Interleukin-18 does not influence infarct volume or functional outcome in the early stage after transient focal brain ischemia in mice.

RESEARCH Open Access
Interleukin-18 does not influence infarct volume
or functional outcome in the early stage after
transient focal brain ischemia in mice
Stefan Braeuninger*†, Christoph Kleinschnitz†, Guido Stoll
Abstract
Interleukin-18 (IL-18) is a proinflammatory cytokine of the interleukin-1 family which is upregulated after cerebral
ischemia. The functional role of IL-18 in cerebral ischemia is unknown. In the present study, we compared infarct
size in IL-18 knock-out and wild-type mice 24 hours and 48 hours after 1-hour transient middle cerebral artery
occlusion (tMCAO). Moreover, the functional outcome was evaluated in a modified Bederson score, foot fault test
and grip test. There were no significant differences in infarct size or functional outcome tests between wild-type
and IL-18 knock-out mice. These data indicate that the early inflammatory response to cerebral ischemia does not
involve IL-18, in contrast to other interleukin-1 family members such as interleukin-1.
Background
There is increasing evidence that inflammatory pro-
cesses play a detrimental role in ischemic stroke [1,2].
On the other hand, the postischemic immune response
may also be beneficial with respect to neuroprotection
and tissue remodelling. The proinflammatory cytokine
IL-18 is an interleukin-1 family member identified as
and named interferon-g-inducing factor [3,4]. The
effects of IL-18 are complex and pleiotropic involving
activation of T cells and NK cells in autoimmune disor-
ders (for review, see Reddy [5]). In the central nervous
system, IL-18 can locally be produced by activated
microglia [6,7]. Increased IL-18 serum levels have been
detected within 24 hours in patients with acute ischemic
stroke [8], and elevated IL-18 plasma levels at 48 hours
were associated with unfavorable clinical outcome at 3
months [9]. Moreover, in hypoxic-ischemic brain injury
in neonatal rats, an early IL-18 activation (already within
hours) and a progressive increase for at least 14 days
have been described [10]. At 3 days after hypoxia-ische-
mia, IL-18 deficiency has been shown to ameliorate
infarct volume and grey matter injury [10] as well as
white matter injury [11] in neonatal mice. These studies
suggest that IL-18 may play a pathophysiological role in
stroke development. To elucidate a functional role of
IL-18 in cerebral ischemia we investigated infarct size
and functional outcome 24 hours and 48 hours after
tMCAO in adult mice with IL-18 deficiency.
Methods
Animal studies were conducted in accordance with
institutional guidelines and approved by the appropriate
authorities (Regierung von Unterfranken). Wild-type
and IL-18 knock-out mice that had been generated [12]
and backcrossed onto BALB/C mice [13] as described
were kindly provided by Drs. X-Q Wei and FY Liew,
Glasgow, Scotland, and bred and raised in our labora-
tory animal facility. A total of 35 wild-type and 29 IL-18
knock-out animals (plus additional 11 wild-type and 10
knock-out mice for laser-Doppler flowmetry and ink
perfusion) were used.
Mice weighting 18-24 g were subjected to transient
focal cerebral ischemia in the right middle cerebral
artery (MCA) territory for 1 hour using the intraluminal
suture MCA occlusion method [14]. In brief, mice were
anesthetized with 2% to 2.5% enflurane in a 70% N2O/
30% O2 mixture. A servo-controlled heating blanket was
used to maintain core body temperature close to 37°C
throughout surgery. A silicon rubber-coated 6.0 nylon
monofilament (Doccol, Albuquerque, NM) was inserted
into the right common carotid artery and advanced via
the internal carotid artery to occlude the origin of the
* Correspondence: braeuninge_s@klinik.uni-wuerzburg.de
† Contributed equally
Department of Neurology, Julius-Maximilians-Universitaet Wuerzburg, Josef-
Schneider-Strasse 11, D-97080 Wuerzburg, Germany
Braeuninger et al. Experimental Translational Stroke Medicine 2010, 2:1
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© 2010 Braeuninger et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

MCA, causing focal ischemic brain injury in the right
MCA territory. The occluding filament was removed
after 1 hour to allow reperfusion. Animals were sacri-
ficed 24 hours or 48 hours after tMCAO. Brains were
harvested and 2 mm-thick coronal slices were sectioned
in a mouse brain matrix. After staining with 2% 2,3,5-
triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St.
Louis, MO) in PBS, the pale infarctions were readily dis-
cernable from the brick-red non-ischemic areas and pla-
nimetric measurements were obtained using the ImageJ
software package (available at http://rsb.info.nih.gov/ij/;
National Institutes of Health, Bethesda, MD). The calcu-
lated lesion volume was corrected for brain swelling as
described by Ginsberg et al. [15].
Additionally, we assessed the functional outcome in
representative subsets of the animals. Immediately after
recovery from anesthesia, and 24 hours and 48 hours
later, a modified Bederson score [16] was determined
according to the following scoring system: 0, no deficit; 1,
forelimb flexion; 2, as for 1, plus decreased resistance to
lateral push; 3, unidirectional circling; 4, longitudinal
spinning or seizure activity; 5, no movement. 24 hours
and 48 hours after surgery, the foot fault test and grip test
were performed. The foot fault test was done as described
by Gibson et al. [17], with the following modifications:
Mice were placed on an elevated grid with 1.44 cm2 open-
ings and allowed to take 25 paired steps. Animals not
moving spontaneously for at least 25 steps were excluded.
The number of foot faults of the ipsilateral and contralat-
eral limbs was counted. Foot faults were given as the per-
centage of contralateral (left) limb foot fault errors of all
errors made. The grip test, also known as string test, was
adopted from Moran et al. [18], with modified scoring
system. For this test, the mouse was placed midway on a
string between two supports and rated as follows: 0, falls
off; 1, hangs onto string by one or both forepaws; 2, as for
1, and attempts to climb onto string; 3, hangs onto string
by one or both forepaws plus one or both hindpaws; 4,
hangs onto string by fore- and hindpaws plus tail
wrapped around string; 5, escape (to the supports).
Laser-Doppler flowmetry (Moor Instruments, Axmin-
ster, United Kingdom) was used to monitor cerebral
blood flow in 6 IL-18 +/+ and 5 IL-18 -/- animals
before surgery (baseline), immediately after tMCAO,
and 5 minutes after removal of the occluding monofila-
ment (reperfusion). For this, a flexible laser-Doppler
probe was positioned perpendicular to the exposed skull
2 mm posterior and 6 mm dexterolateral to the bregma,
corresponding to the laser-Doppler probe position in
murine MCA occlusion reported by Connolly et al. [19].
These animals were not included in the infarct size and
functional outcome evaluations, because laser-Doppler
flowmetric measurements in addition to tMCAO inevi-
tably leads to prolonged operation times.
To exclude anatomic differences that could cause dif-
ferent susceptibility to tMCAO in wild-type and knock-
out mice, we studied the cerebrovasculature in 5 IL-18
+/+ and 5 IL-18 -/- mice. Animals were deeply anesthe-
tized with CO2 and transcardially perfused with 4% paraf-
ormaldehyde and then with black ink (T25; Edding,
Ahrensburg, Germany). After brain removal and over-
night fixation in 4% paraformaldehyde, the circle of Willis
was visualized under a dissecting microscope. Special
attention was paid to the posterior communicating
arteries, whose level of plasticity was rated as described
by Murakami et al. [20]: 0, absent; 1, capillary anastomo-
sis; 2, small truncal vessel; 3, patent. A posterior commu-
nicating artery with a score of 0 or 1 is regarded as
hypoplastic, and with a score of 2 or 3 as normal.
Corrected infarct volumes, data from the functional
outcome tests and from the posterior communicating
artery score, and laser-Doppler flow measurements were
statistically analyzed in two-tailed Mann-Whitney U tests
using Prism 4 (GraphPad Software, San Diego, CA).
Results
Ink perfusion was performed in IL-18 wild-type and
knock-out mice to visualize the complete circle of
Willis. No gross anatomic differences were noted that
could influence stroke outcome (Figure 1). The score
assessing formation of the posterior communicating
arteries of both hemispheres, which are pivotal in collat-
eral blood flow between the anterior and posterior cir-
culation, did not differ significantly in wild-type and
knock-out mice (median of 2 in both groups; p = 0.91).
Moreover, Laser-Doppler flowmetry ensured technical
accuracy and similar basic characteristics in IL-18 +/+
and IL-18 -/- mice, since it did not show any significant
differences between wild-type and knock-out animals.
After right MCA occlusion, there was a similar substan-
tial reduction of right hemispheric cerebral blood flow
(median, 16.15% and 16.4%, respectively; p = 0.93). The
blood flow recovered to a median of approximately 50%
of baseline blood flow already within minutes after
removal of the occluding intraluminal monofilament
(median, 48.9% and 50.9%, respectively; p = 1.00).
We next assessed the influence of IL-18 on infarct size
and on functional outcome. There were no significant
differences in edema-corrected infarct size on standar-
dized TTC-stained brain slices between wild-type and
IL-18 knock-out mice after 24 hours (median, 79.25
mm3 and 86.1 mm3, respectively; interquartile range,
41.9 - 90.3 mm3 and 38.1 - 95.6 mm3, respectively; p =
0.51) or 48 hours (median, 93.05 mm3 and 85.2 mm3,
respectively; interquartile range, 75.3 - 104.6 mm3 and
73.9 - 98.7 mm3, respectively; p = 0.36). These results
are presented in Figure 2A. The functional outcome
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Figure 1 Ink perfusion. Images of a BALB/C wild-type mouse brain (A) and of a brain from an interleukin-18 knock-out mouse on a BALB/C
background (B) with ink-perfused cerebrovasculature.
Figure 2 Infarct volumes and functional outcome. Infarct volumes (A) and functional outcome scores (B: modified Bederson test score; C:
foot fault test; D: grip test score) of interleukin-18 wild-type and knock-out mice. The results are diagrammed as whisker boxes with medians.
Boxes represent interquartile ranges and whiskers indicate extreme values. The p values resulting from Mann-Whitney U tests are given; all p
values were greater than 0.05 and were thus considered insignificant. Abbreviations: wt, wild-type animals; ko, interleukin-18 knock-out animals;
n, number of animals; 0 h, 0 hours (postoperatively after recovery from anesthesia); 24 h and 48 h, 24 hours and 48 hours after 1-hour middle
cerebral artery occlusion.
Braeuninger et al. Experimental Translational Stroke Medicine 2010, 2:1
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scores were not significantly different in IL-18 +/+ as
compared to IL-18 -/- mice, too (Fig. 2B, C, D).
Discussion
As principal finding, we show that deficiency of IL-18
does not protect mice from ischemic brain damage after
tMCAO. These findings are surprising given the
reported upregulation of IL-18 blood levels in stroke
patients [8,9] associated with adverse clinical outcome
[9] and the profound impact of IL-18 in experimental
neonatal stroke [10,11]. However, two recent nested
case-control studies have not confirmed an association
of IL-18 with increased risk of stroke in older people
[21] or with recurrent stroke [22]. Moreover, our data
are in accordance with a previous study by Wheeler et
al. showing no differences in infarct size at 24 hours
between wild-type and IL-18 -/- mice on a C57BL/6
background subjected to 15 and 30 minutes of tMCAO
which leads to smaller infarcts than 1 hour occlusion
time [23]. We extend this previous study by applying a
longer tMCAO time (1 hour) leading to infarcts invol-
ving the entire MCA territory, by assessing functional
outcome and by following infarct development up to 48
hours. Recently, IL-18 expression and activation has
been described already at 24 hours in a thromboembolic
murine stroke model [24]. In contrast, in the rat, IL-18
mRNA expression was increased later at 48 hours, and
peaked between 7 and 14 days [25]. IL-18 has been loca-
lized to microglia/macrophages within ischemic lesions
[25]. The structurally similar interleukin-1b reached a
peak already within 16 hours and was rapidly downregu-
lated subsequently [25]. Thus, unlike interleukin-1b, IL-
18 seems to be associated with the mid-stage inflamma-
tory response to ischemic brain lesions. Accordingly,
similar findings were reported for traumatic brain injury
in mice: IL-18 was significantly elevated at 7 days, but
not within 4 hours to 24 hours, after experimental
closed head injury as compared to sham treatment [26].
Inhibition of IL-18 by IL-18-binding protein resulted in
improved neurological recovery by 7 days, while brain
edema at 24 hours was not reduced [26]. The cytokine
response in neonatal rodents subjected to hypoxic-
ischemic brain injury may differ. Here, a significant
mRNA elevation for IL-18 has been reported to occur
already at 3 hours, but also progressively increased until
day 14 [10]. In summary, our findings in adult IL-18
knock-out mice support the notion that IL-18 is not
functionally relevant for early stroke development, but
may play a role in late-stage neuroinflammation after
stroke which awaits further elucidation.
Acknowledgements
IL-18 knock-out mice and corresponding wild-type mice were kindly
provided by Prof. Liew, Glasgow, Scotland. We thank Gabi Koellner for
excellent technical assistance.
Authors’ contributions
SB and CK wrote the paper and conducted the experiments. GS designed
the study and reviewed the paper. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 15 October 2009
Accepted: 5 January 2010 Published: 5 January 2010
References
1. Stoll G, Jander S, Schroeter M: Inflammation and glial response in
ischemic brain lesions. Prog Neurobiol 1998, 56:149-171.
2. Stoll G, Jander S, Schroeter M: Detrimental and beneficial effects of injury-
induced inflammation and cytokine expression in the nervous system.
Adv Exp Med Biol 2002, 513:87-113.
3. Okamura H, Tsustsui H, Komatsu T, Yatsudo M, Hakura A, Tanimoto T,
Torogoe K, Okura T, Nukada Y, Hattori K, Akita K, Namba M, Tanebe F,
Konoshi K, Fukuda S, Kurimoto M: Cloning of a new cytokine that induces
IFN-g production by T cells. Nature 1995, 378:88-91.
4. Bazan JF, Timans JC, Kastelein RA: A newly defined interleukin-1?. Nature
1996, 379:591.
5. Reddy P: Interleukin-18: recent advances. Curr Opin Hematol 2004, 11:405-
410.
6. Conti B, Park LCH, Calingasan NY, Kim Y, Kim H, Bae Y, Gibson GE, Joh TH:
Cultures of astrocytes and microglia express interleukin 18. Mol Brain Res
1999, 67:46-52.
7. Prinz M, Hanisch U-K: Murine microglial cells produce and respond to
interleukin-18. J Neurochem 1999, 72:2215-2218.
8. Zaremba J, Losy J: Interleukin-18 in acute stroke patients. Neurol Sci 2003,
24:117-124.
9. Yuen C-M, Chiu C-A, Chang L-T, Liou C-W, Lu C-H, Youssef AA, Yip H-K:
Level and value of interleukin-18 after acute ischemic stroke. Circ J 2007,
71:1691-1696.
10. Hedtjärn M, Leverin A-L, Eriksson K, Blomgren K, Mallard C, Hagberg H:
Interleukin-18 involvement in hypoxic-ischemic brain injury. J Neurosci
2002, 22:5910-5919.
11. Hedtjärn M, Mallard C, Arvidsson P, Hagberg H: White matter injury in the
immature brain: role of interleukin-18. Neurosci Lett 2005, 373:16-20.
12. Wei X-Q, Leung BP, Niedbala W, Piedrafita D, Feng G-J, Sweet M, Dobbie L,
Smith AJH, Liew FY: Altered immune responses and susceptibility to
Leishmania major and Staphylococcus aureus infection in IL-18-deficient
mice. J Immunol 1999, 163:2821-2828.
13. Jiang H-R, Wei X-Q, Niedbala W, Lumsden L, Liew FY, Forrester JV: IL-18 not
required for IRBP peptide-induced EAU: studies in gene-deficient mice.
Invest Ophthalmol Vis Sci 2001, 42:177-182.
14. Clark WM, Lessov NS, Dixon MP, Eckenstein F: Monofilament intraluminal
middle cerebral artery occlusion in the mouse. Neurol Res 1997, 19:641-
648.
15. Ginsberg MD, Becker DA, Busto R, Belayev A, Zhang Y, Khoutorova L, Ley JJ,
Zhao W, Belayev L: Stilbazulenyl nitrone, a novel antioxidant, is highly
neuroprotective in focal ischemia. Ann Neurol 2003, 54:330-342.
16. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H: Rat
middle cerebral artery occlusion: evaluation of the model and
development of a neurologic examination. Stroke 1986, 17:472-476.
17. Gibson CL, Bath PMW, Murphy SP: G-CSF reduces infarct volume and
improves functional outcome after transient focal cerebral ischemia in
mice. J Cereb Blood Flow Metab 2005, 25:431-439.
18. Moran PM, Higgins LS, Cordell B, Moser PC: Age-related learning deficits
in transgenic mice expressing the 751-amino acid isoform of human b-
amyloid precursor protein. Proc Natl Acad Sci USA 1995, 92:5341-5345.
19. Connolly ES Jr, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ: Procedural
and strain-related variables significantly affect outcome in a murine
model of focal cerebral ischemia. Neurosurgery 1996, 38:523-532.
Braeuninger et al. Experimental Translational Stroke Medicine 2010, 2:1
http://www.etsmjournal.com/content/2/1/1
Page 4 of 5

20. Murakami K, Kondo T, Epstein CJ, Chan PH: Overexpression of CuZn-
superoxide dismutase reduces hippocampal injury after global ischemia
in transgenic mice. Stroke 1997, 28:1797-1804.
21. Stott DJ, Welsh P, Rumley A, Robertson M, Ford I, Sattar N, Westendorp RG,
Jukema JW, Cobbe SM, Lowe GD: Adipocytokines and risk of stroke in
older people: a nested case-control study. Int J Epidemiol 2009, 38:253-
261.
22. Welsh P, Lowe GDO, Chalmers J, Campbell DJ, Rumley A, Neal BC,
MacMahon SW, Woodward M: Associations of proinflammatory cytokines
with the risk of recurrent stroke. Stroke 2008, 39:2226-2230.
23. Wheeler RD, Boutin H, Touzani O, Luheshi GN, Takeda K, Rothwell NJ: No
role for interleukin-18 in acute murine stroke-induced brain injury. J
Cereb Blood Flow Metab 2003, 23:531-535.
24. Abulafia DP, de Rivero Vaccari JP, Lozano JD, Lotocki G, Keane RW,
Dietrich WD: Inhibition of the inflammasome complex reduces the
inflammatory response after thrombembolic stroke in mice. J Cereb
Blood Flow Metab 2009, 29:534-544.
25. Jander S, Schroeter M, Stoll G: Interleukin-18 expression after focal
ischemia of the rat brain: association with the late-stage inflammatory
response. J Cereb Blood Flow Metab 2002, 22:62-70.
26. Yatsiv I, Morganti-Kossmann MC, Perez D, Dinarello CA, Novick D,
Rubinstein M, Otto VI, Rancan M, Kossmann T, Redaelli CA, Trentz O,
Shohami E, Stahel PF: Elevated intracranial il-18 in humans and mice
after traumatic brain injury and evidence of neuroprotective effects of
il-18-binding protein after experimental closed head injury. J Cereb Blood
Flow Metab 2002, 22:971-978.
doi:10.1186/2040-7378-2-1
Cite this article as: Braeuninger et al.: Interleukin-18 does not influence
infarct volume or functional outcome in the early stage after transient
focal brain ischemia in mice. Experimental & Translational Stroke Medicine
2010 2:1.
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