1411
Reduction of Central Nervous System
Reperfusion Injury in Rabbits
Using Doxycycline Treatment
Wayne M. Clark, MD; Frank A. Calcagno; Walter L. Gabler, DDS, PhD;
John R. Smith, PhD; Bruce M. Coull, MD
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Background and Purpose Activated leukocytes appear to
potentiate central nervous system reperfusion injury, and
agents that block leukocyte adhesion have shown neuroprotective efficacy in experimental models. Doxycycline, a tetracycline antibiotic, inhibits leukocyte function in vitro, presumably through divalent cation binding. We used a model of focal
central nervous system reperfusion injury to determine the
efficacy of doxycycline treatment in preserving neurological
function.
Methods Rabbits randomly received 10 mg/kg IV doxycycline 30 minutes before ischemia (pretreatment group) or 45
minutes after ischemia (posttreatment group) or received
phosphate-buffered saline vehicle (control group) followed by
10 mg/kg q 8 hours times two. The average length of reversible
spinal cord ischemia required to produce paraplegia (PM) at 18
hours was calculated for each group.
L
eukocyte appearance in central nervous system
(CNS) ischemic tissue has previously been considered to represent a pathophysiological response to existing injury. Recent evidence suggests that
leukocytes may also be directly involved in the pathogenesis and extension of ischemic injury.1-4 Leukocytes
appear to play an important role in the potentiation of
reperfusion injury, with leukocyte adhesion to the endothelium felt to be the critical initial step.5 We have
previously found that monoclonal antibodies which
block leukocyte adhesion have neuroprotective efficacy
in an experimental CNS reperfusion model, 67 and beneficial results have been reported in other animal stroke
models with agents that block leukocyte function.8
Recently we have shown that doxycycline, a member
of the tetracycline family of antibiotics, inhibits in vitro
human leukocyte superoxide synthesis, degranulation,
and adherence to protein-coated surfaces.9'10 These
inhibitory effects on leukocyte function appear to be
produced by doxycycline's binding of the divalent catReceived October 26, 1993; final revision received January 11,
1994; accepted February 28, 1994.
From the Oregon Stroke Center, Department of Neurology
(W.M.C., F.A.C., B.M.C.), and Departments of Biologic Structure
and Function and Oral Molecular Biology, School of Dentistry
(W.L.G., J.R.S.), Oregon Health Sciences University, and the
Department of Neurology (B.M.C.), Veterans Administration
Medical Center, Portland, Ore.
Correspondence to Dr Wayne M. Clark, Department of Neurology L226, Oregon Health Sciences University, 3181 SW Sam
Jackson Park Rd, Portland, OR 97201.
C 1994 American Heart Association, Inc.
Results For the control group (n=13), the P^ was 22.8±2.2
minutes; for the pretreatment group (n=14), 35.5±2.4 minutes
(P<.01; /=3.8); and for the posttreatment group (n=13),
31.4±4.2 minutes (not significant; /=1.6). Doxycycline also
attenuated postischemic decreases in in vivo leukocyte counts
and inhibited in vitro leukocyte adhesion. Therapeutic doxycycline levels at 24 hours were confirmed in the plasma and
spinal cord.
Conclusions This significant protective effect suggests that
doxycycline, a safe and readily available agent, may play a role
in reducing clinical central nervous system reperfusion injury.
(Stroke. 1994^5:1411-1416.)
Keywords • leukocytes • cerebral ischemia •
doxycycline
reperfusion
See Editorial Comment, page 1416
ions Mg 2+ and Ca 2+ ." By inhibiting leukocyte function,
doxycycline treatment may reduce CNS reperfusion
injury and improve neurological function.
In the present study, we used a selective model of
CNS ischemia in rabbits to test whether treatment with
doxycycline would improve functional outcome. We also
assessed the effects of doxycycline on rabbit leukocyte
function to determine a potential neuroprotective
mechanism.
Materials and Methods
Male New Zealand White rabbits weighing 2 to 3 kg were
used. The rabbit was selected because previous antileukocyte
agents have shown neuroprotective efficacy in this species. All
animal procedures were approved by the Oregon Health
Sciences University Internal Animal Care Use Committee.
The rabbit spinal cord ischemia model has been previously
described in detail.12 Under halothane anesthesia, rabbits have
a snare ligature occluding device placed around the abdominal
aorta just below the left renal artery. The end of the occluder
is left accessible through the skin. The rabbit is allowed to
recover for a minimum of 2 hours. To induce ischemia, the
occluder is tightened and clamped. All rabbits are completely
paraplegic within 2 minutes. The animals show no evidence of
discomfort, and the procedure appears to be painless, based
on lack of previously measured changes in heart rate, blood
pressure, or circulating catecholamine levels. At the end of a
variable predetermined occlusion period, the device is
undamped and removed, and the skin is closed. Animals are
exposed to varying durations of ischemia. Animals with shorter
occlusion times tend to regain function, whereas those with
longer occlusion durations remain permanently paraplegic.
1412
Stroke
Vol 25, No 7 July 1994
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Each animal is evaluated 18 hours later for degree of neurological impairment by an observer who is blinded to the
treatment or ischemic duration. Each animal is scored as 0,
paraplegic (no hindlimb movement), or 1, functional (normal/
paretic). The animals are then killed at 24 hours. In the
present study, a 10-cc blood sample was obtained before
surgery (baseline) and at 24 hours after ischemia for complete
blood count and doxycycline level determinations measured by
spectrophotofluorometer.13 Spinal cords were removed at 24
hours for measurement of doxycycline levels.
After full recovery from surgery, animals were randomly
assigned to one of three groups: the pretreatment group, which
received 10 mg/kg IV doxycycline (Sigma Chemical Co) 2
mg/mL dissolved in phosphate-buffered saline (PBS; Sigma)
30 minutes before ischemia, followed by 10 mg/kg BID every
8 hours; the posttreatment group, which received 10 mg/kg IV
doxycycline 45 minutes after the onset of ischemia, followed by
10 mg/kg q 8 hours times two; or the control (untreated
ischemic) group, which received PBS equivalent to that in the
pretreatment group. Preliminary studies found that 10 mg/kg
doxycycline produced therapeutic levels with an approximate
12-hour half-life in rabbits. The animals were observed continuously and evaluated at 18 hours by an investigator blinded
to group. A 30-minute pretreatment period was chosen because it was effective in previous antiadhesion antibody leukocyte studies and it allows time for maximal leukocyte uptake. A
45-minute posttreatment period was chosen to determine the
effects of doxycycline treatment during reperfusion because
the aorta of all animals is undamped by 45 minutes.
To determine whether doxycycline modifies rabbit leukocyte
function, in vitro leukocyte adhesion was assessed in two
separate experiments. To determine whether in vitro concentrations of doxycycline affect both rabbit neutrophil and
mononuclear cell (MNC) function, an 80-cc blood sample was
obtained from a donor rabbit during euthanasia. Rabbit
neutrophils and MNC were isolated by using a modified
dextran sedimentation and Percoll density gradient centrifugation method.14'15 The final purity of the two isolated cell
populations was 97% neutrophils and 95% MNC respectively
by morphological and nonspecific esterase staining evaluation.
After hypotonic red cell lysis, cells were resuspended in
EDTA-free PBS to a working concentration of 1 x10 6 cells per
milliliter.
The wells of microliter plates were coated by adding 200 jiL
albumin (5 mg/^L) and incubating for 2 hours at 37°, followed
by three rinses with distilled water and air drying. Test systems
consisted of 5 x 103 cells per well, 0 to 25 uglmL doxycycline
and 2x10"' mol/L formyl-methionyleucylphenylalanine, 100
ng/mL phorbol 12-myristate 13-acetate, or buffer alone (PBS).
To account for nonspecific leukocyte-substrata binding, one
half of the wells of each test system contained 5 mmol/L
iodoacetamide. The optical density values of iodoacetamideinhibited adherent cell systems were subtracted from those of
adherent cells of noninhibited systems to gfve specific adhesion
values.
Cells, test agents, and buffer were placed in wells, and the
plates were incubated at 37° for 30 minutes before the addition
to the activator. The plates were then incubated for an
additional 3 hours, at which time the media and nonadherent
cells were removed using a standardized technique." One
hundred microliters of a PBS solution containing 0.25% rose
bengal was added to each well and allowed to incubate for 5
minutes.16 The stain was removed, and the wells were washed
three times with 200 fiL saline. Finally, 200 fiL of an ethanolPBS (1:1) solution was added to each well to release the
cell-bound stain, and the plates were incubated at 37° for at
least 60 minutes. The optical density values of the released
stain were measured at a 550-nm wavelength. The results were
expressed as a percentage of the total number of cells initially
added (percent adhesion). The data are presented as the
arithmetic mean of at least eight wells.
In a second, separate experiment, we investigated whether
the in vivo doxycycline dose used in our therapeutic study was
sufficient to inhibit neutrophil adhesion. A 20-cc blood sample
was obtained at baseline and 1 hour after administration of a
10-mg/kg dose of doxycycline in five rabbits. Neutrophils were
isolated as previously described. Neutrophil adherence to
laminin, a basement membrane protein, was determined by a
modification of the method used by Bohnsack et al17 that we
have previously described.18 After a standardized washing of
nonadherent cells, a myeloperoxidase activity assay was used
to determine cell adherence.19 The myeloperoxidase activity in
the cell adhesion wells was compared with a standard curve
generated by known cell concentrations derived from each
rabbit. The number of adherent cells present in the coated
plates was calculated by comparing optical density readings
with those of the known cell concentration values. The results
were expressed as a percentage of the total number of cells
initially added (percent adhesion).
Statistical Analysis
We constructed quantal dose-response curves from the
neurological function ratings at 18 hours. This analysis involves
iterative fitting of a logistic function. The derivation of this
type of analysis has been described in detail,20 and its utility as
a pharmacological screen has been demonstrated.21'22 At each
point on the abscissa the percentage of paraplegic animals in
the group is calculated and plotted as the ordinate. The total
increases from 0% at the shortest duration of occlusion to
100% at the longest. The resulting sigmoid curve represents
the effect of a range of durations on neurological outcome.
The point at which 50% of the animals are paraplegic is
calculated. This point, termed P w , is the average length of
ischemia that produces impairment in 50% of the animals. An
agent that is effective in reducing neurological damage will
shift the curve (and Pso) to the right; ie, the animals will on
average tolerate a longer period of ischemia. To determine
significance, / tests were performed (P<.05).
For the adhesion and hematologic values, differences among
groups were assessed by ANOVA; when there was a significant
difference (P<.05) among groups, the probability value was
determined by Bonferroni-corrected post hoc t tests.
Results
The results of doxycycline treatment in the spinal
cord ischemia model are presented in Table 1. The
average length of ischemia that produced impairment
(Pso) in the control group (n = 13) was 22.8±2.2 minutes
(mean±SE); in the pretreatment group (n = 14),
35.5±2.4 minutes (P<.01; t=3.8); and in the posttreatment group (n=13), 31.4±4.2 minutes (not significant;
t=1.6). Thus, pretreatment with doxycycline but not
delayed treatment produced a significant reduction in
ischemic injury in this model. The Figure shows the
quantal dose-response curves for these gToups. Doxycycline treatment showed no effects on respiration or
rectal temperature. We did not assess blood pressure or
pulse in this study.
Complete blood count and plasma doxycycline levels
were obtained at baseline and at 24 hours. Doxycycline
levels at 24 hours were as follows: pretreatment group
(n = 10), plasma 0.35±0.08 /ig/mL and spinal cord
1.16±0.27 /xg/mg; and posttreatment group (n=9),
plasma 0.20±0.03 jtg/mL and spinal cord 1.76±0.43
fig/vng (not significant between groups). In addition, a
peak 1-hour plasma doxycycline level of 1.7 ^g/mLwas
found in two animals. Table 2 shows hematologic variables. There was a significant hematocrit drop from
baseline in all three groups due to surgical blood loss
Clark et al
Doxycycline Treatment in Stroke
1413
TABLE 1. Efficacy of Doxycycline Treatment in the
Rabbit Spinal Cord Ischemia Model
Neurological Status
Ischemlc
Duration, mln
Normal or Paretlc, n
Paraplegic, n
0
Control group
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14
1
16
1
0
18
1
0
20
0
1
23
1
0
24
1
0
26
0
1
28
0
1
30
0
1
32
0
1
34
0
1
36
0
1
38
0
1
uoxycyoine ou-minut6i pretreatrr
20
22
1
1
0
24
1
0
26
1
0
27
1
0
28
1
0
30
0
1
0
32
1
0
34
1
0
35
1
0
36
1
0
37
0
1
38
0
1
40
0
1
control
1
/
/
45 min. post t*
1
111
'" ' '
30 win. pn In
DURATION IN MINUTES
Quantal dose-response curves for doxycycline treatment in
rabbit model of spinal cord ischemia: percentage of paraplegic
animals versus duration of spinal cord ischemia. Midpoint of
curve, ET50, Is ischemic duration that causes 50% of rabbits to
be paraplegic. Error bars indicate the SE of the ETM.
and phlebotomy. For total leukocyte count, both the
control and posttreatment groups showed a significant
decrease at 24 hours compared with baseline. This
postischemic leukocyte count drop was not seen in the
group pretreated with doxycycline.
The results of the effects of in vitro doxycycline on
neutrophil and MNC adhesion are shown in Table 3.
Treatment with doxycycline from 0 to 25 /zg/mL did not
alter unstimulated neutrophil or MNC adhesion. However, doxycycline significantly inhibited specific adhesion in both neutrophils and MNC in a dose-dependent
fashion. Results of in vivo doxycycline treatment on
neutrophil adhesion to laminin are shown in Table 4.
There was a trend toward reduced adhesion in unstimulated cells in animals (n=5) treated with doxycycline,
whereas adhesion of stimulated neutrophils was significantly inhibited.
Discussion
In this study we found that pretreatment with doxycycline improved neurological function in a CNS reperfusion model. A similar benefit was not seen when
treatment was delayed until after the onset of reperfusion. We also found that doxycycline treatment signifi-
Doxyctydine 45-minute posttreatment group
18
1
0
TABLE 2. Hematologlc Profile at Baseline and 24 Hours
by Group
19
1
0
20
1
0
22
0
1
Group
24
1
0
Control (n=11)
26
1
0
28
1
0
30
0
1
32
0
1
34
1
0
36
1
0
37
0
1
39
0
1
n, number of animals. For each duration of ischemia, the
number of animals exhibiting each neurological grade is shown.
Descriptor
Hematocrtt, %
Leukocyte, x i o 3
Baseline
40.4+2.1
7.6±1.9
24 hours
35.0±2.4*
5.8±2.0*
Baseline
41.9±3.1
8.1±1.4
24 hours
37.8±4.4*
8.7±3.3
Baseline
38.5 ±1.8
8.1±1.9
24 hours
33.3±2.3*
5.3+2.0*
Pretreatment (n=12)
Posttreatment (n=11)
Values are mean±SD.
*P<.05 different from corresponding baseline values.
1414
Stroke
Vol 25, No 7 July 1994
TABLE 3. Effect of In Vitro Doxycycline on Adherence of FMLP- or PMA-lnduced Neutrophlls and MNC to Albumin
Neutrophlls
Mononuclear Cells
Doxycycllne,
jig/mL
Unstimulated
PMA*
0 (control)
2±2
6
Unstimulated
PMA
FMLP
98±10
FMLPt
18±11
4±3
41+5
15±3
3±2
54±&t
5±4$
9±4
31 ±9*
7±1t
12
6±3
37±6*
6±4*
9±5
7±&t
0±4*
25
2±2
9±3*
0±3*
8±3
7±6*
7±2*
Values are mean±SE percent adhesions. Three-hour incubation. Values represent percent of total: the average optical density (OD)
(n=8) remaining after the subtraction of the average OD of iodoacetamide-treated cells divided by the average ODof 5xi0 5 cellsxi00.
PMA indicates phorbol 12-myristate 13-acetate; FMLP, forrnyl-methionyleucylphenylalanlne.
*PMA=50 ng/mU tFMLP<=1 x1(T 8 mol/L; tP<.05 different from corresponding 0 doxycycline values.
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cantly inhibited both neutrophil and mononuclear cell
in vitro adhesion and prevented the postischemic decrease in total leukocyte count that is seen in this model.
The results of this study may seem paradoxical in that
an antibiotic is being given to block leukocyte function.
The explanation is that although all tetracyclines appear
to have anti-inflammatory properties, they have a far
greater ability to inhibit bacterial replication.9 Tetracyclines produce bacteriostatic effects through inhibition
of protein synthesis. However, the ability of tetracyclines to inhibit leukocyte function appears to be unrelated to their antimicrobial effects, because studies
using a modified nonantibacterial tetracycline still
found anti-inflammatory effects.23
The degree of neurological protection seen with
doxycycline pretreatment is similar to that which we
have previously observed in this model using specific
antiadhesion molecule monoclonal antibodies.6'7 Although there was a strong trend toward neuroprotection
when doxycycline treatment was delayed for 45 minutes,
it did not reach statistical significance. The possibility
that we did not detect a real treatment effect because of
inadequate sample must be considered (type II error).
Based on the assumption that the observed difference in
PJO values between the delayed treatment and control
groups is real, if we had chosen to design the experiment
to have a power of 90% and set the a error at the usual
5%, we would have needed 17 animals in each group to
find a statistically significant difference.24 The 45minute delay in treatment was chosen so that all animals
would be treated during the reperfusion period. Consequently, any leukocyte adhesion during the initial reflow
period would have occurred before the start of doxycycline treatment. In our previous studies demonstrating
neuroprotection in this model, the antiadhesion monoclonal antibodies were given only pretreatment. In a
similar spinal cord model, Lindsberg et al23 found
beneficial effects when antiadhesion treatments were
TABLE 4. Effect of In Vivo Doxycycllne Administration
on Neutrophil Adhesion to Lamlnln
Group
Unstimulated
FMLP
Untreated (n=5)
4.7±1.1
18.3±3.5
Posttreatment (n=5)
1.5±1.5
9.3±2.9*
Values are mean±SE percent. FMLP indicates formylmethtonyleucylphenylalanine.
*P<.05 different from FMLP-stimulated samples from animals
that did not receive doxycycline treatment by Bonferroni-corrected post hoc comparison.
given 30 minutes after ischemia. In our study, it is
possible that a shorter posttreatment period might have
produced neuroprotection. The peak plasma and spinal
cord levels of doxycycline found in this experimental
study are within the peak doxycycline plasma levels (1 to
3 /xg/mL) recommended in clinical treatment. However,
since doxycycline appears to produce a dose-dependent
inhibition of leukocyte function, higher treatment doses
may have produced greater therapeutic effects.
We found a significant decline in total leukocyte
count in both untreated animals and in animals whose
treatment was delayed until after reperfusion was initiated. One possible explanation for these findings is that
during initial reperfusion a population of leukocytes is
sequestered in the microcirculation so that the leukocytes are no longer present in the 24-hour sample. This
finding would fit with the theory that leukocytes adhere
to endothelium and are trapped in the microcirculation
early during the reperfusion period, contributing to the
"no-reflow phenomenon."26'27 A similar drop in leukocyte counts was not seen in the group pretreated with
doxycycline, suggesting that pretreatment with doxycycline may be preventing in vivo leukocyte adherence and
sequestration.
The results of our in vitro studies provide further
evidence that doxycycline may be inhibiting leukocyte
adherence. We found that doxycycline, in concentrations similar to that used in our treatment study, produces inhibition of specific monocyte and neutrophil
adhesion in rabbits. We have previously found that
doxycycline, along with other tetracyclines, produces
specific inhibition of human mononuclear cell and neutrophil adhesion.9 This inhibition is believed to be due
to the ability of the tetracyclines to bind the divalent
cations Ca2+ and Mg2+, because the addition of these
cations prevents this inhibition.11 Specific leukocyte
adhesion to the endothelium is predominantly mediated
by a leukocyte membrane glycoprotein receptor complex termed CD-18.28 This receptor comprises three
beta subunits that require Ca2+ and Mg2+ to associate.29
By preventing CD-18 reception function, doxycycline
may also be inhibiting adhesion in a similar fashion to
specific monoclonal antibodies that are directed against
these adhesion receptors. We have previously found
that in vitro tetracycline treatment also inhibits other
neutrophil functions, including oxygen free radical generation, degranulation, and collagenase activity.9 Leukocyte granule contents, reactive oxygen metabolites,
and elastase release have been found to injure endothelium and potentiate ischemic injury.30-31 The ability of
Clark et al
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doxycycline treatment to suppress these functions may
also have been important in producing the neuroprotective effects seen in our study.
We found that the dose of doxycycline used in this
study produced plasma levels of doxycycline that were
sufficient to inhibit in vitro leukocyte function. Doxycycline appears to be sequestered in the CNS, based on
our finding of relatively higher levels in the spinal cord.
Because doxycycline binds Ca2+ and Mg2+, an alternative explanation for the observed neuroprotective effects is that doxycycline is decreasing the locally available Ca2+ in the ischemic CNS tissue. This could directly
inhibit the excitatory ischemic cascade that is felt to be
detrimental to CNS tissue during ischemia.32 Further
work with in vitro neuronal or microglial cultures is
needed to investigate this theory.
To our knowledge, this is the first investigation of
doxycycline treatment in CNS ischemia. We have recently demonstrated that doxycycline treatment is also
protective in a rat model of hepatic reperfusion injury.33
In this study, both pretreatment (1 hour) and posttreatment (1 hour into reperfusion) with 10 mg/kg doxycycline reduced hepatic injury as assessed by serum
alanine aminotransferase levels.
We conclude that treatment with doxycycline reduces
CNS ischemic injury in this reperfusion model, with
inhibition of leukocyte adhesion a potential mechanism.
These findings support the role of leukocytes as active
participants in CNS injury. Doxycycline is a safe and
readily available clinical therapeutic agent. In our
model it appears to have therapeutic potential equal to
and would be much less expensive than specific monoclonal antiadhesion antibodies. Finding an agent that
could safely reduce CNS reperfusion injury would have
widespread clinical benefit when used after thrombolysis or as a pretreatment in clinical situations in which
the risk of stroke is high.
Acknowledgments
Supported in part by a National Stroke Association/Allied
Signal Inc. Award (W.M.C.), a National Institutes of Health
Clinician Investigator Development Award (W.M.C.), NINDS
2PO1 NS17493-08 (B.M.C.), and the Kettering Foundation
(W.L.G.).
References
1. Hallenbeck J, Dutka A, Tanishima T, Kochanek P, Kumaroo K,
Thompson C, Obreovitch P, Contreras T. Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during
early postischemic period. Stroke. 1986;17:246-253.
2. Giulian D, Robertson C. Inhibition of mononuclear phagocytes
reduces ischemic injury in the spinal cord. Ann Neurol. 1990;27:33-42.
3. Pozzilli C, Lenzi G, Argentino C. Imaging of leukocyte infiltration
in human cerebral infarcts. Stroke. 1985;16:251-256.
4. del Zoppo G, Schmid-Schonbein G, Mori E, Copeland B, Chang C.
Polymorphonuclear leukocytes occlude capillaries following
middle cerebral artery occlusion and reperfusion in baboons.
Stroke. 1991;22:276-283.
5. Hallenbeck J, Dutka A. Background review and current concepts
of reperfusion injury. Arch Neurol. 1990;47:1245-1254.
Doxycycline Treatment in Stroke
1415
6. Clark W, Madden K, Rothlein R, Zivin J. Reduction of central
nervous system ischemic injury by monoclonal antibody to intercellular adhesion molecule. J Neurosttrg. 1991;75:623-627.
7. Clark W, Madden K, Rothlein R, Zivin J. Reduction of CNS
ischemic injury using leukocyte adhesion antibody treatment.
Stroke. 1991;22:877-883.
8. Kochanek P, Hallenbeck J. Polymorphonuclear leukocytes and
monocytes/macrophages in the pathogenesis of cerebral ischemia
and stroke. Stroke. 1992;23:1367-1379.
9. Gabler W, Creamer H. Suppression of human neutrophil function
by tetracyclines. J Periodont Res. 1991;26:52-58.
10. Gabler W, Smith J, Tsukuda N. Comparison of doxycycline and a
chemically modified tetracycline inhibition of leukocyte functions.
Res Commun Chem Pathol Pharmacol. 1992;78:151-160.
11. Gabler W, Tsukuda N. The influence of divalent cations and
doxycycline on iodoacetamide-inhibitable leukocyte adherence.
Res Commun Chem Pathol Pharmacol. 1991;74:131-140.
12. Zivin J, DeGirolami U, Hurwitz E. Spectrum of neurological deficits
in experimental CNS ischemia. Arch Neurol. 1982;39:408-412.
13. Gabler W. Fluxes and accumulation of tetracyclines by human
blood cells. Res Commun Chem Pathol Pharmacol. 1991;72:39-51.
14. Lynch J, Lotner G, Betz S, Henson P. The release of a plateletactivating factor by stimulated rabbit neutrophils. J Immunol. 1979;
123:1219-1224.
15. Boyum A. Isolation of mononuclear cells and granulocytes from
human blood. Scand J Clin Lab Invest. 1968;97:77-82.
16. Gamble J, Vadas M. A new assay for the measurement of the
attachment of neutrophils and other cell types to endothelial cells.
J Immunol Methods. 1988;109:175-184.
17. Bohnsack J, Akiyama S, Damsky C, Knape W, Zimmerman.
Human neutrophil adherence to laminin in vitro. J Exp Med. 1990;
171:1221-1237.
18. Clark W, Coull B, Corliss L, Beamer N, Austin T, de Garmo P,
Briley D. Role of leukocyte adhesion in clinical stroke. J Stroke
Cerebrovasc Dis. 1992;2:80-84.
19. Bath P, Booth R, Hassall D. Monocyte-lymphocyte discrimination
in a new microtitre-based adhesion assay. J Immunol Methods.
1989;118:59-65.
20. Zivin J, Waud D. Quantal bioassay and stroke. Stroke. 1992;23:
767-773.
21. Madden K, Clark W, Zivin J. Delayed therapy of experimental
ischemia with competitive /V-methyl-D-aspartate antagonists in
rabbits. Stroke. 1993;23:1068-1071.
22. Zivin J, Venditto J. Experimental CNS ischemia: serotonin antagonists reduce or prevent damage. Neurology. 1984;34:469-474.
23. Golub L, McNamara T, D'Angelo G, Greenwald R, Ramamurthy
N. A non-antibacterial chemically-modified tetracycline inhibits
mammalian collagenase activity. J Dem Res. 1987;66:1310-1314.
24. Snedecor G, Cochran W. Statistical Methods. Ames, Iowa: The
Iowa State University Press; 1980:103-105.
25. Lindsberg P, Siren A, Feuerstein G, Hallenbeck J. Postischemic
antagonism of neutrophil adherence has an acute therapeutic
effect on functional recovery in the deteriorating stroke model in
rabbits. J Cereb Blood Flow Metab. 1991;11:S754.
26. Schmid-Schonbein G. Capillary plugging by granulocytes and the
no-reflow phenomenon in the microcirculation. FASEBJ. 1987;46:
2397-2401.
27. Ames A, Wright L, Masayoshi K, Thurston J, Majno G. Cerebral
ischemia. The no-reflow phenomenon. Am J Pathol. 1968;52:437-443.
28. Albelda S, Buck C. Integrins and other adhesion molecules.
FASEBJ. 1990;4:2868-2990.
29. Arnaout M. Structure and function of the leukocyte adhesion
molecule CD/11/CD18. Blood. 1990;75:1037-1050.
30. Harlan J. Neutrophil mediated vascular injury. Acta Med Scand.
1985;715:123-129.
31. Janoff A, Zweifach B. Production of inflammatory changes in the
microcirculation by cationic proteins extracted from lysosomes.
J Exp Med. 1964;120:747-764.
32. Rothman S, Olney J. Glutamate and the pathophysiology of
hypoxic-ischemic brain damage. Ann Neurol. 1986;19:105-lll.
33. Smith J, Gabler W. Doxycycline suppression of ischemia/
reperfusion-induced hepatic injury. Inflammation. In press.
Reduction of central nervous system reperfusion injury in rabbits using doxycycline
treatment.
W M Clark, F A Calcagno, W L Gabler, J R Smith and B M Coull
Stroke. 1994;25:1411-1415
doi: 10.1161/01.STR.25.7.1411
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