoids. A recent EULAR
convention (Buttgereit, da Silva et al. 2002) produced a dosage advisory that is based on
the fact that 100 mg prednisolone results in virtually a complete GR saturation in a
normal adult person, or equal to a tissue concentration in the order of 10-5 M. The
recommended doses are:

Low dose: < 7.5 mg prednisolone/day, equal to 42 % receptor occupation

Medium dose: > 7.5 but 90 mg prednisolone/day, equal to > 63 % receptor occupation.
High dose: > 30 mg but 5100 mg prednisolone/day, equal to 100% receptor saturation.
Very high dose: 2100 mg prednisolone/day.

Extrapolation from this recommendation suggests that, according the classical order of
glucocorticoids, dexamethasone therapy in horses at a dose of 0.1 mg/kg would be

categorized as a high dose equal to 35-42 mg prednisolone per person. For several

 

glucocorticoids, plasma and local tissue concentrations in horses have been reported
(table 4). Even when adjustment for molar mass is taken into account, it appears that
intra-articular concentrations exceed for a considerable time the KB and EC50
concentration ranges that produce genomic effects. From the potencies for non-genomic
effects, methylprednisolone acetate would therefore likely be a better clinical choice than
triamcinolone acetonide. Although plasma concentrationss are at best in a low activity
range, it is likely that lipophility of these drugs causes adequate tissue concentrations and

receptor occupation.

Table 4: Serum and intra-articular concentrations of glucocorticoids in horses. Serum and local
concentrations are in ng/ml. Molar concentrations can be estimated by dividing the values with the molar
masses Mw. The Mw for these lucocorticoids is in the range of 392-434.

 

 

 

 

 

 

CONCENTRATION (nglml)
DRUG DOSE APPLICATION "Etta-
serum articular
Peak 24-h 48-h 24-h 96-h
DEX ’ ’ 0.01 mglkg IV once 20-35 <2.5
0.04 mglkg IV q24h 1.9
4 5 6 0.27 pg/kg Eye q8h 0.1-0.5 <0.06
TAA ’ ’ IV once
3 x 6 mg IA once 4.3 2 <10 7500 10
7 30 mg IA once 2.4 1.8
PREIgNSL 2.2 mg/kg IV once 1700 <10
MPA 100 "'L IA once <10 <10 20000 <100

 

 

 

 

 

 

 

 

Data interpretated from 1(Chen, Zhu et al. 1996), 2(Hoffsis and Murdick 1970),3(Spiess, Nyikos et al.
1999),4 (French, Pollitt et al. 2000), 5(Chen, Sailor et al. 1992), 6 (Koupai-Abyazani, Yu et al.
l995),7(Peroni, Stanley et al. 2002) and 8(Lillich, Bertone et al. 1996)

Summary

Glucocorticoids are potent anti-inflammatory drugs. Recent research shows that their
mode of action is via genomic and non-genomic pathways. Genomic pathways are the
result of transactivation or transrepression of transcription and take several hours to
become effective. Non-genomic effects involve post translational modifications of

cellular proteins and have rapid effects. The potency of glucocorticoids is associated with

20

affinities of the glucocorticoid receptor for the glucocorticoid and lipophility of a

glucocorticoid.

21

CHAPTER 2
GLUCOCORTICOID THERAPY FOR RECURRENT AIRWAY OBSTRUCTION

IN THE HORSE

Recurrent airway obstruction

Recurrent airway obstruction (RAO) in the horse is a lower airway disease of the middle-
aged and geriatric horse (Couetil and Ward 2003). Similar to human asthma a genetic
predisposition is suspected (Gerber and Bailey 1995; Marti et al. 1991; Marti et al. 2003).
The disease is characterized by recurrent attacks of respiratory distress when horses are
stabled indoors for considerable periods of time (Thomson and McPherson 1984;
Vandenput et a1. 1998). The clinical Signs during such an RAO crisis include coughing,
pronounced nostril flaring, an increased abdominal component to the respiration and an
increased respiratory rate. Crackles and wheezes are audible upon auscultation of thorax
(Dixon et a1. 1995; Gerber 1968; Robinson et al. 2003; Robinson et al. 2001; Robinson et
al. 2000). In more severe cases, the breathing can become very labored with an obvious
expiratory push and horses can become hypoxemic (Dixon 1978; Vandenput et al. 1998).
The association with a challenging stall environment suggests a hypersensitive
inflammatory response to inhaled allergens that are present in this indoor milieu. This is
supported by studies that Show that organic substances such as thermophilic spores and
endotoxin indeed can trigger clinical exacerbations of RAO (Derksen et al. 1988; Pirie et
al. 2003a; Pirie et al. 2003b; Pirie et al. 2002; Robinson et al. 1996; Woods et al. 1993).
Straw bedding and roughage are the primary sources for organic dusts and therefore

control of the environment is mandatory. Indeed changing the bedding to wood shavings

22

or newspaper shred, and wetting the hay can decrease the amount of exposed allergens
with consequent attenuation or subsidence of the clinical signs (Kirschvink et al. 2002;
Thomson and McPherson 1984; Vandenput et al. 1998; Woods et al. 1993). Similarly
removal of these horses to an outdoors environment can cause the horses to go into
remission with complete resolution of the Signs within 3 days (Jackson et al. 2000;
Vandenput et al. 1998). New exposure to a challenging stall environment or hay will

however result in rapid reoccurrence of the signs.

Inflammatory mediators in RAO

The inhaled allergens are known stimuli to epithelial cells and local resident macrophages
to produce inflammatory cytokines (Hammond et al. 1999; Jackson et al. 2004; Liden et
al. 2003; MacKay et al. 1991). These mediators of inflammation cause a massive
neutrophil influx into the lower airway of the horse (F airbaim et al. 1993) accompanied
by airway bronchoconstriction and increased mucus production (Robinson et al. 2003;
Robinson et al. 2001; Robinson et al. 1996). The bronchoconstriction and mucus
production cause a narrowing of the airway lumen, with an increase in airway resistance
as a result, hence the respiratory distress. Increased airway wall thickening due to
inflammatory edema or mucus cell hyperplasia/metaplasia are considered additional
causes for airway lumen obstruction (Robinson et al. 2003; Robinson et al. 1996). It is
also thought that repeated episodes eventually can result in remodeling and scarring of
the airway and lung tissue (Nevalainen et al. 2002). The production of the inflammatory
mediators is the result from up regulated expression of their genes. The activation of

these genes is under control of transcription factors such as NF-KB and AP-l. Indeed in

23

 

recent studies on RAO-affected horses the level of NF-KB activity in cells from bronchial
epithelial brushings and in cells from bronchoalveolar fluid (BALF) was higher
compared to that of healthy horses exposed to the same environmental challenges during
crisis as well as remission (Bureau et al. 2000b; Sandersen et al. 2001). The level of NF-
KB activity in turn correlated well with the level of clinical airway obstruction (Bureau et
al. 2000a). In addition a recent study reported that during the acute phase of a crisis (table
1) the expression and concentration] of the key regulatory cytokines interleukin-16 (IL-
18) and tumor necrosis factor-or (TNF-or) is Significantly higher in the BALF of RAO
horses compared to BALF of healthy horses (Giguere et al. 2002). Additionally,
depending on the timing of the BALF samples, a Significantly greater expression of the
interleukins IL-4, IL-8, IL-13 and interferon-y (INF-y) in RAO-affected horses has been

reported (table 2.1) (Ainsworth et al. 2003; Bowles et al. 2002; Giguere et al. 2002).

Tabel 2.1: Expression and levels of cytokines In BALF cells over time compared between control
and RAD-affected horses
EXPRESSION LEVEL EXPRESSION LEVEL EXPRESSION

 

Data from Ainsworth D.M. et al Vet Immunol Immunolopathol 2003;83-91
2 Data from Giguere S. et al Vet Immunol Immunolopathol 2002;147—158
V A 2 Significant change within RAO horses compared to baseline

Long term exposure to a challenging environment results in increased expression of INF-
‘y and IL-8. However the elevations in expression are not always significantly higher
compared to initial remission values (e.g. IL-8, IL-13), or do not always result in

significantly increased levels of active protein product (e.g. TNF-or, IL-16, IL-4) in the

24

BALF (table 1). Overall during remission or crisis it appears that in RAO-affected horses
only INF-y is persistently elevated when compared to healthy control horses, while the
expression of other cytokines such as IL-13 actually is decreased (table 2.1). Therefore
up regulation of expression of other cytokines such as RANTES or inflammatory
products like leukotriene LTB4 (Lindberg et al. 2002; Lindberg et al. 2004), nitric oxide
(Costa et al. 2001; Hammond et al. 1999) or matrix metalloproteinase MMP-9
(Nevalainen et al. 2002; Raulo et al. 2000) might have more pathophysiologic

Significance.

Treatment of allergic airway disease: Bronchodilators

Lower airway inflammation, bronchoconstriction, increased mucus production
and airway remodeling are also clinical features of human asthma. It is thus no surprise
that the treatment of RAO in horses has been following the many of the methods used to
combat human asthma. Modern treatment of RAO consequently focuses on
bronchodilation for improvement of ventilation during crisis, and control of the
inflammatory cascade in order to prevent new attacks and stop airway remodeling
(Robinson et al. 2001; Tesarowski et al. 1994; Thomson and McPherson 1983).
Additionally mucolytics and expectorantia have been used in order to break up and
increase the clearance of mucus. Anticholinergic drugs such as atropine (0.02 mg/kg IV),
glycopyrolate (0.007 mg/kg IV) or ipratropium (360 rig/kg IH q6h) and methylxanthine
derivates have Shown to be successful bronchodilators for treating exacerbations of RAO
in the horse (Robinson et al. 2001). However Bz-agonists, likely as a result of their

massive use in the treatment of human asthma, have become a more popular application.

25

Various fiz—agonists have been used with success in the treatment of RAO in horses. The
reports include oral (clenbuterol; 0.8-3.2 jig/kg q12h), intravenous (clenbuterol; 0.8-3.2
ug/kg q12h) and inhalation (albuterol 360-720 jig/kg q3h, fenoterol;1000 mg, pirbuterol
600-1200 ug/kg q12h) applications (Derksen et al. 1999; Erichsen et al. 1994; Mair
1996; Mazan et al. 2003; Robinson et al. 2000; Rush et al. 1999; Tesarowski et al. 1994;
Tomeke er al. 1998). AS an additional benefit it appears that BZ-agonists stimulate the
mucociliary escalator (Dixon 1992; Turgut and Sasse 1989). It is likely that the resulting
improvement in clearance of obstructive mucus aids in improvement of ventilation. The
continued use of fiz-agonists has been associated with a decrease in the number of 62-
agonist receptors on equine lymphocytes (Abraham et al. 2002). It is thus likely that,
similar with the use of these drugs in human asthma (Nishikawa et al. 1996), this also
happens in the equine lower airway and renders these drugs less effective on the long

term.

Treatment of allergic airway disease: Glucocorticoids in Asthma

Glucocorticoids were introduced for the treatment of human asthma in the late fifties
(Blanchon et al. 1956; Turiaf et al. 1956). However it was the introduction of inhalation
therapy one decennium later (Kravis and Lecks 1966), in combination with a better
understanding of these drugs, that resulted in their exponential use. Genomic and non-
genomic mechanisms form the basis of the potent anti-inflammatory actions of
glucocorticoids. The magnitude of these effects, as discussed earlier in chapter I, are
dependent on many factors including the intrinsic properties of the glucocorticoid, the

number and affinity of the glucocorticoid receptors (GR) in the tissue, the genome of the

26

 

tissue type and the local tissue concentrations of the glucocorticoid. It appears that
number and affinity of GRS between tissues or Species is relatively constant (Damon et
al. 1985; Rohdewald et al. 1985), as is the affinity of the GRS for an individual
glucocorticoid (table 1, chapter 1 ). In addition, with the exception of cortisol and
prednisolone, it appears that the modern glucocorticoids are insensitive to metabolism
and inactivation by 11-[3—HSD-2 (Diederich et al. 1998; Feinstein and Schleimer 1999; Li
et al. 1997). Therefore the type of airway cell, the intrinsic potency of a glucocorticoid
for a desired effect (e.g. inhibition of LTB4, , table 2 chapter 1 ), dose and the
pharmacokinetics of that glucocorticoid (e. g. inhalation vs. oral) are the main factors that
determine the success of a chosen glucocorticoid in the treatment of asthma. Indeed
reported anti-inflammatory effects by glucocorticoids in human asthma include 1)
increased apoptosis and a lower influx of eosinophils, the key inflammatory cell type in
asthma (Debierre-Grockiego et al. 2003; Druilhe et al. 2003; Walsh et al. 2003) 2)
decreased NF-KB expression in lung (A549) epithelial cells (Roumestan et al. 2003) 3)
decreased expression of mucus genes (MUC-S) in goblet cells or lung (A549) epithelial
cells (Fergie et al. 2003; Lu et al. 2005) 4) decreased cytokine production by stimulated
alveolar macrophages (Leung et al. 2004) and 5) decreased levels of cytokines and other
inflammatory mediators such as NO in tracheal epithelial lining fluid (TELF), BALF or
breath condensate (Eum et al. 2003; Gelb et al. 2004; Hoshino et al. 1999; Lehtimaki et
al. 2001; Leung et al. 2004; Pelaia et al. 2003; Umland et al. 2002; Yuan et al. 2003) and
6) decreased MMP-9 expression in bronchial biopsies of asthmatics (Hoshino et al.
1999). On the other hand glucocorticoids have been ineffective in down regulating

expression of 1) NF-KB in bronchial biopsies and alveolar macrophages of asthmatic

27

patients or human airway (BEAS-2B) epithelial cells (Hart et al. 2000; Newton et al.
1998) 2) mucin expression (MUC-S) in bronchial biopsies from asthmatics (Groneberg et
al. 2002) 3) iNOS in human bronchial epithelial cells (Donnelly and Barnes 2002) and 4)
MMP-9 expression in human alveolar neutrophils (Cundall et al. 2003). Thus while it is
clear that glucocorticoids down regulate key pathways relevant to the inflammation in
human asthma, the exact interaction between them and different cell types still needs
further Study. Nevertheless several important clinical observations regarding
glucocorticoid therapy for asthma have been made. For instance, it appears that the
addition of a glucocorticoid to fiz-agonist therapy results in additional improvement of the
airway resistance compared to the use a Bz-agonist alone. This effect is significant within
1-2 hours, has a duration of 10 hours (Storr et al. 1987) and results in lower hospital
readmission rates (Littenberg and Gluck 1986; Scarfone et al. 1993). It is likely that this
additive effect can be explained by lesser degrees of mucus production and inflammatory
airwall thickening and based, on the speed of onset, might include non—genomic actions.
The addition of glucocorticoids to Bz-agonist therapy also antagonizes Bz-agonist induced
reduction in fig-agonist membrane receptors (Mak et al. 1995a; Mak et al. l995b).
Glucocorticoid therapy, therefore contributes to successful Bz-agonist therapy with the
use of lower needed doses. Despite apparent poor suppression of MMP-9 expression of
alveolar neutrophils it appears that longer term inhalation glucocorticoid therapy still
results in decreased remodeling and scarring of the lung (Lee et al. 2004; Trigg et al.

1 994).

28

 

 

Treatment of allergic airway disease: Glucocorticoids in RAO

The successful use of glucocorticoids in the treatment of human asthma was soon
followed by the introduction of these drugs for the treatment of RAO in horses. The first
studies reported on the application 25 mg dexamethasone-alcohol via repeated
intramuscular injections (Gerber 1969; Muylle and Oyaert 1973). Much later this was
followed by clinical efficacy studies on the intramuscular application of the longer acting
formulations of dexamethasone-21-isonicotinate (Robinson et al. 2002) and
triamcinolone acetonide (Lapointe et al. 1993), oral applications of prednisone (Jackson
et al. 2000; Robinson et al. 2002; Traub-Dargatz et al. 1992) and inhalation therapy with
beclomethasone (Ammann et al. 1998; Rush et al. 1998a; Rush et al. 1998b) and
fluticasone-diproprionate (FP)(Giguere et al. 2002). All these studies, with the exception
of all reports regarding oral application of prednisone, Showed significant improvement
in parameters of lung functions after at least 3 days of treatment. The improvement was
ofien as good as remission values [FP, 2000 pg, IH, q12h for 21 days; (Giguere et a1.
2002)], [DEX, 0.1 mg/kg, IV q24h for 3 days; (Robinson et al. 2002)] or when compared
to maximal bronchodilation with atropine [DEX, 0.04 mg/kg, IV q24h for 7 days;
(Picandet et al. 2003)]. Recently dexamethasone treatment (0.1 mg/kg q24h, 7 days) was
reported to result in significant improvement of mucus production and cough frequency
(Robinson et al. 2003). The failure of prednisone has been attributed to the poor oral
availability and the lack of conversion of this drug to the active metabolite, prednisolone,
in the liver (Peroni et al. 2002).

The improvements in lung function, mucus production and cough by glucocorticoids in

horses with RAO are likely the result of down regulation of the inflammatory response.

29

However Similar to the studies of human asthma at the moment it is unclear which

inflammatory pathways are down regulated. Currently it is not known what the effect of

glucocorticoid therapy is on the up regulation of expression of the transcription factor

NF-KB during an exacerbations of RAO. So far only one study has reported on cytokine

profiles in BALF after glucocorticoid therapy in horses with exacerbations of RAO

(Giguere et al. 2002). In this study 2000 pg fluticasone propionate q12h for 3 weeks

resulted in a decreased expression of INF-y compared to healthy control horses or RAO

Tabel 2.2: Effect of fluticasone-
diproprionate treatment (TX) on
cytokine expression in cells from BALF
of horses in crisis of RAO compared to
no treatment (NO-TX) or healthy
controls.

 

CYTOKINE EXPRESSION

 

 

TX vs CONTROL] rx vs NO-TX

 

 

 

 

    

 

 

,, -‘ _ . TNF-a
lL-1B fl IL-1B
IL-4 ‘ ‘- '
IL-5

IL-8 || lL-8

 

 

 

. - ' . . . .
I: - y...

I POST 3 WEEKS r=r=1

1FP 2000 pg IH Q12H

 
   

horses that received no treatment (table 2.2).
Additionally IL-4 was Significantly decreased
compared to no treatment. The decrease in
INF-y and IL-4 did correlate with the
improvement of lung function, however many
of the other up regulated cytokines were not
down regulated. Similarly glucocorticoid
therapy of horses in a RAO crisis has not
consistently resulted in a decrease of
neutrophilic inflammation in the lower airway.
Most studies report significant improvement of

neutrophil percentages in BALF of RAO

horses in crisis when compared to untreated RAO controls (Giguere et al. 2002; Lapointe

et al. 1993; Robinson et al. 2003; Robinson et al. 2002; Rush et al. 1998a). However in

most of these reports, the earlier improvement in respiratory parameters did not associate

with a Significant temporal decrease of the neutrophil response (Giguere er al. 2002;

30

 

 

’1‘“ ‘th at

Lapointe et al. 1993; Robinson et al. 2003; Robinson et al. 2002; Rush et al. 1998a).
Contrary to their effect on eosinophils in human asthma, it appears that glucocorticoids
actually inhibit apoptosis of neutrophils (Cox 1995; Liles et al. 1995; Nittoh et al. 1998;
Zhang et al. 2001; Zhang et al. 2002). This effect is dose-dependent and varies in potency

amongst different glucocorticoids (table 2.3).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2.3: Glucocorticoid potencies for Maximal inhibition is achieved at
apoptosis
HUMAN NEUTROPHIL APOPTOSIS concentrations of 10'6 M and in the
-9
GLUCOCORTICOID ECSO (10 M) order of 50 to 90 % (Liles et al.
-9

1 (10 M) 1995; Zhang et al. 2001; Zhang et
CORTISOL 38 i 5
PREDNISOLONE1 13 i 15 al. 2002). AS a consequence

1
DEXAMETHASONE 8 :l: 4 neutrophils will reside longer in
BECLOMETHASONE1 20 a; 0.3 . . .
1 srtes of rnflammatron. Although
BUDESONIDE 0.8 :l: 0.2
FLUTICASONE1 0.6 1 01 reports on apoptosis of equine
2

MOMETHASONE 0-17 1' 0-03 neutrophils by glucocorticoids are
1 Zhang, X. at al: Life sciences 2002 (71) ; . . _ . _ . _ .
£523.15“ ' lacking, It IS likely that Inhibition
36253-29, X. et al: EurJ Pharmacol 2001 (431); of apoptosis is a key factor for the

poor temporal decrease of BALF neutrophils. This delay of neutrophil apoptosis iS also
associated with prolonged or increased activity of these cells. So in human neutrophils
the MMP-9 activity is poorly inhibited (Cundall et al. 2003) while superoxide production
actually is up regulated (Cox 1995). Functional changes have also been documented for
equine neutrophils after exposure to glucocorticoids and include enhanced stimulated
migration (Morris et al. 1988) and increased superoxide production (Guelfi and Kraouchi

1989). Based on the dramatic improvement in clinical response it is therefore likely that

31

yet to define down regulation of other cell types, or inflammatory pathways, by

glucocorticoids have more relevance in RAO.

Clinical efficacy of glucocorticoids in RAO

The clinical data indicate that glucocorticoids are valuable drugs for the treatment of
RAO in the horse. Despite these data, comparative reports on ideal dose and dose-interval
are still lacking. So far the improvement of lung function in RAO affected horses by
glucocorticoids has only been reported from 24-h on afler inhalation therapy (Rush et al.
1998b) or 3 days or more after parenteral application of various glucocorticoids in
different doses (Ammann et al. 1998; Giguere et al. 2002; Lapointe et al. 1993; Picandet
et al. 2003; Robinson et al. 2003; Robinson et al. 2002). However genomic and non
genomic mechanisms, and human clinical studies (Storr et al. 1987) indictate that a faster
response is more than likely. Ideally the therapy should be at a low dose to minimize
adverse effects, be rapidly effective and easily applicable with a view towards
maintenance therapy. Additionally the dose and dose interval Should be flexible so it can
be adjusted pending clinical improvement and environmental Situation. In human
medicine these requirements are all easily met by inhalation therapy through which a
relativly small amount of potent glucocorticoid can be deposited at the needed location,
and with minimal induction of systemic side effects. Although reported successful in the
treatment of RAO, inhalation therapy of glucocorticoids in horses is cumbersome, as well
as relatively expensive, due to the need of special delivery devices. It also appears that
even low doses of the relativly weak inhalation steroid beclomethasone still results in

systemic adrenocortical depression (Rush er al. 1998c), thereby taking away an important

32

 

 

rational for this type of application. Contrary to prednisone, the reported oral
bioavailability in fasted horses for the glucocorticoids prednisolone and dexamethasone is
quite good and in the order of 61% (Cunningham et al. 1996; Peroni et al. 2002).
Therefore this way of application should have promise as a flexible, easily administrable

and cheap method of treatment for RAO in the horse.

Summary chapter 2

Based on the previous discussion of the literature on glucocorticoid treatment of RAO in
the horse it seems important that future studies focus on: 1) which cell types and
mediators are down regulated and correlate with clinical improvement; 2) how fast do
glucocorticoids result in clinical improvement of lung function; 3) is oral application as
efficacious a parenteral application, and what is the influence of roughage on this; and 4)
what is the ideal dose for achieving all these effects. Therefore the following chapter will
report on a study to address points 2,3,4 regarding the treatment of RAO in horses with

dexamethasone.

33

CHAPTER 3:
EFFICACY OF ORAL AND INTRAVENOUS DEXAMETHASONE IN HORSES

WITH RECURRENT AIRWAY OBSTRUCTION

Cornelis J. Cornelisse, N. Edward Robinson, Cathy E.A. Berney, Christina A. Kobe,

Daniel T. Boruta and Frederik J. Derksen

Pulmonary Laboratory, Department of Large Animal Clinical Sciences, College of

Veterinary Medicine, Michigan State University, East Lansing, MI 48824-1314 USA.

Corresponding author:

Cornelis Jan Cornelisse

Department of Large Animal Clinical Sciences
College of Veterinary Medicine

Michigan State University

East Lansing, MI 48824-1314

Telephone: 517-353-9710

Fax: 517-432-1042

email: comelis@cvm.msu.edu

Keywords: horse; dexamethasone; RAO; pulmonary function

Word count: 4324 (including title page and summary)

34

Summary

Reasons for performing the study: Although the efficacy of dexamethasone for the
treatment of recurrent airway obstruction (RAO) has been documented, the
speed of onset of effect and its duration of action are unknown, as is the efficacy
of orally administered dexamethasone with or without fasting.

Objectives: To document the time of onset of effect and duration of action of a
dexamethasone solution when administered intravenously or orally with and
without fasting.

Methods: Protocol 1 used eight RAO-affected horses with airway obstruction in a
crossover design experiment that compared the effect of intravenous (IV)
administration of saline and dexamethasone (0.1 mg/kg) on pulmonary function
over 4 hours. Protocol 2 used six Similar horses to compare, in a crossover
design, the effects of dexamethasone (0.1 mg/kg) IV, dexamethasone (0.164
mg/kg) P0 with and without prior fasting, and dexamethasone (0.082 mg/kg)
P0 with fasting.

Results: Dexamethasone IV caused significant improvement in lung function within
2 hours with a peak effect in 4-6 hours. Similarly oral dexamethasone was
effective within 6 hours with a peak effect at 24 hours when administered orally
at a dose of 0.164 mg/kg prior to feeding. The duration of effect was for all
dexamethasone treatments statistically significant for 30 hours when compared

to saline and tended to longer duration of effect when used orally.

35

 

Dexamethasone given orally at a dose of 0.164 mg/kg to fed horses had
comparable mean effects as dexamethasone at a dose of 0.082 mg/kg PO given
to fasted horses indicating that feeding decreases bioavailability.

Conclusions: Intravenously administered dexamethasone has a rapid onset of action
in RAO-affected horses. The oral administration of a bioequivalent dose of the
same solution to fasted horses is as effective as IV administration and tends to
have longer duration of action. Fasting horses before oral administration of
dexamethasone improves the efficacy of the treatment.

Potential relevance: The oral administration to fasted horses of a dexamethasone
solution intended for IV use provides an effective treatment for RAO-affected

36

Introduction

Recurrent airway obstruction (RAO) in horses is caused by exposure to organic dust that
can be present in the horse's environment. Because stables contain a great deal of organic
dust, stabling horses is one of the major predisposing factors associated with this disease.
The exposure to organic dust can result in a lower airway inflammation characterized by
increased mucus production, contraction of the bronchial smooth muscle and thickening
of the airway wall. Airway obstruction that impairs breathing is the result.
Glucocorticoids (GCS) have been used successfully to relieve the Signs of RAO in horses
(Lapointe et al. 1993, Ammann et al. 1998, Rush, et al. 1998, Robinson et al. 2002,
Robinson et al. 2003). Most of the effects of GCS are attributed to the down-regulation of
the cellular transcription machinery for mediators of inflammation, either directly via
glucocorticoid response elements (GRES) or indirectly via NF-KB pathway (Jaffuel et al.
2000, Sandersen et al. 2001, Giguere et al. 2002). The down-regulation of inflammation
results in reduction of excess fluid and secretions, as well as decreased production of
bronchoconstrictor mediators.

An easy and economical way for the horse owner to administer GCS would facilitate
treatment of horses known to be suffering from RAO. In general, intermittent bolus
dosing and applications that bypass the systemic circulation are preferred over
systemically long-acting formulations of GCS in order to minimize potential metabolic
side effects. Oral GC administration provides an inexpensive and convenient alternative
for therapy of RAO and allows flexibility and control over dose and dose interval. Oral

bioavailibility for GCS in fasted horses has been reported for prednisolone and

37

dexamethasone and is in the order of 61% of the total administered dose (Cunningham et
al., 1996, Peroni et al. 2002). Despite the widespread use of parenteral and oral
dexamethasone for treatment of RAO, the earliest measurements of its efficacy have been
after 3 days of administration. The speed of onset and duration of action of
dexamethasone in the equine airways has not been determined. Furthermore, feeding
could negatively influence oral bioavailability and consequently efficacy. For these
reasons, we investigated the following questions: 1) How rapid is the onset of action of
dexamethasone? 2) Is a Single oral dose (P0) of a dexamethasone solution as efficacious
as a single intravenous (IV) dose in the treatment of RAO in horses? 3) Is the efficacy of
orally administered dexamethasone affected by roughage feeding?

Increasingly veterinary clinicians are using a dexamethasone solution intended for
intravenous use as an oral application because it is convenient to administer and
inexpensive. Although owner and veterinary reports suggest satisfactory results there are
no clinical studies of efficacy. Therefore the efficacy of this dexamethasone solution was

investigated.

Materials and methods

The study used two protocols, which were approved by the All-University Committee for
Animal Use and Care at Michigan State University. The aim of protocol 1 was to
examine the speed of onset of action of dexamethasone on change in pleural pressure
(APplmax) while protocol 2 studied the efficacy of orally and intravenously administered

dexamethasone as measured by the onset, duration and magnitude of effect on several

38

respiratory parameters. Both protocols used RAO-affected horses from the Pulmonary

Laboratory herd that normally are kept at pasture.

Protocol 1

Eight horses (seven mares, one gelding, 13 to 31 years old, 384 to 583 kg) affected with
RAO were used in the protocol. These included Six Quarter Horses, one Hanoverian and
one Arabian. At the start of the experiment the horses were brought in from pasture,
bedded with straw and fed hay that was of poor quality in order to induce lower airway
obstruction. When horses started to Show clinical signs of lower airway obstruction,
pulmonary function was measured. When the maximal change in pleural pressure during
tidal breathing (APplmax ) was 25 cm H20 or more (measured without a facemaSk), the
horses were randomly assigned to one of the following treatments: 1) saline IV or 2) 0.1
mg/kg bwt dexamethasone IV. The horses remained instrumented and cross-tied in the
stall over a 4-hour period, during which APplmax was measured at 10, 30, 60, 90, 120,
150, 180, 210 and 240 minutes after injection. Immediately after the 240-minute
measurement, atropine (0.02 mg/kg bwt. IV) was given to induce maximal
bronchodilation. Fifteen minutes later a final pulmonary function measurement was
made. The treatments were separated by a minimum of 14 days and followed a crossover

study design.

39

Protocol 2

Six horses (five Quarter Horses and one Hanoverian) that were affected with RAO were
used. All were mares that were 13 to 31 years old and weighed between 404 and 555 kg.
At the start of the experiment the horses were brought in from pasture and lung function
was measured to confirm that they were in remission (APplmax < 15 cm H20 with
facemask). The horses were thereafter bedded on straw and fed poor quality hay. Prior to
feedings in the morning, pulmonary function measurements were made if clinical signs
suggested an acute exacerbation of RAO. When the APplmax was 25 cm H20 or more
(with facemask), the horses were randomly assigned to one of the following five
treatments: 1) 0.1 mg/kg bwt. dexamethasone IV, 2) an equivalent volume of saline IV, 3)
0.082 mg/kg bwt. dexamethasone PO (low-dose non-fed; LDNF), 4) 0.164 mg/kg bwt.
dexamethasone PO (high-dose, non-fed; HDNF) or 5) 0.164 mg/kg bwt. dexamethasone
PO 1 hour after roughage feeding (high-dose fed; HDF). The intravenous dexamethasone
and saline groups were fed roughage 1 hour prior to treatment while for the HDNF and
LDNF groups a minimum of 1 hour was allowed after treatment before the horses were
fed. Subsequently pulmonary function was measured at 6, 24, 30, 48 and 72 hours after
treatments. The horses remained in the stall during that period. The treatments were

separated by a minimum of 14 days and followed a randomized crossover design.

Instrumentation and measurements
For both protocol 1 and 2, APplmax was estimated by measurement of esophageal pressure

with a latex condom sealed over the distal end of a polypropylene catheter (3 mm inside

40

diameter, 4.4 mm outside diameter, 240 cm long). The balloon was passed via the nareS
to the distal third of the esophagus and connected via a pressure transducer (Validynel,
Model DP/45-35) to either a portable physiograph (Dash 112) in protocol 1 or a respiratory
function computer with data acquisition software (Buxco BioSystem XA3) for protocol 2.
The final position of the catheter was adjusted to obtain the maximal APplméx during tidal
breathing and was thereafter secured by taping it to the halter. For protocol 2 airflow rate
also was measured by use of a pneumotachograph (No. 5 F1eisch4) fitted in a facemask
that was placed over the horse's muzzle. The system was sealed with a rubber shroud and
tape. The pneumotachograph was connected to a transducer (Validynel, Model DP/45-22)
and the respiratory function computer. The flow Signal was integrated to provide tidal
volume. Prior to measurements, volume was calibrated by means of a 2-liter syringe and

pressure by a water manometer.

Drugs

A commercial solution of dexamethasone5 intended for IV use was chosen for both oral
and IV administration. The IV dose (0.1 mg/kg) was chosen because it has been
demonstrated to be effective for treatment of RAO (Rush, Raub et al. 1998; Robinson,
Jackson et al. 2002). The higher oral dose (0.164 mg/kg) was based on the IV dose
adjusted to be equipotent based on published bioavailability. The lower oral dose was 50
percent of the high dose. The saline dose was standardized to a volume equal to that of

the injected dexamethasone formulation.

41

Data analysis

Data were collected over a 2-minute period or a minimum of 10 breaths. For protocol 1
the APplmax was calculated from the difference between peak inspiratory and peak
expiratory pressure of at least 10 breaths. Swallows and sighs were not included. Data of
at least 10 breaths were averaged in protocol 2. The measured parameters included
APplmax , tidal volume (VT ), respiratory rate (R), minute ventilation (me), peak
inspiratory flow (PIF) and peak expiratory flow (PEF). The APplmax, flow and VT during
breathing were additionally processed to calculate pulmonary resistance (R1,) and dynamic

compliance (Cdyn).

Statistical analysis
Data were analyzed with the aid of statistical software6 by two-way ANOVA for repeated
measures with time and treatment as main effects. The Student Newman-Keul's test was

used for post hoc comparison between means. A significance level of 0.05 was chosen.

Results

Protocol 1

There was no Significant difference between the baseline values of APplmax for the 2
treatments, indicating comparable levels of obstructive disease. There were significant
effects of time and treatment as well as a significant time x treatment interaction. Post hoc

comparison showed Significant differences in APplmax between dexamethasone IV and

42

saline that occurred between 120 and 240 minutes after treatment. Additionally, at these
times, APplmax was also significantly less than at baseline (Fig 3.1). Following
dexamethasone IV the decrease in APplmax corresponded to an improvement of 36% and
59% at 120 min and 240 min, respectively. The APplmax after atropine treatment was
significantly different from 240 minutes in both the saline as well as the dexamethasone

treated group.

Protocol 2

Baseline values (0 hours) were not significantly different among all treatment groups,
indicating a similar level of airway obstruction at the start of the experiments. During
remission, there were no differences among treatments with the exception of Cdyn, which
was significantly greater for HDF than for saline or HDNF.

There were Significant main effects of treatment on APplmax, PIF, Cdyn and RR and
significant effects of time were on APpImax, Cain, PIF, PEF, RR, TV and Vmin. Significant
treatment x time interactions were detected for APplmax, Cdyn, RL, PIF, PEF and RR (Fig
3.2, Table 3.1). For all variables measured, there were differences between saline and
dexamethasone treatments but no differences among the treatments themselves.

All of the dexamethasone treatments resulted in a Significant decrease of APplmax at
6, 24 and 30 hours when compared to saline. At 48 and 72 hours, the APplmx after HDNF
continued to be statistically Significantly better than after saline administration .
Compared to saline treatment, all dexamethasone treatments resulted in a significant

improvement of Cdyn at 6 hours. This continued to be the case for dexamethasone IV and

43

HDNF at 24 hours, while HDNF was still effective at 30 hours. Treatment with
dexamethasone IV resulted in a Si gnificant reduction of RL at 6 and 24 hours. Treatments
HDF and HDNF caused Significant improvement in R1, at 30 hours while HDNF was also
effective at 48 hours. All dexamethasone treatments resulted also in significantly lower
PIF at 6 hours when compared to the saline treatment. This effect continued at 24 hours
for HDNF and LDNF, whereas at 30 hours only HDNF caused a Significant improvement
over saline. All dexamethasone treatments resulted in a Significant improvement of PEF
at 6 hours when compared to saline. Treatment with dexamethasone IV, HDF and LDNF
caused significant improvement of the respiratory rate (R) at 6 hours. All treatments
were better than saline at 24 and 30 hours. HDNF and LDNF as well HDF, HDNF and
LDNF were still effective at 48 and 72 hours, respectively.

In the time within treatment comparisons (Fig 3.2, Table 3.1), the APplmax was, in the
case of dexamethasone IV, significantly less than baseline values at 6 and 24 hours and
also not significantly different from remission values at these times. Additionally, the
APplmax after HDNF was Significantly less than baseline at 24 hours and it was also not
different from remission at this time. Also in the case of RL, dexamethasone IV at 6 and
24 hours resulted in values Similar to those in remission and Significantly different from
baseline. At 6 hours the treatments dexamethasone IV and LDNF resulted in PEF values

as good as remission and significantly different from baseline.

44

Discussion
Glucocorticoids are well accepted as treatment for exacerbations of asthma in people.
They additionally appear to have an important role in maintenance therapy and can
prevent remodeling of the lung in the long term (Trigg et al. 1994, Hoshino et al. 1999).
The effects of GCS are attributed to the down-regulation of the cellular transcription
pathways responsible for inflammation, which is reported to take several hours to become
fully effective (Schimmer et al. 1996). Despite a relative slower onset of action compared
to bronchodilators, GCS are a very useful adjuvant therapy in treating exacerbations of
asthma. In asthmatic patients the addition of prednisone or prednisolone to the initial
bronchodilator therapy results in lower hospital admission rates compared to placebo
when reassessed 4 hours after admission to an ER (Littenberg et al. 1986, Scarfone et al.
1993). The addition of a Single treatment with an oral or intravenous GC to these patients
results in increased peak expiratory flow rates within 1 to 2 hours compared to a placebo
(Storr et al. 1987). The duration of this additive difference was approximately 10 hours.
Protocol 1 Showed that dexamethasone in horses with RAO has a Similar time course
of the effect as GCS in human patients. Intravenous dexamethasone administration
resulted in a significant improvement of APplmax beginning 120 minutes after
administration. The average improvement at this time was 36.5%. After 4 hours, the
APplmax Showed approximately 59% improvement to a APplmax of 17.71 cm H20. This
improvement is comparable to the lowest value after dexamethasone IV in protocol 2
found at 6 hours (56% compared to the within-treatment baseline). These observations

suggest therefore that significant effects of down-regulation of transcription of

45

inflammatory mediators occurs within 2 to 6 hours after dexamethasone administration in
the horse. Although maximal effect was achieved between 4 to 6 hours, atropine given at
the end of protocol 1 still resulted in improvement of APplmax, indicating that maximal
bronchodilation had not been achieved.

Protocol 2 showed that orally administered dexamethasone solution was as effective
as dexamethasone IV. The oral bioavailability of GCS is around 61 % in horses that are
fasted or receive the drug with some grain. This results in peak blood levels within 1-2
hours (Cunningham et al. 1996, Peroni et al. 2002). This rapid absorption is the reason
that all oral treatments caused Significant improvement in APplmax by 6 hours. Based on
the measurement of APplmax, all dexamethasone treatments were effective for up to 30
hours. Our data further suggest that 0.164 mg/kg dexamethasone administered before
feeding had beneficial effects for up to 72 hours. Although no Significant differences were
found between the dexamethasone treatments themselves, the means of our data suggest
that oral applications of dexamethasone, especially HDNF, prolongs duration of action in
comparison to IV administration. Indeed, if a p <0.1 is chosen in the case of APplmax the
HDNF would be better than HDF and LDNF at 24 hours, and better than HDF or DEX IV
at 30 and 48 hours. This therefore strongly suggests that oral application results in a
longer duration of action and that feeding impairs bioavailability. Although prolonged
benefits of orally administered dexamethasone were observed for up to 72 hours, some of
these effects would not be clinically visible to the horse owner. Published studies from
our laboratory Show that at a clinical score > 5 clinicians can confidently detect signs of

RAO. This clinical score of 5 correlates with a APplmax of 24.5 i 1.1 cm H20 when

46

 

“T31

 

measured while horses wore a facemask (Robinson et al. 2000). Translated to an owner's
point of view, our data suggest that dexamethasone IV and HDNF provide acceptable
improvement for up to 24 hours in horses that are in a severe exacerbation of RAO.

The APplmax is determined by changes in both airway caliber (indicated by Cdyn and
RL) and breathing strategy (indicated by PIF, PEF and RR) (Robinson et al. 1999). Both
Cdyn and RL are indicators of the severity airway obstruction, with Cdyn reflecting
inhomogeneous ventilation of the more peripheral airways, and R1, the resistance in the
larger airways. Consistent with previous reports (Lapointe et al. 1993, Ammann et al.
1998, Rush et al. 1998, Robinson et al. 2002) this study showed that dexamethasone
improved Cdyn as well RL in horses with RAO. The additive beneficial effects of
dexamethasone on Cdyn and RL contributed to the significant overall effect on APplmax.
During airway obstruction, horses with heaves try to maintain me. For this they alter
breathing strategy, either via changes in V], R, APplmax or combinations thereof and this
consequently affects flow rates (Petsche et al. 1994). Protocol 2 showed that horses in all
treatment groups maintained constant me and VT. However, after the dexamethasone
treatments similar Vmin and VT could be generated with lower RR, PIF, and PEF. Hence
the reductions in respiratory rate and airflow added to the significant reduction in APplmax.
Our investigation did not identify the reasons for the lower respiratory rate. One
possibility is improved gas exchange and a reduction in the severity of hypoxemia.
However, Derksen et al. (1982) demonstrated that the increased respiratory rate that
followed aerosol antigen challenge of sensitized ponies was due to vagal inputs from the

inflamed lung. It is possible therefore that dexamethasone, by reducing inflammation,

47

decreased the activation of pulmonary vagal afferents that participate in the regulation of

respiratory rate.

Conclusion

Intravenous and oral administration of a dexamethasone solution is effective in treating
exacerbations of RAO in horses. The effect of dexamethasone becomes clinically relevant
within 6 hours post administration. An oral dose of 0.164 mg/kg is clinically effective for

up to 24 hours in horses that have not eaten roughage in the previous 12 hours.

48

Table 3.1. Effect of intravenous and orally administered dexamethasone on pulmonary
function parameters in RAO-affected horses. Measurements were made before horses
were stabled (-1), at baseline when horses had airway obstruction (0), and 6, 24, 30, 48

and 72 hours after treatment. Data are mean i se. n = 6. Cdyn = dynamic compliance
(l/cm H20), RL = pulmonary resistance (cm Hzo/l/S), PIF = peak inspiratory flow (l/S),
PEF = peak expiratory flow (l/S), RR = respiratory rate (breaths/min). See text for
definition of treatments. Treatment (Tx) within time comparison: (p<0.05). a Significantly

different from saline IV. Time within treatment comparison: Si gnificantly different from I
baseline (0 h) and not significantly different from remission (REM).

 

49

 

jibe

Tx

Cont

RI.

PF

PEF

RR

 

REM

fl; IV

0.97 1 0.14

0.95 1 0.11

3.72 1 0.33

3.98 1 0.47

12.65 1 1.67

 

DEX-IV

1.11 10.10

0.76 1 0.19

4.29 1 0.62

 

HDF

1.32 10.08a

0.80 1 0.12

4.27 1 0.61

13.39 1 2.11

 

4.34 1 0.78

4.45 1 0.65

13.32 1 2.21

 

HDNF

0.94 1 0.05

0.85 1 0.12

4.22 1 0.54

4.35 1 0.37

14.49 1 1.51

 

LDNF

1.17 1 0.22

1.07 1 0.19

3.60 1 0.30

3.40 1 0.21

12.35 1 1.83

 

SAL IV

0.36 1 0.11

2.22 1 0.11

7.21 1 0.72

6.47 1 0.54

23.96 1 2.1L

 

DEX-IV

0.40 1 0.06

2.36 1 0.64

6.20 1 0.81

6.82 1 0.39

23.05 1 1.3

 

HDF

0.41 1 0.06

2.03 1 0.24

5.80 1 0.92

6.fi30 1 0.30

21.62 1 0.68

 

HDNF

0.39 1 0.09

2.35 1 0.34

6.21 1 0.89

6.31 1 0.49

20.35 1 2.68

 

LDNF

0.36 1 0.07

2.51 1 0.50

6.46 1 0.76

6.49 1 0.89

20.40 1 1.34

 

SALIV

0.24 1 0.05

2.81 1 0.35

8.80 1 0.77

6.96 1 0.62

26.36 1 2.21

 

DEX-IV

0.61 1 0.09a

Latina“

3.96 1 0.20a

4.7210241:b

20.02 1 2.26a

 

HDF

0.57 1 0.11a

1.62 1 0.18

4.48 1 0.70a

5.01 1 0.33a

19.32 11.128

 

HDNF

05710403

1.79 1 0.32

5.04 1 0.86a

5.55 1 0.86a

21.45 1 4.05

 

LDNF

0.50 10.06a

2.52 10.61

4.17 1 0.618

4.5910511"ID

17.00 1 1.68a

 

24

SAL IV

0.24 1 0.08

2.68 1 0.49

8.57 1 0.49

6.48 1 0.3_6_

 

DEX-IV

0.57 10.08a

initntb

4.97 1 0.35

6.06 1 0.11

28.04 1 1.94
19.33 11.508

 

HDF

0.45 1 0.13

2.22 1 0.84

6.79 1 1.29

6.34 1 0.67

20.63 1 1.251'

 

HDNF

0.66 10.033

1.53 1 0.20

4.74 1 0.72a

5.00 1 0.64

16.94 1 2.42‘I

 

LDNF

0.51 1 0.16

2.34 1 0.59

4.87 1 0.56a

5.30 1 0.47

17.58 11.18‘I

 

30

SAL IV

0.22 1 0.07

3.47 1 0.54

9.51 1 0.92

6.71 1 0.32

28.55 1 3.14

 

DEX-IV

0.47 1 0.08

2.44 1 0.90

7.09 1 0.84

6.85 1 0.37

20.10 11.44a

 

HDF

0.41 1 0.11

2.16 10.66a

6.87 1 0.98

6.89 1 0.47

21.26 11.54a

 

HDNF

0.56 10.06a

1.75 1 0.27a

5.16 1 0.57a

5.39 1 0.23

16.20 11.05a

 

LDNF

0.47 1 0.11

2.43 1 0.57

7.35 1 1.01

7.1111.01

18.22 11.14a

 

48

SJ’LIV

0.25 1 0.09

3.06 1 0.26

7.62 1 1.32

 

DEX-IV

0.38 1 0.13

2.69 1 0.71

6.40 1 0.64

26.96 1 3.53

 

6.9; 1 0.50

6.95 1 0.24

23.02 1 2.75

 

HDF

0.43 1 0.12

2.1 3 1 0.44

7.81 1 1.06

7.03 1 0.61

2 .7 11.24

 

HDNF

0.54 1 0.14

1.56 10.23a

6.061 0.94

5.69 1 0.61

19.29 1 2.958

 

LDNF

0.46 1 0.09

2.15 1 0.31

6.47 1 1.23

6.57 1 0.80

18.77 11.11a

 

SA_L IV

0.15 1 0.03

2.44 1 0.44

8.68 1 0.63

6.65 1 0.31

29.53 1 3.05 .

 

 

DEX-N

Q71 1 0.07

2.50 1 0.38

6811012

6.49 1 0.40

24.96 11.222

 

HDF

0.43 1 0.11

2.32 1 0.53

6.26 1 0.78

6.65 1 0.42

20.15 1 1.55a

 

HDNF

0.47 1 0.12

1.99 1 0.32

6.79 1 1.56

6.00 1 0.34

21.50 1 4.458

 

 

LDNF

 

0.29 1 0.08

 

2.60 1 0.36

7.18 1 0.88

6.23 1 0.48

 

20.92 1 1.87a

 

 

50

 

 

 

 

60

APi=~L~le (cm H20)

 

 

 

 

 

 

  

 

 

 

 

 

 

 

10 30 60 90 120 150 180 210 240 255
Time (min)

Fig 3.1: Effect of intravenous saline and intravenous dexamethasone on the maximal change in pleural
pressure during tidal breathing (APplmax) in RAO-affected horses. Saline or dexamethasone (0.1 mg/kg IV)
was administered immediately after the time 0 measurement. Atropine (0.02 mg/kg IV) was administered
immediately after the time = 240 min. measurement period. Data are mean i se. n = 8. * = Significant
difference between treatments within a measurement period. ’4 = Significantly different from atropine within
treatment. (p<0.05).

I:] SALINE IV
DEX IV (0.1 mg/kg)

51

 

 

 

 

 

significantly

6.*

 

 

 

 

 

 

 

 

 

 

80

change in pleural pressure (APplmax ) in RAO-affected horses. Measurements were made
before horses were stabled (-1), at baseline when horses had airway obstruction (O), and
significantly different form baseline (0 h) and not significantly different from remission

Fig 3.2. Effect of intravenous and orally administered dexamethasone on the maximal

n
m.
+—
m
e
m
e
m
m
a
D
m m)
m m m
me o.“ H
mm )m w n.
mu . umbx. m
a.m 0» m. m. m
u...“ 0 V I...“ 1 8
.mw < 1 w... o. 0.
2e 1m.“ v o. (m.
m...“ mmmmmm
mm m S D H H L
1 — fi 8 a
m m. m o 4,...“ m
01. .
3.m m AV
6f 83354 u m m i \
6...m#.m 7“

 

52

CHAPTER 4
GLUCOCORTICOID THERAPY AND EQUINE LAMINITIS:

FACT 0R FICTION?

Cornelisse, C.J., Robinson, N.E.
Pulmonary Laboratory, Department of Large Animal Clinical Sciences, College of

Veterinary Medicine, Michigan State University, East-Lansing, MI 48824, USA

Introduction
Is administration of glucocorticoids associated with laminitis in horses? That is the core
question in the paper by McCluskey and Kavenagh regarding the prevalence of equine
laminitis after parenteral treatment of 205 cases with triamcinolone acetonide (TMC).
ls there clinical evidence for the connection between glucocorticoids and laminitis?
Since the introduction of glucocorticoids into equine medicine more than 30 years
ago, their use has been accompanied by fear of induction of laminitis. Despite this fear,
real information regarding glucocorticoid use and laminitis is scarce. In a handful of older
reports in the veterinary and lay literature that implied an association between laminitis
and prior glucocorticoid use, it was unclear if the glucocorticoid was really the cause of
the problem. For instance, some reports included horses with chronic obstructive
pulmonary disease (Gerber 1970; Muylle and Oyaert 1973), animals that are usually
older and could have had Cushing’s disease. Horses in other reports may have had
coexisting gastrointestinal disease (Lose 1980) or what is now known as peripheral

Cushing’s syndrome (PCS) (Frederick and Kehl 2000). Both cushingoid disorders, as

53

well gastrointestinal diseases, are often associated with laminitis. The lack of credible
reports makes it difficult to get a good impression of the risk of equine laminitis
associated with glucocorticoid use. There is some information, however, that suggests
that this risk is not very high. Out of a combined total of 526 horses from three studies
that looked at multiple risk factors in referred cases of acute or chronic laminitis (Hunt
1993; Slater et al. 1995; Cripps and Eustace 1999), only three were attributed to
glucocorticoid-induced laminitis. Even more convincing is the lack of support for a high
prevalence of steroid induced laminitis in published reports of adverse drug reactions. For
instance, the 1993 and 1994 reports of the Australian Veterinary Association Adverse
Drug Reaction Subcommittee (Maddison 1994; Maddison 1996) did not mention any
case of glucocorticoid-induced equine laminitis. Similarly, the current FDA-CVM
Adverse Drug Experience web pages (FDA 2002) mention only 5 “possibly” drug-related
cases of laminitis over the period 1987-2000. These were associated with a variety of
parenterally used glucocorticoids, including dexamethasone (DEX), betamethasone,
TMC, and methyl-prednisolone. The lack of information regarding actual numbers of
sales for these products, as well as the likelihood that underreporting exists, make it
difficult to determine the exact prevalence of glucocorticoid-induced laminitis. However,
the inclusion of owner reports in the FDA cases does suggest that glucocorticoid—induced
laminitis is a relatively rare occurrence.

In addition to the scarcity of reports of laminitis induction, there are the multiple
studies in which glucocorticoids were administered for longer periods, or at higher doses
without mention of the induction of laminitis. Laminitis was not reported as a

complication during or after treatment with either parenteral administration of TMC (0.09

54

— 0.2 mg/kg) (LaPointe et al. 1993; Lepage et al. 1993; French et al. 2000) or DEX at
clinical (0.1 mg/kg for up to 10 days) (Rush et al. 1998; Robinson et al. 2002) or even
higher doses (1 mg/kg for 9 days) (Tumas et al. 1994).

How might glucorticoids predispose to the development of laminitis?

Despite the lack of credible clinical evidence that glucocorticoid therapy causes
laminitis, the association of the condition with both Cushing’s disease and peripheral
cushingoid syndrome in obese horses (Johnson 1999) suggests that chronic exposure to
glucocorticoids may be an important modulating factor. The classic theories on the
pathogenesis of laminitis focus on either 1) a primary disturbance of perfusion of the
laminae followed by secondary events that lead to laminar injury, or 2) a disturbance in
the proliferation and differentiation of the keratinocyte by toxic or metabolic substances,
or 3) increased matrix metallo-protease (MMP) activity induced, for example, by
endotoxins or streptococcal exotoxins (Grosenbaugh et al. 1991; Weiss 1997; Pollitt and
Daradka 1998; Adair et a1. 2000; Mungall et al. 2001; Wattle 2001). Current
understanding of the molecular mechanisms of glucocorticoid action can be implicated in
any of the above theories.

The activation of the cellular glucocorticoid receptor is dependent on cortisol, the
active form of the glucocorticoid molecule. The net balance of the combined activity of
two kinetically distinct isoforms of the enzyme IIB-hydroxy steroid hydrogenase (1 1-13
HSD) determines the local tissue level of this cortisol. The 11-13 HSD-1 isoform
primarily converts inactive cortisone into cortisol whereas ll-B HSD-2 catalyzes the
reverse reaction. It appears that the net balance between the 11-13 HSD isoforms for 9-

alpha fluoro glucocorticoids like DEX and TMC favors a decreased breakdown of these

55

drugs (Best et al. 1997; Diederich et al. 1998) and hence a prolonged tissue activity. In
certain types of obesity in humans increased activity of ll-B HSD-1 is associated with
insulin resistance (Masuzaki et al. 2001; Paulmyer—Lacroix et al. 2002), which in turn is
associated with an impaired production of nitric oxide (NO) by endothelial cells
(Masuzaki et al. 2001; Paulmyer-Lacroix et al. 2002; Steinberg and Baron 2002; Trovati
and Anfossi 2002). Production of NO has a direct vasodilator effect on equine digital
arterial vascular smooth muscle and impairment of its production could cause reduced
blood flow to the foot (Baxter 1995; Cogswell et al. 1995; Schneider et al. 1999).
However, regulation of digital blood flow is not simply under the control of one mediator
but is due to a complex interplay between vasoconstrictors such as endothelin (ET) and
vasodilators such as NO (Cardillo et al. 1999; Miller et al. 2002). Tipping the balance
toward vasoconstriction by reduction of NO production could play a part in the onset of
laminitis. A similar combination of mechanisms is thought to be responsible for the high
prevalence of peripheral vascular disease in humans with obesity and diabetes (Miller et
al. 2002).

In rats, chronic administration of low doses of dexamethasone induces
hypertension on the basis of decreased NO production. The hypertension is preceded by a
decreased sensitivity to insulin, and both can be blocked by metformin, a drug that
increases peripheral insulin sensitivity and cellular glucose uptake (Severino et al. 2002).
Thus, glucocorticoids decrease NO production and cause an impaired vasodilator reserve.
The observations from obese insulin-resistant humans and rats could be in agreement
with l) a decreased lamellar perfusion model as the initiating event of laminitis, 2)

exaggerated contractile responses of equine digital blood vessels after administration of

56

 

glucocorticoids (Eyre et al. 1979), and 3) a reduced production of endothelium derived
nitric oxide that contributes to an impaired vasodilator reserve (Baxter 1995; Schneider et
al. 1999). Additionally endotoxins impair the vasodilator reserve of equine digital arteries
(Baxter 1995) and increased ET expression has been documented in laminitic hoof tissue
(Katwa et al. 1999). It could therefore be the case that glucocorticoids potentially
function as modulators of laminitis induction in endotoxemic diseases and in cases of
chronic laminitis. However, in two large studies of horses with Cushing’s disease, of
which a large percentage also had laminitis, a single intramuscular dose of DEX (10 mg)
did not induce or exacerbate the laminitis (Dybdal et al. 1994; Schott personal
communication 2002).

How do glucocorticoids affect keratinocytes and how might that be a factor in
laminitis? Keratinocytes and fibroblasts are responsible for the production and quality of
the connective tissues in the skin whereas the spatial and temporal activity of MMPs is
important in wound healing. Glucocorticoids can alter proliferation and differentiation
properties of keratinocytes and fibroblasts, resulting in decreased collagen synthesis and
thus a weakened structural supporting matrix. Therapy with glucocorticoids leads to skin
atrophy and impaired wound healing. Indeed, 3 days of topical glucocorticoid treatment
results in down regulation of the collagen synthesis in human skin (Haapasaari et al.
1996; Oikarinen et al. 1998) and clinically significant thinning of the skin can be found
after 1-2 weeks of daily topical fluorinated glucocorticoids (Korting et al. 2002). Because
alterations in the proliferation and differentiation of keratinocytes are features of equine
laminitis, it is therefore possible that glucocorticoids can influence the integrity of the

connective tissues and contribute to the onset of laminitis, especially if used long term. It

57

seems unlikely however that increased basement membrane degradation due to increased
MMP activity per se contributes to this since there is no evidence that glucocorticoids up
regulate MMP activity (Kylmaniemi et al. 1996; Madlener et al. 1998).

Based on data obtained from horses with cushingoid diseases, it seems likely that
chronic exposure to glucocorticoids at the local tissue level could be a very important
factor in the induction of laminitis. The relative refractoriness of fluoro-glucocorticoids
like TMC and DEX to 11-13 HSD-2 inactivation could therefore mean that basement
membrane keratinocytes are always exposed to an activated form of glucocorticoids.
Although the reported half-life of intravenous TMC in the horse is shorter (1.39 —l.58 h)
(French et al. 2000) than that of DEX (2.63 h) (Cunningham et al. 1996), TMC appears
to have a longer GR-binding half-life (Rohdewald et al. 1986). Additionally, different
glucocorticoids have different transcriptional potencies in different tissues. This
consequently results in effects of different magnitude in different tissues, and bypasses
the classical order of glucocorticoid potencies (Jaffuel et al. 2001). For example, TMC
actually has equipotent diabetogenic effect as dexamethasone by inducing similar
increases in the activity of the gluconeogenic enzyme tyrosine aminotransferase (TAT) in
a hepatoma tissue cell line.

Drug formulation and mode of application have significant effects on elimination
rate and final tissue levels. Acid esterification of drugs (e.g., acetate, propionate) results
in a slower systemic absorption of the drug compared to the alcohol or sodium
phosphate/succinate analogs (Schimmer and Parker 1996). Current formulations of TMC

in use in veterinary medicine are the long-acting acetonide esters.

58

The 205 cases reported by McCluskey and Kavenagh were treated by local
application in the joints, tendon sheaths, and bursae. This is likely associated with low,
short duration systemic levels of glucocorticoids, and thus poses a smaller risk for
additional side effects compared to systemic application. Indeed, 18 or 30 mg TMC by
the intra-articular route results in serum levels that are detectable for less than 48-102
hours with peaks between 4 and 12 hours respectively. The lower dose results in cortisol
suppression for less than 120 hours (Chen et al. 1992; Koupai-Abyazani et al. 1995),
compared to 336 hours (Slone et al. 1983) after a similar intramuscular dose.
Additionally, in reports spanning a total of 472 horses that were treated intra—articularly
or intra-bursally with different glucocorticoids, including TMC, and followed over a
longer period after administration, no cases of laminitis were mentioned (Verschooten et
al. ; Houdeshell 1969; Swanstrom and Dawson 1974; Vemimb et al. 1977; Frisbie et al.
1997). This therefore supports the relative safety of intra-articular application of
glucocorticoids in the horse.

In conclusion, current research is getting closer to unraveling the mechanisms by
which glucocorticoids work. Besides the effects of formulation and route of
administration, it appears that the newer glucocorticoids have long activity at the tissue
level. Their genomic effects are tissue dependent and do not necessarily follow the
classical ordering of glucocorticoids. Their effects can be associated with vascular
dysfunction and interfere with keratinocyte proliferation/differentiation as well as matrix
integrity, all mechanisms that possibly can initiate laminitis. A summation of factors
suggests that TMC might have longer tissue activity and have stronger diabetogenic

effects than DEX. However, regarding the induction of laminitis, a paucity of reported

59

 

cases and a multitude of clinical studies suggests that the risk for this after parenteral,
especially intra-articular use, is not high. Based on experiences of horses with a
cushingoid disorder, it seems that the most likely risk factor might be prolonged
treatment. Additionally prior diseases such as endotoxemia, prior laminitis that can affect
vascular function and/or keratinocyte proliferation/differentiation might modulate this

risk as well. However evidence for this based on sufficient numbers is currently lacking.

60

 

CHAPTER 5
EFFECTS AND MECHANISMS OF GLUCOCORTICOID ACTIONS

ON MICROCIRCULATION

The microcirculation

The viability and function of cells in the tissues is dependent on an adequate blood supply
for the delivery of nutrients, hormones and oxygen, and the uptake and removal of
cellular products. The blood supply at the tissue level is regulated by the
microcirculation, i.e. the blood vessels from the first order arterioles via capillaries into
first order venules (Boron and Boulpaep 2003). The activity level of the cells fluctuates,
and therefore an adaptive microcirculation is needed to guarantee an optimal blood flow.
Regulation of blood flow is via alteration in the tone of vascular smooth muscle cells
(VSMC). These VSMC are distributed throughout the wall of the arterioles and venules.
Of particular importance are the precapillary sphincters, i.e. small conglomerates of
VSMC, which strategically are placed in front of the capillary entrance. The tone of the
precapillary sphincters determines the amount of blood flow to the downstream capillary
bed. In some vascular beds, like the skin and laminae of the equine hoof, direct arterio-
venous shunts (A-V shunts) can short circuit blood supply if needed (Hood, Amoss et al.
1978; Robinson 1990; Hood, Grosenbaugh et a1. 1993). Strong precapillary tone with
additional bypass of the blood via A-V shunts will consequently minimize the capillary
blood flow. The tone of VMCS is under control of systemic and local regulatory systems

that work together to adjust and optimize the blood flow to the tissues.

61

Systemic regulation

Systemic regulation of the microvascular blood flow is under control of the autonomic
nervous system. Sympathetic and some parasympathetic nerve endings that are enmeshed
around the blood vessels walls communicate on a local level with VSMC and endothelial
cells. The sympathetic nerve endings release norepinephrine, a catecholarnine that
activates adrenergic Oil-receptors on VSMC. Transduction of the Oil-receptor signal is
through a G-protein coupled pathway and results in contraction of the VSMC, hence
vasoconstriction. Activation of the enzyme myosin light chain kinase (MLCK) plays a
central role in VSMC contraction (Boron and Boulpaep 2003). Sympathetically
controlled release of epinephrine into the blood stream can on the other hand cause
VSMC relaxation via an adrenergic flz-receptor mediated decrease in MLCK activity. The
parasympathetic regulation of blood flow is under control of 1) acetylcholine (ACH), a
muscarinic agonist and 2) nitric oxide (NO) which acts as an activator for soluble
guanylyl cyclase in VSMCs. Both are released from the terminal nerve endings (Boron
and Boulpaep 2003). ACH causes vasodilation via two pathways. Activation of M2-
receptors on VSMCs in the vascular wall, but not the precapillary sphincters, results in a
G-protein coupled pathway that results in a decrease of MLCK activity. ACH
additionally activates M3-receptors on endothelial cells, which results in the production of
various vasoactive substances by these cells. This includes a G-protein mediated increase
in activity of the enzyme nitric oxide synthase (NOS). NOS catalyzes the conversion of
arginine into citrulline and NO. The nitric oxide from the terminal nerve ending and that
of the endothelial cell diffuse into nearby VSMC and activates a soluble guanylyl cycle.

A downstream pathway causes a decrease in MLCK activity with vasodilation as a result.

62

Other vasodilators that are released by the endothelial cell in response to ACH
stimulation include prostaglandins (PGIz) (Kellogg, Zhao et al. 2005) and endothelium-
derived hyperpolarizing factor (EDHF) (Buus, Simonsen et al. 2000). Additional

systemic regulation of vascular tone includes hormones like angiotensin 11.

Local regulation

The local adjustment of blood flow is regulated via several mechanisms that involve a
complex interplay between local stimuli, VSMCs and endothelial cells. This
autoregulatory system on vascular tone can override that of the autonomic system, and
therefore optimize blood supply to local demand. The VSMCs respond to local changes
in P02, C02, pH and shear stress. Changes that accompany increases in metabolism (e.g.
increased PCOz, decreased P02) are associated with vasodilation, while increases in shear
stress cause vasodilation via stress sensitive channels on the VSMCs (Boron and
Boulpaep 2003). However the local stimuli also influence the production and paracrine
release of several vasoactive compounds by endothelial and vascular smooth muscle cells
(Morawietz, Talanow et al. 2000). These include the earlier discussed vasodilators NO,
P012 and EDHF by endothelium, but also the potent vasoconstrictor endothelin-1 (ET-1)
by endothelial cells and VSMCs. The net production of endothelin-1 is dependend on the
intracellular expression of preproendothelin, a precursor molecule. Preproendothelin is
intracellularly converted by endothelin converting enzyme into the smaller active
endothelins (ET-1, ET-2, and ET-3). The endothelins activate ETA- and ETB-receptors on
VSMCs and ETB receptors on endothelial cells. Their activation involves a G-protein

coupled pathway. Activation of ETA- and ETB-receptors on VSMC by ET-l results in

63

vasoconstriction. On the other hand the activation of ETB-receptors on endothelial cells
results in production and release of NO, which consequently is associated with relaxation
of nearby VSMCs, and thus vasodilation (Cardillo, Kilcoyne et al. 2000). Normally in the
vasculature the ETA- receptors outnumber ETB-receptors, but receptor type and density
differs between vascular beds (e.g. systemic vs. pulmonary), gender and species (Brain,
Crossman et al. 1989; Crossman, Brain et al. 1991; Warner 2001). The endothelin-1
' induced vasodilation is usually seen at lower ET-l concentrations and more common in
females (Kellogg, Liu et al. 2001). The overall response of ET-1 is however a prolonged
vasoconstriction of arteries and veins (Haynes, Clarke et al. 1991; Warner 2001; Katz,
Marr et al. 2003). The function of ET-2 and ET-3 is less clear. They are produced in
much smaller amounts and have less affinity for ETA-receptors and equipotent affinity for
ETB-receptors. Their role seems less prominent. The endothelins might additionally be
involved in potentiation of angiotensin-II and norepinephrine mediated vasoconstriction
(Webb, Dickinson et al. 1992; Wenzel, Ruthemann et al. 2001). ETA receptors have been
detected in equine digital, intestinal and pulmonary vasculature (Benamou, Marlin et al.
2001; Venugopal, Holmes et al. 2001; Benamou, Marlin et al. 2003; Katz, Marr et al.
2003). The detection of ETB-receptors in equine digital vasculature so far has been
unsuccessful. There is some evidence that NO also can influence ET-l production. It
appears that blocking of NOS with inhibitor L-NAME abolishes a decrease in
preproendothelin expression from shear stress compared to no treatment (Morawietz,

Talanow et a1. 2000). In this way NO would also stimulate vasodilation.

64

 

Vascular effects of glucocorticoids.

The genomic and nongenomic effects of glucocorticoids form the basis for their broad
range of effects. This includes modification of the vascular tone. For instance topical
application of glucocorticoids on human skin results in local blanching (Ahluwalia and
Flower 1993; Goldsmith, Bunker et al. 1996). Studies with laser Doppler imaging
associated the blanching with a decrease in dermal blood flow (Sommer, Veraart et al.
1998)and venoconstriction (Andersen, Milioni et al. 1993). Similarly systemic
administration of glucocorticoids has been associated with hypertension (Wallerath,
Witte et al. 1999; Severino, Brizzi et al. 2002; Di Filippo, Rossi et al. 2004; Wallerath,
Godecke et a1. 2004). Some of these effects could be related to changes in electrolyte
balance and plasma volume from residual mineralocorticoid action of some
glucocorticoids (Kenyon, Brown et al. 1990). However modification of the local vascular
regulation appears to be the major mechanism by which glucocorticoids alter vascular
tone. Increased vasomotor tone is induced via several mechanisms.

1) Glucocorticoids up regulate the expression of alpha and beta adrenergic receptors
in many tissues, including on the surface of VSMC (Haigh and Jones 1990; Mak,
Nishikawa et al. 1995). This results in a greater and potentiated reaction to the
catecholamines norepinephrine and epinephrine (Eyre, Elmes et al. 1979; Stepp
and Frisbee 2002)

2) Glucocorticoid therapy results in higher circulating levels of ET-1 (Di Filippo,
Rossi et al. 2004), and this has been associated with hypertension. A
glucocorticoid induced increase in expression of preproendothelin forms the basis

for this effect (Koshino, Hayashi et al. 1998; Morin, Asselin et al. 1998;

65

 

3)

4)

5)

Provencher, Villeneuve et al. 1998). In vitro studies suggest that VSMC, and not
endothelial cells, are the primary source for this rise in ET-l levels (Kanse,
Takahashi et al. 1991). These studies also showed that the rise of ET-1 is
associated with a decrease in ETA-receptor numbers on the VSMCs(Clozel,
Loffler et al. 1993; Koshino, Hayashi et al. 1998). This negative feedback
mechanism resulted consequently in an attenuated vasoconstrictive response by
exogenous ET-l. (Nambi, Pullen et al. 1992; Roubert, Viossat et al. 1993;
Telemaque-Potts, Kuc et al. 2002).

Glucocorticoid therapy has been associated with decreased circulating levels of
nitrate, a breakdown product of NO (Wallerath, Witte et al. 1999; Rogers, Bonar
et al. 2002; Severino, Brizzi et al. 2002; Di Filippo, Rossi et al. 2004; Wallerath,
Godecke et al. 2004). This effect is mediated through a decrease in NOS-
expression (Kunz, Walker et a1. 1996; Wallerath, Witte et al. 1999; Rogers, Bonar
et al. 2002; Wallerath, Godecke et al. 2004) and an increased post translational
degradation of this enzyme (Rogers, Bonar et al. 2002).

Glucocorticoids increase the number of angiotensin-II receptors (AT-1) on
VSMCs (Sato, Suzuki et a1. 1994; Provencher, Saltis et al. 1995; Ullian, Walsh et
al. 1996) and increases pulmonary angiotensin converting enzyme (ACE) activity
(Ialenti, Calignano et al. 1986; Dasarathy, Lanzillo et al. 1992). Angiotensin-II is
a potent vasoconstrictor. Consequently these effects have been associated with
glucocorticoid induced hypertension (Ullian, Walsh et al. 1996).

Glucocorticoids are associated with the induction of type II insulin resistance

(Severino, Brizzi et al. 2002; Binnert, Ruchat et al. 2004). Clinical Type II

66

 

diabetes is associated with hypertension, increased vascular complications (Tooke
1999; Chaturvedi, Stevens et al. 2001), and a rise in non esterfied fatty acids
(NEFA) (Barbera, Fierabracci et al. 2001). Insulin appears to have a vasodilatory
action that primarily is mediated via NO production (Steinberg, Brechtel et al.
1994; Steinberg and Baron 2002). The downstream insulin receptor pathway
includes the activation of insulin receptor substrate-1 (IRS-l),
phosphatidylinositol-3-kinase (PI3K) and the serine/threonine protein kinase
AKT/PKB. Blocking of P13K abolishes insulin mediated NO production, while
AKT has shown to be important in the activation of NOS. Glucocorticoids cause a
decrease in the expression of IRS-l, PI3K and PKB (Ducluzeau, Fletcher et al.
2002). It could therefore be possible that this consequently results in an
impairment of insulin mediated NO production, and hence an augmented
impairment in NO mediated vasodilatation. Additionally recent research indicates
that elevated levels in NEFAs could cause impairment of a vasodilatory response
of the vasculature. This is hypothesized to involve NO and NO-independent
pathways including interference with PI3K, shear stress induced NO production
and muscarinic receptors (Steinberg and Baron 2002). Hyperinsulinemia also
results in increased ET-l levels and ETA-receptor numbers (Piatti, Monti et al.
1996; Piatti, Monti et al. 2000; Katakarn, Pollock et al. 2001). It appears that
blocking of ETA-receptors normalizes the vasodilator responses to insulin in
insulin resistant subjects (Miller, Tulbert et al. 2002). This therefore indicates that

hyperinsulinemia is associated with enhanced endothelin activity.

67

“"1

I - L, I ..Iu.'_l

From the above it is clear that glucocorticoids alter both the vasoconstrictive and
vasodilating side of the vasomotor balance. Recent research indicates that the
glucocorticoid-mediated alterations on both systems are closely related. For instance
studies in rats showed that glucocorticoid (prednisolone) induced hypertension can be
blocked by the simultaneous administration of a nitric oxide donor (Severino, Brizzi
et al. 2002; Di Filippo, Rossi et al. 2004). This effect was associated with an absence
in rise of ET-1 levels (Di Filippo, Rossi et a1. 2004). On the other hand the
administration of an ETA-receptor antagonist during glucocorticoid therapy was also
successful in blocking the induction of hypertension (Di Filippo, Rossi et a1. 2004).
Additionally in genetically altered mice that are homozygotus negative for NOS, no
changes in blood pressure are observed with glucocorticoid therapy (Wallerath,
Godecke et al. 2004). Therefore current literature suggests that glucocorticoid
induced reductions in NO partially are related to increased ET-l levels. The increased
ET-l results in a greater vasoconstrictor response that is not compensated by a
properly working vasodilator system. An autoregulated feedback mechanism that
causes a decrease in the ETA-receptors numbers on VSMC attempts to attenuate this

effect instead.

Implications

Glucocorticoids have vascular effects that interfere with both the autonomic
(systemic) and local regulation of vascular tone. Both the vasoconstrictive and
vasodilatory responsiveness are affected. The net effect of glucocorticoids therapy is

an increased vascular tone. It is likely that such an effect in tissues with A-V shunts

68

will result in an even more significant decrease of capillary blood perfusion. Bypass
of blood flow through A-V shunts has been implicated as cause for clinical laminitis
in the horse. Therefore a potentiated vasoconstrictor response, or impaired
vasodilatory response, or both from glucocorticoid administration to horses could be a
vascular mechanism that leads to laminitis. The in vivo vascular responsiveness
during glucocorticoid therapy has not been investigated in the horse. The following
two studies were designed to answer the question if glucocorticoid therapy in horses

alters local vasoconstrictior and vasodilator responsiveness in the horse.

69

CHAPTER 6
A THERMOGRAPHIC STUDY OF THE IN VI V0 MODULATION OF
VASCULAR RESPONSES TO PHENYLEPHRINE AND ENDOTHELIN-l BY

DEXAMETHASONE IN THE HORSE

C.J. Cornelisse*, N.E. Robinson, C.A.E. Berney, S. Eberhardt, J.G. Hauptman and

F..1. Derksen

Department of Large Animal Clinical Sciences, College of Veterinary Medicine,

Michigan State University, East Lansing, MI 48823-1314, USA.

Corresponding author:

Dr. N. Edward Robinson

Department of Large Animal Clinical Sciences
College of Veterinary Medicine

Michigan State University

East Lansing, MI 48824-1314

Telephone: 517-353-5978

Fax: 517-353-2935

Email: robinson@cvm.msu.edu

Keywords: horse; dexamethasone; endothelin; intradermal; phenylephrine;
thermography; vascular function

Word count: 5,962

70

 

 

Summary

Reasons for performing the study: Glucocorticoids modulate many physiologic
mechanisms, including those hypothesized to be involved in the development of
laminitis. A vascular etiology has been implicated as a cause for laminitis and in
vitro studies of equine digital vessels have shown an augmented constrictive
response to catecholamines after addition of a glucocorticoid to the medium.
However in viva compensatory mechanisms can still be present and the net
responses could therefore be different. Up to now the in viva modulation of
constrictor responses of vasculture by glucocorticoids has not been investigated
in the horse.

Objectives: To study the in viva effects of glucocorticoid therapy on vasoconstrictor
responsiveness in the horse. For this purpose the skin was used as a model for
the laminar vascular bed of the equine hoof. The vascular responses to
intradermal injections of various concentrations of vasoconstrictors were
investigated by means of thermography in horses treated with either
dexamethasone or saline.

Methods: In a blinded randomized cross-over design, nine horses were treated with
either dexamethasone (0.1 mg/kg IV q24h) or saline IV for six days. On day six,
the changes in local average skin temperature before (baseline) and after
intradermal injections (at times 20, 60, 120 and 180 minutes) of the
catecholamine phenylephrine (PHE; at 10“, 10's, 10‘, 10'7 and 10'8 M),

endothelin-1 (ET-1; at 10“, 10‘, 10”, 10‘8 and 10" M) or endothelin-l plus a

71

L1“

 

blocker (BQ-123, RES-701 and L-NAME) were investigated with a
thermograph.

Results: Baseline skin temperatures were significantly lower in the dexamethasone
group, suggesting an increase in vasomotor tone. This was accompanied by a
greater and potentiated effect of PHE as demonstrated by the left shift in both
the dose-response curve and 50% decrease in ECso. DEX did not potentiate ET-
1, but the interplay with the lower baseline temperature still resulted in
significantly lower skin temperatures at each ET—l concentration. The different
ET-l blockers had no effect on the ET-l decrease in temperature in either
treatment group.

Conclusions: Our results show that dexamethasone therapy in horses causes a
significant decrease in skin perfusion, most likely due to an increase in basal
vasoconstrictor tone. This is accompanied by a potentiated vasoconstrictor
response to catecholamines. As a consequence of the increase in basal vascular
tone, the responses to other potent vasoconstrictors such as ET-l can be greater
without the need for potentiation of the response to these substances.
Thermography proved to be a useful technique to study in vivo vascular dose-
responses in the skin, and their modulation by the application of a systemic
drug.

Potential relevance: Based on the similarities between blood flow in the skin and the
laminar vascular bed it seems likely that glucocorticoid therapy can result in
additional compromise of perfusion of the equine hoof during disease states

that are already characterized by hypoperfusion and/or increased levels of

72

circulating catecholamines. Thus, during such pathophysiologic states,
glucocorticoid therapy could, according to the vascular model of laminitis, tilt

the balance in favor of laminitis.

73

Introduction

Glucocorticoids are potent anti-inflammatory drugs that are used successfully in equine
medicine for treatment of various diseases, including inflammatory/allergic airway
disease, lameness and immune-mediated syndromes. Unfortunately, laminitis has been
implicated as a possible severe adverse effect from the use of these drugs in horses
(Gerber 1970; Ryu et al. 2004). Several mechanisms regarding glucocorticoid-induced
laminitis have been proposed (Eyre et al. 1979; Pass et al. 1998; French et al. 2000;
Cornelisse and Robinson 2004), including the derailment of vascular function and
disturbances in energy and metabolic pathways.

In the vascular model, enhanced constriction of pre- and/or post-capillary
sphincters is thought to cause decreased blood flow to the laminae, with most of the
blood bypassing this tissue via arterio-venous shunts and thereby predisposing the hoof to
laminitis (Robinson et al. 1976; Hood et al. 1978). Support for vascular effects by
glucocorticoids being the cause can be inferred from several observations: 1) topical
application of glucocorticoids on human skin causes vasoconstriction as indicated by
blanching in the skin (Brown et al. 1991; Andersen et al. 1993; Noon et a1. 1996;
Sommer et al. 1998), 2) glucocorticoids potentiate vasoconstrictor responses to
catecholamines (Eyre et al. 1979), and 3) glucocorticoids induce insulin resistance
(French et al. 2000) and insulin resistance has been associated with an increased risk for
vascular disease as well as with potentiated responses to catecholamines (Tooke 1999;
Tooke and Goh 1999; Stepp and Frisbee 2002).

Despite these observations, so far only one study has reported on the direct effects

of glucocorticoids on equine vasculature. The in vitro study reported additional

74

vasoconstriction of partly precontracted equine digital blood vessels (arteries and veins)
immediately after addition of a glucocorticoid to the medium, and detected enhanced
contraction to the catecholamines epinephrine and norepinephrine 30 minutes later (Eyre
et al. 1979). However, in vivo the vascular tone is the net result of a balance between
vasodilator and vasoconstrictor mechanisms, both regulated by a variety of different local
vasoactive mediators (Hocher et al. 2004; Mather et al. 2004). Therefore, conclusions
regarding vascular tone on the basis of enhancement of one system without knowing the
status of the opposite system can be misleading.

Recent studies in human subjects have indicated complex interactions at the local
vascular level between vasoconstrictors such as norepinephrine and endothelin—1 (ET-1),
and the vasodilator nitric oxide (NO). It appears that stimulation of the ETA- or ETB-type
receptors that are present on vascular smooth muscles cells results in contraction,
whereas stimulation of ETB receptors on endothelial cells results in NO production with
subsequent vascular smooth muscle cell relaxation. Additionally, blockade of ETA
receptors attenuates the vasoconstrictor response to norepinephrine and angiotensin II
(Wenzel et al. 2001). Glucocorticoid therapy is associated with hypertension (Severino et
al. 2002; Di Filippo et al. 2004) and affects all the local systems that are involved in the
regulation of vascular tone. This includes the up-regulation of pulmonary angiotensin
converting enzyme (Ialenti et al. 1986), increases in vascular smooth muscle angiotensin-
I receptor density (Provencher et al. 1995) and the down-regulation of transcription of
endothelial nitric oxide synthase (Di Filippo et al. 2004; Wallerath et al. 2004), the key
enzyme involved in NO production. Glucocorticoids have also been associated with

increased levels of ET-1 (Kanse et al. 1991; Roubert et al. 1993; Kato et al. 1995;

75

 

Koshino et al. 1998; Morin et al. 1998; Di Filippo et al. 2004). All of these effects
suggest a net shift towards increased vasoconstriction. However, glucocorticoids cause a
50 to 60% decrease in ETA receptor density on vascular smooth muscle cells (V SMC),
resulting in an attenuated response to ET-l (Nambi et al. 1992; Roubert et al. 1993;
Provencher et al. 1995; Koshino et al. 1998) and suggests the presence of autocrine
feedback mechanisms that modulate the in viva responses.

Direct in vivo interventional studies on the local vasculature of the laminae in
hoof are restricted by the limited access to this tissue. However, skin tissue has the same
ontogenic origin and has many identical vascular properties as the laminar tissue in the
hoof, including A-V shunts (Robinson et al. 1975; Johnson et al. 2004). The aim of the
present study was to investigate the in viva vascular effects of glucocorticoid therapy in
the horse with the skin as a model for local circulation in the hoof. The responses to local
intradermal injections of the vasoactive substances phenylephrine (PHE; vasoconstrictor),
endothelin-1 (ET-1;vasoconstrictor), BQ-123 (ETA-receptor antagonist), RES-701 (ETB-
receptor antagonist) and the nitric oxide synthase antagonist L-NAME were investigated
in horses treated with either dexamethasone or saline. The local perfusion in the skin is
reflected in the skin surface temperature. The change in local dermal circulation by the
injected substances was therefore measured as changes in surface temperature with a
thermograph. Our hypothesis was that glucocorticoid therapy enhances in vivo vascular

responsiveness to vasoconstrictors.

Material and methods

Preparation of solutions for intradermal injections

76

 

Stock solutions were prepared freshly every 48 hours out of weighed amounts of
endothelin-1 (ET-1“), endothelin receptor antagonists (BQ-123a, RES-701“) and the nitric
oxide synthase antagonist L—NAMEb. The drugs were dissolved in DMSOc and
subsequently further diluted in phosphate-buffered saline (PBS). The stock solutions
were refrigerated at 10°C until further processing into solutions of ET-1, antagonists
alone, or ET-l plus an antagonist. The final working solutions (RX) therefore contained
ET-l (10“), 108, 107, 106, 10'5 M) with or without one of the antagonists (BO-123, RES-
701 [10’6 M] or L-NAME [10'4 M]). Control solutions included either BQ-123, RES-701
(10‘6 M) or L-NAME (10“1 M). The final dilution of the solvent DMSO in all solutions
was 1210,000. Additionally, fresh solutions of phenylephrined (108, 107, 106, 10'5 to 10“1
M) in PBS were prepared. All solutions were equilibrated to room temperature (22°C)
prior to injection. The concentrations of ET-1, antagonists, and PHE were based on in
vitro and in vivo vascular dose-response studies (Elliott et al. 1994; Lawrence and Brain
1994; Pang et al. 1998; Lipa et al. 1999; Katugampola et al. 2000; Venugopal et al. 2001;

Benamou et al. 2003; Katz et al. 2003; Bowen et al. 2004; Wilson et al. 2004).

Horses

Nine recurrent airway obstruction-affected mares (age 16-27 years, weight 442-571 Kg)
(Robinson et al. 1999) were used in the study. During the study, horses were in clinical
remission. While on pasture the horses were treated at 08.00 with either dexamethasonec
(0.1 mg/kg IV q24h) or an equivalent volume of saline (SAL IV q24h) for 6 consecutive
days. The treatments were administered according to a randomized cross-over design and

were blinded to the investigator. The wash-out period between treatments was at least 2

77

 

weeks. The horses were stabled on shavings in a temperature-controlled environment 18
hours prior to the start of the experiments in order to acclimatize, and were fed a
commercial pelleted diet. Immediately after stabling, skin test areas (25 by 20 cm) over
both lateral shoulders were clipped and subsequently cleaned with alcohol. A 5 by 5 grid
was drawn on the area with a silver sharpie pen so that the lines were visible on

thermography images.

Study protocol

During the experiments, the horses were free standing in their temperature-controlled
stall (22°C). In each test area, a top, middle and ventral row of the grid was used for
intradermal injections, leaving an unused row between each treatment row. In each of the
three treatment rows one of the solutions of ET-1, ET—l plus antagonist, PHE, or controls
was injected craniad to caudad in decreasing concentration (CONC). The control row
additionally included DMSO 1:10000 and an empty needle stick. The order of the rows
was randomized between horses, but identical within a horse between the treatments. A
total volume of 0.1 ml was injected intraderrnally. A 24-mrn diameter circular adhesive
bandagef was applied between two of the rows for purposes of distance calibrations.
Earlier pilot studies had determined that the bandage size was similar to the
vasoconstricted area of 0.1 ml intradermal PHE at 10“1 M. A mounted thermal imager
(Microscan 75153) was used to measure the skin temperature in the injected areas. The
emissivity of the imager was set at 0.98 with a sensitivity of 03°C, and the camera was
adjusted for background temperature and humidity. The imager was kept at a distance of

70 cm perpendicular to the study area. Digital temperature images were captured before

78

' 10'....¢'Lm A. [1“

 

Fl

(baseline at TM = 0 min) and 20, 60, 120 and 180 minutes after intradermal injections.
During the study, environmental temperature and humidity data also were collected. The
study was approved by the All-University Committee for Animal Use and Care at

Michigan State University.

Image and data analysis

The digital images were analyzed with complementary software (Microspec 2.7)8. First,
the total number of pixels in the area covered by the bandage was determined.
Subsequently, the average temperature (TAVG) of circular areas of similar pixel size was
measured over the individual injection locations. A mixed factorial procedure for
repeated measures (SAS statistical sofiwareh) was used for analysis of 1) baseline skin
temperature between treatments; 2) PHE and the needle stick as control; and 3) ET-l with
its antagonists and DMSO. When significant effects (p < 0.05) were observed for the
main effects and their interactions, the Bonferoni test was used to determine differences
between means. Additionally, at each time in a series, the temperature difference between
the control and a given concentration of agonist was calculated and this difference was
used to construct a concentration-response curve. T-tests were then used to compare these
temperature differences between saline and dexamethasone treatments at each agonist
concentration. Similarly, in each horse the estimated dose of ET-1 and PHE that
produced 50% of the maximal response (EC50) was determined by iteration for times 60
and 120 minutes. From the mean EC50 the clinical potency for ET-1 and PHE was
compared within the SAL or DEX treatment with a paired t-test. The environmental data

were compared with a t-test.

79

 

Results

Baseline skin temperature

A mixed procedure for repeated measures showed that the baseline skin temperature after
DEX (33.58 i 0.05 °C) was significantly (P < 0.0001) lower compared to baseline values

after SAL (34.33 i 0.05 °C) (Figure 6.1 and Figure 6.3).

PHE-series

Significant differences were found for main effects of treatment (TX; p = 0.0004), time
(TM; p < 0.001) and PHE concentration (CONC; p < 0.0001) (Table 6.1). Additionally,
significant differences were detected for the TM*CONC (p < 0.0001), TX*CONC (p =
0.0002) and TX*TM*CONC (p < 0.0001) interactions. Within DEX and SAL treatments
there was no difference in local skin temperature prior to PHE injections. PHE injections
caused a concentration-dependent decrease in skin temperature that was maximal 60—120
minutes after injection. The decrease became significant at 10’5 M and 104 M (Table
6.1). Post hoc analysis of the TX*CONC interactions showed that at PHE concentrations
of 108, 107, 10'6 and 10'5 M, skin temperature was significantly lower after DEX than
after SAL treatment (Figure 6.1). Figure 6.2 shows the concentration-response curves to
PHE at 20, 60 and 120 minutes after injection. DEX treatment shifted the concentration-

response curve to the left with a significant greater efficacy at 10'5 M PHE.

80

E T -1, antagonists and E T -1 plus antagonist combination (RXS) series

Statistical analysis showed significant main effects of treatment (TX; p = 0.0043), time
(TM; p < 0.001) and concentration (CONC; p < 0.0001) (Table 6.2). Significance was
detected for the TX*CONC (p = 0.001), TM*CONC (p < 0.0001) and
TX*RX*TM*CONC (p < 0.0003) interactions. Antagonists alone had no effect on skin
temperature (p = 0.33). The ET-1 and ET-l plus antagonist combinations caused a
concentration-dependent decrease in local skin temperature that was maximal 60-120
minutes after injection (Figure 6.3). None of the antagonists, BQ-123, RES-701 or L-
NAME had any effect on the reduction in skin temperature caused by ET-l.
Dexamethasone treatment significantly reduced skin temperature at each concentration of

ET-1 but did not potentiate the response to ET—l or any of the antagonist combinations.

Relative clinical potency of PHE and E T -1

During saline treatment at 60 minutes, the EC50 (in —LOG[M]) for ET-1 and PHE were
6.87 and 4.67, respectively, and were significantly different from each other (p < 0.0001).
Treatment with dexamethasone potentiated the effect of PHE significantly (p = 0.0019),
as demonstrated by the left shift of the EC50 to 5.68, whereas the EC50 for ET-1 was
unchanged at 7.09 (p = 0.29) (Figure 6.4”). Similarly, during saline treatment at 120
minutes the EC50 for PHE was 4.49 and was significantly (p = 0.00002) different from
that of ET-1 at 6.88. Dexamethasone treatment shifted the EC50 of PHE significantly (p =

0.039) to 5.33 while that for ET-1 was unchanged at 6.71 (p = 0.49).

81

.-. _J ”i

 

Environment

Statistical analysis detected no significant difference between the treatments for

environmental temperature (DEX: 22.4 1 0.1 °C, SAL: 22.1 1 0.2 °C) and humidity

(DEX: 73.3 1 1.7 %, SAL: 69.6 .1 1.7 %). These values were within those reported for

the therrnoneutral zone of the horse (Morgan 1998).

Discussion

Thermography proved to be a successful technique to measure the in vivo dose response
of vasoconstrictor substances that were injected in the skin. Because these responses
reflected the net result between vasoconstricting and vasodilating systems it was possible
with this technique to investigate effects that are closer to the net physiological outcome.
It was therefore also possible to study the modulation of the responses by systemic
dexamethasone treatment.

The treatment with dexamethasone resulted in significantly lower baseline
temperatures and significantly lower temperatures of the skin after intradermal injections
of PHE and ET-l. This was accompanied by potentiation of PHE with a shift of 1 log
concentration to the left for the EC50 of PHE (Figure 6.2, 6.4a). PHE had additionally a
greater maximal effect compared to ET-l (Figure 6.1b, 6.3b). Recent studies on vascular
smooth muscle cells have shown that dexamethasone causes an increase in 011-
adrenoceptor gene transcription and al-adrenoceptor second messenger production. The
result, respectively a greater al-adrenoceptor density and stronger coupling to G-proteins,
would consequently cause an increased sensitivity of vascular smooth muscle to

catecholamines (Haigh and Jones 1990; Sakaue and Hoffman 1991; Liu et al. 1992) . It is

82

likely that this effect therefore explains the potentiated effect for PHE in our
dexamethasone group. The increased vasomotor tone would subsequently have resulted
in a lower baseline skin temperature. Increased vasomotor tone has indeed been
implicated as the cause for dermal blanching after topical application of glucocorticoids
(Brown et al. 1991; Andersen et al. 1993; Noon et al. 1996; Sommer et al. 1998).

While it is clear that DEX potentiated the effect of the catecholamine PHE, that
may not be the only cause of the decrease in skin temperature. For example, recently it
was shown that glucocorticoid-associated hypertension in rats is associated with
decreased NO production (Wallerath et al. 1999; Severino et al. 2002) and increased
plasma ET-l concentrations (Di Filippo et al. 2004). Pretreatment with an ET-antagonist
(Di Filippo et al. 2004) or NO donor (Severino et al. 2002; Di Filippo et al. 2004)
abolishes this hypertensive effect, indicating the importance of these systems. Although
we did not measure ET-1 and NO concentrations in the blood it could be possible that an
increase in ET-l or decrease in NO concentrations did contribute to an increase in
vasomotor tone. Our study did show the presence of endothelin receptors in the equine
dermal vasculature. In vascular smooth muscle cells of rats and rabbits the in vitro
glucocorticoid-induced increase of ET-1 production was accompanied by a decrease in
ET-l receptor density. This consequently resulted in an attenuated response to exogenous
ET-l (Nambi et al. 1992; Roubert et al. 1993; Provencher et al. 1995; Koshino et al.
1998). We explored the effect of DEX on the response to ET-1 and found neither
attenuation nor potentiation of its effect (Figure 6.3, Figure 6.4). The reason for this is

unclear but could be similar to that for bovine smooth vascular muscle cells for which in

83

k1

 

vitro no change in endothelin receptor density could be demonstrated after glucocorticoid
addition to the medium (Kanse et al. 1991).

We were unable to detect specific effects related to the coinjection of endothelin
antagonists BQ-123 and RES-701, or the NOS blocker L-NAME. This was surprising
because, in other species, although measured with different techniques, the coinjection of
these drugs caused changes in dermal perfusion (Clough 1999; Katugampola et al. 2000;
Shastry et al. 2000; Wenzel et al. 2001). It is possible that differences between species or
technique could account for our inability to detect antagonism by these drugs. Indeed, up
to now, blocking of ETB-receptors in the different vascular beds of the horse has been
unsuccessful (Benamou et al. 2003; Katz et al. 2003). It has also been reported that the
blockade of ETB-receptors in other species did not always result in an alteration of
vasomotor tone (Pang et al. 1998; Lipa et al. 1999; Leslie et al. 2004). It could on the
other hand be possible that the alteration in vasomotor tone caused by the blockers
resulted in temperature changes that were below the limit of detection of thermography.
Coinjection of endothelin antagonists or L-NAME, even when injected in surplus to ET-
1, resulted in maximal dermal flow changes in the order of 40 to 50% (Clough 1999;
Katugampola et al. 2000; Shastry et al. 2000; Wenzel et al. 2001). With a reported
correlation range from 0.3 to 0.79 between the change in dermal perfussion and a change
in skin temperature (Harrison et al. 1994; Bommyr et al. 1997; Clark et al. 2003;
Yosipovitch et al. 2004) it is thus possible that the antagonistic effects of BQ—123, RES-
701 and L-NAME were not detected with thermography. A slower diffusion into the skin
or a different time to become effective as causes for the failure to detect antagonism by

these blockers seems however unlikely because we used doses and time windows similar

84

"“4"“!

 

to those used in other coinjection protocols (Clough 1999; Katugampola et al. 2000;
Shastry et al. 2000; Wenzel et al. 2001).

This study was conducted because of the common belief that dexamethasone
treatment can be complicated by the development of laminitis, and our findings support
that hypothesis. Dexamethasone caused an in vivo decrease of baseline dermal
temperature that was likely due to reduced perfusion and this effect was accompanied by
a potentiated vasoconstrictor response to catecholamines. The combination of reduced
blood flow and a magnified vasoconstrictor response to activation of alpha-adrenoceptors
could lead to a reduction in blood flow in situations in which circulating catecholamines
concentrations are increased. Even though the response to ET-1 was not potentiated by
dexamethasone, the additive effect of a DEX-induced reduction in blood flow to the
vasoconstriction caused by ET could still result in a clinically significant hypoperfusion.
The many anatomic and physiologic similarities between the dermal and laminar vascular
beds suggest that dexamethasone therapy could cause a significant decrease in the
laminar perfusion during disease states that are characterized by hypoperfusion and/or
increased circulating levels of catecholamines. Thus, during pathophysiologic states such
as colic or septic metritis, glucocorticoid therapy could, according to the vascular model
of laminitis, possibly tilt the balance towards the direction of laminitis. However, the
vascular changes induced by DEX may be of little clinical significance for diseases such
as heaves, or skin and joint disorders which have minimal involvement of the systemic
circulation or are unaccompanied by increased concentrations of circulating

catecholamines.

85

 

[S’sIA-flflo u .U'c' .-

I

Table 6.1: Average temperatures (TAVG) of local skin areas injected with various
concentrations (CONC) of phenylephrine over TIME in horses treated with either

dexamethasone (DEX) or saline (SAL). we Same scripts are significantly different
within main effect.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(Tm in °C)

MAIN
EFFECT VALUE Mean :1: se P < 0.05
TREATMENT DEX 32.85 :1: 0.12 a
TREATMENT SAL 33.89 :1: 0.12 a
TIME (MIN) 0 33.85 1 0.14 a,b
TIME (MIN) 20 33.37 3: 0.14 a
TIME (MIN) 60 33.06 :1: 0.14 b,c
TIME (MIN) 120 33.02 :1: 0.14 a,d
TIME (MIN) 180 33.56 a 0.14 c,d
CONC (M) control 33.78 1 0.13 a

-8
CONC (M) 10 33.79 a: 0.13 b .
CONC (M) 10"7 33.70 1 0.13 c
CONC (M) 10'6 33.56 1 0.13 d

-5
CONC (M) 10 33.20 a: 0.13 a,b,c,d,e

.4
CONC (M) 10 32.21 :1: 0.13 a,b,c,d,e

 

 

 

86

Table 6.2: Average temperatures (TAVG) of local skin areas injected with various
concentrations (CONC) of ET-1 over TIME in horses treated with either

dexamethasone (DEX) or saline (SAL). a'CSarne scripts are significantly different
within main effect.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. 0
MAIN (Tm; In C)
EFFECT VALUE Mean :1; se 1) < 0.05
TREATMENT SAL 34.03 1 0.32 a
TREATMENT DEX 33.08 1 0.32 a
TIME (min) 0 33.99 1 0.31 a,b,c
TIME (min) 20 33.62 1 0.31 b
TIME (min) 60 33.38 1 0.31 a
TIME (min) 120 33.14 1 0.31 b,c
TIME (min) 180 33.66 1 0.31 c
CONC (M) 0 33.86 10.30 a
-9

CONC (M) 10 33.81 1 0.30 b
CONC (M) 10'8 33.76 1 0.30 c
CONC (M) 10'7 33.50 1 0.30 a,b,c
CONC (M) 10“5 33.31 1 0.30 a,b,c
CON (M) 10’5 33.10 1 0.30 a,b,c
Rx DMSO 33.57 1 0.30
Rx 130-123 33.59 1 0.30
RX RES-701 33.46 1 0.30
Rx L-NAME 33.61 1 0.30

 

 

 

 

87

 

 

 

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u
."
.
u
o
,-
0"
.0

ll

.‘Il

.-
.U‘
,,,,
..-
. ,..
0‘.

.....
........
.........

\.
33 9 '\. §
.\ /.
\, .
32 - I. _/-'/

 

T1176 1 °C)

 

 

 

 

31-
30 I I I I I I I I . I
0 20 40 60 80 100 120 140 160 180 200
TIME (min)

 

 

Figure 6.1a: Temperatures (TAVG; mean and sem) of local skin areas injected with
a control (needle stick) or various concentrations of phenylephrine (PHE) over
TIME in horses treated with either saline (1a) or dexamethasone (1b). The sem
represents the comparisons between TX but within TM x CONC.

 

 

 

—0— Control

--8— PHE(10'°M)
—+— PHE(10'7M)
—-<>—— PHE(10'°M)
........ .W PHE(1o-5M)
—~-v——~- PHE(1O4M)

 

 

 

88

 

Fig. 6.1b

>
‘.'. \
.1 \ \ \ /
d-‘ 4' \ \ \ /
d... N /
33 _ 6. / -" ..- ’ ‘.
\.l \\ “a ‘ ..u— '_ ”’
_. ‘1. I
. .- ‘~‘ ‘\ ’I’
\" " ‘~~_——— ‘. v”
—— "

Tm; ( °C)

32 1 K.-..
\'-... u
31 _ \.\.O_%/.

 

 

 

30 I I I I I I I I I
o 20 40 60 80 100 120 140 160 180 200
TIME (min)

 

 

Figure 6.1b: Temperatures (TAVG; mean and sem) of local skin areas injected with a
control (needle stick) or various concentrations of phenylephrine (PHE) over TIME in
horses treated with either saline (1a) or dexamethasone (1b). The sem represents the
comparisons between TX but within TM x CONC.

 

 

—0— Control

—-8— PHE(10‘°M)
——A—— PHE(10"M)
——<>—— PHE(10'°M)
........ ..W. PHE (10-5 M)
—-—---v—--- PHE(10"M)

 

 

 

89

 

 

 

Fig. 6.2a

    

o
Q ...... -O"'

 

 

 

10'9 10'8 10‘7 10" 10'5 10" 1O"3
[PHE] in (M)

 

 

Figure 6.2a: Temperature differences (ATM/G) between control (needle stick) and
various concentrations of phenylephrine [PHE] in horses treated with saline (open
circle, dotted line) or dexamethasone (closed circle, solid line) at time 20 minutes.

* significantly different (P < 0.05).

 

9O

 

 

 

 

 

Fi .6.2b
g Q
*
2 .
G
E.
‘>’
151 -
<
0 ‘1 O ................. O .......

 

10'9 10'8 10'7 10'6 10'5 10'4 10‘3
[PHE] In (M)

 

 

Figure 6.2b: Temperature differences (ATAVG) between control (needle stick) and
various concentrations of phenylephrine [PHE] in horses treated with saline (open
circle, dotted line) or dexamethasone (closed circle, solid line) at time 60 minutes.

* significantly different (P < 0.05).

 

 

91

 

ATAVG (°C)

 

 

”9.6.21:
2 3
1 _
O ................. O ................ O .......
0 s

 

 

 

10"9 10‘8 10'7 10'6 10'5 10" 10'3
[PHE] in (M)

 

 

 

Figure 6.2c: Temperature differences (ATM/G) between control (needle stick)
and various concentrations of phenylephrine [PHE] in horses treated with saline
(open circle, dotted line) or dexamethasone (closed circle, solid line) at time 120

minutes.* significantly different (P < 0.05).

 

 

92

 

(I. -. .

 

35.0 *

 

 

 

 

Fig. 6.3a: ET-1, SAL

34.5

34.0 . '\ ‘D‘:-~...:‘~--__ ,¢;~’/
co \\ -~. .....
v '\\ 46
0. 33.5 4 ' \ \ ‘_ /./
5 b1 _. -3 \ \ 4’

33.0 ..

32.5 -

I SE = 0.37
32.0 I T I I I I I I I
0 20 40 60 80 100 120 140 160 180 200
TIME (MIN)

 

 

Figure 6.3a: Average local skin temperatures (TAVG) of areas injected with various
concentrations of ET-1 (see legend) without (a,b) or with either antagonists BQ-123
(c,d) or RES-701 (e,f) or L-NAME (g,h) over TIME in horses treated with either
saline (SAL) or dexamethasone (DEX). The sem for comparison between TX but
within RX xTM x CONC values is 0.35.

 

 

 

 

—0— Control

......... ........... ET-1(10'9M)
——¥—— ET-1(10‘°M)
—--—1:i—~-- ET-1(1O'7M)
——A— ET-1(10'°M)
—-—O—~— ET-1(10'5M)

 

 

 

93

11511.“? v...'- 1

TEMP (°C)

35.0

 

 

 

 

 

 

Fig. 6.3b: ET-1, DEX
34.5 -
34.0 -
33.5
33.0 1
32.51 _ “' ->_\ //,/-/
32.0 T j I I I I j I I
0 20 40 60 80 100 120 140 160 180 200
TIME (MIN)

 

Figure 6.3b: Average local skin temperatures (TAVG) of areas injected with
various concentrations of ET-1 (see legend) without (a,b) or with either
antagonists BQ-123 (c,d) or RES-701 (e,f) or L-NAME (g,h) over TIME in
horses treated with either saline (SAL) or dexamethasone (DEX). The sem for

 

comparison between TX but within RX xTM x CONC values is 0.35.

 

 

 

—0— Control

......... 4...”... ET_1(1O-9M)
-—¥-— Er-1(10'°M)
_.._D_-- E'r-1(10"M)
—-A— ET-1(10'6M)
—-—O—-— ET-1(10'5M)

 

 

 

94

 

--1. 1"" fl

 

TEMP (°C)

 

 

 

 

 

 

.(D 11..

I A

\\ ., ...._., .. --

33.51 “\L__"_\‘\ ‘3' / .fi
0»... _____ O~ \\ / /-/ r.
.\_ \ / /. _.
.L

32.5 -
32.0 I I T I I I F I I
0 20 40 50 80 100 120 140 160 180 200
TIME (MIN)

 

 

Figure 6.3e: Average local skin temperatures (TAVG) of areas injected with
various concentrations of ET-1 (see legend) without (a,b) or with either
antagonists BQ-123 (c,d) or RES-701 (e,f) or L-NAME (g,h) over TIME in
horses treated with either saline (SAL) or dexamethasone (DEX). The sem for
comparison between TX but within RX xTM x CONC values is 0.35.

 

 

 

—-0— Control

......... .H...“ ET_1(10-9 M)
—-—¥-— ET-1(10'°M)
—~—1:}—~~ ET-1(10"M)
—-A— ET-1(10'°M)
-—~—<}4— ET-1(10'5M)

 

 

 

95

35.0

 

Fig. 6.3d: ET-1 + BQ-123, DEX

34.5 a

34.0 -

 

TEMP (°C)

 

 

 

 

0 20 40 60 80 100 120 140 160 180 200
TIME (MIN)

 

 

Figure 6.3d: Average local skin temperatures (TAVG) of areas injected with
various concentrations of ET-1 (see legend) without (a,b) or with either
antagonists BQ-123 (c,d) or RES-701 (e,f) or L-NAME (g,h) over TIME in
horses treated with either saline (SAL) or dexamethasone (DEX). The sem
for comparison between TX but within RX xTM x CONC values is 0.35.

 

 

 

 

—0— Control

......... +........ ET_1(10-9 M)
--¥-— ET-1(10°"M)
—--43—---- ET-1(10"M)
—-A— ET-1(10'°M)
—-—O—-— ET-1(10‘5M)

 

 

 

96

TEMP (°C)

 

 

 

 

 

 

 

‘ID.. {a
. \A ./" E
33.5 'l O \ "D / /
\'~__‘O'\. \ \ // _/'
33.0 . "TO" .
32.5 -
32.0 I I I I I I If I I
0 20 4O 60 80 100 120 140 160 180 200
TIME (MIN)

 

 

Figure 6.3e: Average local skin temperatures (TAVG) of areas injected with various
concentrations of ET-1 (see legend) without (a,b) or with either antagonists BQ-
123 (c,d) or RES-701 (e,f) or L-NAME (g,h) over TIME in horses treated with
either saline (SAL) or dexamethasone (DEX). The sem for comparison between
TX but within RX xTM x CONC values is 0.35.

 

 

 

---—0— Control

......... +........ ET_1(10-9 M)
—-+—— ET-1 (101M)
—~-CI-~- ET-1(10"M)
-—4— ET-1(1O‘6M)
—-—O—-- ET-1(10‘5M)

 

 

 

97

 

Fig. 6.3f: ET-1 + RES-701, DEX

 

 
   

TEMP (°C)
8
0|

 

 

 

33.0 . '\.\{\ \§'\“-+....,_. //j

\. \\\'\\\ .......... .////// O

O\ ‘_ \ ‘\"\:~F:‘_——'V:f/'// _/'/
325~ T~o1\g\“-—=14n/ /./-/
“T ~41"
32.0 ' 1 T I I T I I I
0 20 40 60 80 100 120 140 160 180 200
TIME (MIN)

 

Figure 6.3f: Average local skin temperatures (TAVG) of areas injected with
various concentrations of ET-1 (see legend) without (a,b) or with either
antagonists BQ—123 (c,d) or RES-701 (e,f) or L-NAME (g,h) over TIME in
horses treated with either saline (SAL) or dexamethasone (DEX). The sem for
comparison between TX but within RX xTM x CONC values is 0.35.

 

 

 

 

+ Control

......... 4......... ET_1(1o-9 M)
—-v—— ET-1(10'°M)
—~4:I—--- ET-1(10"M)
—-A— ET-1(10*‘M)
—-—0-— ET-1(1O'5M)

 

 

 

98

a'fifl‘

 

 

TEMP (°C)

 

 

 

 

 

 

. \ 1. _ ..A/
33.0 . \. ~/
\O\.\ /./ /

\ ~ ~ __ --O __
32.5 ~
32.0 I I I I I I I I l

0 20 40 60 80 100 120 140 160 180 200
TIME (MIN)

 

 

Figure 6.3g: Average local skin temperatures (TAVG) of areas injected with
various concentrations of ET-1 (see legend) without (a,b) or with either
antagonists BQ-123 (c,d) or RES—701 (e,f) or L-NAME (g,h) over TIME in
horses treated with either saline (SAL) or dexamethasone (DEX). The sem
for comparison between TX but within RX xTM x CONC values is 0.35.

 

 

 

——O—— Control

......... +....... ET_1(10-9 M)
-—¥-—— ET-1(10'°M)
—---—I:I---- ET-1(10"M)
——4— ET-1(10'°M)
—-—O—-— ET-1(10'5M)

 

 

 

99

TEMP (°C)

 

Fig. 6.311: ET-1 + L-NAME, DEX

 

 

 

 

 

0 20 40 60 80 100 120 140 160 180 200
TIME (MIN)

 

 

Figure 6.3b: Average local skin temperatures (TAVG) of areas injected with
various concentrations of ET-1 (see legend) without (a,b) or with either
antagonists BQ-123 (c,d) or RES-701 (e,f) or L-NAME (g,h) over TIME in
horses treated with either saline (SAL) or dexamethasone (DEX). The sem for
comparison between TX but within RX xTM x CONC values is 0.35.

 

 

+ Control

......... .......... ET-1 (10-9 M)
-—w--— ET-1(10‘°M)
—--13—~-- ET-1(10"M)
—-A— ET-1(10'6M)
—-—O—-— ET-1(10'5M)

 

 

 

100

 

TAVG (°C)

35

 

 
      

 

 

 

Fig. 6.4a; PHE
34 -
33 _ % .............. %W”.
32 ‘ %
SAL: EC5O = 4.67 (2.14 x 10'5 M) -------
31 DEX: EC50 = 5.68 (2.09 x 10" M)
10'10 10'9 10'8 10'7 10'° 10'5 10" 10‘3

CONC (M)

 

 

Figure 6.4a: Dose-temperature (TAVG) curves of PHE 60 minutes after
intradermal injection in horses that are treated with either saline (SAL; solid
symbol and line) or dexamethasone (DEX; open symbol, dotted line).
Effective concentration required to produce 50% of the maximal response.

 

 

101

Tm (°C)

35

 

Fig. 6.4b; ET-1

34~

33-

 

32*

SAL: E050 = 6.87 (1.35 x 10'7 M)
DEX: ec5o = 7.09 (3.13 x 10" M)

 

 

 

31 1 . I
10'10 10‘9 10'8 10'7 10" 10'5 10"
CONC (M)

 

 

Figure 6.4b: Dose-temperature (TAVG) curves of ET-1 60 minutes after
intradermal injection in horses that are treated with either saline (SAL; solid
symbol and line) or dexamethasone (DEX; open symbol, dotted line).
Effective concentration required to produce 50% of the maximal response.

 

102

 

35

 

Fig. 6.4c; SAL

341
8
a.

333‘
I'-

32-

 

ET-1: EC50 = 6.87 (1.35 x 10'7)
PHE: 5050 = 4.67 (2.14 x 10'5)

 

31 I I I
10'10 10'9 10'8 10‘7 10'6 10'5 10" 10'3
CONC (M)

 

 

 

 

Figure 6.4c; The relative potencies between PHE and ET-l are depicted as
dose-temperature (TAVG) curves of PHE 60 minutes after intradermal injection in

horses that are treated with saline (solid symbol and line). EC 50: Effective
concentration required to produce 50% of the maximal response.

 

 

103

CHAPTER 7

A THERMOGRAPHIC STUDY OF THE IN VIVO MODULATION OF

METHACHOLINE INDUCED VASODILATION BY DEXAMETHASONE IN

THE HORSE.

104

Summary

Reasons for performing the study: Glucocorticoids modulate many physiologic
pathways, including those that are hypothesized to be involved in the
development of laminitis. A vascular etiology has been implicated as a cause for
laminitis. In vitro studies of equine digital vessels and studies in other species
indicate that glucocorticoids can impair endothelial production of nitric oxide,
a potent local vasodilator. Neuronal acetylcholine, an endothelial M3-receptor
agonist, is considered the primary stimulus for local nitric oxide production. An
impaired production of nitric oxide can tilt the vascular balance towards
increased vasomotor tone. In horses such an effect could therefore result in
decreased perfusion of the laminar tissue of the hoof. Up to date no in viva
vascular studies on the modifying effect of vasodilation by glucocorticoids has
been investigated in the horse.

Objectives: To study the in vivo effects of glucocorticoid therapy on nitric oxide
induced vasodilation in the horse, and its possible implications for laminitis.
For this purpose the skin was used as a model for the laminar vascular bed of
the equine hoof. Responses to intradermal injections with synthetic M3-agonist
methacholine were investigated by means of thermography in horses that were
treated with either dexamethasone or saline. The effect of the treatments on
MCH was further studied with the coinjection of substances that either
attenuate (L-NAME) or aggravate effects of MCH (L-arginine, BQ—123). A

nitric oxide donor (SNAP) was studied as well.

105

 

Methods: In a blinded randomized cross over design, ten horses were treated with
either dexamethasone (0.1 mg/kg IV q24h) or saline IV for six days. On day six,
the changes in local average skin temperature before (baseline) and after
intradermal injections (20 minutes) that contained MCH (5 x 104M), L-
arginine (10’3 M), L-N AME (10'3 M), BQ-123 (10" M), or phenylephrine (PHE;
10“ M). A nitric oxide donor (SNAP; 3.75 x 10“ M), saline and placebo needle
stick were injected as well. The horses were studied in a cold environment in
order to induce baseline vasoconstriction. Skin on both shoulder areas was
studied for duplicate measurements.

Results: Baseline skin temperatures were significantly lower in the dexamethasone
group indicating an increase in vasomotor tone. There was a significant
increase in skin temperature over time which affected the overall sensitivity of
the study. Some significant effects were detected from various intradermal
injections. The data were, however, inconsistent and difficult to interpret.

Conclusions: Our results show that dexamethasone therapy in horses causes a
significant decrease in skin perfusion. This effect appears greater than under
normal room temperatures. The study was unable to answer the question if

dexamethasone impairs nitric oxide mediated vasodilation.

106

Introduction

Glucocorticoids are potent drugs that with great success have been used for
allergic and inflammatory airway diseases in the horse. However glucocorticoid therapy
in the horse has been associated with laminitis, a debilitating disease of the equine hoof
(Cornelisse and Robinson 2004). Several pathophysiologic mechanisms have been
proposed in the etiology of clinical laminitis. This includes an vascular model with
decreased perfusion of the laminar tissue in the hoof (Robinson, Scott et al. 1976; Hood,
Amoss et al. 1978).

Vascular tone at local tissue level is the net result from a delicate interaction
between parasympathetic and sympathetic tone, and the local production of vasoactive
substances such as nitric oxide (NO) and endothelin-1 (ET-1). Increased acetylcholine
release from parasympathetic nerves stimulates M3-receptors that are present on
endothelial cells. In response endothelial cells produce via increased activation of the
enzyme nitric oxide synthase increased amounts nitric oxide. The nitric oxide relaxes the
locally present vascular smooth muscles cells, which results in local vasodilation.
Glucocorticoids are known to modulate the production of nitric oxide and ET-l (Morin,
Asselin et al. 1998; Provencher, Villeneuve et a1. 1998; Wallerath, Witte et al. 1999;
Severino, Brizzi et al. 2002; Di Filippo, Rossi et a1. 2004). Therefore these drugs can
modulate the net balance between locally present vasodilators and vasoconstrictors.
Clinically glucocorticoids have indeed been associated with hypertension due to change
in vasomotor tone (Wallerath, Witte et al. 1999; Severino, Brizzi et al. 2002). A

glucocorticoid induced imbalance between locally active levels of vasodilating and

107

vasoconstricting substances could therefore be a vascular mechanism that results in
equine laminitis.

The aim of the present study was to investigate the in vivo effects of
glucocorticoid therapy on vascular tone in the horse with emphasis on the vasodilatory
action of nitric oxide. The skin was used as a vascular model because it has many
anatomical similarities to the equine laminar vascular bed and is easy to study. Local
nitric oxide production was stimulated with methacholine (MCH), a more stable form of
Mg-agonist. Additional coinjection with other vasoactive substances was used to estimate
the relative importance of nitric oxide in equine vascular tone. These substances included
L-arginine (ARG; nitric oxide precursor), L-NAME (nitric oxide synthase antagonist),
BQ-123 (ETA-receptor antagonist) and phenylephrine (PHE; vasoconstrictor). The local
perfusion in the skin is reflected in the skin surface temperature. The change in local
dermal circulation by the injected substances was therefore measured as changes in
surface temperature with a thermograph. Our hypothesis was that glucocorticoid therapy

impairs in vivo mediated vasodilation of the MCH from impaired nitric oxide production.

Material and methods

Preparation of solutions for intradermal injections

Fresh stock solutions of weighed amounts of endothelin receptor-A antagonists BQ-123a ,
nitric oxide precursor L-arginineb (ARG) and the nitric oxide synthase antagonist L-
NAMEc were prepared every 48 hours. For this the drugs were dissolved and further

diluted in phosphate buffered saline (PBS). The stock solutions were refrigerated at 10 °C

108

 

until further processing. A solution of methacholined(MCH), a synthetic M3- receptor
agonist, was freshly prepared in PBS prior to the experiments. Fresh MCH and stock
solutions were combined for intradermal injections. Final working solutions (RX)
therefore included BQ-123 (10“ M), ARG (10'3 M) and L-NAME (10'3 M) alone or with
MCH (5 x 10'4 M). Additional RX included freshly prepared control solutions in PBS of
nitric oxide donor SNAPe (3.75 x 10’4 M) and ail-agonist phenylephrinef (PHE; 10“1 M).
All solutions were kept in a water bath with a controlled temperature of 22°C prior to
injection. The concentrations of MCH, BQ-123, ARG, L-NAME, SNAP and PHE were
based on on expected maximal effects (Clough, Bennett et a1. 1998; Lipa, Neligan et al.
1999; Katugampola, Church et a1. 2000; Newton, Khan et a1. 2001; Milosevic, Janosevic
et al. 2002; Katz, Marr et a1. 2003; Vivancos, Parada et a1. 2003; Wilson, Monahan et al.

2004)

Horses

Ten recurrent airway obstruction- affected mares (age 16-27 years, weight 442-571 Kg)
(Robinson, Derksen et al. 1999) were used in the study. During the study, horses were in
clinical remission. They were fed a commercial pelleted diet. While on pasture the horses
were treated (TX) at 08.00 am with either dexamethasoneg (DEX 0.1 mg/kg IV q24h) or
an equivalent volume of saline (SAL IV q24h) for 6 consecutive days. The treatments
were administered according a randomized cross over design and were blinded to the
investigator. The wash out period between treatments was at least 2 weeks. Horses were
18-hours prior to the start of the experiments transported to a separate outside pen that

had free communication with a covered study facility that was open to the environment.

109

Immediately after arriving at this study facility, skin test areas (25 by 20 cm) over both
lateral shoulders were clipped and subsequently cleaned with alcohol. A 3 by 4 grid was
drawn on the area with a silver sharpie pen so that the lines were visible on thermography

images.

Study protocol

The studies were performed in an cold environment in order to archieve baseline
vasoconstriction. Therefore, during the winter months January and February, horses were
studied while standing in an examination stock that was centrally placed in the covered
study facility with open communication to the outdoor environment. Starting on the left
side, 0.1 ml of solutions containing MCH alone or MCH with BQ-123, ARG or L-
NAME, and control solutions containing BQ-123, ARG, L-NAME, SNAP and PHE were
injected intradermally with l-minute intervals between each injection. Additional
controls included 0.1 ml PBS and an empty needle stick. A 24-mm diameter circular
bandageh was applied for purposes of reference size. The location of all injections and
circular bandage was the same amongst all horses and treatments (figure 7.1), but
randomized in time order within treatment for each horse. Thermographic pictures of the
injection sites were taken at before (TIME= baseline), and 20 minutes after each injection
with a mounted thermal imager (Microscan 7515i). The emissivity of the imager was set
at 0.98 with a sensitivity of 0.7 °C, and the camera was adjusted for background
temperature and humidity. The imager was kept at a distance of 70 cm perpendicular to
the study area. The injection protocol and thermographic data collection were repeated on

the right side in an identical manner. Earlier pilot studies had determined that the circular

110

bandage size was similar to the vasoconstricted area of 0.1 ml intradermal PHE at 10'4 M.
During the study, environmental temperature and humidity data at the open study facility
were collected. The study was approved by the All University Committee for Animal Use

and Care at Michigan State University.

Image and data analysis

The digital therrnograph images were analyzed with complementary software (Microspec
2.7)i. First the total number of pixels in the area covered by circular bandage was
determined. Subsequently the average temperature (TAVG) of circular areas of similar size
(CIRCLE-1) and 1.5 times the size (CIRCLE-1.5) were measured over each individual
injection at baseline and 20 minute values. By electronically deleting CIRCLE-1
temperature pixels out of the CIRCLE-1.5 image it was additionally possible to calculate
the TAVG of the area between both circles (Tm/GL5). In this area it was less likely to find
residual effects related to injection or absorption of a small intradermal volume. A mixed
factorial procedure for repeated measures (SAS statistical software’) was used for
analysis of main effects TX, TM and RX on TAVGI or TAVGl.S of the left or the right side,
and for the averaged value of both sides combined . When significant effects (p < 0.05)
were observed for the main effects and their interactions, the Bonferoni test was used to
determine differences between means. The strength of association for TAVG data (SAL
and DEX combined) between the left side and right side was determined by simple linear
regression at times 0 and 20 minutes. The environmental data were compared with a

simple t-test.

111

Results

Intradermal injections (RX)

There were significant main effects of treatment (TX) and TIME on TAvm and TAVGM
when analyzed for the left side, right side, or both sides combined (table 7.1). The
dexamethasone treatment resulted on both sides, or when combined, in a significant
decrease of skin temperature for both TAVG! and Tax/01.5 (table 7.2). On both sides, and
when combined, TAVG) as well TAVGIS increased significantly between baseline and 20
minutes later (table 7.2). A significant TIME x TX interaction was detected on the left
side (table 7.1). TAVGI as well TAVGlS were significantly lower with dexamethasone
treatment than with saline. After 20 minutes, both temperatures had increased and were
no longer different from one another i.e. temperature increased more with dexamethasone
treatment (table 7.2). The various intradermal injections (RX) resulted in significant
effects only on Tavms and only on the right side (table 7.1). Post hoc analysis of RX
showed that injections of MCH plus NAME (mean i se: 30.81 i 0.36) resulted in
significantly higher Tax/(31.5 compared to injections with MCH (29.35 :t 0.36, p=0.006),
NAME-C (29.47 :1: 0.36, p=0.019), SAL (29.51 :1: 0.36, p=0.027) or PHE (29.51 i 0.36,
p=0.020). On the right side TX x RX and TIME x RX interactions were additionally
significant for TAVGM. The post hoc analysis of TX x RX interaction during
dexamethasone treatment revealed a significantly higher TAVGLS for MCH plus NAME
(29.81 :t 0.55) compared to SAL (27.14 i 0.55, p=0.0015), SNAP (27.66 at 0.55,
p=0.049) or MCH (27.0 d: 0.55, p=0.0012). MCH and SAL were additionally lower than
NDL (29.46 i 0.55, p=0.013). The many TIME x RX interactions that were significant in

the post hoc analysis showed that 20 minute values of TAvol.5-R of MCH, MCH plus

112

NAME, MCH plus BQ, NDL, SAL and SNAP were significantly higher than their

baseline values (table 7.3).

Strength of association between left and right side

Simple linear regression detected a poor association between left and right side at
baseline time for both rm. (r2 = 0.0023, p=0.62) and T m1, (r2=0.0018, p=0.65). At
baseline (mean 3: sem) TAVG was consistently lower on the left side (29.18 i 0.17 °C)
than on the right side (30.18 i 0.14 °C) (Fig 7.4a, 7.4c). The association between the left

and right side was much stronger at 20 minutes and significant for both TAVG) (r2 = 0.65,

p=0.001) and TAvol.5 (r2 = 0.66, p=0.001) (fig 7.4b, 7.4d).

Environment

There was a significant difference (p=0.007) in environmental temperature when horses
were treated with dexamethasone (2.3 i 1.4 °C, mean i se) compared to when they were
treated with saline (7.0 i 0.53 °C). Differences in environmental humidities were not

significant (p=0.07) (table 7.4)

Discussion

Dexamethasone resulted in a significantly lower baseline temperature of the skin. This
effect was similar but more pronounced (approximately 2 °C difference between saline
and dexamethasone treatment) compared to the findings in the vasoconstriction study
(approximately 1 °C, chapter 5). Based on the observations in the vasoconstriction study

it is likely that a greater response to various endogenous vasoconstrictors (ET-1, PHE)

113

resulted in a decreased of the baseline skin temperature observed here. Several studies
have reported that exposure to cold results in increased sympathetic activity with higher
circulating norepinephrine levels (Hiramatsu, Yamada et al. 1984; Jansky, Sramek et al.
1996). Clinically this is associated with hypertension and dermal. vasoconstriction
(Jansky, Sramek et a1. 1996). A potentiated response to endogenous catecholamines by
dexamethasone is thus likely to result in an even more increased basal vasomotor tone
under cold environmental conditions.

Over TIME temperature increased significantly on both sides of the horse and this
complicated the analysis of the data. On the left side this resulted in a significant
interaction with treatment (table 7.1, table 7.2). The observations that the horses became
more anxious and agitated (stamping, pushing, and attempts to back out the stock) during
the experiments can possibly explain the overall general increase in temperature at 20
minutes. The result, an increase in body heat production likely caused skin temperature to
rise over the study period on the left side and increased baseline temperature on the right
side (table 7.2, figure 7.4a, figure 7.4c). Consequently a more pronounced temperature
change by TIME was present on the left side that caused a blunting of the response to
injections (RX) and a significant TX x TIME interaction. However ongoing anxiety had a
smaller effect on the right side. This resulted for ”I‘M/(31,5 in a significant effect by the
injections (RX), and significant TX x RX and TIME x RX interactions. The effects of RX
and the interactions on the right side for T Av015 are however hard to explain because
some of the significant findings were opposite to what was expected. For instance in RX,
significantly higher temperatures were detected for MCH plus NAME and not MCH

alone or MCH combined with BQ-123 (ETA-blocker) or L-arginine (ARG: nitric oxide

114

donor). Similarly in the TX x RX interaction during dexamethasone treatment skn
temperature afte MCH was also lower than MCH plus NAME while not different from
saline but lower than NDL. MCH alone or when combined with ARG or BQ-123 had
however a significant effects over TIME, as did controls SAL and NDL (table 3). Overall
the significant findings in this study for only TAVG15 and only on the right side suggest
that besides TX and TIME other factors must have confounded the effects of RX. These
factors remain unexplained from these experiments but likely related to study design (e. g.
injections too close together, fixed injection locations, dose of MCH and the other
substances) or assumptions that were in hindsight possibly different than expected (e. g.
same baseline temperatures in different injection areas, same response of skin in different
injection areas, a 20 minute interval for measurement, combination of substances does
not interfere with ability of intradermal diffusion). Additionally it appeared that the
environmental temperatures during the experiments on the horses that were treated with
dexamethasone were significantly higher compared to experiments on horses treated with
saline (table 6.4). Although this was a relative small difference it could have resulted in a
different (underestimated) baseline perfusion of the skin with different responses to
intradermal injection due to better diffusion and uptake by increased perfusion in the
skin.

The aim of this study was to investigate the in vivo modulation of vasodilation by
glucocorticoids, with emphasis on NO mediated effects of MCH. The study reinforced
the findings of the earlier vasoconstriction study that dexamethasone treatment results in
a lower baseline temperature. This effect appeared amplified in a cold environment,

likely associated with increased levels of circulating catecholamines and increases in

115

sympathetic tone. Some significant effects were detected from intradermal injection of
MCH alone or with other substances. The data were inconsistent and difficult to interpret
likely due to confounding in the experimental design. The study was unable to answer the

question if dexamethasone impairs nitric oxide production.

116

Table 7.1: Effects of treatment (TX), TIME, injections (RX) and their
interactions on TAVG) and Tax/515 of the left (L) side, right side (R) or both sides
combined (L+R).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 7.1 P-VALUE
EFFECT rm,

TAve1. L TAvo1. R TAvo1. L+R
TX 0.0001 <0. 001 <. 0001
TIME <.0001 <0.001 <.0001
RX 0.465 0.477 0.131
TIME x TX 0.015 0.207 0.063
TX x RX 0.994 0.993 0.988
TIME x RX 0.212 0.206 0.503
TIME x TX x RX 0.986 0.987 0.934
EFFECT TAVG1.5

1'Avcs1.5. TAVG1.5.

L R TAvg1.s. L+R
TX <.0001 0.021 <.0001
TIME <.0001 <.0001 <.0001
RX 0.830 <.0001 0.845
TIME x TX 0.031 0.052 0.077
TX x RX 0.998 0.002 0.997
TIME x RX 0.488 <.0001 0.680
TIME x TX x RX 0.997 0.055 0.999

 

 

 

 

117

 

Table 7.2: Influence of main effects treatment (TX), TIME, injections (RX) and TIME
xTX interactions on TAvol and TAVGl.S (both in 0C) for left (L) side, right side (R) or

both sides combined (L+R).

a,b,c,# $

’ significantly different.

 

Table 7.2

   

MTLFT” "—

 

EFFECT

VALUE

mean

TAVGI

“RIGHT" ”fl "

 

error

p<0.05

mean error

  
 

 

 

TX

DEX

29.124

0.418

a

30.675 0.322

 

TX

SAL

31.069

0.418

32.468 0.322

 

TIME

0 MIN

28.066

0.421

b

30.788 0.322

 

TIME

20 MIN

32.127

0.414

b

32.356 0.322

 

TIME*TX

DEX

0 MIN

26.602

0.476

c,#

29.746 0.356

 

TIME*TX

SAL

0 MIN

29.530

0.476

c,$

31.829 0.356

 

TIME*TX

DEX

20 MIN

31.646

0.463

31.604 0.356

 

TIME*TX

 

SAL

 

20 MIN

32.608

 

0.463

 

 

 

 

33.107 0.356

 

 

 

 

Table 2

TAVG1.5

 

EF

GH

 

EFFECT

VALUE

mean

error

mean error

p<0.05

 

TX

DEX

29.215

0.394

31.548 0.723

 

TX

SAL

31.278

0.394

28.264 0.886

 

TIME

0 MIN

28.131

0.397

28.752 0.577

b

 

TIME

20 MIN

32.362

0.390

31.059 0.577

b

 

TIME*TX

DEX

0 MIN

26.694

0.454

30.568 0.730

NA

 

TIME*TX

SAL

0 MIN

29.568

0.454

26.936 0.894

NA

 

TIME*TX

DEX

20 MIN

31.736

0.441

32.527 0.730

NA

 

 

TIME*TX

 

SAL

 

20 MIN

 

32.988

 

0.441

 

 

 

 

29.591 0.894

NA

 

 

 

 

 

118

Table 7.3: Effect of TIME x RX interaction on Tavms (inoC) on the right side.

 

 

 

 

 

 

 

 

 

 

 

 

Table 7.3 BASELINE TIME = 20
p.

RX mean i so mean 1' 86 value
MCH 27.45 i 0.53 31.26 i 0.53 <.0001
MCH+ARG 28.35 i 0.53 31.79 2': 0.53 <.0001
MCH+BQ 28.30 i 0.53 32.12 i 0.53 <.0001
NDL 29.38 i 0.53 31.60 :t 0.53 0.01
SAL 28.44 i 0.53 30.58 i- 0.53 0.01
SNAP 28.62 i 0.53 30.63 :t 0.53 0.03

 

119

 

 

.-. -;v—-— '0'"

Table 7.4; Mean and se of the enviromental temperature (°C) and humidity (0%)
in the study facility when studying horses treated (TX) that were treated with
dexamethasone (DEX) or saline (SAL).

 

 

 

 

 

 

 

 

 

 

Table 7.4 Temperature Humidity
TX mean se mean se
SAL 7.00 0.53 72.50 2.78
DEX 2.32 1.45 64.60 3.05
p-value 0.0073 0.0700

 

 

120

Figure 7.1: a) Locations of intradermally injected vasoconstrictors 20 minutes
post administration. b) Thermographic image of figure 1a. See main text for

 

121

36

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

Fig. 7-233TAVG1-L; SAL
34 ~ I
I I I ,
32 — : ‘ I .
A 30 1 T __ "i
A: i , 1.1 ‘ , B
2' 28 “ I )1 it i. .4
g i
.l v.
'_< 26 ' pi ’ ‘ n :1 2
. p ' .
24 — L , E
I ,
22 1 - :1 t, I t
.‘i )6 1 H ‘
20 T I $1 “I I I l é I
\. \. .0 O. .0 G .0 ?
‘30 5% 9“ “C: 9090*6 P36 «(p3 P356 “\ka 15$“
0‘ “,0 \A c,\‘\
RX “‘
36
Fig. 7.2bz'rm,,_; DEX
34 _
T 1)— I
32 J I . .
A F I ‘i I
o 30 - :1 . i ‘ __
0" 1 i‘ I I
. 1 .
g 28 1 1 i 1 ii i" K F
> ' . i ' i
< _( . . " S
'- 26 i! h j t i“ *1 r'.
“it I ’ h
24 7 _. i 1 . '
22 -
20 ‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9» p,\«\.\€o\'\ .090. .030 5‘36 p?
a s v 8) 9&0" “$6” mpggfilx as
RX \8
Figure 7.2a,b: Effect of the interaction between treatment (DEX or SAL) ,TIME ( baseline:
Black bar, or 20 minutes: grey bar) and injected solutions (RX: MCH, MCH +ARG,

MCH+BQ, MCH+NAME, ARG, BQ, NAME, PHE, SALINE, SNAP,NDL) on TAVG) of the
left side (a:SAL, szEX). Data as mean and se. See text for abbreviations.

122

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

36
Fig. 7.2czTAVG1.R; SAL
34 a I 11, I I I I I 1' j
32 . ", " I 5 " I "I . I
‘ . : I 1' “I
A 30 - t t i "
°~:. . * . * " 1‘ :
a 28- i l . a . 9
“‘ “ . ‘ i 1 ~
i- 26 . ' . If i a (
24 ‘ I ' t I i.
22 I I "' I I .7 . 1
L. III ’ 4i 5. ‘ .
20 i l i i l l l l l: l
G" 0.0 “()6 P36 *90 we as. “9» “\6 Sp.» “p?
6.8 6 “‘0‘“ “930“,,“ “a“ RX 5
36
Fig. 7-2diTAVG1-R ; DEX
3H
32 — I I I I I I I I ‘5
a so - , " ‘ )1 . ‘
2.,
E 28 - .
g . r
l—< 26 - i ,1
24 ~ I
22 ~ I
i
20 . __

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0 0.0 ()6 955 *go. “(8 $0 “9» Q98; 61"" “P3
“I 9 IIGWIWQICNINQ“ mr 5
Figure 7.2c,d: Effect of the interaction between treatment (DEX or SAL) ,TIME ( baseline:
Black bar, or 20 minutes: grey bar) and injected solutions (RX: MCH. MC H +ARG,
MCH+BQ. MCH+NAME, ARG, BQ, NAME, PHE. SALINE. SNAP,NDL) on TWO) of the
right side (c:SAL, dzDEX). Data as mean and se. See text for abbreviations.

123

 

36
Fig. 7-293TAVG1-L+R ; SAL
32 ~ __’ --%
30 -

28—

O
TAVG1-L+R( c)

26-

24‘ p

 

 

22. I

20 . II

“9" $R\'Q\’\ a“ c“6§’\:\*&?€§r :Egg ec‘ipN‘efiR?
RX ‘ICI‘ w“

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

36

 

Fig. 7-2fiTAVG1-L4-R; DEX
32 - I
30 -
28 -

26-l

TAVG1-L+R (°C)

24~

 

22*

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

20 T I ' 1 ' I
go» sP~\’9\'\ EC”. 66‘3“, $.60ng ‘ 86:8,?“QIE, “F?
RX ‘10“ W“

 

Figure 7.2e,f: Effect of the interaction between treatment (DEX or SAL) ,TIME ( baseline: Black
bar. or 20 minutes: grey bar) and injected solutions (RX: MCH, MCH +ARG, MCH+BQ,
MCH+NAME, ARG, BQ, NAME, PHE, SALINE, SNAP,NDL) on TAVGI of the left + right side
(6: SAL, f: DEX. Data as mean and se. See text for abbreviations.

124

TAVG1.5-L (°C)

TAVG1.5-L (°C)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

36
Fig. 7-3a5TAVG1.5-Li SAL
34 n
I T I I
32 ‘
-- "r- "'I "it
30 - i i ‘ t
it ‘ I
28 - I 1 I
26- ' . i
24 - ’ .
- I
22 - i i“ i ,
2° ' ' e“ a i
o» p.» \.\ .0 0- .0
V‘ 5 \& 6“G“*6§?‘GY\
RX
36
34 J Fig. 7-3b3TAVG1.5-Li DEX
I I I
I i I 5 I , T I
30 i i l i I i
.. ~ ,‘ I ‘ a i .—
28 - x
26 - ‘
. > i 1
24 c ‘ _ “ I L
22 « l j r
I . .
mf'eia'c'id'c'ev
“0» 5M“ 0 . 2.90 G, 95’ e ‘5‘ “F
V" 9&0“ fiofi*p‘\&g\v§k 5

RX
Figure 7.3a,b: Effect of the interaction between treatment (DEX or SAL) , TIME (
baseline: Black bar, or 20 minutes: grey bar) and injected solutions (RX: MCH, MCH
+ARG, MCH+BQ, MCH+NAME, ARG. BQ, NAME, PHE, SALINE, SNAP,NDL) on
TAVGU of left side (a: SAL, b: DEX). Data as mean and se. See text for abbreviations

125

TAVG1.5-R (°C)

TAVG1.5-R (°C)

36

 

Fig. 7-3CZTAVG1.5-Ri SAL
34 .t

I N I i E I I 1-
30 - , it "
28 a H j I. it
26 ~ A ‘ it

24-

TTVEF

22 - , i i i .
. i i
20 , . ll- . a

“19°90” :ngwo‘w‘f‘fioxwy west ”set?

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

36

 

Fig. 7-3d3TAVG1.5-R; DEX
34 _

32— L E I

284
26~
24-

22-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

20 i i i
acfigoc' “((13 MM fig“ %pfi‘faec’x “0" “‘6 ”as“?
$0“

 

Figure 7.3c,d: Effect of the interaction between treatment (DEX or SAL) . TIME (
baseline: Black bar, or 20 minutes: grey bar) and injected solutions (RX: MCH, MCH
+ARG, MCH+BQ, MCH+NAME, ARG, BQ, NAME, PHE, SALINE, SNAP,NDL) on
TAVGLS of right side (c: SAL, d: DEX). Data as mean and se. See text for abbreviations

126

O
TAVG1.5-L+R ( C)

O
TAVG1.5-L‘I-R ( C)

36

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 7.38: TAVG1.5-L+R; SAL
34 ~ 1—
I I I I I if I I
32 ~ —— T T __ r —— y " —— "a -- "F V
i
30 - I ’ , l a
, ti 5 ,. k
28 d ' w J PI 4
L L B
26 ‘ s u I
t: A H
P
24 - . j M p
t i
22 - L i‘ ~ at ‘ q,
r t” . i‘ I
20

“0» 5p.» ?“e\ho“90’o *90'955’0 F90“e£ ”2““
“O“RXRAGYV‘ “go“k“

36

 

34 Fig 7-3f1TAVG1.5-L+Ri DEX

32~ I I I I I
30« t i
28-
26-

24-

 

22*

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

20

 

9» p.\«\.\€c,\‘\ .09 6.09.6 .G‘afi k?

\* ‘5 V w egcw' %c“*%p.g‘§*“k s“
RX “‘

Figure 7.3e,f: Effect of the interaction between treatment (DEX or SAL) , TIME ( baseline:

Black bar, or 20 minutes: grey bar) and injected solutions (RX: MCH, MCH +ARG,

MCH+BQ, MCH+NAME, ARG, BQ, NAME, PHE‘ SALINE. SNAP,NDL) on TAVG. 5 of
left * right side (e: SAL, f: DEX). Data as mean and se. See text for abbreviations

127

36

 

 

Fig 7a: TAVG1: Baseline
34 - ., . .
Q . 0
324 ”' .o'u‘io’.
O . *.. ~. . ‘ 0.. ..O
O O .
30 d o o . I ‘ .
m. _ W—fi—f
§28- ' %"g'. . o: . .
a: O .
b- ‘ .. on . e.
26 O . . Q .
‘ U o
24 ~ . 0.. .
22 ~ ' O ' .
0
O
20

 

 

 

16 18 20 22 24 26 28 30 32 34 36
TAVG1'L

Figure 7.4a: Linear regressions between temperatures from left and right at
either baseline or at 20 minutes. TAvcii 3) Baseline or b) 20 minues.

128

Figure 7.4b: Linear regressions between temperatures from left and right at

 

 

 

- Fig. 7b: TAVG1, 20 minutes
_ . c O o_____
o o
o- 1. am; “'3 "
‘ o .. F””""o
o... __..; o ‘ o.
_ . . o It... '3
o o o -__f’ §
- I . ..
_ e
O
C
. O
O
- o
22 24 26 28 30 32 34
TAVGi'L

either baseline or at 20 minutes. TAVGI: 3) Baseline or b) 20 minues.

129

 

36

 

 

 

 

 

 

Fig. 7c: TAVG1.5: Baseline
34 ‘ Q . Q ‘0 .. . . .
. o o o $ '
32 e o :’:O.l..‘.'..o.0'.
30 ‘ . o .. .0 O z 4
q _ W _
I528 - O .0 ’... . : O. . O
> o
+5 - 1' ’a' 3
26 o
’ o .0 .
24 '4 . . . .
’ o
22 d o
o
20 . 1 . fl . . . ,
18 20 22 24 26 28 30 32 34 36
TAVG1.5'L

Figure 7.4c: Linear regressions between temperatures from left and right at
either baseline or at 20 minutes.T Avg”: 0) Baseline or b) 20 minutes

130

TAvc1.5 'R

 

Fig. 7d: TAVG1.5. 20 min . .
34 .
32 -
30 a O
O
28 ~ f,
/,
26 - ,z/
x” O I
24 d . .
O o
22 T '
20 ‘ I l I r

 

 

 

 

Figure 7.4c: Linear regressions between temperatures from left and right at

either baseline or at 20 minutes. T AVG] _5: c) Baseline or b) 20 minutes

131

36

GENERAL CONCLUSIONS

The aims of my Ph.D studies were to investigate the clinical effects of glucocorticoid
therapy in the horse. My first study investigated the efficacy of oral and intravenous
dexamethasone for the treatment of RAO in horses. The purpose of this study was to
come up with a dose that would be efficacious, safe and easily applicable under practice
conditions. This included keeping the horses in a challenging environment. The question
of how fast these drugs result in clinical improvement, and thus owner satisfaction was
also studied. My research on horses in RAO showed, based on analysis of many lung
function parameters, that one time oral application of an inj ectable dexamethasone
solution at doses of 0.164 mg/kg with or without food, or at a dose of 0.082 mg/kg
without food, is at least as efficacious as the standard intravenous dose of 0.1 mg/kg
when compared to placebo treatment. All these dexamethasone applications resulted in a
statistical improvement of lung function within 6 hours and had effects for at least 30
hours (as determined by the intrapleural pressure change APPLMAx). Oral applications
tended to have longer duration of effect. A clinical relevant effect based on the cut off
APpLM AX for detection of heaves showed that dexamethasone IV and oral dexamethasone
at a dose of 0.164 mg/without food were the most efficacious with duration of effect for
up to 24 hours. Intravenous dexamethasone results in clinical and significant
improvement within 4 hours, although its effect is not as good as complete
bronchodilation by atropine. The practical implications of this study are therefore that an
owner can treat an exacerbation with on oral dose of O. l 64 mg/kg. They can expect

clinical improvement within 4-6 hours and a dosing interval under challenging conditions

132

in the order of 24-30 hours. On a daily scheme when drug administration before food can
be controlled, or in combination with improvements in the environment, interpolation
suggests that even lower doses and dose intervals are likely to be beneficial.

Because laminitis has been implicated as an adverse effect of glucocorticoid
therapy in the horse, my second and third study investigated the in vivo effects of
dexamethasone treatment on local vascular function. In these studies the skin was studied
as a representative vascular model with many similarities to that of the laminar vascular
bed of the equine hoof. The in vivo responses to various intradermally injected
concentrations of vasoconstricting (study 2) and vasodilating (study 3) substances were
measured with a thermo graphic imager in horses that were treated either intravenous
saline or dexamethasone. Dexamethasone treatment resulted in a significant lower
baseline temperature compared to saline suggesting an increased vasomotor tone. This
was accompanied by a greater and potentiated response to the all-agonist phenylephrine.
Additionally, the interplay with the lower baseline temperature resulted in a greater
response to vasoconstrictor endothelin-1. Study 3 confirmed and reemphasized the
finding of the lower baseline temperature by dexamethasone. The greater decrease in
baseline temperature was likely due to higher circulating levels of catecholamines under
cold experimental conditions. Unfortunately the statistical analysis of the data on nitric
oxide controlled vasodilation from intradermal injections of methacholine was difficult to
interpret. The reasons for this were a rise in skin temperature over time due to anxiety of
the horses, likely in combination with other experimental confounding factors.
Conclusion regarding impaired vasodilatory capacity of the equine vasculature during

glucocorticoid treatment could therefore not be made. Based on the similarities between

133

blood flow in the skin and the laminar vascular bed, study 2 and study 3 indicate that
glucocorticoid therapy can result in additional compromise of perfusion of the equine
hoof during disease states that are already characterized by hypoperfusion and/or
increased levels of catecholamines. Thus during such pathologic states, glucocorticoid
therapy could, according the vascular model of laminitis, tilt the balance in favor of
laminitis.

The results of these studies, in combination with the literature reviews of
glucocorticoid actions on RAO and the risk for laminitis, indicate that these drugs are of
great benefit for the treatment of RAO, a disease not associated with circulatory risk

factors.

134

APPENDIX A
FOOTNOTES

(Manufacturers’addresses)

Chapter 3

'Validyne, Northridge, California, USA

2Dash, Astro-Med Inc., West Warwick, RI, USA

3Buxco Electronics Inc., Sharon, Connecticut, USA

4Fleisch, OEM Medical, Richmond, Virginia, USA

5Dexamethasone solution (2 mg/ml): Vedco, Inc., St Joseph, Missouri, USA

6Sigmastat 2.0, SPSS Inc., Chicago, IL, USA

Chapter 6

a H-6995 (ET-1), H-1252 (BQ-123), H-2508 (RES-701): Bachem California, Torrance Ca
90505

b DMSO America, Lawrence, MA 01843

c N5751 (L-NAME): Sigma—Aldrich, St Louis, MO 63178

d Phenylephrine-HCL 1%: American Regent Laboratories, NY 11967

e Dexamethasone solution: Vedco, St Joseph MO 64507
fNexcarem: 3M Health Care, St Paul, MN 55144

g Microscan 7515 and Microspec 2.7: MikronR , Oakland, NJ 07436

h SAS 9.0: SAS Institute Inc, Cary, NC 27513

135

Chapter 7

a H-1252 (BQ-123): Bachem California, Torrance Ca 90505

b (L—ARGININE): Sigma-Aldrich, St Louis, MO 63178

C N5751 (L-NAME): Sigma-Aldrich, St Louis, MO 63178

d Methacholine: Sigma-Aldrich, St Louis, MO 63178 Laboratories, NY 11967
c ALX-420-00 (SNAP) : Alexis,

f Phenylephrine-HCL 1%: American Regent

g Dexamethasone solution: Vedco, St Joseph MO 64507
h Nexcarem: 3M Health Care, St Paul, MN 55144

‘ Microscan 7515 and Microspec 2.7: MikronR, Oakland, NJ 07436

j SAS 9.0: SAS Institute Inc, Cary, NC 27513

136

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