Effect of intravenous lidocaine administration on laminar
inflammation in the black walnut extract model of laminitis
J. M. WILLIAMS*, Y. J. LIN†, J. P. LOFTUS‡, R. R. FALEIROS, J. F. PERONI§, J. A. E. HUBBELL, W. R. RAVIS†and J. K. BELKNAP
Sciences,Auburn University,Auburn,Alabama 36849;‡Department of Veterinary andAnimal Sciences, University of Massachusetts,Amherst,
Massachusetts 01003; and§Department of Large Animal Medicine, University of Georgia, Athens, Georgia 30602, USA.
Keywords: horse; laminitis; lidocaine; cytokine; leucocyte emigration; real time-quantitative PCR (RT-qPCR)
complication of horses suffering from sepsis/endotoxaemia-
related events. Laminitis in horses and organ injury in human
sepsis are both reported to involve inflammatory injury to the
laminae/organs including early activation of endothelium and
leucocytes leading to emigration of neutrophils into the tissue
interstitium. In the black walnut extract (BWE) model,
systemic inflammatory events coincide with marked increase
in laminar mRNA concentrations of inflammatory genes
including proinflammatory cytokines (i.e. IL-1b, IL-6),
COX-2, chemokines (i.e. IL-8) and endothelial adhesion
molecules (i.e. ICAM-1 and E-selectin). In models of human
sepsis, i.v. lidocaine has been reported to decrease leucocyte
proinflammatory cytokines and chemokines.
Objectives: To evaluate the effect of i.v. lidocaine therapy on the
inflammatory processes documented to occur in the BWE
model of laminitis.
Methods: Twelve horses were administered BWE and treated
immediately with either lidocaine (1.3 mg/kg bwt bolus,
followed by 0.05 mg/kg bwt/min CRI, n = 6) or saline (n = 6)
for 10 h. At 10 h post BWE administration, laminar samples
were obtained under general anaesthesia for assessment of
proinflammatory gene expression (using RT-qPCR) and
leucocyte emigration (via CD13 immunohistochemistry).At 0,
3 and 10 h post BWE administration, skin samples were
obtained for assessmentof
Results: No significant differences between groups were noted
for inflammatory gene mRNA concentrations (IL-1b, IL-6,
IL-8, COX-2) or for number of leucocytes present within the
laminar interstitium or skin dermis. Increased (P<0.05)
laminar E-selectin mRNA concentrations were present in the
LD group (vs. SAL group).
Conclusions: Continuous administration of i.v. lidocaine does
not inhibit inflammatory events in either the laminae or skin
in the horse administered black walnut extract.
Potential relevance: This work questions the use of continuous
i.v. administrationof lidocaine
inflammatory therapy for systemic inflammation.
as aneffective anti-
Lidocaine hydrochloride has been used extensively in human and
veterinary medicine to provide local and regional anaesthesia, and
to treat and prevent ventricular arrhythmia (Mama and Steffey
2001; Plumb 2005). Systemically, its use in veterinary medicine,
particularly in equine medicine, has been as a treatment for post
operative ileus (Brianceau et al. 2002; Cohen et al. 2004; Malone
et al. 2006). Lidocaine has also been reported to have inhibitory
effects on a broad spectrum of systemic inflammatory processes
(Hollmann and Durieux 2000). It has been shown to inhibit
neutrophil adhesion and migration (Schmid et al. 1996), reduce
free radical production by neutrophils, and inhibit the phagocytic
activity of leucocytes (Siminiak and Wysocki 1992; Hyvonen and
Kowolik 1998). Lidocaine reportedly decreases gene expression of
various cytokines (i.e. IL-1b, IL-6, IL-8) in epithelial cells,
endothelial cells and neutrophils (Sinclair et al. 1993; Lahav et al.
2002; Lan et al. 2005) and attenuates cytokine-induced endothelial
injury (de Klaver et al. 2003) and endothelial cell expression of
extravasation (i.e. ICAM-1) (Lan et al. 2005).
Laminitis is a serious disease process in which laminar injury
occurs as a remote organ injury in animals suffering from a wide
array of septic conditions. Acutely, laminitis occurs secondary to
numerousdisease states affecting
respiratory, reproductive, endocrine or musculoskeletal systems
carbohydrate overload or extract from black walnut heartwood
(BWE) (Garner et al. 1975; Galey et al. 1991). Using the BWE
model, researchers have detailed both systemic and local
inflammatory processes in the laminae in the early developmental
stages of the disease process, with many similarities to
inflammatory events leading to organ injury in human sepsis.
These BWE-induced inflammatory events include activation of
circulating leucocytes (Hurley et al. 2006), activation of adhesion
molecule expression by the laminar endothelium (Loftus et al.
2007)and upregulation of
proinflammatory mediators (Fontaine et al. 2001; Belknap et al.
2007; Loftus et al. 2007). This inflammatory process in the BWE
model is characterised by increased laminar mRNAconcentrations
of proinflammatory cytokines/chemokines (IL-1b, IL-6 and IL-8),
increasedlaminar mRNA and
of the gastrointestinal,
laminar geneexpression of
*Author to whom correspondence should be addressed.
[Paper received for publication 10.04.09; Accepted 15.06.09]
EQUINE VETERINARY JOURNAL
Equine vet. J. (2010) 42 (3) 261-269
© 2010 EVJ Ltd
cyclooxygenase-2 (COX-2), and an influx of inflammatory
leucocytes (neutrophils and monocytes) into the dermal laminae at
both the developmental stage of laminitis and at the acute onset of
clinical signs (Waguespack et al. 2004a,b; Black et al. 2006;
Blikslager et al. 2006; Belknap et al. 2007; Loftus et al. 2007;
Faleiros et al. 2009).
Due to the reported inhibitory effects of systemic lidocaine on
the majority of inflammatory events reported to occur in the
laminae in the BWE laminitis model, and its proposed use as an
anti-inflammatory therapy in human inflammatory conditions
including systemic inflammation in sepsis (Siminiak and Wysocki
1992; Sinclair et al. 1993; Schmid et al. 1996, Hyvonen and
Kowolik 1998; Hollmann and Durieux 2000; Lahav et al. 2002; de
Klaver et al. 2003; Lan et al. 2005), the effect was evaluated of a
constant rate infusion (CRI) of lidocaine on multiple inflammatory
processes that occur in the BWE model of laminitis. It was
hypothesised that a CRI of lidocaine would markedly decrease
laminar inflammatory events including endothelial activation,
leucocyte emigration into the laminar tissue, and proinflammatory
cytokine and chemokine mRNA concentrations.
Materials and methods
Twelve healthy Standardbred horses (age 3–12 years) with no
apparent forelimb foot abnormalities or forelimb lameness (as
determined by lameness evaluations and hoof tester application)
were obtained from an outbred population. The horses were
quarantined and kept together for 2 weeks at pasture at a private
boarding facility. They were then moved to the Ohio State
University Finley Farms facility and kept in quarantine for 2 weeks.
While quarantined, physical examinations including assessment of
rectal temperature, heart rate, respiratory rate, abdominal sounds
and digital pulses were performed daily. The horses were moved
subsequently to the Galbreath Equine Center at the Ohio State
University for completion of the study.
All animal protocols were approved by the Institutional Animal
Care and Use Committees of the Ohio State University. The health
status of the horses was evaluated by physical and lameness
examination on the day of experiment to ensure no concurrent
illness that would affect peripheral white blood cell count and to
ensure no forelimb lameness. An indwelling catheter was placed
into each jugular vein, one for blood sample collection and the
other for constant rate infusion of lidocaine or saline. A blood
sample was obtained for haematology and plasma was used for
determining baseline lidocaine concentrations. Skin samples were
obtained with a 6 mm diameter skin biopsy punch from the cranial
aspect of the right neck (0 h time point) and caudal aspect of the
right neck (3 h time point) following local skin anaesthesia with
1 ml of mepivacaine per site. The BWE was made by soaking 2 g
black walnut heartwood shavings/kg bwt in 6 l deionised water as
previously described (Belknap et al. 2007). Six litres of BWE were
administered via nasogastric tube. Horses were assigned randomly
to one of 2 treatment groups, with 6 horses in each group: 1)
lidocaine 1.3 mg/kg bwt loading dose administered over 15 min,
followed by 0.05 mg/kg bwt/min constant rate infusion; or 2) 0.9%
saline administered as a bolus and constant rate infusion at
administrators of lidocaine/saline were blinded to the contents of
each therapy, and therefore calculated each administration as if it
Physical examinations were performed by observers blinded to
treatment and included assessment of vital parameters, abdominal
and rolling, flank watching, Flehmen response), weight shifting.
described above at 0, 3 and 10 h post BWE administration and were
snap frozen immediately in liquid nitrogen in addition to formalin
xylazine (1.1 mg/kg bwt, i.v.) and anaesthetised using diazepam
(0.1 mg/kg bwt,i.v.)andketamine(2.2 mg/kg bwt,i.v.).Thehorses
were intubated orotracheally and a deep surgical plane of
anaesthesia maintained with isofluorane during sample collection
(see below). Intravenous constant rate infusions of lidocaine or
saline were continued throughout the anaesthetic procedure until
euthanasia with pentobarbital sodium containing phenytoin sodium
(Beuthanasia-D)1(20 mg/kg bwt, i.v.).
basedupon lidocaine administration.The
Each distal limb was removed rapidly by disarticulation of the
metacarpo- and metatarsophalangeal joints after placement of a
tourniquet, and 1.5 cm thick sagittal sections of the digit were
immediately cut with a band saw. The laminae were dissected
rapidly from the hoof and third phalanx, and some sections were
snap frozen immediately in liquid nitrogen, while the others were
placed in 10% formalin. The processing time from disarticulation
to placement of samples in formalin or liquid nitrogen was
approximately 5 min. All horses were subjected to euthanasia with
pentobarbital sodium (1 ml/4.54 kg bwt) while under anaesthesia
at the end of the protocol. Samples fixed in formalin were
transferred to 70% ethanol 24 h after collection and stored at 4°C
until paraffin-embedded. Flash frozen samples were kept in liquid
nitrogen and transferred to -80°C approximately 30 min after
collection. All skin samples were frozen in liquid nitrogen or fixed
in formalin in an identical manner to the laminar samples.
Formalin-fixed laminar and skin samples were paraffin-embedded
and sectioned. Sample slides were created with multiple
immunohistochemical methods were performed as previously
described (Lunn et al. 1998; Black et al. 2006; Faleiros et al.
2009). Briefly, slides were deparaffinised and antigen retrieval
performed using citrate buffer. An anti-equine CD13 monoclonal
antibody (courtesy of Dr D. Paul Lunn, Department of Clinical
Sciences, College of Veterinary Medicine and Biomedical
Sciences, Colorado State University, Fort Collins, Colorado, USA)
was used as a marker to assess the presence of leuococytes
(neutrophils and monocytes) in the laminae in order to compare
results to those previously reported in the BWE model (Black et al.
2006), while a mouse anti-human calprotectin monoclonal
antibody (MAC387)2was used as a marker to assess the presence
of leucocytes (neutrophils and monocytes) in the skin (calprotectin
© 2010 EVJ Ltd
262Effect of i.v. lidocaine administration on laminar inflammation
staining more effectively detected skin leucocytes compared to
CD13 in a pilot experiment).
Localisation of CD13 and calprotectin antigens was performed
via routine IHC protocols as previously described (Black et al.
2006; Faleiros et al. 2009), using a commercial immunoperoxidase
kit (Vectastain Elite ABC Kit)3and 3′3 diaminobenzidine
hydrochloride (DAB- Peroxidase Substrate Kit)3. Slides were
assessed by an investigator blinded to the horse and treatment
group from which the sections were obtained. Laminar CD13-
positive cell counts were performed by centring the 40x objective
on a laminar vessel in the primary dermal lamina, and all positively
stained cells within the field were counted. Thirty (10 fields per
section ¥ 3 sections per slide ¥ 1 slide per horse) 40x fields were
counted from each horse and averaged yielding one CD13-positive
cell count per horse. Skin calprotectin-positive cell counts were
performed by taking 0.54 ¥ 0.46 mm sized photocaptures (Image
Scope)4of each sample. Twenty photocaptures were obtained for
each sample, and counts were performed electronically by Image
J5. The blinded observer ensured that cells highlighted by the
program were appropriately labelled leucocytes. The counts were
then averaged together to yield one count per time point per horse.
Plasma lidocaine concentrations
Venous blood samples were obtained for measurement of plasma
lidocaine concentrations at 0, 3 and 10 h post BWE administration.
Plasma lidocaine concentrations were determined by an analytical
method modified from a procedure previously described by Rofael
and Abdel-Rahman (2002). An internal standard (ketamine) and
borate buffer (pH 9.0) were added to plasma samples followed by
extraction with a mixture of isopropanol-chloroform (1:9, v/v).The
mixture was vortexed for 1 min and then centrifuged at 1000 g for
10 min at 4°C. The organic layer was aspirated and dried under a
gentle stream of air. The residue was reconstituted with mobile
phase and analysed with a Waters HPLC system6.
The aqueous portion (65%) of mobile phase consisted of
100 mmol/l monobasic phosphate with 30 mmol/l triethylamine
dissolved in distilled water. The organic portion (35%) of the
mobile phase consisted of 60% acetonitrile and 40% of methanol
(v/v). The HPLC system was equipped with a Phenomenex
precolumn (SecurityGuard)7and a C-18 reversed phase column
(Luna 5 mm, C18, 100A, 100 ¥ 4.6 mm)7. The mobile phase flow
rate was 1.5 ml/min and UV detector (Jasco UV 2075 plus)8was
performed at 210 nm.
The resolution times for ketamine and lidocaine were 9 and
12.5 min, respectively. A linear equation was obtained for plasma
concentrations of 40–2808 ng/ml (correlation coefficient: 0.9994).
The extraction efficiency for lidocaine from plasma was 97%. Both
intra- and interday assay variations were <10%. The limit of
detection in plasma was 10 ng/ml and the limit of quantification
was 40 ng/ml.
RNA isolation and cDNA synthesis
Total RNA was extracted from the forelimb laminae of each horse
(Absolutely RNA Miniprep)9. PolyA mRNA was then isolated
(mRNA Extraction Kit)10and used to make cDNA via reverse
transcription (Retroscript)11. The cDNA was used to perform a
real-time quantitative polymerase chain reaction (RT-qPCR) to
determine mRNA concentration of different genes of interest.
Real time-quantitative PCR (RT-qPCR) procedure and
A real-time thermocycler10was used and quantification with
external standards was performed with the fluorescent format for
SYBR Green I dye as previously described (Waguespack et al.
2004a,b). Previously designed and created primers for IL-1b, IL-6,
IL-8, COX-2, E-selectin and 3 housekeeping genes (b-actin,
b2-microglobulin and glyceraldehyde-3-phosphate dehydrogenase)
were used for RT-qPCR (Belknap et al. 2007; Loftus et al. 2007).
All PCR reactions were performed in glass capillaries7in 20 ml
volumes (5 ml sample cDNA and 15 ml 1.33 X PCR master
mixture). The master mixture was created and the PCR reaction
was performed as per previously described for similar genes of
interest (Belknap et al. 2007). Standard curves were performed for
each gene product and water included as a negative control.Aserial
dilution of cDNA from each gene of interest was performed as
previously described (Belknap et al. 2007) to generate standard
curves. These curves were used for quantification of both the target
genes and housekeeping genes in each cDNA sample for the
normalisation process. Standards and laminar cDNAwere prepared
in separate capillaries but always amplified during the same PCR
run. The reactions were performed in duplicate for the individual
laminar cDNA samples from each horse.
Real-time qPCR was performed for 3 housekeeping genes. The
resulting data for these genes were assessed by use of the geNorm
computer program9. The 2 housekeeping genes that were reported
as acceptable and received the best score from geNorm,
b2-microglobulin and b-actin, were used to make a normalisation
factor. The amplification data obtained by the RT-qPCR for the
different genes were divided by the normalisation factor of the
housekeeping genes in the same sample in order to normalise
the RT-qPCR data for each gene of interest.
Once RT-qPCR sample data were divided by the normalisation
factor, a D’Agostino and Pearson omnibus test was performed
(which confirmed a normal distribution of the data sets), followed
by a 2-tailed unpaired t test on the data of each individual gene of
interest (IL-1b, IL-6, IL-8, COX-2, E-selectin) from each horse for
the 2 treatment groups. Values of P<0.05 were considered
significant. Laminar and skin leucocyte counts were tested for
normal distribution with a D’Agostino and Pearson omnibus test,
and a 2-tailed unpaired t test was performed between the 2 groups
(SAL and LD). Values of P<0.05 were considered significant.
The temperatures of all horses in the study ranged from 36.9–40.
3°C. The average high temperature was higher (P = 0.0053) for the
SAL group (39.4°C ? 0.26) vs. the LD group (37.8°C +/- 0.12).
Two horses within the LD group exhibited signs of colic vs. 5
horses in the SAL group. All horses in the SAL group had
decreased gastrointestinal sounds vs. 4 horses in the LD group. One
horse (LD group) exhibited signs of ataxia lasting approximately
30–45 min in duration. Only one horse in the SAL group was
subjectively considered “bright, alert and responsive” vs. 4 horses
in the LD group.
© 2010 EVJ Ltd
J. M. Williams et al.
The lidocaine concentrations (mean ? s.e.) for the LD group at
time points 0 h, 3 h and 10 h were 0 ? 0 mg/ml, 0.820 ? 0.117
mg/ml and 0.951 ? 0.192 mg/ml, respectively. Concentrations for
Horse 6 (LD6) were well below the reported therapeutic minimum;
therefore, the data for this horse were excluded from this study.
Concentrations of all other horses in the study were within
acceptable therapeutic ranges and thus included in the study.
Peripheral leucocyte counts
the study at time point 0 h (before administration of BWE or
systemic saline/lidocaine) was 6.16 ? 0.39 ¥ 109cells/l. The mean
? s.e.WBC count for all horses included in the study at the 3 h and
10 h time point was 2.98 ? 0.28 ¥ 109cells/l and 7.83 ? 0.65 ¥ 109
between the 0 h and 3 h time points and an increase (P<0.0001) in
WBC count between the 3 h and 10 h time points for all horses
receiving BWE. When comparing the averages of the WBC counts
between the LD and SALgroups at these time points, there were no
differences (0 h: P = 0.95, 3 h: P = 0.59 and 10 h: P = 0.81).
CD13-positive cells were present in the laminar interstitium of all
horses at the 10 h time point (Fig 1) in numbers comparable to the
results previously reported (Black et al. 2006). The number of cells
present in the laminae of BWE treated horses at the 10 h time point
in that study were increased (P<0.05) when compared to the
laminae of the control (non-BWE treated) group. There was no
difference (P= 0.91) between the number of CD13-positive cells in
the laminae of horses treated with saline vs. lidocaine at the 10 h
timepoint (Fig 1). Aspreviously
immunohistochemistry to label skin leucocytes (Black et al. 2006),
a small number of calprotectin-positive leucocytes were identified
surrounding superficial dermal vessels in the skin immediately
prior to BWE administration (0 h) in both the saline and lidocaine
calprotectinpositive cells surrounding the superficial dermal
vessels in the skin at the 10 h time point in both the saline and
lidocaine treated groups (Fig 2) when compared to the 0 h samples.
There was no difference (P = 0.28 and P = 0.81) between the
number of calprotectin-positive leucocytes in the skin dermis of
horses treated with saline vs. lidocaine at either the 3 h or 10 h time
points, respectively (Fig 2).
groups.There wereincreased numbers of
Laminar gene expression
To ensure that a similar inflammatory response occurred in the
laminae in the current BWE protocol as that reported previously in
this model, we compared laminar IL-6 mRNA concentrations
administration) of the current study, and non-BWE treated control
animals from a previous study (archived samples from previous
BWE protocol [animals administered water instead of BWE],
n = 5). There was a 491-fold increase (P = 0.015) in mRNA
concentration of IL-6 in the laminae of the SAL group (BWE-
treated, administered i.v. saline instead of lidocaine) when
compared to laminar samples from the archived non-BWE treated
controls (administered water),
administered in the present study effectively induced laminar
inflammation. When comparing the mRNA concentrations of
cytokines IL-1b, IL-6 and IL-8 in the laminae of SAL vs. LD
groups, there was no difference (P = 0.3876, P = 0.3505 and
P = 0.3596, respectively) (Figs 3a–c). There was no difference
(P = 0.7668) in mRNA concentration of COX-2 in the laminae of
SALvs. LD groups (Fig 3d). There was an increase (P= 0.0345) in
laminar mRNAconcentration of the endothelial adhesion molecule
E-selectin in the LD group as compared to the SAL group (Fig 3e).
indicatingthat the BWE
Lidocaine has been investigated as a possible anti-inflammatory
medication in multiple human diseases, including studies assessing
ischaemia/reperfusion injury and ulcerative colitis (Bjorck et al.
2002; Yokoyama and Onishi 2005). In several studies, there is
agreement that lidocaine modifies the normal immune response of
Treatment groups (10 h)
CD13-positive cells/40x field
Fig 1: Cell counts of CD13-positive cells (leucocytes) in laminae of
salinetreated (SAL) and lidocaine-treated (LD) horses 10 h after
nasogastric administration of black walnut extract (BWE). The bar located
within the middle of the each column represents the mean of the data set.
0 h0 h3 h
3 h 10 h 10 h
Calprotectin-positive cells/0.25 mm2
Fig 2: Cell counts of calprotectin-positive cells (leucocytes) surrounding
superficial dermal vessels in laminae of saline-treated (SAL) and lidocaine-
treated (LD) horses 0, 3 and 10 h after nasogastric administration of black
walnut extract (BWE). The bar located within the middle of the each column
represents the mean of the data set.
© 2010 EVJ Ltd
264 Effect of i.v. lidocaine administration on laminar inflammation
a variety of cell types (Hollmann et al. 2001; Lan et al. 2005). The
collective results of numerous in vitro studies evaluating the effects
of lidocaine or lidocaine-related local anaesthetics on leucocytes
indicate an inhibitory effect of lidocaine on neutrophil expression
of surface integrins CD11b and CD18 (important in adhesion to
endothelial surface, Martinsson et al. 1997), adhesion of leucocytes
to injured venules (Giddon and Lindhe 1972; MacGregor et al.
1980; Martinsson et al. 1997), tissue migration of macrophages
(Dickstein et al. 1985), and LPS-stimulated secretion of IL-1a and
leucotriene B4 from peripheral blood mononuclear cells (Sinclair
et al. 1993).
Furthermore, in an in vitro study by Lan et al. (2005), lidocaine
attenuated the expression of cytokines/chemokines IL-1b, IL-6 and
IL-8, as well as the expression of ICAM-1 (intracellular adhesion
molecule-1) by activated endothelial cells (HUVECs). These
antiinflammatory effects of lidocaine are further supported by the
inhibition of secretion of IL-8 and IL-1b by TNF-a-stimulated
intestinal epithelial cells exposed to lidocaine (Lahav et al. 2002).
In vivo effects of lidocaine were examined in rabbits in which
lidocaine attenuated increases in serum concentration of IL-8 and
IL-6 following i.v. administration of endotoxin (Taniguchi et al.
In horses, lidocaine CRI has been assessed as a treatment for
gastrointestinal injury using an intestinal mucosal healing model of
equine small intestinal ischaemia/reperfusion (Cook et al. 2008). In
this study, the effects of lidocaine with and without concurrent
treatment with flunixin meglumine were examined on the intestinal
wall recovery following ischaemic injury to the gastrointestinal
tract. The results of the study indicated that systemic lidocaine
attenuates a flunixin meglumine-induced increase in LPS
permeability of ischaemic-injured jejunum (Cook et al. 2008). In a
more recent report by the same investigators, the attenuation does
not appear to be a direct inhibition of inflammatory events, as
lidocaine alone had no effect on ischaemia-induced neutrophil
extravasation (Cook et al. 2009a). It is possible that lidocaine’s
effect is only to limit sodium influx into ischaemia-injured cells
mRNA concentrations (copies/NF)
mRNA concentrations (copies/NF)
mRNA concentrations (copies/NF)
mRNA concentrations (copies/NF)
mRNA concentrations (copies/NF)
Fig 3: Laminar mRNA concentrations of IL-1b (a), IL-6 (b), IL-8 (c),
COX-2 (d) and E-selectin (e) obtained via RT-qPCR from saline- (SAL) and
lidocaine-treated (LD) horses 10 h after nasogastric administration of
black walnut extract. The bar located within the middle of the each column
represents the mean of the data set.
© 2010 EVJ Ltd
J. M. Williams et al.
(Sheu and Lederer 1985); excess intracellular sodium due to Na/K
ATPase pump dysfunction in ischaemic epithelial cells reportedly
reestablishment of barrier function in an injured epithelial lining
(Rajasekaran and Rajasekaran 2003). A more rapid return of the
mucosal barrier function due to lidocaine-mediated change in
permeability, but would also remove a stimulus for neutrophil
extravasation as the injured tissue returned to a normal state.
In equine medicine, a constant rate i.v. infusion of lidocaine is
commonly used as a therapy to enhance motility or prevent the
development of ileus in cases recovering from gastrointestinal
surgery (Brianceau et al. 2002; Malone et al. 2006). However,
despite its use as a pro-motility agent, the mechanisms of action of
systemic lidocaine have not yet been elucidated. The proposed
roles of systemic lidocaine that may contribute to its use for
motility are that of an analgesic, a direct prokinetic, or an
antiinflammatory agent (Cook and Blikslager 2008). The
gastrointestinal pain was challenged and determined to have no
effect on visceral pain (Robertson et al. 2005). When tested as a
direct prokinetic, lidocaine had no effect on contraction of the
pylorus or midjejunum when placed topically (Nieto et al. 2000),
and did not have a different effect on the myoelectrical activity of
post operative jejunum as compared to the administration of saline
when administered systemically (Milligan et al. 2007). Therefore,
it has been suggested that the reported promotility effects of
systemic lidocaine are due to its potential as an anti-inflammatory
agent (Cook and Blikslager 2008). As discussed above, the results
of the current study do not support any efficacy of lidocaine as a
systemic anti-inflammatory therapy in the horse, and question any
direct anti-inflammatory effect in any tissue including the GI tract.
Anecdotally, systemic lidocaine has been used clinically for
equine cases suffering from laminitis (Belknap 2005). This action
theoretically has merit given the aforementioned results of
lidocaine and inflammation and more recent research that has
indicated that similar to organ failure in human sepsis, early
inflammation may lead to a series of events that culminate in failure
of the laminae (Belknap et al. 2007). In the current study, laminar
IL-1b, IL-6 and IL-8 concentrations were assessed due to the facts
that: 1) these cytokines/chemokines are commonly increased in
human sepsis studies (Cohen 2002; Boontham et al. 2003; Bhatia
and Moochhala 2004; Frantz et al. 2005); 2) the 3 cytokines/
chemokines have been found to be consistently increased in
laminae in the BWE model of laminitis at a similar time point
(Belknap et al. 2007); and 3) the 3 cytokines have been reported to
be significantly decreased in studies using i.v. lidocaine (Lahav
et al. 2002; Lan et al. 2005). Although TNF-a is also usually
increased in human sepsis-related inflammation studies, it was not
assessed as it has never been increased in laminar tissues in
laminitis models (Belknap et al. 2007; Loftus et al. 2007).
There wereno differences
concentrations of IL-1b, IL-6 and IL-8 between the LD and SAL
groups, indicating no effect of a lidocaine CRI on expression of
these inflammatory mediators. However, it was necessary to
confirm that the lack of difference was not due to a lack of response
of all animals to the BWE that was administered. Therefore, the
increased laminar IL-6 mRNA concentrations in the SAL group
from the current study (administered BWE followed by a CRI of
saline) compared to archived non-BWE treated control laminar
samples documented that the horses in this study underwent a
similar inflammatory response as previously reported in this model.
Furthermore, all horses receiving BWE in this study (SAL and LD
groups) became leucopenic, a response consistent with systemic
inflammation in the BWE model. Therefore, the lack of difference
in cytokine mRNA concentrations between SAL and LD groups in
the current study are due to a lack of efficacy of lidocaine, not due
to a lack of inflammatory response of the horses to the BWE
administration. The lack of effect of lidocaine in decreasing
laminar IL-1b and IL-6 mRNAconcentrations is corroborated by a
previous study in HUVECs in which lidocainemediated attenuation
of IL-1b and IL-6 gene expression by activated endothelial cells
was only achieved at doses exceeding the toxic in vivo threshold
and thus deemed clinically unsafe (Lan et al. 2005). At doses
within the therapeutic range, lidocaine had no effect on the
expression of IL-1b or IL-6 (Lan et al. 2005).
An important event in human sepsis-related organ injury that is
also present in the equine laminae is a prominent emigration of
leucocytes through an activated endothelium into the tissue
interstitium (Cohen 2002; Mizgerd 2002; Strassheim et al. 2002;
Annane et al. 2005; Nourshargh and Marelli-Berg 2005; Black
et al. 2006). In human sepsis, leucocytes entering the extravascular
tissues produce multiple cytokines, reactive oxygen species and
matrix metalloproteases (MMPs), which ultimately can result in
organ failure (Maier 2000; Aldridge 2002; Annane et al. 2005).
Therefore, extravasated leucocytes are likely to be a primary source
of injurious inflammatory molecules reported to be present in
proinflammatory cytokines (Johnson et al. 1998; Fontaine et al.
2001; Belknap et al. 2004; Kyaw-Tanner and Pollitt 2004;
Waguespack et al. 2004b; Black et al. 2006). Increased mRNA
concentrations of the endothelial adhesion molecule, E-selectin, are
present in affected laminae in the BWE model of laminitis at the
time point laminar tissue was assessed in the current study (Loftus
et al. 2007). E-selectin mediates the initial attachment/rolling of
neutrophils and other leucocytes to venules at sites of inflammation
(Abbas et al. 2000).
In the current study, there was an increase (P<0.05) in the
expression of laminar E-selectin in the LD group compared to the
SAL group; this was the only significant difference observed
between LD and SAL groups in any of the parameters assessed in
this study. Importantly, this increase in laminar E-selectin mRNA
concentration in the LD group implies that the systemic
administration of lidocaine has an inflammatory/activating effect
on the endothelium. This in vivo finding is corroborated by a recent
report in which ex vivo exposure of equine neutrophils to lidocaine
did not inhibit neutrophil migration or adhesion at a therapeutic
concentration, and actually increased neutrophil adhesion and
transendothelial migration at higher concentrations of lidocaine
(Cook et al. 2009b). Several properties of lidocaine have been
investigated aspossible causes
inflammatory effect. Lidocaine is an acidic local anaesthetic
(Catterall and Mackie 2005), which could possibly be an irritant to
cells due to its pH. However, the acidity of lidocaine was
determined to not be a factor in its role as an initiator of
inflammation in the in vitro study (Cook et al. 2009b).
Other factors, such as the presence of the preservative methyl
paraben in 2% lidocaine solutions have been determined to be
unlikely sources of the inflammatory effects seen at that
concentration (Cook et al. 2009b).Although the current study does
not define the reason for a proinflammatory effect of systemic
lidocaine, the lidocaine-induced increase in laminar E-selectin
matrix metalloproteases and
ofthe drug’s observed
© 2010 EVJ Ltd
266Effect of i.v. lidocaine administration on laminar inflammation
expression does indicate that the reported ex vivo effect of lidocaine
on adhesion and transendothelial migration of neutrophils may be
due to activation of the endothelium. Finally, the similar laminar
leucocyte counts in the LD and SAL groups in the current study
indicate no inhibitory effect of lidocaine on overall leucocyte
emigration (Black et al. 2006).
a similar dermal/epidermal relationship as the laminae, and have
been reported previously to reflect a similar increase in leucocyte
emigration as that observed in the laminae (Black et al. 2006).
Importantly, the skin represents a tissue not within the digit and
therefore allows us to assess the effect of lidocaine CRI on systemic
activation. Skin leucocyte counts were increased significantly at
10 h when compared to the 0 h time point for both groups; this
finding is also consistent with that previously reported (Black et al.
2006). However, there was no difference in skin leucocyte counts
between the LD and SAL group. Therefore, when comparing the
SAL group to the LD group, there was no significant difference in
in either skin or laminae at any similarly compared time point.
Lidocaine appeared to have an antipyretic effect (significantly
decreased maximum rectal temperature in LD vs. SAL horses) in
the current study. Due to the lack of differences observed in all of
the inflammatory parameters evaluated in this study, we do not
believe that the lower temperatures observed in the LD group were
due to systemic anti-inflammatory effects of lidocaine. Throughout
the literature, there are reports of the effects of systemic lidocaine
causing the opposite effect, hyperthermia (Tatsukawa et al. 1992;
Crandall et al. 2002). It has been speculated that lidocaine-induced
hyperthermia can occur due to suppressed uptake of calcium ions
from the sarcoplasmic reticulum (Fujioka et al. 1988), leading to
increased intracellular calcium concentration and muscular
contraction (Britt 1974). However, other studies failed to induce
hyperthermia in any of the animals administered i.v. lidocaine
(Wingard and Bobko 1979; Harison and Morrell 1980). In one
study, perfusion of the caudal hypothalamus with a sodium-rich
perfusate resulted in a rise in body temperature (the opposite effect
occurred with calcium) (Feldberg and Myers 1963); lidocaine may
therefore block an effect such as this by limiting influx of sodium
into the cellular constituents of the hypothalamus. Considering
these data, it may be speculated that the temperature differences
observed were due to a combination of effects of black walnut
extract and lidocaine on the central thermoregulatory centres of the
Plasma lidocaine concentrations were above the published
therapeutic and below the toxic dose for all horses included in the
LD group at the time of laminar sampling (10 h time point) (Meyer
et al. 2001; Brianceau et al. 2002; Cook et al. 2008). The average
lidocaine concentration for the LD group at the 3 h time point was
slightly below the therapeutic minimum (0.82 mg/ml vs. 0.9 mg/ml)
(Meyer et al. 2001; Brianceau et al. 2002; Cook et al. 2008). This
is the only time in which samples were taken and evaluated for
leucocyte emigration when lidocaine levels were below therapeutic
levels. The reason for the slightly lower lidocaine concentrations at
the 3 h time point is not known. However, as all horses included in
the study received the standard clinical dose (1.3 mg/kg bwt bolus
of lidocaine, followed by 0.05 mg/kg bwt/min constant rate
infusion) controlled by titrated infusion pumps and signs of
lidocaine toxicity were observed in one horse in the present study,
it is likely that these are the concentrations commonly achieved in
the clinical setting and, therefore, the data for the 3 h time point are
still clinically relevant. The one horse in which the lidocaine
plasma concentrations were below the appropriate therapeutic dose
at sampling of the laminae due to an administration error was
eliminated from the study. The elimination of this horse decreased
the sample size of the LD group to 5 horses, a number used in
previous laminitis studies (Belknap et al. 2007).As is the case with
many equine studies, the strength of the study would be increased
with a larger sample size. However, due to the overwhelming lack
of difference between the 2 groups, there was not enough
justification to add more horses to the study.
In summary, this study indicates that systemic lidocaine at
concentrations achievable in the clinical setting does not have anti-
inflammatory properties in regards to systemic inflammation
documented in the black walnut extract model of laminitis. In fact,
the data suggest that systemic lidocaine may possibly have a
deleterious effect on systemic inflammatory disease by activating
the endothelium. In conclusion, the present data suggest that
lidocaine should not be used clinically for treatment of laminar
inflammatory events that may lead to laminar destruction and
failure in laminitis. Furthermore, when evaluating the laminar and
skin results of this study in light of several other reports (discussed
above) questioning the anti-inflammatory effects of lidocaine,
lidocaine may not be an effective agent for addressing local or
systemic inflammation in any species and its use as such a
treatment is open to question.
1Schering Plough, Union, New Jersey, USA.
2Abcam, Cambridge, Massachusetts, USA.
3Vector Laboratories, Burlingame, California, USA.
4Aperio Technologies, Vista, California, USA.
5National Institutes of Health, Bethesda, Maryland, USA.
6Waters Corporation, Milford, Massachusetts, USA.
7Phenomenex, Torrance, California, USA.
8Jasco, Easton, Maryland, USA.
9Stratagende, Inc., LaJolla, California, USA.
10Roche Molecular Biochemical, Indianapolis, Indiana, USA.
11Ambion Inc., Austin, Texas, USA.
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Author contributions The initiation, conception and planning for
this study were by J.M.W., J.F.P., J.A.E.H. and J.K.B. Its execution
was by J.M.W., Y.J.L., J.P.L., R.R.F., J.A.E.H. and J.K.B., with
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R.R.F., W.R.R. and J.K.B.
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Rossdale & Partners Foal Care Course Notes
Publisher: Whorl Publishing, January 2010 Category: Course Notes Binding: Ring bound
Course notes from the 3 day course held in January 2010
Subjects covered: Neonatal medicine: challenges of the current economic climate; The healthy neonatal foal. Routine
examinations and preventive medicine; Management of the high risk pregnant mare; Resuscitation and stabilisation of
the foal in the field; Immaturity; Assessment of the critically ill foal; Immunological problems of the foal; Infections of the
neonate and sepsis; Neonatal nutrition and the orphan foal; Neurological conditions of the neonate; Assessment and
treatment of abdominal pain in the neonatal foal; Therapeutics - what to use and how much; Diagnostic imaging and
advances in CT; Foals - sedation and anaesthesia; Fluid therapy and electrolyte disturbances; Supportive foal care in general
practice; What to carry in your foal kit and essential hospital equipment; Septic arthritis and osteomyelitis; Clinical
pathology for the neonatal and young foal; Post mortem examination; Rhodococcus equi - an overview and what’s new; Diagnostic techniques for
R. equi; Conformation and limb deformities; Conformational deformities - how your farrier can help; Surgical techniques and shockwave therapy
for limb deformities and DOD; Non infectious lameness in the foal; Rotavirus: treating the individual and managing an outbreak; Lawsonia
intracellularis: an emerging disease in the UK; Vaccination: immunological issues; Vaccination: practical considerations.
Anaesthesia Course Notes
Publisher: Whorl Publishing, November 2009 Category: Course Notes Binding: Ring bound
Course notes from the 2 day course held in November 2009
Subjects covered: Pre-operative assessment: Focus on cardiovascular (and respiratory) system(s); The risks of general anaesthesia TIVA versus
inhalational; What can be done standing?: How to...... perform standing chemical restraint; How to...... do local anaesthetic blocks – head, flank,
limbs; Monitoring: Haemodynamic impact of general anaesthesia – should we be measuring arterial blood pressure (ABP) or cardiac output
(CO)?; How to...... use arterial blood pressure monitors - non invasive, - invasive ; How to...... use lithium dilution cardiac output (LiDCO); Intra-
operative hypoxaemia – why does it occur; can we treat it; can we prevent it?; Anaesthetic management: Peri-operative fluid therapy, with focus
on the colic horse; Partial intravenous anaesthesia (PIVA) – balanced anaesthesia for the 21st century?; What place for desflurane?; Analgesia:
How to...... provide balanced analgesia; How to...... epidural catheters; Potential complications: Overview of potential anaesthetic pitfalls for colic
surgery, Caesarean sections and laparoscopies; How to...... be prepared for anaesthetic accidents and emergencies; Recovery: Post-anaesthetic
myopathies/neuropathies – current knowledge; Other problems, but are they all in the head?; Different options for recovery.
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