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Supplementary Methods
Study design
All studies described below were approved by the Animal Care and Use
Committee of the Clinical Center at the National Institutes of Health. Thirty-eight
purpose-bread beagles (12-28 month, 10-12 kg) were studied prospectively over 96 hours
comparing dexamethasone and desoxycorticosterone administered together (DOC +
DEX) to placebo. In one study, animals were challenged with increasing doses of S.
aureus (n=20) to achieve a final control mortality of 80-90%. In a second study, animals
were given the same corticosteroid regimen but challenged only with the high mortalityproducing dose of S. aureus (1.5 x 109 cfu/kg; n=18) to directly address the hypothesis
that corticosteroids are efficacious in severe sepsis with high lethality. These studies were
analyzed together to use all available data from animals receiving this regimen (total n =
38; Statistical Methods). Bacteria challenges were administered intrabronchially at time 0
(T0) as previously described [1], and DOC + DEX or placebo treatment was started
according to randomization allocation (n=19 for each group). Corticosteroids were started
immediately after bacterial challenge in a therapeutic design to increase our chances of
finding an effect. Corticosteroid dosing was designed to be comparable to physiologic
stress doses in humans [2] (Corticosteroid Therapy). Technicians and veterinarians
responsible for randomizing and caring for animals and for performing all measures were
blinded to the study design. A maximum of four animals were randomized in any study
week.
Study Protocol
2
Prior to infectious challenge, animals had a tracheostomy, external jugular vein
introducer, and percutaneous femoral artery and urinary catheters placed under general
anesthesia [1]. Following these procedures, animals were weaned off anesthesia;
continuous sedation (fentanyl, midazolam, and medetomidine) and mechanical
ventilation via the tracheostomy tube were initiated; and a balloon-tipped pulmonary
arterial thermodilution catheter was placed via the introducer. At time 0 (T0), animals
underwent bronchoscopy and inoculation of the right lower (caudal) lobe with S. aureus
(Bacterial Preparation and Inoculation).
During the first 4 h after S. aureus inoculation, phenylephrine was titrated to
maintain mean arterial pressure (MAP) >80 mmHg as sedation was optimized and sepsis
developed. After 4 h, when symptoms of sepsis were more fully developed,
phenylephrine administration was discontinued and intravascular hemodynamic measures
and blood samples were obtained (Physiological and Laboratory Measurements).
Treatment for sepsis was then initiated with fluid boluses and norepinephrine (NE)
infusion titrated based on algorithms driven by measurements of pulmonary artery
occlusion pressure (PAOP) and MAP, respectively (Fluid and Vasopressor Support).
Fractional inspired oxygen concentration (FiO2), respiratory rate (RR), and positive end
expiratory pressure (PEEP) levels were titrated based on algorithms incorporating
scheduled arterial blood gas measurements to determine partial pressure of CO2 and
oxygen saturation (SaO2) as previously described [1]. Oxacillin (30 mg/kg IV q 8 h),
dosed based on standard veterinary practice [3] was started 4 h after bacterial inoculation
and continued until the end of the 96 h experiment. Conventional intensive care unit
support employed during the ventilation of critically ill large animals was administered as
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previously described [1]. Animals alive at 96 h were considered survivors and then
euthanized (Beuthanol; 75 mg/kg IV) while still sedated.
Corticosteroid therapy
Beginning at 0 h, 19 of 38 animals received a continuous infusion of DEX (0.014
mg/kg/h IV), a synthetic glucocorticoid with no mineralocorticoid activity, coadministered with DOC acetate, a mineralocorticoid with no glucocorticoid activity [4].
DOC was given as an intravenous injection (0.2 mg/kg dissolved in DMSO infused over
40 min.) simultaneously with a subcutaneous (SQ) injection (0.2 mg/kg dissolved in
sesame oil). The SQ injection was repeated daily. These doses of DEX (equivalent to 10
times the glucocorticoid activity of daily maintenance therapy for adrenal insufficiency in
canines [5]) and DOC (equivalent to the mineralocorticoid activity of daily maintenance
therapy for adrenal insufficiency in canines [6]) were previously studied in this model
[7]. These doses are comparable in glucocorticoid and mineralocorticoid potency to
stress-dose hydrocortisone (300 mg/day) typically used to treat patients with septic shock
(i.e. equivalent to 10 times the daily unstressed production of cortisol in humans [8]).
Bacterial Preparation and Inoculation
S. aureus isolates, sensitive to oxacillin [minimal inhibitory concentration (MIC)
= 0.5µg/mL + 2% saline], were prepared and administered into a right lower (caudal)
lobe, subsegmental airspace via a bronchoscope as previously described [1]. The dose of
S. aureus (1.05-1.5 x 109 cfu/kg suspended in 10 mL of 2% saline) was the same for all
animals in an individual 96 h experiment, but doses in the first study were started at 1.05
4
x 109 cfu/kg and increased incrementally by 0.15 x 109 cfu/kg after 4-6 animals were
studied with the objective of finding a S. aureus dose that produced control mortality
rates of 80-90%.
Sedation and Analgesia Management
Animals were monitored by a clinician or trained technician at the bedside
throughout the study. Midazolam (0.2 mg/kg loading dose, 50 µg/kg/min infusion IV)
sedation and fentanyl (5 µg/kg loading dose, 0.7 µg/kg/min infusion IV) analgesia were
titrated based on an algorithm as previously described [1]. Medetomidine infusion (2-5
mg/kg/min) was used to supplement sedation as needed according to pre-established
criteria.
Physiological and Laboratory Measurements
Hemodynamic parameters [MAP, mean pulmonary artery pressure (PAP), PAOP,
central venous pressure (CVP), heart rate (HR), and cardiac output (CO)], arterial and
mixed venous blood gases, complete blood counts (CBC), serum chemistries, and serum
cortisol and aldosterone levels were measured as previously described [1, 9] . Plasma was
collected to measure cytokine (IL-6, IL-10) levels in duplicate using the Searchlight
multiplex array system according to the manufacturer's instructions (Pierce
Biotechnology, Rockville, IL); cultures (blood, femoral site, tracheal aspirates, and urine)
were collected; and urinary output was measured as previously described [1]. Oxacillin
levels were determined from serum obtained from S. aureus-challenged animals in this
and other studies (n = 48 animals total) that received a similar regimen of oxacillin
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treatment (30 mg/kg IV q 8 h starting 4 h after bacterial challenge; see Study Protocol
above) using a commercially-available bioassay (Focus Diagnostics, Cypress, CA). The
MIC to oxacillin of the S. aureus isolates used in these experiments was measured using
microdilution plates and a Trek Diagnostic automated reader system (Sensititre ARIS 2X,
Trek Diagnostics, Cleveland, OH).
Fluid and Vasopressor Support
Animals received continuous maintenance fluids (Normasol-M + 27 mEq K+/L, 2
mL/kg/h, IV) beginning at T0. To simulate clinical hemodynamic support and equalize
initial volume status in all animals, up to three fluid boluses (0.9 % NaCl, 20 mL/kg)
were administered at 20 min intervals based on pre-established algoriths, if PAOP
measured at 4 h was < 10 mmHg. For a MAP < 80 mmHg after the three fluid boluses, a
NE infusion was initiated at 0.2 µg/kg/min and adjusted incrementally (0.2 to 0.4 to 1.0,
to a maximum of 2.0 µg/kg/min) at 5-min intervals to maintain MAP between 80 and 110
mmHg throughout the 96 h study. At subsequent times (T6, T8, T10, T12, and every 4 h
thereafter), an additional IV fluid bolus (20 mL/kg) was administered, if PAOP < 10
mmHg.
Statistical Methods
Across two studies, the same corticosteroid regimen was investigated using
identical protocols at different doses of S. aureus [1.05 x 109 cfu/kg (n = 6), 1.2 x 109
cfu/kg (n = 6), 1.35 x 109 cfu/kg (n = 4) and 1.5 x 109 cfu/kg (n = 22)] as described in
Bacterial Preparation and Inoculation, above. We then combined these two studies to
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utilize all available data. Likelihood ratio test in a Cox proportional hazard model was
used to assess if there was an interaction between challenge dose and treatment on
survival. Notably, corticosteroid therapy had significantly different effects when given
with higher- vs. lower doses of S. aureus. Bacterial challenge doses were thus divided
into two groups (the two higher doses in one and the two lower doses in another) that
minimized the within-group variation. These groups are subsequently referred to as the
high- and lower-dose groups, respectively.
Survival times were compared between the two treatment groups using exact log
rank tests (StatXact, Cytel Software Corp., Cambridge, MA), with stratified tests applied
where applicable to account for potential week effect. Baseline characteristics within
each treatment group were summarized using the mean and standard error and were
compared between treatment groups using an F test. For variables except those for
bacteriology, all values for individual animals available from each of 4 time periods ([0-4
h], (4-12 h], (12-30 h] and (30-96 h] hours) were averaged based on convention
established in a similar prior study [7]. To evaluate shock reversal, we standardized MAP
and NE using Z-scores and then calculated a “shock reversal” score based on the
difference of the MAP Z-score and NE Z-score, for which a higher score indicates
improvements in shock reversal, as done previously [7]. To evaluate pulmonary function,
we constructed a “lung injury” score based on the first principal component of A-aO2,
plateau pressure, PAP, SaO2, and RR, also as done previously [7]. Measures of IL-6 and
IL-10 were analyzed using a log10 transformation to stabilize variance and satisfy the
assumption of normality. Linear mixed models (SAS PROC Mixed) were used to
compare the effects (as change from baseline when possible) of different treatments.
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Repeated measurements of each animal and the actual pairing of animals within each
week were accounted for in the model. Standard residual diagnostics were used to
confirm model assumptions. Pearson correlation was used to assess the association
between log10 (IL6) values and survival times. Figures 2-4 were generated based on
model estimated mean values during each period for each treatment.
We used logistic regression to compare the probability of S. aureus-positive
cultures from the blood, S. aureus-positive cultures from tracheal aspirates, and non-S.
aureus colonization and/or possible superinfections from tracheal aspirates, urine and the
skin around the femoral artery catheter site between treatments. Generalized Estimating
Equations (GEE; SAS PROC GENMOD) was used to account for the correlation induced
by multiple tests at each time and multiple times for each animal. Time (0-10 h, 24 h, 4896 h) was included in the model to account for the possibility of changing infection rates
over time.
SAS version 9.2 (Cary, NC) was used for all analyses except those noted above
(i.e. StatXact). All p-values are two-tailed and considered significant if p ≤ 0.05.
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Supplementary References
1. Minneci PC, Deans KJ, Hansen B, Parent C, Romines C, Gonzales DA, Ying SX,
Munson P, Suffredini AF, Feng J, Solomon MA, Banks SM, Kern SJ, Danner RL,
Eichacker PQ, Natanson C, Solomon SB (2007) A canine model of septic shock:
balancing animal welfare and scientific relevance. Am J Physiol Heart Circ Physiol
293:H2487-500
2. Minneci PC, Deans KJ, Banks SM, Eichacker PQ, Natanson C (2004) Meta-analysis:
the effect of steroids on survival and shock during sepsis depends on the dose. Ann Intern
Med 141:47-56
3. Plumb DC (2005) Plumb's veterinary drug handbook. In: , 5th edn. PhrmaVet;
Distributed by Blackwell Pub., Stockholm, Wis.; Ames, Iowa, pp 578-80
4. Barrett KE, Ganong WF (2010) Ganong's review of medical physiology. McGraw-Hill
Medical, New York
5. Kintzer PP, Peterson ME (1997) Treatment and long-term follow-up of 205 dogs with
hypoadrenocorticism. J Vet Intern Med 11:43-49
6. Lynn RC, Feldman EC, Nelson RW (1993) Efficacy of microcrystalline
desoxycorticosterone pivalate for treatment of hypoadrenocorticism in dogs. DOCP
Clinical Study Group. J Am Vet Med Assoc 202:392-396
7. Hicks CW, Sweeney DA, Danner RL, Eichacker PQ, Suffredini AF, Feng J, Sun J,
Behrend EN, Solomon SB, Natanson C (2011) Efficacy of selective mineralocorticoid
and glucocorticoid agonists in canine septic shock. Crit Care Med
9
8. Minneci PC, Deans KJ, Eichacker PQ, Natanson C (2009) The effects of steroids
during sepsis depend on dose and severity of illness: an updated meta-analysis. Clin
Microbiol Infect 15:308-318
9. Sweeney DA, Natanson C, Banks SM, Solomon SB, Behrend EN (2010) Defining
normal adrenal function testing in the intensive care unit setting: a canine study. Crit Care
Med 38:553-561
Variable
Aldosterone
log(pmol/L)
ALT (U/L)
AST (U/L)
BUN (mg/dL)
CO (L/min)
Chloride
(mmol/L)
Cortisol
log(nmol/L)
Creatinine
(mg/dL)
CVP (mmHg)
Hemoglobin
(g/dL)
Potassium
(mmol/L)
Sodium
(mmol/L)
PAOP (mmHg)
WBC (K/uL)
Treatment
Group
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
Control
DOC+DEX
0-4 h
1.20±0
1.40±0.20
77.3±29.2
178± 96
25.2±7.8
35.0±7.7
4.83±1.35
6.33±1.69
1.69±0.16
1.63±0.34
116±1
117±2
1.36±0.17
1.67±0.21
0.65±0.08
0.65±0.04
5.83±1.38
7.00±1.10
12.1±0.63
13.2±1.2
3.53±0.13
3.40±0.13
147±1
148±1
7.33±1.76
9.33±1.26
8.75±0.76
8.15±1.36
Lower Dose Bacteria
>4-12 h
>12-30 h
2.66±0.22 2.36±0.53
2.64±0.35 3.32±0.12
123±54 82.3±34.7
161±79
136±61
32.6±4.7 18.5±0.5
29.8±5.3 13.8±2.9
3.75±0.48 5.50±2.25
4.83±0.99 4.50±1.94
1.77±0.20 1.43±0.32
1.71±0.13 1.40±0.20
121±1
125±2
120±1
119±2
2.58±0.09 2.17±0.21
2.50±0.06 2.28±0.25
0.72±0.05 0.80±0.17
0.70±0.06 0.79±0.08
4.04±0.73 4.70±0.66
6.33±1.12 6.48±0.64
14.3±0.3 17.1±1.7
13.9±0.5 16.0±1.1
3.48±0.04 3.53±0.08
3.50±0.04 3.70±0.08
145±1
143±2
145±1
145±2
8.17±0.60 11.6±2.4
9.29±0.45 10.7±0.7
3.64±0.90 3.82±1.78
4.25±0.77 5.48±2.22
>30-96 h
1.46±0.26
2.75±0.45
91.7±27.2
122±35
17.9±5.7
22.6±5.3
4.11±1.87
5.88±2.18
1.54±0.05
1.46±0.12
123±1
123±1
1.77±0.13
1.56±0.31
0.64±0.06
0.70±0.11
7.65±1.15
8.27±1.40
12.6±0.3
13.0±1.1
3.43±0.13
3.77±0.15
145±2
143±1
10.8±1.1
11.9±0.8
8.97±1.54
9.16±2.09
0-4 h
1.66±0.12
1.57±0.13
22.3±10.1
34.8±7.1
32.0±9.9
24.0±1.6
6.75±1.25
10.8±0.9
1.80±0.11
1.59±0.13
118±1
119±1
1.88±0.13
1.88±0.12
0.58±0.05
0.75±0.06
7.15±0.80
7.23±0.76
12.0±1.4
12.2±1.8
3.41±0.09
3.32±0.08
146±0
147±1
9.92±0.59
8.69±0.86
6.10±0.99
5.30±0.74
High Dose Bacteria
>4-12 h
>12-30 h
2.74±0.10 3.15±0.18
2.54±0.17 3.08±0.13
45.9±4.1 33.9±2.1
85.2±28.3 48.1±11.4
40.8±7.0 39.6±10.2
58.9±24.1 28.4±3.9
8.35±1.04 10.1±2.1
8.96±0.95 10.2±1.2
1.59±0.14 1.22±0.21
1.45±0.15 1.10±0.09
118±1
117±1
119±1
121±1
2.61±0.04 2.44±0.11
2.61±0.08 2.33±0.18
0.86±0.10 0.80±0.11
0.80±0.07 1.09±0.35
4.69±0.42 4.74±0.43
5.21±0.60 5.56±0.62
15.7±0.6 18.5±0.8
15.0±0.5 16.2±0.6
3.32±0.08 3.94±0.11
3.41±0.08 3.80±0.09
144±1
144±2
146±1
145±1
9.21±0.44 10.2±0.7
8.81±0.44 10.0±0.6
1.85±0.36 2.69±1.22
3.04±0.72 5.01±2.08
>30-96 h
2.43±0
2.61±0.21
25.0±0
52.4±9.8
12.0±0
78.6±31.3
11.3±0
14.3±1.9
1.30±0
0.31±0.12
123±1
122±2
1.70±0
1.51±0.10
1.17±0
0.95±0.12
5.68±1.14
7.81±0.75
12.8±0
12.6±0.9
4.18±0.22
4.27±0.29
147±1
144±1
10.8±2.1
11.9±0.5
12.3±0
8.50±2.43
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Table E1A. Supplemental laboratory values.
ALT: Alanine amino-transferase; AST: aspartate amino-transferase; BUN: blood urea nitrogen; CO: cardiac output; CVP: central
venous pressure; PAOP: pulmonary artery occlusion pressure; WBC: white blood cell count.
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Variable
Survival
(logHR 0-96 h)
Shock score
(>30-96 h)
Lung score
(>30-96 h)
IL6 levels
(log10 at 24 h)
Positive blood
culture
(log OR 0-96 h)
DOC + DEX (n=13)
vs. Controls (n=13)
DEX alone (n=12)
vs. Controls (n=6)
p-value (DOC +
DEX vs. DEX
alone)
-1.20 (0.66)
-1.64 (1.16)
0.74
1.56 (0.60)
2.19 (0.75)
0.37
-2.71 (0.98)
-1.52 (1.21)
0.30
-0.69 (0.25)
-0.18 (0.33)
0.13
1.22 (0.82)
0.83 (0.80)
0.52
Table E1B. Comparison of effects of DOC + DEX vs. DEX alone [7] in high dose (1.3 –
1.5 x 109 cfu/kg) S. aureus pneumonia (estimate ± SEM).
HR = hazards ratio; OR = odds ratio
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Supplementary Figure Legends
Figure E1. Effects of DOC + DEX on individual components of the shock reversal
and lung injury scores. Group I: When comparing the high vs. lower doses of S. aureus,
DOC + DEX had different effects on shock reversal (p = 0.02 for interaction): from 12 to
96 h DOC + DEX improved mean shock reversal scores compared to controls after high
dose S. aureus challenge (p = 0.009), but did not significantly alter shock reversal after
lower dose challenge (p = ns). Group II: DOC + DEX improved mean lung injury scores
from 12 to 96 h with high- (p = 0.02) but not low-dose (p = ns) bacterial challenges.
However, the differences in effect of DOC + DEX on lung injury score at high vs. lower
doses of S. aureus challenge did not reach statistical significance (p = ns).
Figure E2. Pearson's correlation for IL-6 levels at T10 h and T24 h vs. survival
times. Group I: IL-6 levels at 10 h after infection were negatively correlated with
survival (r = -0.46 p =0.004; panel A). This correlation showed a similar strong negative
correlation in subgroup analysis regardless of dose of bacteria or treatment group
examined (B1-4). Group II: IL-6 levels at 24 h after infection were negatively correlated
with survival (r = -0.83, p< 0.0001; A). This correlation showed a similar strong negative
correlation in subgroup analysis regardless of dose of bacteria or treatment group
examined (panels B1-4).