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Microvascular blood flow and stasis in transgenic sickle mice Utility of a dorsal skin fold chamber for intravital microscopy.

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American Journal of Hematology 77:117–125 (2004)
Microvascular Blood Flow and Stasis in Transgenic
Sickle Mice: Utility of a Dorsal Skin Fold Chamber for
Intravital Microscopy
Venkatasubramaniam S. Kalambur,1 Hemchandra Mahaseth,2 John C. Bischof,1
Miroslaw C. Kielbik,2 Thomas E. Welch,2 Åsa Vilbäck,2 David J. Swanlund,1
Robert P. Hebbel,2,3 John D. Belcher,2,3 and Gregory M. Vercellotti2,3*
1
Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota
2
Department of Medicine, University of Minnesota, Minneapolis, Minnesota
3
Vascular Biology Center, University of Minnesota, Minneapolis, Minnesota
Vascular inflammation, secondary to ischemia–reperfusion injury, may play an essential
role in vaso-occlusion in sickle cell disease (SCD). To investigate this hypothesis, dorsal
skin fold chambers (DSFCs) were implanted on normal and transgenic sickle mice
expressing human a and bs/bs-Antilles globin chains. Microvessels in the DSFC were
visualized by intravital microscopy at baseline in ambient air and after exposure to
hypoxia–reoxygenation. The mean venule diameter decreased 9% (P < 0.01) in sickle
mice after hypoxia–reoxygenation but remained constant in normal mice. The mean RBC
velocity and wall shear rate decreased 55% (P < 0.001) in sickle but not normal mice after
hypoxia–reoxygenation. None of the venules in normal mice became static at any time
during hypoxia–reoxygenation; however, after 1 hr of hypoxia and 1 hr of reoxygenation,
11.9% of the venules in sickle mice became static (P < 0.001). After 1 hr of hypoxia and
4 hr of reoxygenation, most of the stasis had resolved; only 3.6% of the subcutaneous
venules in sickle mice remained static (P = 0.01). All of the venules were flowing again
after 24 hr of reoxygenation. Vascular stasis could not be induced in the subcutaneous
venules of sickle mice by tumor necrosis factor alpha (TNF-a). Leukocyte rolling flux and
firm adhesion, manifestations of vascular inflammation, were significantly higher at
baseline in sickle mice compared to normal (P < 0.01) and increased 3-fold in sickle
(P < 0.01), but not in normal mice, after hypoxia–reoxygenation. Plugs of adherent leukocytes were seen at bifurcations at the beginning of static venules. Misshapen RBCs were
also seen in subcutaneous venules. Am. J. Hematol. 77:117–125, 2004. ª 2004 Wiley-Liss, Inc.
Key words: sickle cell disease; intravital microscopy; skin fold chambers; blood flow;
stasis; vaso-occlusion
INTRODUCTION
Sickle cell disease (SCD) is characterized by recurring
acute vaso-occlusive episodes resulting in damage to
multiple organs [1]. The substitution of valine for glutamate at the sixth position of the b chain of hemoglobin
[2] results in the polymerization of sickle hemoglobin and
the sickling of RBCs under deoxygenated conditions.
The polymerization of deoxygenated sickle hemoglobin
is the primary event in the molecular pathogenesis of
SCD and is responsible for the vaso-occlusive phenomena that are the hallmarks of the disease [3]. The repetitive vaso-occlusive episodes cause tissue ischemia and
ª 2004 Wiley-Liss, Inc.
Contract Grant Sponsor: NHLBI; Contract grant numbers: HL67367,
HL55552
*Correspondence to: Gregory M. Vercellotti, M.D., University of
Minnesota, Department of Medicine, Division of Hematology,
Oncology and Transplantation, 420 Delaware St. SE, MMC 480,
Minneapolis, MN 55455. E-mail: verce001@umn.edu
Received for publication 12 May 2003; Accepted 13 April 2004
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/ajh.20143
118
Kalambur et al.
reperfusion injury, resulting in endothelial cell activation
and inflammation [4,5]. Sickle patients have multiple
indicators of an inflammatory response including elevated white counts [6–9], C-reactive protein levels
[10–13], cytokines [14–17], as well as activated monocytes [13,18], neutrophils [19–21], platelets [18,22–28],
and endothelial cells [29,30] in circulation.
Transgenic mice expressing human a and bS globins
provide unique models to study abnormalities in microvascular blood flow in SCD. Their RBCs change shape
upon de-oxygenation [5,31], and they display blood
flow abnormalities including adhesion of RBCs, rolling
and adhesion of leukocytes, and stasis in post-capillary
venules [4,31–33]. Upon pathological analysis, there
is tissue damage in multiple organs including the kidney, liver, lung, and spleen [34–36]. Like human SCD
patients, transgenic bS mice display numerous signs of
an inflammatory response such as elevated white
counts and enhanced activation of NF-kB, a transcription factor critical for the inflammatory response [37].
In addition, there are significant increases in the expression of endothelial cell adhesion molecules and the
acute phase response protein serum amyloid P-component (SAP) [37]. Sickle mice exhibit biochemical footprints consistent with excessive reactive oxygen species
(ROS) generation, even at ambient air [5]. These footprints are exaggerated further after exposure to
hypoxia–reoxygenation resulting in enhanced activation of NF-kB and a pro-adhesive endothelial cell phenotype which is conducive to the formation of vascular
obstructions [4,5].
We have incorporated the DSFC model [38] to
observe blood flow changes in the subcutaneous tissue
of normal and transgenic sickle mice. This DSFC
model allows repeat visualizations of the same vessels
over several hours, days, or even weeks and analysis of
vessel diameters, RBC velocities, vascular stasis, and
leukocyte rolling and adhesion after administering proinflammatory insults such as hypoxia–reoxygenation
and TNF-a. The role of inflammation and the molecular events leading to the formation and resolution of
vascular obstructions in SCD are still largely speculative. Because vaso-occlusion is a critical event in the
pathology of SCD, we have used the DSFC and intravital microscopy to examine blood flow in the subcutaneous microcirculation of bS/S-Antilles mice and
examined the effects of pro-inflammatory insults on
leukocyte interactions with the endothelium and the
genesis and resolution of vascular stasis.
MATERIALS AND METHODS
Mice
All animal experiments were approved by the University of Minnesota’s Institutional Animal Care and
Use Committee. We utilized female bS/S-Antilles
transgenic mice as our mouse model for SCD [36].
The bS/S-Antilles mice are homozygous for deletion of
the mouse b major globin locus [bMDD] and express
human a, bS, and bS-Antilles globin transgenes. These
mice were produced by breeding aHbS[bMDD] mice
[39] into a mouse line expressing human bS-Antilles on
the mouse homozygous b major deletional background
[35]. bS-Antilles contains, in addition to the bS mutation
at b6, a second mutation at b23 (Val!Ile). bS-Antilles has
low oxygen affinity and decreased solubility under
deoxygenated conditions, resulting in a more severe
form of SCD. In the bS/S-Antilles mice, approximately
42% of the b globins expressed are human bS and 36%
are human bS-Antilles. These transgenic bS/S-Antilles mice
are on the C57BL/6 genetic background.
Normal female mice (C57BL/6) obtained from
Jackson Laboratory (Bar Harbor, ME) were used as
normal controls for the bS/S-Antilles mice. The mice used
in these studies were between 8 and 12 weeks of age.
The mice weighed 20–30 g and were housed in specific
pathogen-free housing to prevent common murine
infections that could cause an inflammatory response.
All the mice were maintained on a standard chow diet.
Hematocrits, Reticulocytes, and White Counts
Blood was collected from the tail vein of unanesthetized mice after warming the animals under a heat
lamp. Heparinized whole blood was collected in capillary tubes for manual measurement of packed RBC
volume (hematocrit) and for reticulocyte staining and
counting in methylene blue. Reticulocytes, identified by
their DNA staining, were expressed as a percentage of
RBCs. For white counts, the RBCs were immediately
lysed by diluting whole blood 20-fold in 2% acetic acid
containing 30 mg/mL EDTA. Leukocytes were counted
on a hemocytometer four times and averaged.
Anesthetic Preparation
Anesthesia was administered to all mice before surgery and intravital microscopy. The anesthetic cocktail,
administered intraperitoneally, consisted of 0.2 mL
of xylazine (20 mg/mL) and 0.6 mL of ketamine (100
mg/mL) mixed with 9 mL of normal saline. We injected
0.2–0.4 mL of this anesthetic cocktail into each mouse. All
controlled substances used were approved by Research
Animal Resources at the University of Minnesota.
DSFC Implantation
The experimental model used in this study was originally developed for the rat dorsal skin fold [38] and modified for mice. After the mouse was anesthetized, a
suitable location for chamber placement was selected,
Stasis in Sickle Cell Disease
and the hair on that location was shaved off with
a clipper. The remaining hair was removed from the
dorsal skin with Nair Hair Remover (Church & Dwight,
Princeton, NJ). The smooth skin was washed twice with
betadine and dried. A sterile field was maintained
throughout the surgical procedure. One sterilized chamber piece containing a round window was placed on one
side of the dorsal skin fold. Another matching chamber
piece without a window was placed on the opposite side
of the skin fold, holes for three connecting screws were
made through the skin fold and the assembly was tightened together with screws and nuts. A circle of cutaneous tissue and fascia was carefully cut away from the
skin fold inside the DSFC window exposing the blood
vessels of the subcutaneous tissue adjacent to the striated
muscles of the opposing skin fold. Antibiotic ointment
(bacitracin zinc/polymyxin B sulfate/neomycin sulfate)
was used on the edges of the wound inside the DSFC
window. A glass window was placed in the chamber to
cover the exposed tissue and secured with a snap ring.
Finally, the chamber was sutured to the skin. After
surgery, the animals were housed in barrier cages inside
a humidified incubator maintained at 32 C with unrestricted access to food and water. To minimize the risk of
infection, all mice were given the antibiotic amoxicillin in
their drinking water (250 mg per 5 mL of water) after
DSFC implantation and throughout the experiment.
The mice had a mild inflammatory response to the
DSFC implantation, as evidenced by elevated levels of
serum amyloid P component (SAP) [40] 3 days after
surgery (data not shown). SAP returned to pre-surgical
baseline levels 4–7 days after DSFC implantation.
Venule diameters, RBC velocity, vascular stasis, and
leukocyte rolling and adhesion measurements, presented
below, were measured on days 4–7 after DSFC implantation, and measurements in normal and sickle mice were
made on the same days. Similar stasis and leukocyte
rolling values were obtained on days 4–7 (data not
shown) in both sickle and normal mice, indicating that
the days chosen for data collection did not influence the
results. Figure 1 shows a close-up of an implanted DSFC
with the vasculature visible through the window.
Intravital Microscopy and Analysis
Four to seven days after DSFC implantation,
anesthetized mice were placed on a specially constructed microscope stage where individual flowing
venules were selected at random and their relative locations were noted with a hand drawn map of the microscopic field. Figure 2 shows a mouse mounted on the
special microscope stage for intravital microscopy. The
DSFC was illuminated with a 30-W halogen bulb.
Microscopic observations were carried out in bright
field using a Nikon Labophot-2 microscope (Fryer
119
Fig. 1. Close up of a dorsal skin fold chamber (DSFC)
implanted on a transgenic sickle mouse. Subcutaneous blood
vessels of the opposing dorsal skin fold are visible through
the chamber window.
Fig. 2. Mouse with an implanted DSFC mounted on the
microscope stage prior to intravital microscopy of microvessels inside the DSFC window.
Corp., Minneapolis, MN) fitted with a Hamamatsu
Newvicon television camera (North Central Instruments, Minneapolis, MN) and a JVC S-VHS video
recorder (JVC Company of America, Aurora, IL).
The video images were subsequently digitized from
the videotapes using ImagePro (Leeds Precision Instruments, Minneapolis, MN). Mean vessel diameters were
measured offline using a digital image splitting device
[41]. Mean RBC velocities (VRBC centerline) were measured online using an optical Doppler velocimeter [42]
obtained from Texas A&M University Microcirculation Research Institute. Wall shear rates were calculated using the formula 8Vmean/D, where D is the
vessel diameter and Vmean is the mean velocity obtained
from the equation Vmean ¼ VRBC centerline/1.6, as previously reported [43]. Each vessel was observed online
for blood flow. Venules with no observable blood flow
were reported as static.
120
Kalambur et al.
For measurement of leukocyte rolling and adhesion,
the leukocytes were labeled in vivo with 100 mL of
0.02% rhodamine 6G (Sigma Chemical Co., St. Louis,
MO) in sterile saline administered intravenously via tail
vein injection [44]. The DSFC was illuminated with a
mercury lamp and rhodamine signal-enhancing filters
(excitation l ¼ 480–520 nm and emission l ¼ 535–
585 nm) were used to view the fluorescence. A Hamamatsu silicon-intensified transmission camera (North
Central Instruments) fitted to the Nikon Labophot-2
microscope was used to detect the fluorescent signal.
Each vessel was recorded on videotape for approximately 2 min. Rolling leukocytes were measured offline
using the videotape and were defined as those leukocytes that distinctly roll along the endothelial surface of
venules [4]. The rolling flux was determined as the total
number of leukocytes rolling through a given section of
vessel per minute. A leukocyte was considered adherent
if it remained stationary for at least 30 sec.
rank sum test. The proportions of venules exhibiting
stasis at each time point were compared using a z-test.
RESULTS
The hematocrits (mean ± SE) of sickle (46.3 ± 3.0%,
n ¼ 3) and normal (45.6 ± 0.8 %, n ¼ 7) mice were
similar, although the number of reticulocytes in sickle
mice (10.1 ± 1.0%, n ¼ 4), expressed as percent of
RBCs, was 3.5-fold higher than normal mice (2.9 ±
0.7%, n ¼ 3, P < 0.01). Mean white counts in sickle mice
([14.3 ± 1.1] 103/mL, n ¼ 12) were 1.4-fold higher than
normal mice ([8.7 ± 0.6] 103/mL, n ¼ 14, P < 0.001).
A scatter plot of RBC velocities as a function of
diameter within the venules (Fig. 3A) and the arterioles (Fig. 3B) of normal mice with implanted DSFCs
at baseline ambient air conditions are compared with
published values for the cat mesentery and rabbit
Experimental Protocol
Four to seven days after the DSFC implantations,
anesthetized normal and bS/S-Antilles (sickle) mice were
visualized separately and the venule diameters, mean
RBC velocities, stasis, and leukocyte rolling and
adhesion were measured as described above. After
baseline measurements in ambient air, the mice were
transferred to a special chamber and subjected to 1 hr
of hypoxia (7% O2/93% N2) followed by reoxygenation in ambient/room air. Additional measurements
of blood flow parameters were made at various times
(0–24 hr) after hypoxia during the reoxygenation
phase. Some anesthetized mice with DSFCs were
injected intraperitoneally with recombinant mouse
TNF-a (25 mg/kg body weight, R&D Systems, Minneapolis, MN) in ambient air. Vascular stasis and
leukocyte rolling and adhesion were measured as
described above 0–8 hr after TNF-a injection.
Histology of Dorsal Skin
In some sickle mice, dorsal skin samples were taken
for histological analysis after 1 hr of hypoxia and 1 hr
of reoxygenation. Skin samples were fixed overnight
in formalin, cut into 5-mm sections, embedded in
paraffin, mounted on slides, and stained with hematoxylin and eosin before microscopic examination.
Statistics
All statistical analyses were performed with SigmaStat 2.0 for Windows (SPSS Inc., Chicago, IL). Comparisons of data from normal and transgenic sickle
mice in ambient air and after hypoxia–reoxygenation
were made using Student’s t-test or a Mann–Whitney
Fig. 3. Scatter plots of mean RBC velocities (mm/sec)
versus mean diameters (mm) in venules (A) and arterioles
(B) in normal mice with DSFCs compared to other animal
model systems. Values were obtained from subcutaneous
venules (n = 60) and arterioles (n = 42) in normal mice with
implanted DSFCs. For comparison, data from the mesentery
of cats (n = 48 venules and n = 44 arterioles) and the
omentum of rabbits (n = 32 venules and n = 34 arterioles)
were taken from the literature [45].
Stasis in Sickle Cell Disease
121
TABLE I. Hypoxia–Reoxygenation Reduces Mean Diameters, RBC Velocities, and Wall Shear Rates in the Subcutaneous
Venules of Sickle, But Not Normal, Mice
Venule measurementsa
Diameter (mm)
RBC velocity (mm/sec)
Wall shear rate (1/sec)
Mouse model
Number
of venules (n)
Baseline ambient
air
Normal
Sickle
Normal
Sickle
Normal
Sickle
21
43
21
43
21
43
37.7 ± 4.0
33.2 ± 3.9
3.4 ± 0.3
4.2 ± 0.4
507 ± 50
979 ± 122B
!
1 hr hypoxia + 1 hr
reoxygenation
37.3 ± 3.7
30.1 ± 3.8AA,B
4.3 ± 0.7
1.9 ± 0.3AAA,BBB
627 ± 88
450 ± 74AAA,B
a
Venule diameters, RBC velocities, and wall shear rates at baseline and after 1 hr of hypoxia and 1 hr of reoxygenation in normal and sickle mice.
Values are presented as mean ± SE.
AA
P < 0.01 and AAAP < 0.001 when measurementstaken after hypoxia–reoxygenation are comparedtothose made at ambientair within the same venules.
B
P < 0.05 and BBBP < 0.001 when the measurements made in sickle mice are compared to those made in normal mice under the same treatment conditions.
omentum [45]. The RBC velocities in venules and
arterioles of normal mice with DSFCs compare
favorably with values of blood flow in cat mesentery
and rabbit omentum model systems.
The DSFC affords the advantage of examining the
same venules at multiple time points before, during, and
after a vaso-active intervention such as hypoxia–reoxygenation. Table I summarizes the blood flow measurements in normal and transgenic sickle mice in venules
inside the subcutaneous DSFC microcirculation at
ambient air baseline and after 1 hr of hypoxia and 1 hr
of reoxygenation treatment. The mean venule diameter
remained constant after hypoxia–reoxygenation in normal mice but decreased 9% (P < 0.01) in the sickle mice
after hypoxia–reoxygenation. In the normal mice, there
was a non-statistically significant 26% increase in the
mean RBC velocity after hypoxia–reoxygenation, but in
sickle mice the mean RBC velocity decreased 55% (P <
0.001) after hypoxia–reoxygenation. Similarly, the calculated wall shear rates increased an average of 24% in
normal mice (not significant) after hypoxia–reoxygenation, but decreased 54% (P < 0.001) in sickle mice after
hypoxia–reoxygenation. Baseline wall shear rates in
sickle mice in ambient air were 93% greater than normal
mice under the same conditions (P < 0.05). But, wall
shear rates in sickle mice dropped precipitously to 72%
of the wall shear rates in normal mice after hypoxia–
reoxygenation (P < 0.05).
We used the DSFC model to examine the genesis
and resolution of microvascular stasis after hypoxia–
reoxygenation in the venules of sickle and normal mice
(Fig. 4). All of the selected venules were flowing at
ambient air baseline (H,R ¼ 0,0) in normal (n ¼ 80)
and sickle mice (n ¼ 178). None of the venules in
normal mice became static at any time during
hypoxia–reoxygenation, and none of the venules in
sickle mice became static during the 1 hr of hypoxia
or during the first 15 min of reoxygenation (H,R ¼ 1,0).
However, after 1 hr of hypoxia and 1 hr of reoxygenation (H,R ¼ 1,1), 11.9% of the venules in sickle mice
Fig. 4. Stasis occurs in the subcutaneous venules inside the
DSFC of transgenic sickle mice, but not normal mice, after 1 hr
of hypoxia and 1 hr of reoxygenation. Subcutaneous venules
inside DSFCs implanted on normal and sickle mice
were visualized by intravital microscopy. Flowing venules
were randomly selected at ambient air baseline
[H(ypoxia),R(eoxygenation) = 0,0]. After baseline
examination of venules, the mice were exposed to 1 hr of
hypoxia (7% oxygen and 93% nitrogen) and the same venules
were re-examined for blood flow/stasis at the end of hypoxia
during the first 10 min of reoxygenation (H,R = 1,0). Similarly,
the same venules were re-examined for blood flow after 1 hr of
hypoxia and 1 hr of reoxygenation (H,R = 1,1) and after 1 hr of
hypoxia and 4 hr of reoxygenation (H,R = 1,4). Venules without
any visible blood flow were counted as static and those with
blood flow were counted as flowing. Each bar represents the
percentage of static venules seen in the DSFC window. The
total number of venules (n) examined was 178 in sickle mice
and 80 in normal mice at each time point. **P < 0.001 and *P =
0.01 in sickle mice compared to the same venules at ambient
air baseline.
became static (P < 0.001 compared to baseline). After
1 hr of hypoxia and 4 hr of reoxygenation (H,R ¼ 1,4),
most of the stasis had resolved; only 3.6% of the
122
Kalambur et al.
TABLE II. Leukocyte Rolling and Adhesion in the Subcutaneous Venules of Normal and Sickle Mice at Ambient Air Baseline
and After 1 hr of Hypoxia and 1 hr of Reoxygenation*
Treatment
Baseline at ambient air
1 hr hypoxia + 1 hr reoxygenation
Mouse model
Total number
of venules (n)
Leukocyte rolling
flux (leukocytes/min)
Leukocyte adhesion
(leukocytes/100 mm)
Normal
Sickle
Normal
Sickle
17
16
13
25
2.5 + 0.6
13.4 ± 3.5BB
3.0 + 1.2
38.7 ± 6.1AA,BBB
0.7 + 0.3
2.6 ± 0.5BB
1.3 + 0.4
8.1 ± 1.0AAA,BBB
*Values are presented as means ± SE.
AA
P < 0.01 and AAAP < 0.001 when leukocyte rolling and adhesion values after hypoxia–reoxygenation are compared to those at baseline
ambient air within the same mice.
BB
P < 0.01 and BBBP < 0.001 when values in sickle mice are compared to those in normal mice under the same treatment conditions.
subcutaneous venules in sickle mice remained static (P ¼
0.01 compared to baseline, n ¼ 111 venules examined)
and all of the venules were flowing again after 24 hr of
reoxygenation (data not shown). The static venules in
sickle mice had a smaller mean diameter than the nonstatic venules. The mean baseline diameter of the
venules that became static in sickle mice was 39.0 ±
18 mm (mean ± SE), while the non-static venules had a
mean baseline diameter of 47.5 ± 50.2 mm.
TNF-a was injected intraperitoneally (25 mg/kg
body weight) into sickle mice to see if the pro-inflammatory cytokine could induce stasis in a manner
similar to hypoxia–reoxygenation treatment. None
of the subcutaneous venules in sickle mice became
static 0–8 hr after TNF-a injection despite significant
increases in leukocyte rolling (data not shown).
Leukocyte rolling flux and leukocyte firm adhesion
were measured in the subcutaneous venules of normal
and sickle mice at ambient air baseline and after 1 hr of
hypoxia and 1 hr of reoxygenation (Table II). Leukocyte
rolling flux and adhesion were significantly higher in
the venules of sickle mice compared to those in normal
mice at both ambient air baseline (P < 0.01) and after
hypoxia–reoxygenation (P < 0.001). Hypoxia–reoxygenation increased leukocyte rolling flux (P < 0.01)
and adhesion (P < 0.001) in transgenic sickle mice
approximately 3-fold; however, in normal mice,
hypoxia–reoxygenation had little effect on leukocyte
rolling and adhesion. Rhodamine fluorescent staining
of leukocytes revealed that there was an overabundance
of firmly adherent leukocytes at sites of vascular stasis
(Fig. 5A). Venules that become static are filled with
adherent leukocytes at bifurcations near the ‘‘head’’ or
beginning of the static venule (Fig. 5A). In data not
shown, a video taken of the same venule in Fig. 5A
shows that the venule is plugged with adherent leukocytes near a bifurcation that is continuous with a larger
flowing venule. The leukocytes are labeled with rhodamine 6G and appear as white fluorescent balls. The video
also shows some of the leukocytes become dislodged as
the static vessel attempts to re-establish flow.
Fig. 5. (A) Fluorescent image of a static venule with adherent
leukocytes after hypoxia-reoxygenation in the DSFC
implanted on a sickle mouse. Leukocytes were labeled
in vivo with intravenous rhodamine 6G and appear as white
balls in the figure. Adherent leukocytes can be seen at a bifurcation plugging the ‘‘head’’ or beginning of a static venule. The
static venule is continuous with a larger flowing venule. The
image was captured after 1 h of hypoxia (7% O2/93% N2) and 1 h
of reoxygenation in room air. (B) Histology of venule in the
dorsal skin of transgenic sickle mice after 1 hr of hypoxia and
1 hr of reoxygenation. Dorsal skin samples were taken for
histological analysis after the sickle mice were exposed to 1 hr
of hypoxia and 1 hr of reoxygenation when approximately 12%
of the venules were static. Skin samples were fixed overnight
in formalin, cut into 5-mm sections, embedded in paraffin,
mounted on slides, and stained with hematoxylin and eosin
before microscopic examination. The figure shows a venule
with a suspected vascular obstruction. White arrowheads
point to leukocytes that appear to be adherent to the vascular
endothelium and white arrows point to misshapen RBCs
inside the venule. [Color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
Stasis in Sickle Cell Disease
Dorsal skin samples were taken for histological analysis from sickle mice after exposure to 1 hr of hypoxia
and 1 hr of reoxygenation when approximately 12% of
the subcutaneous venules become static. Figure 5B
shows hematoxylin and eosin staining of a subcutaneous venule with a suspected vascular obstruction.
White arrowheads point to leukocytes that appear to
be adherent to the vascular endothelium, and white
arrows point to misshapen RBCs inside the venule.
DISCUSSION
The development of transgenic murine models to
study SCD provides an opportunity to understand the
molecular and cellular events during the genesis and
resolution of vaso-occlusion. Characterizing and understanding the molecular basis for vaso-occlusion in SCD
is of critical importance to devising strategies to prevent
and treat it. By its nature, vaso-occlusion and its subsequent resolution promote ischemia–reperfusion
injury, which can induce ROS production, inflammation, and organ pathology. These studies utilized
DSFCs in conjunction with intravital microscopy to
examine the evolution of vascular stasis after hypoxia–
reoxygenation in transgenic mice expressing human a
and bS/S-Antilles globins. These studies show that vascular
stasis, induced by hypoxia–reoxygenation, is a rapid
process of relatively short duration; stasis occurs in the
subcutaneous venules within 1 hr of reoxygenation and
resolves largely by 4 hr and completely within 24 hr. The
stasis is accompanied by a significant increase in leukocyte rolling and firm adhesion to the endothelium of
venules with plugs of adherent leukocytes present at
bifurcations at the ‘‘head’’ or beginning of the static
venule continuous with larger flowing venules.
Intravital microscopy of the subcutaneous microcirculation in the murine DSFC model provides blood
flow measurements that are comparable to vascular
beds in other animal models. The DSFC is advantageous to examine temporal changes in the same vessels,
and the effects of interventions such as hypoxia–reoxygenation and TNF-a in transgenic bS mouse models.
The DSFC allows animals to be observed over hours,
days, or even weeks, without sacrificing the animal
after several hours as is required in the cremasteric,
mucosal–intestinal, and omentum preparations. In
addition, unlike the cremasteric model, the DSFC
model allows the study of the microcirculation in both
male and female mice. Such sex comparisons are warranted given the reported disparities in nitric oxide
production in males and females [46].
The relationship of RBC velocities to venular and
arterial diameters in normal mice with a DSFC was
similar to the cat mesentery and rabbit omentum
models as well as diameters and velocities in venules
123
of the mouse cremasteric model [4]. Placing the chamber on the mice does cause a mild inflammatory
response that subsides over time as the wound heals
as indicated by SAP values (data not shown). A
surgically induced inflammatory response is also a
concern in the other intravital microscopy models as
well. However, in the bS/S-Antilles and normal mice, the
percentage of static venules and leukocyte rolling at
baseline and after hypoxia–reoxygenation were similar on days 4–7 after DSFC implantation (data not
shown). This indicates that any inflammatory
response that may have been present on days 4–7
after DSFC surgery did not have a significant impact
on stasis and rolling in the subcutaneous venules.
In our studies, using a bS/S-Antilles murine model with
generally moderate severity of SCD [36], differences in
blood flow were observed in subcutaneous venules
when sickle mice were compared to normal mice. Of
note, at ambient air baseline, the sickle mice had significantly faster blood flow and higher wall shear rates
than normal mice. This finding contrasts with reports
of slower blood flow in bS/S-Antilles mice in ambient air
in the post-capillary venules of cremasteric muscle preparations [31]. This difference could be due to differences in the vascular beds or perhaps to differences in
the level of baseline vascular inflammation between the
two models. In preliminary studies in our laboratory,
adaptive, cytoprotective genes such as heme oxygenase1 are induced in SCD and enzymatic products from
heme destruction such as carbon monoxide and biliverdin have vasodilatory and anti-inflammatory properties that may compensate and protect the vascular
endothelium and alter blood flow in the absence of an
inflammatory stress such as hypoxia. After hypoxia–
reoxygenation, a pro-inflammatory insult, the venules
of sickle mice with DSFCs had markedly slower blood
flow and lower wall shear rates than normal mice;
a similar finding was reported for the cremasteric muscle preparation [4]. Unlike normal mice, sickle mice
were extremely sensitive to hypoxia–reoxygenation.
Dramatic reductions in blood flow in response to
hypoxia–reoxygenation were accompanied by 3-fold
increases in leukocyte rolling and firm adhesion.
In addition to increased leukocyte rolling and
adhesion, sickle mice also responded to hypoxia–
reoxygenation treatment with the development of stasis in the subcutaneous venules inside the DSFC after
1 hr of reoxygenation. The stasis in sickle mice was
significantly reduced after 4 hr of reoxygenation
and completely absent after 24 hr of reoxygenation.
There were no signs of stasis in any normal animals.
Histological analysis of the dorsal skin from sickle
mice after hypoxia–reoxygenation revealed leukocytes
and misshapen RBCs that appear to be adherent to the
endothelium in suspected static venules.
124
Kalambur et al.
Previous studies of the blood flow dynamics in bS
mice were performed in the mucosal–intestinal [32] and
cremasteric [4,31,33] vascular beds. Intravital microscopy studies of blood flow in the cremasteric muscle
preparation of bS mice showed adhesion of RBCs and
leukocytes in the post-capillary venules [31], and studies
in the mucosal–intestinal microcirculation detected
sludging and decreased blood flow velocities in venules
of all diameters in bS mice compared to normal [32].
Some of these studies induced inflammation in the
cremasteric venules by exposing the animals to
hypoxia–reoxygenation [4] or by administration of the
pro-inflammatory cytokine TNF-a [33]. Hypoxia–
reoxygenation increased endothelial ROS production,
leukocyte rolling flux and leukocyte firm adhesion and
decreased leukocyte rolling velocities and venular
blood flow in bS mice compared to normal [4]. In
severe, but not milder, mouse models of SCD, TNF-a
induced stasis in the cremasteric venules and was fatal
in a high percentage of mice transplanted with bone
marrow from the severe bS ‘‘BERK’’ mouse models
[33]. Inhibition of P-selectin binding, but not E-selectin,
in bS mice during hypoxia–reoxygenation essentially
eliminated leukocyte rolling and firm adhesion and
markedly increased RBC velocities and wall shear
rates [4]. Similarly, mice deficient in P- and E-selectin,
transplanted with bone marrow from ‘‘BERK’’ bS mice,
had reduced leukocyte rolling and adhesion and were
protected from TNF-a induced stasis [33]. Thus, these
intravital microscopy studies of blood flow in inflamed
subcutaneous and cremasteric venules of bS and
bS+S-Antilles mice and normal mice transplanted with
bone marrow from bS mice suggest that endothelial
cell activation and the accompanying leukocyte rolling
and adhesion may be linked to vascular stasis in SCD.
However, because we could not induce stasis with the
pro-inflammatory cytokine TNF-a, despite increases in
leukocyte rolling and adhesion seen 4 hr after TNF-a
administration, factors other than adhesion molecule
expression may be involved in stasis. These data suggest
that sickling, and perhaps hemolysis of the red cell,
which occurs during hypoxia, may be required before
stasis can occur; TNF-a induced inflammation, alone,
is not sufficient to induce stasis in less severe models of
SCD such as the bS+S-Antilles.
CONCLUSIONS
In summary, the DSFC model provides an excellent tool for examining vascular stasis, RBC velocity,
and leukocyte interactions with the endothelium in
the subcutaneous microcirculation of transgenic
sickle mice. This model may be relevant in SCD as
the subcutaneous vasculature is frequently occluded
and may be the primary cause of skin ulcerations in
humans with SCD [47]. The development of stasis
and its resolution, as seen in the DSFC, is by its
nature consistent with a model of on-going ischemia–reperfusion injury in transgenic sickle mice and
may be perhaps fundamental to the pathology of
SCD. Vascular inflammation could lead to a vicious
cycle of inflammation and stasis, where inflammation
leads to leukocyte and RBC adherence to endothelium, followed by stasis and temporary local ischemia, resolution, and reperfusion leading to ROS
production, more inflammation, adhesion, and stasis.
The DSFC model in transgenic sickle mice affords a
potential avenue for discovery of the molecular and
cellular events leading to stasis and its resolution as
well as the testing of potential therapies. The model
needs to be tested on other transgenic sickle animals
that have varying expression of human g globins in
conjunction with the expression of human a and bS
globins and varying severity of SCD [48,49]. This
model can also be used to examine the role of fluorescently labeled sickle RBCs in the formation of vascular obstructions. We hope that the insights derived
from this model will be readily translated to humans
with SCD to improve blood flow, minimize vasoocclusion, and prevent organ injury.
ACKNOWLEDGMENTS
We thank Dr. Kenneth Diller, Department of
Mechanical Engineering, University of Texas, Austin,
Dr. Marcos Intaglietta, Department of Bioengineering, University of California, San Diego, and their colleagues for their assistance on intravital microscopy
setup and enhancement. We also thank Stephana
Choong for breeding and characterizing the transgenic
sickle mice used for these studies and Julia Nguyen for
her help with blood collection.
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