Arterial clots were placed and arteriography was performed on days 0,
7, and 14. In the low-dose group, blood was drawn prior to bolus. Asterisk
indicates missing data in the low-dose nitrite group on day 3. Dashed line
in panel D indicates the 2% level, which is the upper limit of normal.
Cerebral arteriograms on day 7 of a control monkey treated with intravenous
saline for 14 days demonstrate severe vasospasm of the M1 segment of the right
middle cerebral artery compared with a monkey treated with nitrite.
The degree of vasospasm of the right middle cerebral artery (RMCA) was
assessed as the area of the proximal 14-mm segment of the RMCA by 3 blinded
examiners using a computerized image analysis system. Arteriographic vasospasm
was quantified relative to each animal’s baseline arteriogram. The horizontal
line at 25% represents the degree of change required to define the presence
of vasospasm in this model. The low-dose infusion group received a 90-mg sodium
nitrite intravenous solution infused over 24 hours with a 45-mg sodium nitrate
bolus daily. The high-dose infusion group received a 180-mg sodium nitrite
intravenous solution infused over 24 hours. The control group received a saline
solution infusion. The statistical significance of the comparisons was P<.001. Mean (SD) values for the control group and the
combined nitrite groups are shown adjacent to the individual values.
The low-dose infusion group received a 90-mg sodium nitrite intravenous
solution infused over 24 hours with a 45-mg sodium nitrite bolus daily. The
high-dose infusion group received a 180-mg sodium nitrite intravenous solution
infused over 24 hours. The control group received a saline solution infusion.
The nitrite levels in cerebrospinal fluid inversely correlated with degree
of right middle cerebral artery vasospasm (R2 = −0.90; 95% confidence interval, −1.24 to −0.73; P<.001). Despite the lower dose of nitrite in the low-dose
infusion group, the actual whole blood nitrite levels were transiently (about
150 min/d) significantly higher than the peak levels in the high-dose group.
This finding explains the strong correlation between nitrite levels and cerebrospinal
fluid and degree of vasospasm for baseline (day 0) and days 7 and 14 for all
animals. The low-dose group had less vasospasm and higher methemoglobin levels,
which confirms a dose-dependent effect of nitrite in cerebrospinal fluid.
The solid line indicates linear regression and the dashed lines indicate the
upper and lower 95% confidence intervals.
Pluta RM, Dejam A, Grimes G, Gladwin MT, Oldfield EH. Nitrite Infusions to Prevent Delayed Cerebral Vasospasm in a Primate Model of Subarachnoid Hemorrhage. JAMA. 2005;293(12):1477-1484. doi:10.1001/jama.293.12.1477
Author Affiliations: Surgical Neurology Branch,
National Institute of Neurological Disorders and Stroke (Drs Pluta and Oldfield),
Laboratory of Chemical Biology, National Institute of Diabetes and Digestive
and Kidney Diseases (Dr Dejam), Pharmacy Department, Clinical Center (Dr Grimes),
Vascular Therapeutics Section, Cardiovascular Branch, National Heart, Lung,
and Blood Institute (Dr Gladwin), and Critical Care Medicine Department (Dr
Gladwin), National Institutes of Health, Bethesda, Md.
Context Delayed cerebral vasospasm causes permanent neurological deficits or
death in at least 15% of patients following otherwise successful treatment
for ruptured intracranial aneurysm. Decreased bioavailability of nitric oxide
has been associated with the development of cerebral vasospasm.
Objective To determine whether infusions of nitrite will prevent delayed cerebral
Design, Setting, and Subjects A total of 14 anesthetized cynomolgus monkeys had an autologous blood
clot placed around the right middle cerebral artery. Cerebral arteriography
was performed before clot placement and on days 7 and 14 to assess vasospasm.
The study was conducted from August 2003 to February 2004.
Interventions A 90-mg sodium nitrite intravenous solution infused over 24 hours plus
a 45-mg sodium nitrite bolus daily (n = 3); a 180-mg sodium nitrite
intravenous solution infused over 24 hours (n = 3); or a control
saline solution infusion (n = 8). Each was infused continuously
for 14 days.
Main Outcome Measures Nitrite, S-nitrosothiol, and methemoglobin
levels in blood and cerebrospinal fluid and degree of arteriographic vasospasm.
Results In control monkeys, mean (SD) cerebrospinal fluid nitrite levels decreased
from 3.1 (1.5) μmol/L to 0.4 (0.1) μmol/L at day 7 and to 0.4 (0.4) μmol/L
at day 14 (P = .03). All 8 control monkeys
developed significant vasospasm of the right middle cerebral artery, which
was complicated by stroke and death in 1 animal. Sodium nitrite infusions
increased the nitrite and methemoglobin levels (<2.1% of total hemoglobin)
in the blood and cerebrospinal fluid without evoking systemic hypotension.
Nitrite infusion prevented development of vasospasm (no animals developed
significant vasospasm; mean [SD] reduction in right middle cerebral artery
area on day 7 after subarachnoid hemorrhage of 8% [9%] in nitrite-treated
monkeys vs 47% [5%] in saline-treated controls; P<.001).
There was a negative correlation between the concentration of nitrite in cerebrospinal
fluid and the degree of cerebral vasospasm (P<.001).
Pharmacological effects of nitrite infusion were also associated with the
formation of S-nitrosothiol in cerebrospinal fluid.
There was no clinical or pathological evidence of nitrite toxicity.
Conclusion Subacute sodium nitrite infusions prevented delayed cerebral vasospasm
in a primate model of subarachnoid hemorrhage.
Intracranial aneurysm rupture affects an estimated 10 individuals in
a population of 100 000 annually.1- 3 Half
survive to reach the hospital and receive surgical and/or endovascular intervention.1,3 However, half of the patients whose
aneurysm is successfully treated develop delayed cerebral vasospasm.4,5 Despite the use of currently available
management modalities (nimodipine and hypertension-hypervolemia-hemodilution
[triple-H] therapy), cerebral vasospasm severely disables or kills half of
the affected patients.3,5- 7 A
growing body of experimental8- 19 and
clinical20- 24 evidence
suggests that decreased availability of nitric oxide in the cerebral artery
wall is associated with vasospasm development. Nitric oxide plays a dominant
role in the dilation of vessels and in the regulation of cerebral blood flow.
Nitric oxide levels are decreased after subarachnoid hemorrhage due
to (1) toxicity of oxyhemoglobin to neurons containing neuronal nitric oxide
synthase (NOS) in the adventitia of the artery17,25;
(2) endogenous inhibition of endothelial NOS15;
and (3) scavenging of nitric oxide by oxyhemoglobin released from the subarachnoid
clot,26 which is a pathophysiological mechanism
that also produces endothelial dysfunction in hemolytic disease.27
The role of nitric oxide in vasospasm is further confirmed by experimental
and clinical studies showing that exogenous delivery of nitric oxide ameliorates
or prevents vasospasm.8,20- 22,28 A
decrease in nitrite levels in cerebrospinal fluid at the time of delayed cerebral
vasospasm has been observed.15,23,24 However,
a clinical application of these findings has been limited by the following
adverse systemic effects of nitric oxide donors: decreased blood pressure,
increased intracranial pressure, subsequent decreased cerebral blood flow,20,29,30 and increased risk
of ischemic stroke.20 Thus, use of nitric oxide
therapy has been restricted to invasive regional intravascular delivery such
as intracarotid administration of nitric oxide gas solutions, ultrashort-acting
nitric oxide donors,8,28 and extravascular
delivery of nitric oxide donors.20,21,31,32
Endogenous levels of nitrite reflect endothelial NOS activity.33 Recent studies34- 37 indicate
a new biological role for the nitrite anion as a storage molecule for nitric
oxide. Nitrite can be reduced to nitric oxide by mammalian xanthine oxidoreductase38,39 and by nonenzymatic disproportionation,40 although these conversion mechanisms are limited
to conditions of hypoxia and tissue acidosis.34
Recent attention has focused on the reaction of deoxyhemoglobin with
nitrite to produce nitric oxide under physiological conditions, which may
contribute to hypoxic vasodilation.34,36,41,42 Nitrite
is converted in the blood to nitric oxide and to potentially vasoactive and
biologically active chemical species, including S-nitrosothiols, N-nitrosamines, and iron-nitrosyl complexes.34,35,42,43 Because
subarachnoid hemorrhage is associated with the presence of both deoxyhemoglobin44 and low pH45- 47 in
the vicinity of arteries surrounded by the subarachnoid clot, we hypothesized
that intravenous nitrite infusions would lead to a release of nitric oxide,
nitros(yl)ation of cerebrospinal fluid proteins in the vicinity of the affected
arteries, and prevent vasospasm. We examined this hypothesis using a well-characterized
primate model of subarachnoid hemorrhage.28,48
The animal protocol was reviewed by the Animal Care and Use Committee
of the National Institute of Neurological Disorders and Stroke and met the
National Institutes of Health guidelines for animal care. The study was conducted
from August 2003 to February 2004.
Fourteen cynomolgus monkeys were studied. After induction of general
anesthesia, the monkeys underwent right frontotemporal craniectomy. The proximal
1.4 cm of the right middle cerebral artery (RMCA) (M1 segment) was exposed
from a bifurcation of the right internal carotid artery to a trifurcation
of the middle cerebral artery. A 5-mL preclotted autologous blood clot was
placed around the artery. In this model of subarachnoid hemorrhage, which
is widely recognized as the best in vivo model,49 cerebral
vasospasm develops in about 95% of the animals and the course of spasm mimics
clinical events.49,50 The animals
were followed up until day 14 after subarchnoid hemorrhage and were killed
at the end of the experiment.
Initial blood and cerebrospinal fluid samples were collected and all
monkeys received cerebral arteriography followed by clot placement around
the RMCA on day 0 (baseline) (Figure 1).
Cerebral arteriography was repeated on days 7 and 14 and cerebrospinal fluid
samples were collected. Because no prior data on subacute nitrite dosing in
primates or humans were available, we extrapolated the dose from rat51 and human34 studies.
The first 3 monkeys received a 90-mg sodium nitrite infusion (Hope Pharmaceuticals,
Scottsdale, Ariz) over 24 hours (low-dose infusion group). After we observed
no toxic effects during the first day, these animals received an additional
45-mg sodium nitrite bolus daily (1.5 mL over 5 minutes), which was administered
at the time the infusion solution was changed, to establish the maximal tolerated
dose and to determine pharmacokinetics.
Before each bolus was administered, blood pressure was measured and
blood was drawn to determine the nitrite and methemoglobin levels reached
during the nitrite infusion. Blood also was drawn 5 minutes after the bolus
was delivered to measure nitrite levels. Because the bolus increased blood
nitrite levels, produced transient hypotension, and increased methemoglobin
levels above normal limits in the first 3 monkeys, the subsequent 3 received
a 180-mg sodium nitrite continuous infusion over 24 hours without a bolus
(high-dose infusion group). Blood and cerebrospinal fluid samples were collected
throughout the study.
The sodium nitrite solutions were delivered at 0.9 μmol/min (low-dose
infusion plus bolus group) and 1.8 μmol/min (high-dose infusion group)
for 14 days via an ambulatory infusion pump (model 404-SP, Medtronic MiniMed,
Northridge, Calif), which was secured in a pocket of the jacket (Lomir Biomedical
Inc, Notre-Dame-de-I’Île Perrot, Quebec) fitted to the monkey.
The pump was connected to polyethylene 50 tubing that was passed subcutaneously
from the midscapular region and inserted into the external jugular vein under
direct visualization. The infusion started 1 hour after craniectomy and clot
placement to model the initiation of treatment after craniotomy for control
of aneurismal hemorrhage in human disease. The pump was reloaded daily with
sodium nitrite or saline (control group). The incidence and degree of cerebral
vasospasm after subarachnoid hemorrhage with nitrite infusion in animals in
the low- and high-dose infusion groups were compared with the 8 control animals
who received intravenous saline.
To assess the incidence and degree of vasospasm, cerebral arteriography
was performed preoperatively, baseline, and on days 7 and 14 after subarachnoid
hemorrhage, as described previously.28 Each
animal underwent at least 2 arteriographies: one before subarachnoid hemorrhage
and the other on day 7 after subarachnoid hemorrhage. Arteriographies were
performed under general anesthesia induced by a 0.5% mixture of isoflurane
and pancuronium. Systemic arterial blood pressure and end expiratory PCO2 of the animals were continuously monitored; both levels
remained stable during the procedure.
A femoral cut down to expose the femoral artery was performed under
aseptic conditions. An F3 polyethylene catheter (Cook Group, Bloomington,
Ind) was advanced under fluoroscopic control to the right internal carotid
artery and 0.75 mL of contrast medium (diatrizoate meglumine and diatrizoate
sodium) was injected by hand. Subtraction images in the anteroposterior projection
were acquired. The area of the proximal 14-mm segment of the RMCA was measured
by 3 examiners who were blinded to the study data, using a computerized image
analysis system (NIH Image 6.14; Figure 2),
which has been used to ensure maximal objectivity of measurements by our laboratory
since 1992.28,52 Arteriographic
vasospasm was quantified relative to each monkey’s baseline arteriogram;
it was defined as more than a 25% decrease in the middle cerebral artery area
in comparison with the presubarachnoid hemorrhage area.
The monkeys were observed daily for changes in weight, eating behavior,
and general physical and neurological status. Daily blood samples and blood
pressure were collected when the infusion solution pump was changed (under
ketamine anesthesia). The blood was collected to assess methemoglobin and
nitrite levels. Cerebrospinal fluid samples were collected via suboccipital
puncture and were collected at the time of arteriography (days 0, 7, and 14).
Blood pressure was measured using a newborn’s cuff (Zimmer Inc, Dover,
Ohio). The remaining plasma and cerebrospinal fluid were stored at –80°C.
For blood nitrite measurements, 100 μL of whole blood were mixed
with 900 μL of a nitrite-stabilizing solution containing 80 mM of potassium
hexacyanoferrate (III), 20 mM of N-ethylmaleimide, 200 μL
of diethylenetriaminepentaacetic acid, and 0.2% of Nonidet P-40 (Abbott Laboratories,
North Chicago, Ill). The nitrite in whole blood was then measured using triiodide-based
reductive chemiluminescence using a nitric oxide analyzer (model 280, Seivers,
Boulder, Colo) as previously described and validated.15,53,54 Cerebrospinal
fluid was used to measure nitrite and S-nitrosothiol
via a stabilization solution containing 10 mM of N-ethylmaleimide
and 2 mM of diethylene triamine pentaacetic acid. To determine the levels
of specific nitric oxide adducts (N-nitrosamines, iron-nitrosylhemoglobin,
and S-nitrosothiols), samples were reacted with and
without 5 mM of mercuric chloride. Nitric oxide–modified protein is
converted to nitrite in the presence of mercuric chloride and N-nitrosamines
when iron-nitrosyl complexes are stable. Both blood and cerebrospinal fluid
samples were treated with a 0.5% solution of acid sulfanilamide, which eliminates
Data are presented as mean (SDs). Degree of vasospasm, levels of nitrite
and methemoglobin, and blood pressure were assessed using 2-tailed t tests and analysis of variance followed by the Tukey test for post
hoc comparisons of mean values. Statview statistical software (version 5.0,
SAS Institute Inc, Cary, NC) was used; statistical significance was defined
Of 14 monkeys, 13 survived the study without complication. One monkey
from the control group with severe vasospasm of the RMCA became somnolent
and died on day 8 after subarachnoid hemorrhage (24 hours after cerebral arteriography).
Autopsy confirmed the presence of ischemic stroke in the territory of the
RMCA. None of the monkeys in the low- or high-dose infusion groups developed
symptoms of stroke or nitrite toxicity due to methemoglobinemia (Figure 1). Transient decreases in blood pressure
(lasting for 15 to 25 minutes) were observed immediately after the nitrite
boluses were delivered in the low-dose infusion group (mean [SD], 97  mm
Hg to 80  mm Hg; P<.001), but no changes in
blood pressure were observed with continuous infusions (prior to receiving
the bolus in the low-dose infusion group and at all measured times in the
high-dose infusion group; Figure 1).
All of the control monkeys who had subarachnoid hemorrhage (n = 8)
developed vasospasm (Figure 2 and Figure 3). The degree of vasospasm peaked on
day 7 (mean [SD] reduction from baseline, 47% [5%] on day 0; range, 41%-54%; P<.001) and significantly decreased on day 14 (–1.9%
[7.5%]; range, –7% to 3%; P<.001 vs day
None of the monkeys treated with nitrite developed significant vasospasm
on day 7 after subarachnoid hemorrhage (mean [SD] reduction from baseline,
8% [9%]; range, –2.1% to 18.7%; P<.001 vs
controls on day 7); these monkeys remained free of vasospasm on day 14 (8.7%
[12.5%]; range, –8.8% to 28.5%). Importantly, the cerebrospinal fluid
nitrite levels were inversely correlated with the degree of vasospasm (R2, –0.90 [95% confidence interval, –1.24
to –0.73; P<.001; Figure 4).
Nitrite levels were measured in blood samples daily and in cerebrospinal
fluid on days 0, 7, and 14 in 3 monkeys in the control group (Figure 1). These monkeys developed moderate to severe vasospasm
after subarachnoid hemorrhage. Blood nitrite levels remained unchanged compared
with presubarachnoid hemorrhage values; however, cerebrospinal fluid nitrite
levels were significantly lower on days 7 and 14 compared with day 0 (P = .03). Cerebrospinal fluid nitric oxide–modified
protein levels, plasma methemoglobin levels, and mean arterial blood pressure
In the 3 monkeys in the low-dose infusion plus bolus group, the levels
of nitrite in blood and cerebrospinal fluid and levels of nitric oxide–modified
protein in cerebrospinal fluid significantly increased compared with levels
on day 0 and compared with controls (P = .02).
Blood methemoglobin levels also increased significantly on days 4, 5, and
6 (P = .01) compared with the levels in
controls. This low-dose infusion group received a bolus of nitrite of 45 mg/d
for 14 days. Following these boluses, the mean (SD) level of whole blood nitrite
increased from 24.7 (13.1) μmol/L to 328.7 (61) μmol/L (P = .009); level of blood methemoglobin increased from 0.9%
(0.4%) to 10.8% (5.1%) (P = .02); and systemic
blood pressure decreased from 97 (3) mm Hg to 80 (3) mm Hg (P<.001). The cummulative nitrite dose in the low-dose infusion with
bolus group was lower than in the high-dose infusion group. The cumulative
nitrite dose in the low-dose infusion plus bolus group produced transiently
higher blood nitrite levels (≈150 μmol/L) resulting in the higher cerebrospinal
fluid nitrite levels, which appeared to exert more potent antivasospastic
The 3 monkeys in the high-dose infusion group had significantly increased
nitrite levels in the blood (P <.001) and cerebrospinal fluid
(P = .01; Figure 1).
This group also had significantly increased levels of nitric oxide–modified
protein in their cerebrospinal fluid compared with levels on day 0 and compared
with controls (P = .04). Levels of methemoglobin
were significantly increased above the baseline level (day 0), but remained
within clinically acceptable limits (<2%) throughout the study.
This study shows that continuous intravenous infusions of nitrite for
14 days prevent delayed cerebral vasospasm after subarachnoid hemorrhage in
a primate model. Moreover, the results suggest that nitrite therapy is safe
and is associated with limited adverse effects. This study suggests that lower
nitrite levels in cerebrospinal fluid correlate with the development of vasospasm
and that nitrite administration repletes these levels and generates cerebrospinal
fluid nitric oxide and S-nitrosothiols, which are
potent vasodilating molecules.55 Widely used
treatments such as nimodipine and hypertension-hypervolemia-hemodilution (triple-H)
therapy do not influence either the incidence or severity of vasospasm despite
possibly improving outcome.3,7,56,57 Angioplasty
and papaverine alone transiently reverse vasospasm, without changing overall
morbidity and mortality.58,59 Thus,
these results suggest a new, safe, inexpensive, and rationally designed therapy
for a disease for which no current preventative therapy exists.
There have been several experimental and clinical trials using nitric
oxide donors delivered intra-arterially, intravenously, intrathecally, and/or
intraventricularly in an attempt to restore regional nitric oxide bioavailability
and prevent or reverse vasospasm.20,22,30,60- 63 However,
using nitric oxide donors in animals and humans has been limited by systemic
hypotensive effects,20,60 nondiscriminative
dilation of the cerebral vasculature resulting in cerebral blood flow “steal
intracranial pressure,20 and decreased cerebral
perfusion pressure. Intracarotid8,28 and
local20,32 delivery of nitric
oxide donor (isolating the nitric oxide effects to the brain vasculature)
eliminate hypotension,8,28,32 increase
intracranial pressure, decrease cerebral perfusion pressure,20 and
demonstrate that local delivery of nitric oxide can prevent vasospasm after
There is little doubt that oxyhemoglobin and deoxyhemoglobin28,65,66 are slowly released
from erythrocytes after aneurysmal bleeding and are directly and/or indirectly17,25,67,68 responsible
for vasospasm.66 We28 and
others69,70 have reported a significant
concentration of deoxyhemoglobin in a subarachnoid clot in the direct vicinity
of vessels in spasm.28 Deoxyhemoglobin levels
peak in the vicinity of cerebral vessels in spasm around day 7 after subarachnoid
hemorrhage.28 At the same time, dysfunction13,15 with preserved expression of endothelial
NOS17,71 and decreased nitrite
levels in cerebrospinal fluid are observed.15,24,72 The
latter is closely associated with the degree of vasospasm.15 While
some researchers have challenged this observation,73,74 our
study demonstrated a similar and marked reduction of nitrite in cerebrospinal
fluid in untreated primates—a result consistent with the hypothesis
that decreased nitric oxide availability contributes to development of cerebral
vasospasm after subarachnoid hemorrhage17 and
nitrite in cerebrospinal fluid can be consumed after subarachnoid hemorrhage.
Gradients of nitrite are present in human circulation from artery to
vein, with increased nitrite consumption occurring with increased metabolic
stress.36 Recently, we reported that intravenous
sodium nitrite infusion selectively opens the blood-tumor barrier via a nitric
oxide–related mechanism, suggesting that nitrite may be a naturally
circulating nitric oxide donor.51,75 Bioactivity
of nitrite has been confirmed in human studies that have shown nitrite infusions
at pharmacological and near-physiological concentrations producing vasodilation
and increasing blood flow.34 This bioactivity
was associated with a reaction of nitrite with deoxyhemoglobin to produce
nitric oxide, iron nitrosylhemoglobin, and to a lesser extent S-nitrosothiols. These results are consistent with a deoxyhemoglobin–mediated
nitrite reaction that ultimately produces methemoglobin and nitric oxide.34,41,42
The same conditions (ie, the presence of deoxyhemoglobin44 and
reduced pH45,47,76- 78)
exist in the subarachnoid after subarachnoid hemorrhage.77,79 Lower
levels of nitrite in cerebrospinal fluid after subarachnoid hemorrhage and
during the development of vasospasm can be evoked by decreased nitric oxide
production by both neuronal17 and endothelial
NOS,15 and by increased nitrite consumption.
The potent vasodilating effects of nitrite34,37,43 are
consistent with reactions between nitrite and hemoglobin to form nitric oxide, S-nitrosothiols, and methemoglobin—chemistries that
not only vasodilate, but also inhibit further nitric oxide scavenging by hemoglobin.
We demonstrate herein that intravenously administrated nitrite in a
primate model increases nitrite levels in the blood and in the cerebrospinal
fluid, reacts in cerebrospinal fluid to produce S-nitrosothiol,
and potently and completely inhibits middle cerebral artery vasospasm after
subarachnoid hemorrhage. This observation provides additional evidence for
the recently appreciated potent bioactivity of nitrite.34,37,43,51,75,80 Considering
the lack of currently available therapies that prevent this devastating complication
of subarachnoid hemorrhage and the safety and efficacy associated with nitrite
therapy in a primate model, these results support the careful implementation
of phase 1 and 2 trials in humans.
However, this study has limitations. The results we observed with relatively
healthy monkeys may not extrapolate to patients who have subarachnoid hemorrhage
and who are cardiovascularly and neurologically unstable. Due to possible
cardiovascular effects that might result in decreased cerebral perfusion pressure
and the unknown effects of prolonged low levels of methemoglobinemia, careful
studies are needed of dosing and adverse effects of sodium nitrite in healthy
volunteers and in at-risk patients. Such studies should elucidate the pharmacokinetics
of sodium nitrite in humans, establish a proper dosage and safety profile,
and offer a new therapeutic modality for patients surviving subarachnoid hemorrhage.
Corresponding Author: Ryszard M. Pluta,
MD, PhD, Surgical Neurology Branch, 10 Center Dr, Room 5D37, Bethesda, MD
Author Contributions: Drs Pluta, Gladwin, and
Oldfield had full access to all of the data in the study and take responsibility
for the integrity of the data and the accuracy of the data analysis. Drs Gladwin
and Oldfield share senior authorship equally.
Study concept and design: Pluta, Dejam, Gladwin,
Acquisition of data: Pluta, Dejam, Grimes,
Analysis and interpretation of data: Pluta,
Dejam, Grimes, Gladwin, Oldfield.
Drafting of the manuscript: Pluta, Dejam, Gladwin,
Critical revision of the manuscript for important
intellectual content: Pluta, Dejam, Grimes, Gladwin, Oldfield.
Statistical expertise: Pluta, Dejam, Grimes,
Administrative, technical, or material support:
Pluta, Dejam, Grimes, Gladwin, Oldfield.
Study supervision: Pluta, Gladwin, Oldfield.
Financial Disclosures: Drs Pluta, Gladwin,
and Oldfield are among multiple inventors on international patent application
022232 by the National Institutes of Health, which was published on January
Funding/Support: This study was performed at
the National Institutes of Health and was independent of any commercial funding
Role of the Sponsor: The National Institutes
of Health had full control of the design and conduct of the study; the collection,
management, analysis, and interpretation of the data; and the preparation,
review, and approval of the manuscript.
Acknowledgment: We are grateful to John Bacher,
DVM, and the Office of Research Surgery Services staff at the National Institutes
of Health for their expert support in this study. We thank Christian Hunter,
PhD, and Xunde Wang, PhD, for their contributions to this project.