ischemic limb preconditioning downregulates systemic inflammatory activation

Ischemic Limb Preconditioning Downregulates Systemic InflammatoryActivation

Andrea Szabo,1 Renata Varga,1 Margit Keresztes,2 Csaba Vızler,3 Istvan Nemeth,4 Zsolt Razga,4 Mihaly Boros1

1Institute of Surgical Research, University of Szeged, H-6720 Szeged, Pecsi u. 6, Hungary, 2Institute of Biochemistry, University of Szeged, Szeged,Hungary, 3Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary, 4Department of Pathology,University of Szeged, Szeged, Hungary

Received 4 September 2008; accepted 19 November 2008

Published online 22 December 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20829

ABSTRACT: We examined local and systemic antiinflammatory consequences of ischemic preconditioning (IPC) in a rat model of limbischemia-reperfusion (I-R) by characterizing the leukocyte-endothelial interactions in the periosteum and the expression of adhesionmolecules playing a role in leukocyte-mediated inflammatory processes. IPC induction (2 cycles of 10 min of complete limb ischemia and10 min of reperfusion) was followed by 60 min of ischemia/180 min of reperfusion or sham-operation. Data were compared with those onanimals subjected to I-R and sham-operation. Neutrophil leukocyte-endothelial cell interactions (intravital videomicroscopy), intravascularneutrophil activation (CD11b expression changes by flow cytometry), and soluble and tissue intercellular adhesion molecule-1 (ICAM-1;ELISA and immunohistochemistry, respectively) expressions were assessed. I-R induced enhanced leukocyte rolling and adherence in theperiosteal postcapillary venules after 120 and 180 min of reperfusion. This was associated with a significantly enhanced CD11b expression(by �80% and 72%, respectively) and moderately increased soluble and periosteal ICAM-1 expressions. IPC prevented the I-R–inducedincreases in leukocyte adherence and CD11b expression without influencing the soluble and tissue ICAM-1 levels. The results showthat limb IPC exerts not only local, but distant antiinflammatory effects through significant modulation of neutrophil recruitment. � 2008

Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:897–902, 2009

Keywords: periosteum; microcirculation; neutrophil leukocyte; CD11b; ICAM-1

Ischemic preconditioning (IPC) significantly increasesthe resistance to the harmful consequences of sub-sequent ischemia.1 Apart from the local effects, theprotection of remote tissues can also be achievedthrough brief periods of arterial occlusions of theintestine, kidneys, and many other organs.2,3 Althoughthe remote effects of intraabdominal IPC may benotable, the extremities provide better opportunitiesfor clinical purposes. IPC of the limbs in humans can beeasily performed, the risk of surgical complications islow, and moreover, the large tissue mass may theoret-ically provide strong defense-triggering IPC signals.Hence, we hypothesized that IPC is associated withpotential therapeutic benefits against the local anddistant effects of limb ischemia.

Transient limb ischemia occurs in orthopedic andtrauma cases, and during surgical interventions (e.g.,tourniquet method or flap-surgery). The local effectsmay be variable, but microvascular disturbances areamong the most frequent signs accompanying theischemic insult in the periosteum.4 Although severallines of evidence have suggested that IPC can effectivelyinfluence the perfusion of different organs,5,6 theperiosteal microcirculatory response in this conditionis as yet largely undefined. Accordingly, our primary aimwas to assess the degree of local microcirculatoryprotection that can be achieved by IPC in a rat modelof experimental limb ischemia-reperfusion (I-R) usingintravital fluorescence microscopic (IVM) examination ofthe periosteum. We additionally aimed to examine thepossibility of systemic inflammatory activation caused

by a local ischemic trigger, and the extent of IPC-inducedprotection in this scenario. The results demonstrate thatnoteworthy antiinflammatory protection can be achievedwith limb IPC, which is primarily due to reduced level ofpolymorphonuclear (PMN) leukocyte activation.

MATERIALS AND METHODSThe experiments were performed in accordance with the NIHGuidelines (Guide for the Care and Use of LaboratoryAnimals) and the study was approved by the Animal WelfareCommittee of the University of Szeged.

Surgical ProcedureIn sodium pentobarbital (45 mg kg�1 ip)-anesthetized, maleSprague-Dawley rats (average weight, 300� 20 g), the peri-osteum of the proximal part of the medial surface of the lefttibia was exposed by means of an atraumatic surgicaltechnique as detailed elsewhere.4

Experimental ProtocolThe experiments were performed in two series. The firstexperiments were used to determine the extent of I-R–inducedmicrocirculatory changes in the tibial periosteum, using IVM.As the fluorescence technique interferes with measurements ofCD11b expression changes, blood samples for the assessmentof adhesion molecule expression (sICAM-1 and CD11b, seelater) were taken in a second series with an identical protocol(see below).

In the first series, the first group (Sham) (n¼ 8) served assham-operated control; the microcirculatory variables wererecorded for 280 min to exclude changes relating solely to theanesthesia and surgery. In a second group (IPCþSham; n¼ 6),sham-operation was combined with IPC (2 cycles of 10 minof complete hindlimb ischemia and 10 min of reperfusion).The complete hindlimb ischemia was induced by placing atourniquet around the proximal femur, with simultaneousocclusion of the femoral artery with a miniclip. In groups 3 (I-R;n¼ 11) and 4 (IPC combined with I-R; n¼ 9), complete hindlimb

JOURNAL OF ORTHOPAEDIC RESEARCH JULY 2009 897

Correspondence to: Andrea Szabo (T: þ36 62 545103; F: þ36 62545743; E-mail: [email protected])

� 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

ischemia was induced for 60 min. The occlusions were thenreleased (time¼ 0 min), and the periosteal microcirculation wasobserved after reperfusion for 60, 120, or 180 min. At the end ofthe experiments, tissue biopsies from tibias together with thesurrounding muscles were taken for the immunohistochemicallocalization of ICAM-1 (see later).

In the second experimental series, identical experimentalgroups were used and blood samples were taken intoEDTA-coated tubes (BD Microtainer K2E, Plymouth,United Kingdom) at baseline, and at 120 and 180 min ofreperfusion, for sICAM-1 and CD11b measurements (n¼ 6–14;see later).

Intravital VideomicroscopyThe microcirculation of the distal tibia was visualized by IVM(Zeiss Axiotech Vario 100HD microscope, Jena, Germany),using fluorescein isothiocyanate (Sigma Chemicals, St. Louis,MO)-labeled erythrocytes7 (0.2 ml), and rhodamine-6G stain-ing (0.2%, 0.1 ml iv; Sigma) for the leukocytes. The IVM imageswere recorded with a charge-coupled device videocamera (TeliCS8320Bi, Toshiba Teli Corporation, Osaka, Japan) attachedto an S-VHS videorecorder (Panasonic AG-MD 830, Tokyo,Japan) and a personal computer.

Video AnalysisFrame-to-frame analysis of the videotaped images wasperformed using image analysis software (IVM, Pictron Ltd.,Budapest, Hungary). Leukocyte-endothelial cell interactionswere analyzed within five postcapillary venules (diametersbetween 11 and 20 mm) per animal. Adherent leukocytes(stickers): Cells that did not move or detach from theendothelial lining within an observation period of 30 s (numberof cells per mm2 of endothelial surface). Rolling leukocytes:Cells moving at a velocity less than 40% of that of theerythrocytes in the centerline (number of cells per second pervessel circumference).

Immune Labeling and Flow Cytometric Analysis forCD11b ExpressionThe surface expression of CD11b on peripheral blood granulo-cytes was determined by whole blood flow-cytometric analysis(a modification of a literature method8).

Assessment of sICAM-1 Levels by ELISAPlasma sICAM-1 levels were determined with the QuantikinesICAM-1-ELISA Kit (R&D Systems Inc., Minneapolis, MN),according to the manufacturer’s instructions.

ImmunohistochemistrySpecimen were decalcified with an electrophoretic apparatus(decalcifying solution: Sakura TDE30; Sakura Finetek Corp.,Torrance, CA), embedded in paraffin, and sections stainedwith mouse monoclonal anti-rat ICAM-1 antibody (BD Phar-mingen, BD Biosciences, San Jose, CA) as primary antibody(1:200; 30 min), followed by a biotinylated goat anti-mouseantibody conjugated to horseradish peroxidase polymer (Envi-sion1 system; Dako, Glostrup, Denmark) for 30 min, with 3,30-diaminobenzidine as chromogen. Semiquantitative analysiswas based on the calculation of percentage of ICAM-1-positivevessels in the periosteal and intramuscular vessels. Thefollowing semiquantitative categories were used (Scores 1–8):

Statistical AnalysisData in all figures are expressed as means� standard errorof the mean (SEM). Data analysis was performed with astatistical software package: SigmaStat version 2.03 (JandelCorporation, San Rafael, CA). Changes in variables withinand between groups were analyzed by one-way ANOVAfollowed by the Bonferroni test. p-Values < 0.05 were con-sidered statistically significant.

RESULTSWithin the macrohemodynamic variables (includingheart rate and mean arterial pressure), no significantdifferences between the different groups or in com-parison with the baseline were found in any phase of theexperimental period (data not shown).

In the Sham group, the numbers of rolling or adherentleukocytes (Fig. 1A, B) did not change significantlythroughout the experiments. As compared with thebaseline values, however, significantly increased num-bers of rolling leukocytes were observed throughout theentire reperfusion period, whereas increased adherentleukocyte counts were found at 120 and 180 min ofreperfusion in the I-R group. IPC alone did not induceleukocyte rolling or sticking. The I-R–induced elevationsin both parameters, however, were completely preventedby IPC during the whole of the reperfusion phase.

The activation of PMN leukocytes, as evidenced by thesurface expression of CD11b, did not change significantlyin response to the sham-operation (Fig. 2). Hence,we included only these time points in further evalua-tions. I-R caused �1.8-fold and 1.72-fold elevations inthis parameter (p< 0.05) at 120 min and 180 min ofreperfusion, respectively. IPC, however, significantlyprevented the I-R–induced increases of CD11b expres-sion by the end of reperfusion (an �1.2-fold changewas noted). In the event of IPC combined withsham-operation, IPC per se did not affect the CD11bexpression.

As assessed by immunohistochemical analysis in theperiosteum, however, tissue ICAM-1 density, exhibitedsignificantly higher values in the I-R group as comparedwith the Sham group, and IPC did not influence thisparameter (Figs. 3B and 4). There was only a moderateincrease in ICAM-1 positivity in the muscle tissue (from0.75�0.49 in the Sham group and to 1.57� 0.29 in theI-R group), and IPC did not appear to influence thesechanges in this structure either (data not shown).

Score % of Positive Vessels Staining

1 <5% Local2 Diffuse3 5%–25% Local4 Diffuse5 25%–50% Local6 Diffuse7 >50% Local8 Diffuse

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Figure 2. Changes in the expression of CD11b adhesion moleculeon the surface appearance of neutrophil leukocytes in response toischemia-reperfusion (I-R), sham-operation (Sham), or IPC (2 cycles of10 min of complete limb ischemia and 10 min of reperfusion), which wasfollowed by 60 min of ischemia/180 min of reperfusion (IPCþ I-R) orsham-operation (IPCþSham). Data are presented as means and SE.*p< 0.05 versus the baseline; xp<0.05 versus the Sham group;#p< 0.05 versus the I-R group. ANOVA followed by the Bonferroni test.

Figure 3. Changes in levels of soluble (A) and tissue (B) ICAM-1expression induced after ischemia-reperfusion (I-R), sham-oper-ation (Sham), or IPC (2 cycles of 10 min of complete limb ischemiaand 10 min of reperfusion), which was followed by 60 min ofischemia/180 min of reperfusion (IPCþ I-R) or sham-operation(IPCþSham). Data are presented as means and SE. xp< 0.05versus the Sham group. ANOVA followed by the Bonferroni test.

Figure 4. Representative longitudinal section of the rat tibiasurrounded by soft tissues (stained with ICAM-1 antibody plushematoxylin). (Upper panel) Tibial epiphysis (EP), cortical bone(CB), bone marrow (BM), knee-joint synovia (S), muscle (M), andtibial periosteum (P) are indicated. (Lower panel) Positive stainingfor ICAM-1 was found in the veins (V), but not in the arteries (A).A significantly higher positivity was found in response to I-R in theperiosteum (a) than in the muscle (c), whereas in the Sham groups,the staining was weak in the periosteum (b) and the muscle (d). Barrepresents 50 mm.

Figure 1. Primary (rolling, A) and secondary (sticking, B)leukocyte-endothelial cell interactions in postcapillary venulesof the tibial periosteum after ischemia-reperfusion (I-R), sham-operation (Sham), or IPC (2 cycles of 10 min of complete limbischemia and 10 min of reperfusion), which was followed by 60 minof ischemia/180 min of reperfusion (IPCþ I-R) or sham-operation(IPCþSham). Values are means and SE. *p<0.05 versus thebaseline; xp<0.05 versus the Sham group; #p< 0.05 versus the I-Rgroup. ANOVA followed by the Bonferroni test.

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DISCUSSIONThis study investigated the antiinflammatory conse-quences of IPC in the tibial periosteal microcirculation,and its effect on the expressions of different adhesionmolecules that are critically involved in PMN adherenceto the endothelium. Although complete vascular occlu-sion disturbs the perfusion of all tissues of the exposedlimb, microcirculatory deterioration predominated inthe periosteum, while the surrounding muscle layersexhibited much lower ischemic sensitivity.9 In thistissue compartment, the I-R–induced increase inleukocyte-mediated reactions in the reperfusion phasewas significantly ameliorated by IPC. Furthermore,systemic activation of the PMNs in response to the I-Rchallenge was similarly prevented by IPC.

In our model, the local injury was manifested insignificant increases in the primary and secondary formsof PMN-endothelial interactions (rolling and firm adher-ence) in the periosteal postcapillary venules duringreperfusion. As the second phase of adherence iscritically mediated by the integrins, we examined theeffects of IPC on the surface expression of the PMN-derived CD11b (the aM part of the CD11b-CD18complex), and its endothelial ligand, ICAM-1. Theresults show that some of the above changes weresignificantly ameliorated by IPC.

Increased CD11b expression seen in this study isregarded as a sign of systemic activation of the PMNleukocytes.10 Similar overexpression of CD11b on thePMNs was observed in humans after the release offorearm occlusion in the systemic circulation, and notin the veins draining the formerly ischemic tissues,suggesting an enhanced local microcirculatory seques-tration of the PMNs.11 From a clinical aspect, theseobservations suggest that tourniquet ischemia notonly induces local inflammatory reactions, but may alsoresult in the initiation of a systemic inflammatoryreaction. The IPC protocol here and in former studiesameliorated systemic PMN activation after a localpostischemic challenge.11 The background of this findingis incompletely understood, but it has been shown thatseveral intracellular and signal transduction pathways(e.g., NF-kB and TNF-a) are influenced by IPC.12

Similarly, it has been suggested that plasma factorsreleased from ischemic body compartments can induceCD11b activation in naive leukocytes.13 In this sense,the TNF-a released from an ischemic region can comprisea potential plasma factor which mediates remoteinjury.12 Indeed, the extent of TNF-a release is reducedin response to IPC in the heart.14 It is also noteworthy,that IPC attenuated NF-kB activation and subsequentlyreduced TNF-a expression, which resulted in the amelio-ration of microcirculatory disturbances and PMNsequestration in the I/R-injured muscle.15 Moreover,reduced PMN priming by IPC can explain some of thebeneficial remote preconditioning effects of limb IPC.As a result, ameliorated lung injury, together with areduced tissue accumulation of PMNs in the lung tissue,was demonstrated after limb ischemia.16

The endothelial ligand ICAM-1 is a counterpart ofCD11b as an inducible transmembrane protein of thePMN migration process. The level of its soluble form(sICAM-1) is proportional to the expression of ICAM-1 onthe cell membranes, and particularly on the endothelialcells,17 and this parameter can therefore be used toassess endothelial activation. Nevertheless, the resultwas unhelpful in that the plasma concentration of thisprotein displayed a very large dispersion.

Clinical studies have indicated rather controversialchanges in sICAM-1 expression. Specifically, not onlypronounced increases,18 but also decreases in sICAM-1expression were reported after human surgical proce-dures.19 In other studies, high sICAM-1 levels could betraced locally, whereas the systemic concentration evenchanged in the opposite direction.20 In our model, I-Raffected a considerable tissue mass and induced anendothelial dysfunction (as seen by PMN-endothelialinteractions) and systemic PMN activation. Hence, it isunlikely that the noxa would not cause injury to theaffected vasculature. Even though we could not observe asignificant increase in the soluble form of the ICAM-1, wecould trace a marked increase in the tissue form (shownby immunohistochemistry in the periosteum). It isnoteworthy, however, that the highest degree of positivestaining was found in the periosteal vessels, and not inthe muscle. This phenomenon can be explained by thegreater sensitivity of the periosteum than that of themuscle.9 It is reasonable to assume that the signal oflocally released ICAM-1 is blunted in the systemiccirculation. Local tissue expression data of ICAM-1clearly demonstrated that the IPC protocol applied inthis study did not influence this parameter.

We believe that our negative ICAM-1 data do notexclude that IPC preserved endothelial integrity. IPC hasbeen shown to alleviate direct endothelial cell injury,21 toexert nitric oxide-dependent microvascular protection,22

and to restore endothelium-dependent vasorelaxation inhumans.11 As I-R injury is a primarily intracellularsequence of oxido-reductive events, some of the protectiveeffects of IPC are related to the modulation of this processtargeting the endothelium directly or indirectly throughPMNs.23 As such, it has been demonstrated that theprotection provided by IPC is also mediated by oxidants,low concentrations of which are necessary for thisadaptation.24 The endothelium regulates this latterreaction, since it produces adhesion molecules whichenhance PMN attachment and consequent PMN-derivedoxidative damage. Although in vitro studies have pointedto reduced ICAM-1 signaling after IPC following anischemic trigger,25 and kidney IPC reduced the expres-sions of ICAM-1 and TNF-alpha and NF-kappaB/DNA-binding activity,26 we could not prove similar effectsin our model. As ICAM-1 expression can be initiated byproinflammatory cytokines and TNF-alpha,27 similarly towhat was seen with CD11b, reduced TNF release by IPCcan make an important contribution to this protection.Clarification of the details of the adhesion processrequires further in-depth investigations.

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Our study demonstrates that local ischemic toleranceinduction ameliorates the systemic consequences oftourniquet ischemia. The use of limbs is one of the mostpromising possibilities of evolving both pre- and post-conditioning ischemic tolerance.28,29 The major benefi-ciary of these approaches is the heart,29 but the kidney,30

distant muscle,31 liver,32 lung,15 or CNS33 can also betargeted. With respect to remote preconditioning, thepresent study has two messages. Firstly, even thoughvascular occlusion is associated with marked local andsystemic inflammatory reactions, brief, repeated periodsof limb ischemia did not have similar effects. Secondly,reduced PMN priming presented an important antiin-flammatory consequence of limb IPC both locally andduring the remote protection in this animal model.Nonetheless, other potential mechanisms of IPC canalso be involved in this protection.34

From a clinical aspect, these observations suggestthat periosteal microcirculation is particularly affectedby limb ischemia, and systemic inflammatory conse-quences of tourniquet ischemia have to be taken intoconsideration during surgical interventions. Limb IPCmay exert potential therapeutic benefit not only byproviding local protection for the periosteal microcircu-lation after tourniquet ischemia, but additionally byinfluencing potentially beneficial, remote processeslinked to PMN activation and modulation of inflamma-tory cell recruitment.

ACKNOWLEDGMENTSA. S. and R. V. contributed equally to the study. Part of thiswork was presented at a congress of the Society of EuropeanSurgical Research in Rotterdam, 2006. This study wassupported by a research grant from the Hungarian ScientificResearch Fund (OTKA K 60752).

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ischemic limb preconditioning downregulates systemic inflammatory activation

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