Reperfusion injury following cerebral ischemia : pathophysiology , MR imaging , and potential therapies Introduction Restoration of blood flow following ischemic stroke can be achieved by means of thrombolysis or mechanical recanalization . However , for some patients , reperfusion may exacerbate the injury initially caused by ischemia , producing a so-called `` cerebral reperfusion injury '' . Multiple pathological processes are involved in this injury , including leukocyte infiltration , platelet and complement activation , postischemic hyperperfusion , and breakdown of the blood -- brain barrier . Introduction In the treatment of acute stroke , restoration of the blood supply can reduce more extensive brain tissue injured by salvaging a reversibly damage penumbra of tissue -LSB- 1 -RSB- . This mechanism provides a rationale for clinical trials which have demonstrated that reperfusion after thrombolysis improves clinical outcome in selected patients with acute stroke -LSB- 2 -RSB- . Reperfusion , however , carries certain risks . Some patients experience disastrous outcomes in the form of fatal edema or intracranial hemorrhage following thrombolysis -LSB- 3 -RSB- . In some animal stroke models -LSB- 4 , 5 -RSB- , reperfusion after a long ischemic period can cause a larger infarct than that associated with permanent vessel occlusion . Thus , while reperfusion may reduce infarct size and improve clinical outcome in some patients , in others it may exacerbate the brain injury and produce a so-called `` cerebral reperfusion injury '' -LSB- 4 , 6 , 7 -RSB- . Cerebral reperfusion injury can be defined as a deterioration of ischemic but salvageable brain tissue after reperfusion . Thrombolysis -LSB- 2 -RSB- and embolectomy -LSB- 8 , 9 -RSB- usually result in reperfusion of the infarcted brain tissue and therefore carry the risk of causing reperfusion injury . Thus reperfusion injury deserves the attention of those interested in the diagnosis and treatment of acute stroke . Strategies to reduce or minimize cerebral reperfusion injury require the understanding of the pathophysiology of cerebral reperfusion injury , and the way the reperfusion injury is visualized by magnetic resonance imaging -LRB- MRI -RRB- . Therapeutic options for preventing or attenuating cerebral reperfusion need to be considered . Cerebral reperfusion injury : mechanisms and correlation with MRI Leukocyte infiltration and secondary ischemia as depicted with DW MRI Leukocytes play an important role in the development of cerebral reperfusion injury . During reperfusion , activated leukocytes interact with endothelial cells and plug capillaries , disrupt the blood-brain barrier -LRB- BBB -RRB- through the release of neutrophil-derived oxidants and proteolytic enzymes , extravasate from capillaries and infiltrate brain tissue , and release cytokines which mediate inflammation . These processes produce an inflammatory cascade , resulting in the deterioration of the salvageable penumbra -LSB- 7 -RSB- . Evidence of the deleterious effects caused by leukocytes is provided by animal studies . For example , Zhang et al. -LSB- 10 -RSB- , using a rat model of 2 hours transient focal cerebral ischemia induced by advancing a nylon monofilament to occlude the middle cerebral artery -LRB- MCA -RRB- , showed that neutrophils accumulate at the site of neuronal injury 6 hours after restoration of cerebral circulation . The neutrophil accumulation occurred earlier and to a greater extent in reperfusion tissue than in tissue permanently deprived of blood supply -LSB- 10 -RSB- . Furthermore , this study showed that the infarct volume increased dramatically between 6 and 24 hours following the start of reperfusion , and that the period of maximal infarct expansion correlated closely with the time course of neutrophil infiltration . The contribution of leukocytes to cerebral reperfusion injury is also supported by the beneficial effects of neutrophil depletion . Bednar et al. -LSB- 11 -RSB- investigated the effect of administering antineutrophil antiserum treatment on brain infarct size in a rabbit model of transient ischemia . The regional cerebral blood flow -LRB- CBF -RRB- of neutropenic rabbits recovered from less than 5 ml/100 g per minute to 20 -- 30 ml/100 g per minute following reperfusion , while in the non-neutropenic rabbits it remained at less than 10 ml/100 g per minute . Correspondingly , the infarct size was significantly smaller in the neutropenic animals . Similar results have been reported in rats treated with antineutrophil monoclonal antibodies -LSB- 12 -RSB- . The evidence from animal experiments points to the role of neutrophils in restricting CBF and increasing infarct size during reperfusion . High-quality evidence of reperfusion-induced secondary ischemic injury has been obtained using diffusion-weighted imaging -LRB- DWI -RRB- in animals . Furthermore , the evolution of reperfusion injury can be depicted by observing dynamic changes with DWI -LRB- Fig. 1 -RRB- -LSB- 13 -RSB- . Fig. 1DWI images of a rat model with 1 hour of MCA occlusion -LRB- MCAO -RRB- followed by 10 hours of reperfusion . These images show the evolution of ADC at different times : before ischemia -LRB- control -RRB- , at the end of MCAO , and at different time points of reperfusion . Note the transient recovery of ADC during the early phase of reperfusion , followed by secondary deterioration -LRB- reproduced with permission from Olah et al. -LSB- 13 -RSB- -RRB- Olah et al. -LSB- 13 -RSB- investigated the dynamics of the apparent diffusion coefficient -LRB- ADC -RRB- in a rat model with 1 hour of MCA occlusion -LRB- MCAO -RRB- followed by 10 hours of reperfusion . They found that the ADC was decreased at the end of ischemia , then significantly improved during the first 2 hours of reperfusion -LRB- Fig. 2 -RRB- , but decreased again at later time points with a secondary increase of hemisphere lesion volume . The ADC change most likely reflects a secondary ischemic injury . Although a potential role for apoptosis can not be excluded , the observed secondary deterioration of ADC did not support apoptosis as a major mechanism , because the ADC decline indicated cell swelling and not loss of cell volume , which is a fundamental and universal characteristic of programmed cell death -LSB- 14 -RSB- . Similarly , Neumann-Haefelin et al. , also using a rat MCAO model , showed that DWI abnormalities reversed transiently during the early reperfusion period but recurred after several hours of restored blood flow -LSB- 15 -RSB- . Likewise , Li et al. , using a rat model with 10 -- 30 minutes of temporary MCAO , observed that the DWI hyperintensity seen during the initial ischemic insult reverted to normal approximately 60 -- 90 minutes after the start of reperfusion , with secondary hyperintensities emerging 12 hours after reperfusion -LSB- 16 -RSB- . The secondary increase of DWI signal intensity observed in these studies is likely a marker of cerebral reperfusion injury -LSB- 15 -RSB- . Fig. 2Temporal evolution of mean relative ADC in the end-ischemic lesion volume in a rat model with 1 hour of MCA occlusion -LRB- MCAO -RRB- followed by 10 hours of reperfusion . Although a significant improvement in ADC attends reperfusion , there is a secondary deterioration likely as a consequence of the reperfusion-related injury -LRB- reproduced with permission from Olah et al. -LSB- 13 -RSB- -RRB- Several interesting characteristics of the ADC changes during reperfusion have been reported in animal studies . First , the time of onset of the secondary reduction of ADC varies amongst different animal studies , ranging from 2 -- 72 hours after reperfusion -LSB- 12 , 16 -- 18 -RSB- . The reason is probably due to the different durations of the initial ischemic insult and differences in the animal model used . Second , the secondary deterioration of ADC can be predicted by early changes of T2 during reperfusion . Olah et al. -LSB- 13 -RSB- , in their rat model study , observed that an increase in T2 value early in the ischemic period predicts a secondary deterioration in ADC after reperfusion . This suggests the possibility of using T2 value clinically to predict the extent of reperfusion injury likely to follow recanalization , and may be useful in assessing patients ' likelihood of benefiting from thrombolysis . Hemorrhagic transformation -LRB- HT -RRB- follows hyperperfusion and disruption of the BBB -LRB- see below -RRB- , but some of the imaging studies are reviewed in this section because there is a close relationship between the ADC changes and the subsequent HT after cerebral ischemia . In a rabbit model of thromboembolic stroke , Adami et al. -LSB- 19 -RSB- compared the percentage of pixels with very low ADC at various time points after the start of ischemia with the subsequent occurrence of HT -LRB- as determined on histopathological sections -RRB- . The authors found that , at each time point , regions populated by pixels with an ADC value below the threshold of 550 × 10 − 6 mm2/s were likely to undergo HT , and that the percentage of pixels below this threshold correlated with the severity of HT . This result has also been confirmed in several human studies . In a group of acute stroke patients treated with intravenous recombinant tissue plasminogen activator -LRB- rtPA -RRB- within 3 hours from symptom onset , Selim et al. -LSB- 20 -RSB- demonstrated that the absolute number of voxels with ADC values ≤ 550 × 10 − 6 mm2/s was much higher in patients with HT than in those without . The authors suggested that , although patients with both small and large DWI lesions have the tendency to develop HT , the volume of ischemic tissue with ADC ≤ 550 × 10 − 6 mm2/s may best predict the risk of intracerebral HT after thrombolysis . ADC values have also been reported to correlate with recanalization and clinical outcome in ischemic stroke patients undergoing local intra-arterial thrombolysis . Taleb et al. -LSB- 21 -RSB- identified two patient groups on angiographic findings : one group with more than 50 % recanalization and the other with less than 50 % . Relative ADC values were found to be higher in the first group of patients . Moreover , all patients in the recanalization group had a Barthel index of 80 or more , whereas eight of ten patients in the nonrecanalization group had a Barthel index of 50 or less . Molecular mechanisms of leukocyte infiltration The mechanisms of leukocyte infiltration are characterized by leukocyte activation , leukocyte -- endothelial interaction resulting in accumulation in the vascular bed , followed by leukocyte extravasation into the interstitial space . Each of these mechanisms is briefly reviewed . The accumulation of leukocytes , along with red blood cells and platelets during reperfusion , may plug capillaries , preventing the restoration of blood flow and resulting in a `` no-reflow '' phenomenon and secondary cerebral ischemia -LSB- 22 -- 27 -RSB- . Del Zoppo et al. -LSB- 27 -RSB- used a microscopic imaging system in a baboon model of transient ischemia . There was significant capillary obstruction caused by leukocytes reperfusing the microvascular bed within 60 minutes after the restoration of cerebral circulation . The `` no-reflow '' phenomenon explains well the better recovery of regional CBF in neutropenic rabbits -LSB- 11 -RSB- . The molecular mechanisms by which leukocytes infiltrate the parenchyma during reperfusion have been elucidated . This multistage process can be conceptually divided as follows : -LRB- 1 -RRB- leukocytes `` rolling '' on the endothelium , -LRB- 2 -RRB- firm adherence of leukocytes to the endothelium , and -LRB- 3 -RRB- transmigration into the tissue -LSB- 1 , 28 -- 31 -RSB- . Superoxide free radicals produced during ischemia and reperfusion cause the upregulation of endothelial P-selectin . Once leukocytes reach the ischemic territory the endothelial P-selectin interacts with its leukocyte counter-receptor P-selectin glycoprotein 1 -LSB- 31 -RSB- . This initial interaction facilitates the low affinity `` rolling '' of leukocytes on the endothelium . Firm adherence is developed with subsequent interaction of the leukocyte β2 integrins CD11a/CD18 and CD11b/CD18 with endothelial intercellular adhesion molecule 1 -LRB- ICAM-1 -RRB- . The transmigration of leukocytes results from the expression of platelet-endothelial cell adhesion molecule-1 -LRB- PECAM-1 -RRB- along the endothelial cell junction . Once the leukocytes infiltrate the parenchyma , they then release various chemical mediators including neutrophil elastase , reactive oxygen species -LRB- ROS -RRB- , leukotrienes , and prostaglandins , resulting in increased microvascular permeability , edema , thrombosis and parenchymal cell death -LSB- 1 , 27 , 32 -RSB- . This process compounds the initial injury that occurred in these tissues during the hypoxic insult . Platelet-mediated reperfusion injury Platelets play a synergistic role with leukocytes in reperfusion injury . Several studies suggest that platelets are activated after cerebral ischemia and reperfusion -LSB- 33 -- 35 -RSB- . Following activation , platelets may adhere to both leukocytes and cerebral microvascular endothelial cells , contributing to the `` no-reflow '' phenomenon and furthering tissue injury . Additionally , activated platelets release a variety of biochemical mediators of tissue injury . Several studies have confirmed that platelet activation occurs in patients with stroke . Zeller et al. -LSB- 36 -RSB- used flow cytometric detection of platelet adhesion molecules as a marker of platelet activation , and reported a substantially greater activation in acute stroke patients than in normal controls . Similarly , van Kooten et al. -LSB- 37 -RSB- , using urinary excretion of 11-dehydro-thromboxane-B2 as a marker of platelet activation , demonstrated a 347 % increase in the marker excretion in ischemic stroke patients compared with matched controls . Chong et al. -LSB- 38 -RSB- investigated changes in platelet -- leukocyte adhesion using a rat model of transient MCA ischemia . The authors reported that platelet -- neutrophil adhesion was significantly increased from 16.6 % in the control group to 39 % in the reperfusion group -LRB- P < 0.01 -RRB- . Platelets in the stroke group expressed high levels of P-selectin . It was hypothesized that enhancement of platelet -- leukocyte adhesion may be mediated by increased P-selectin expression on the platelet membrane . Studies on the role of platelets in coronary reperfusion of the heart suggest that , in addition to causing mechanical obstruction , platelets can cause temporary vasospasm by releasing serotonin , thromboxane A2 , and free radicals . This can cause diminished reperfusion of the ischemic territory -LSB- 33 -RSB- . Further , activated platelets can release mediators that may cause chemotaxis and migration of leukocytes , thus exacerbating the inflammatory cascade -LSB- 33 -RSB- . These mechanisms may also prove to be important in cerebral reperfusion injury after ischemic stroke . Complement-mediated reperfusion injury Experimental studies have shown that complement activation is an important component of reperfusion injury in several organ systems . Complement may be activated during reperfusion through the antibody-dependent classical pathway , the alternative pathway , or the MBL/MASP -LRB- mannan-binding lectin/mannan-binding lectin-associated serine proteases -RRB- pathway -LSB- 39 , 40 -RSB- . Complement activation results in the formation of several inflammatory mediators , including anaphylatoxins C3a , C5a , and the distal complement component C5b-9 -LRB- membrane attack complex , MAC -RRB- -LSB- 39 -RSB- . C5a and the MAC are the best-characterized effectors of tissue damage within the complement system -LSB- 41 , 42 -RSB- . C5a may stimulate leukocyte infiltration into the ischemia and reperfusion injury-affected tissue by its strong chemotaxis action . C5a may also further amplify the inflammatory response by inducing the release of several proinflammatory cytokines , including IL-1 , IL-6 , monocyte chemoattractant protein 1 -LRB- MCP-1 -RRB- and tumor necrosis factor α -LRB- TNF-α -RRB- . MAC can cause cellular membrane injury directly and can increase cell membrane permeability . Furthermore , MAC plays an essential role in mediating the recruitment of leukocytes to the reperfused tissue via local induction of IL-8 . Several agents that inhibit all or part of the complement components such as cobra venom factor -LRB- CVF -RRB- -LSB- 43 -RSB- , C5a monoclonal antibodies -LSB- 44 -RSB- , and Cl esterase inhibitor -LSB- 45 -RSB- , have been shown to significantly attenuate the degree of reperfusion injury in animal models , providing further evidence that complement plays an important role in reperfusion injury . Postischemic hyperperfusion and PWI Postischemic hyperperfusion is recognized as a frequent occurrence in stroke . Several animal studies suggest that the restoration of cerebral circulation consistently results in a hyperperfusion phase . Hyperperfusion may contribute to the development of reperfusion injury by causing brain edema or hemorrhage . Further , following postischemic hyperperfusion , a phase of secondary hypoperfusion can occur , which results in harmful effects on the reperfused tissue -LSB- 1 , 46 , 47 -RSB- . Heiss et al. -LSB- 46 -RSB- observed the effects of hyperperfusion in cats using a model of temporary MCA occlusion of varying durations -LRB- 30 , 60 , and 120 minutes -RRB- . In the 30-minute group , only a transient reactive hyperperfusion -LRB- compared with preocclusion values -RRB- was found . All animals in this group survived during the observation period , and no cerebral injury was found on histological study . In the 60 - and 120-minute groups , the degree of hyperperfusion was significant , reaching up to 300 % of preocclusion values . Most importantly , a clear difference was found between surviving and dying cats . In dying cats , postischemic hyperperfusion was long and severe , large infarcts developed and intracranial pressure increased fatally . In surviving cats postischemic hyperperfusion was transient and less severe , infarcts were smaller and intracranial pressure was significantly lower . A report of a similar study design suggested that the degree of hyperperfusion correlates significantly with occurrence of intracerebral petechial hemorrhages in the damaged cortex -LSB- 47 -RSB- . Perfusion-weighted imaging -LRB- PWI -RRB- provides quantitative and qualitative maps of CBF , cerebral blood volume -LRB- CBV -RRB- , and mean transit time -LRB- MTT -RRB- , allowing identification of regions of relative hypo - and hyperperfusion -LSB- 48 -RSB- . Kidwell et al. -LSB- 48 -RSB- used PW MRI to characterize hyperperfusion in 12 patients following intra-arterial thrombolysis . Hyperperfusion was visualized in 5 of 12 patients . On day 7 , 79 % of voxels with hyperperfusion demonstrated infarction , whereas only 36 % not showing hyperperfusion demonstrated infarction . Despite the voxel-by-voxel association of hyperperfusion with infarction , there were no significant differences in the degree of clinical improvement in patients with regions of hyperperfusion versus those without . It has been reported that reduction in volume of hypoperfused brain on PWI is a marker of response to treatment and improved function in ischemic stroke patients -LSB- 49 -RSB- . Larger future studies are necessary to assess the extent to which both hyperperfusion and hypoperfusion may result in unfavorable clinical outcome . Breakdown of the BBB and contrast-enhanced MRI Breakdown of the BBB during cerebral reperfusion may lead to the development of vasogenic edema , HT and infarction , all contributing to cerebral reperfusion injury . This hypothesis has been supported by several clinical and animal studies -LSB- 5 , 50 -- 52 -RSB- . Sage and Duffy -LSB- 50 -RSB- investigated the increase in BBB permeability after transient global cerebral ischemia in a rat model . Permeability of the BBB was increased at 15 minutes after reperfusion of the brain , and cerebral edema occurred 15 -- 30 minutes after reperfusion . Another study reported a disruption of the BBB after 3 hours of reperfusion , following a 3-hour temporary occlusion -LSB- 5 -RSB- . In contrast , the BBB remained intact after 6 hours of occlusion in a permanent occlusion group . Moreover , the temporary occlusion group had increased ipsilateral hemispheric water content , suggesting that cerebral edema had occurred . In addition to these reports , numerous histological studies with various tracers have established that reperfusion may exacerbate vasogenic edema due to BBB disruption -LSB- 6 , 53 , 54 -RSB- . T2-weighted imaging is useful for detecting vasogenic edema . Neumann-Haefelin et al. -LSB- 15 -RSB- used several MRI techniques to monitor the dynamics of secondary ischemic damage , BBB disturbances , and development of vasogenic edema during the reperfusion phase after focal cerebral ischemia in rats . After 2.5 hours of ischemia , signal intensity on both DWI and T2-weighted images increased rapidly in the previously ischemic region due to BBB disruption -LRB- enhancement on postcontrast T1-weighted images -RRB- and edema formation . More importantly , early BBB damage after reperfusion appeared to be predictive of relatively pronounced increases in T2 lesion size due to vasogenic edema at subacute time points -LRB- 1 to 2 days -RRB- . Using a novel MRI marker that can image BBB disruption , Latour et al. -LSB- 55 -RSB- studied a total of 144 acute stroke patients , to test the association between reperfusion , HT and clinical outcome . Nearly two-thirds -LRB- 63 % -RRB- of the patients had evidence of reperfusion within 1 week . BBB disruption was more common in patients who reperfused -LRB- 45 % -RRB- than in patients who did not reperfuse -LRB- 18 % -RRB- . Both HT and BBB disruption were more common in patients undergoing intra-arterial rtPA therapy -LRB- 31 % and 55 % -RRB- , than those not treated -LRB- 14 % and 25 % -RRB- . In the reperfused group , patients with BBB disruption were more likely to have a poor clinical outcome -LRB- 63 % -RRB- than those without disruption -LRB- 25 % -RRB- . Moreover , early BBB disruption was an independent predictor of HT . This was a key study because it associated early BBB disruption with HT and poor clinical outcome in humans . Contrast-enhanced T1-weighted MRI can detect disruption of the BBB during reperfusion . Under normal physiological conditions , contrast agent does not cross the intact BBB . Mechanisms that cause BBB disturbances , such as reperfusion injury , result in leakage of the contrast agent into the brain . In a recent study , rats were subjected either to permanent MCA occlusion , reperfusion after 1 hour -LRB- early reperfusion -RRB- , or reperfusion after 3 hours of MCAO -LRB- late reperfusion -RRB- -LSB- 56 -RSB- . This protocol allowed monitoring the evolution of BBB disruption during permanent or temporary MCAO . Contrast agent extravasation was found in all animals with permanent MCAO and late-reperfusion animals , but in only 40 % of early-reperfusion animals . Postischemic hyperperfusion was demonstrated in late-reperfusion animals , but not in early-reperfusion animals . Thus , using contrast-enhanced MRI , the disruption of the BBB was correlated with the hemodynamic and biophysical consequences of reperfusion . Fenstermacher et al. -LSB- 57 -RSB- employed the Patlak plot processing of gadolinium-shifted T1 relaxation-time images to obtain quantitative estimations of BBB opening in rats . Following 2.5 hours of reperfusion , the `` blood-tissue distribution '' of gadolinium-T1-shifted protons was serially imaged every 3 -- 4 minutes for 20 -- 30 minutes . After that , the value of blood-to-brain transfer constants -LRB- K1 -RRB- for gadolinium was calculated from the serial changes of the relaxation time via Patlak plots -LSB- 58 -RSB- of MRI-T1 datasets . The K1 for gadolinium in ischemia and reperfusion areas was around 200 times larger than that of normal areas , indicating a huge impairment of BBB function during ischemia and reperfusion . This approach may be utilized to monitor the time-course of BBB function in transient cerebral ischemia . The disruption of the BBB has also been observed in ischemic stroke patients undergoing rtPA therapy . A retrospective study of 213 patients used the postcontrast enhancement of the cerebrospinal fluid -LRB- CSF -RRB- in FLAIR images -LRB- termed hyperintense acute reperfusion marker , HARM -RRB- to characterize early BBB disruption -LSB- 59 -RSB- . BBB disruption was more common in patients with reperfusion -LRB- 45 % -RRB- than in patients without reperfusion -LRB- 18 % , P = 0.006 -RRB- . In multiple logistic regression , reperfusion was the strongest independent predictor of early BBB disruption -LRB- OR 4.09 , 95 % CI 1.28 to 13.1 , P = 0.018 -RRB- . HARM was associated with HT and worse clinical outcome . This is a key study for two reasons : first , the association between reperfusion , BBB disruption and HT was established in humans , and second , it may be possible to reduce the complications of rtPA by targeting adjunctive therapies against BBB disruption . In another recent study , three of the six patients receiving rtPA showed early parenchymal enhancement , and all three of them went on to develop HT , whereas none of the patients without early parenchymal enhancement developed hemorrhage -LSB- 60 -RSB- . The results suggest that early parenchymal enhancement may be significantly correlated with subsequent symptomatic HT and may be helpful in identifying patients at risk after thrombolytic therapy . Potential therapeutic strategies With the progress made in the understanding of the mechanisms in cerebral ischemia and reperfusion injury , an increasing number of strategies have been developed for limiting or preventing further brain damage during reperfusion -LSB- 61 -- 64 -RSB- . Many of these strategies interfere with leukocyte infiltration . Two previously described animal studies indicated that leukocyte depletion with either antineutrophil antiserum treatment or with antineutrophil monoclonal antibody leads to better recovery of regional CBF and significantly reduces infarct size after cerebral reperfusion -LSB- 11 , 12 -RSB- . Similarly , anti-ICAM-1 monoclonal antibodies reduce neutrophil infiltration and lesion size after reperfusion in rats , while they are ineffective in permanent ischemia -LSB- 65 -RSB- , suggesting that ICAM-1 may be an attractive therapeutic target that specifically addresses the problem of reperfusion injury . However , the translation of these promising antileukocyte strategies into effective therapies in humans has been disappointing . A randomized controlled trial -LRB- RCT -RRB- evaluated the use of enlimomab , a murine ICAM-1 antibody , in 625 ischemic stroke patients -LSB- 66 -RSB- . Patients were randomized to receive either enlimomab for 5 days or placebo within 6 hours after stroke onset . At 90 days , the modified Rankin scale score was worse in patients treated with enlimomab . Moreover , the hazard of death was 43 % higher on enlimomab than on placebo . There are several possible explanations for the failure of enlimomab to produce the desired effects . It is possible that the positive results seen with anti-ICAM therapy in animal studies do not reflect the immunological response in human stroke . Alternatively , ICAM-1 therapy may be effective only after reperfusion -LSB- 65 -RSB- , and it is believed that only a minority of patients experience complete reperfusion after ischemic stroke -LSB- 67 -RSB- . Thus , it may have been more appropriate to try enlimomab in patients who received thrombolytic therapy . Another RCT randomized 966 ischemic stroke patients presenting within 6 hours of acute stroke to either placebo or UK-279,276 , a CD11b/CD18 inhibitor . Of these patients , 204 also received tPA when indicated -LSB- 68 -RSB- . The trial was prematurely terminated when an interim analysis showed that the trial would be unable to demonstrate the benefit of this therapy in terms of improvement in Scandinavian Stroke Scale score at 90 days . Some conventional drugs have been shown in animal experiments to provide effective neuroprotection . For example , studies in a rat model have demonstrated that both dextran sulfate and unfractionated heparin can effectively prevent leukocyte infiltration and reduce infarct volume after reperfusion -LSB- 69 , 70 -RSB- . Although the actual mechanism is still unclear , these findings suggest a possible role for these agents alongside other therapeutic modalities that cause recanalization -LRB- i.e. intravenous/intra-arterial rtPA , mechanical thrombolectomy -RRB- -LSB- 69 , 70 -RSB- . Trials investigating this approach in humans have yet to be conducted . Besides these drug therapies , there are two management strategies , brain cooling and conditioned blood perfusion , which are reviewed in some detail because they can modulate several pathways/mechanisms that contribute to reperfusion injury . Both strategies are promising , and interventional neuroradiologists may have a role in administering these therapies in the future . Brain cooling Reports on the role of hypothermia in humans are encouraging . Two large RCTs established conclusively the neuroprotective effects and safety of systemic hypothermia after cardiac arrest -LSB- 71 , 72 -RSB- . A meta-analysis of the results -LSB- 73 -RSB- showed that patients in the hypothermia group were more likely to be discharged with no or only minimal neurological damage -LRB- risk ratio 1.68 ; 95 % CI 1.29 -- 2.07 -RRB- . This translated to six as the a number needed to treat in order to prevent one death or serious neurological deficit . There are no hypothermia efficacy studies in ischemic stroke patients . Several animal studies of temporary MCAO have shown that systemic hypothermia applied up to 3 hours after reperfusion has started is effective in reducing infarct volume -LRB- by 50 -- 90 % -RRB- and reducing animal mortality -LSB- 74 , 75 -RSB- . Moreover , hypothermia exerts its neuroprotective effects partly by suppressing the BBB opening -LSB- 76 , 77 -RSB- and by attenuating neutrophil infiltration in the ischemic penumbra -LSB- 78 , 79 -RSB- . Hence , hypothermia exerts at least some of its neuroprotective effects by modulating mechanisms responsible for reperfusion injury . In most clinical studies , hypothermia is induced by surface cooling with the use of cooling blankets , alcohol applied to exposed skin , or ice bags to groin , axilla and neck . Although whole-body surface cooling is the simplest and most cost-effective option for inducing hypothermia -LSB- 80 -RSB- , it has two major drawbacks . First , it takes several hours to reach the target body core temperature . All studies report a 3 - to 7-hour time period for cooling down to 32 -- 34 °C -LSB- 81 -- 84 -RSB- . Target temperature should be reached as soon as possible to prevent reperfusion injury ; the 3 -- 7 hour time period to reach target temperature is too long and the therapeutic window will be missed for many patients . Theoretical models predict that intracerebral hypothermia can be achieved up to 30 times faster than whole-body hypothermia -LSB- 85 -RSB- . The second drawback of whole-body cooling is the high incidence of complications such as pneumonia , arrhythmias/bradycardias , hypotension and coagulopathies -LSB- 81 -- 83 , 86 , 87 -RSB- . Selective brain cooling may dramatically improve the safety profile of therapeutic hypothermia . Selective brain cooling can be achieved by means of an endovascular catheter that infuses the cerebral vasculature with cold saline . A recent study examined whether cold saline -LRB- 20 °C -RRB- infused intra-arterially into the ischemic territory of rats that had undergone MCA occlusion could selectively induce cerebral hypothermia and reduce brain injury from stroke -LSB- 88 -RSB- . Following cold saline infusion , cerebral temperature fell within 1 min to 33.4 °C , while rectal temperature was maintained above 36 °C at all times . A significantly reduced infarct volume was found in ischemic rats that received a local cooling infusion compared with control animals -LRB- Fig. 3 -RRB- . Further clinical research is needed to explore the therapeutic potential of selective brain cooling and the yet-unknown adverse effects , as well as to establish the extent to which selective brain cooling specifically modulates reperfusion injury . Fig. 3Graph showing percentages of infarct volume in the four ischemic rat groups -LRB- stroke , stroke with local infusion of saline at 20 °C and 37 °C , and stroke with systemic infusion at 20 °C -RRB- . A significantly -LRB- P < 0.001 -RRB- reduced infarct volume was found in ischemic rats that received a local cooling infusion compared with the rats in the other three groups -LRB- reproduced with permission from Ding et al. -LSB- 88 -RSB- -RRB- Conditioned blood reperfusion Conditioned blood reperfusion is a therapeutic strategy in which the ischemic organ is perfused with autologous conditioned blood at the onset of reperfusion . Blood conditioned by filtration is depleted of leukocytes and platelets , both at levels far greater than 99.99 % , and the complement cascade is modulated in such a way as to prevent the deposition of MAC onto ischemic tissue -LSB- 89 -RSB- . Filtering the reperfused blood in this manner may reduce further tissue damage after restoration of circulation -LSB- 89 , 90 -RSB- . Blood can be filtered using the conditioned blood reperfusion application -LRB- CoBRA -RRB- filter -LRB- Pall B-1328 filter ; Pall Corporation , East Hill , N.Y. -RRB- at the onset of reperfusion -LSB- 89 -RSB- . Withdrawn arterial blood can be filtered through the CoBRA filter and returned back to the cerebral circulation via an endovascular catheter . A small feasibility and safety clinical trial of blood filtering during primary percutaneous transluminal coronary angioplasty for acute myocardial infarction demonstrated that integration of the CoBRA filter into an active hemoperfusion system did not delay the procedure or cause any complications -LSB- 89 -RSB- . Furthermore , conditioned blood reperfusion resulted in an acute improvement in ventricular function . Animal models of hypothermic circulatory arrest have demonstrated improvement of neurological outcomes following conditioned blood reperfusion -LSB- 91 -- 93 -RSB- . In one of these studies , pigs received blood filtered using the CoBRA filter -LSB- 92 -RSB- . Each pig received a common carotid artery filtered perfusion for 10 minutes . Pigs receiving conditioned perfusion had significantly better neurological scores than control pigs -LRB- P < 0.005 -RRB- . These findings suggest that multifocal intervention aimed at leukocytes , platelets and complement attenuation can dramatically improve neurological recovery from ischemic insult . Future studies will establish the efficacy of this strategy and the incidence of possible adverse effects , such as coagulapathies and infections . Further studies are needed to evaluate the role of local hypothermia and conditioned blood perfusion in reducing postischemic reperfusion injury . It is possible , yet still speculative , that in the future interventional neuroradiologists may use these approaches following recanalization with thrombolytic agents or mechanical embolectomy to lessen reperfusion injury .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O

magnetic resonance imaging
cerebral ischemia
reperfusion injury
hypothermia
conditioned blood reperfusion


Contribution of P2Y1 receptors to ADP signalling in mouse spinal cord cultures Mixed neuronal and glial cell spinal cord cultures from neonates express ADP sensitive P2Y1 ,12 & 13 receptors . ADP -LRB- 10 μM -RRB- evoked increases in intracellular calcium that were essentially abolished by the P2Y1 receptor antagonist MRS2179 -LRB- 10 μM -RRB- , responses were also absent in preparations from P2Y1 receptor deficient mice however UTP -LRB- 100 μM -RRB- evoked calcium rises were unaffected . ADP also evoked a robust increase in extracellular signal-regulated protein kinase -LRB- ERK -RRB- phosphorylation that was of similar magnitude in the cultures from wild type and P2Y1 receptor deficient mice . These results suggest that ADP acts through P2Y1 receptors to mediate an increase in intracellular calcium but not to stimulate ERK phosphorylation in the spinal cord . Nucleotides released from neurons , glia and damaged cells play a signalling role in the nervous system . P2Y receptors are a family of G-protein coupled nucleotide receptors , and several of the eight subtypes -LRB- P2Y1 ,2,4,6,11,12,13,14 -RRB- are expressed in the nervous system -LSB- 11 -RSB- . P2Y1 receptors have been shown to be involved in calcium signalling in a range of neurons and glial cells -LRB- e.g. -LSB- 3,4,9,12 -RSB- -RRB- . In addition signalling through recombinant P2Y1 receptors stimulates mitogen-activated -LRB- MAP -RRB- kinases -LSB- 10 -RSB- and in native cells P2Y1 receptors have been implicated to play a role in extracellular signal related kinases -LRB- ERK -RRB- activation and stretch induced injury in astrocytes -LSB- 8 -RSB- . In this study the aim was to determine whether ADP evoked calcium and MAP kinase responses in cultured spinal cord neurons and glial cells and use knockout mice to determine the contribution of P2Y1 receptors to ADP evoked signalling . Wild type or P2Y1 receptor deficient C57 neonatal mice -LSB- 7 -RSB- -LRB- 3 -- 7 days -RRB- of either sex were decapitated and the spinal column was removed and washed twice in a calcium/magnesium-free Hanks balanced salt solution -LRB- HBSS -RRB- containing HEPES -LRB- 0.3 M -RRB- , sodium pyruvate and penicillin/streptomycin -LRB- 50 μg/ml each -RRB- . The spinal cord was then cut into small pieces and placed in ice cold HBSS . The tissue pieces were then placed in a HBSS-trypsin solution -LRB- 2.5 % -RRB- and incubated at 37 °C for 30 min . The tissue was then transferred to a HBSS-trypsin inhibitor solution -LRB- 1 mg/ml -RRB- for 5 min before being replaced with 1 ml Neurobasal media containing glutamine and B27 supplement . Mechanical trituration was used to dissociate the spinal cord cells . The resulting cell suspension was seeded onto poly-d-lysine-coated 16 mm coverslips and maintained at 37 °C in a humidified incubator with a 95 % O2/5 % CO2 atmosphere . After 2 h an additional 2 ml of Neurobasal media was added and cells left to grow for 2 -- 5 days . Spinal cord cultures were ester loaded with a calcium sensitive dye , Fluo-3 acetoxymethyl ester -LRB- Fluo-3-AM , final concentration 1 μM -RRB- for 30 min at 37 °C in a humidified incubator . The coverslips were then washed in extracellular solution -LRB- in mM ; 150 NaCl , 10 HEPES , 10 Glucose , 2.5 KCl , 2.5 CaCl2 , 1 MgCl2 , pH 7.3 -RRB- to remove any extracellular dye and maintained at room temperature . Coverslips were mounted in a perfusion chamber on the stage of a Fluoview FV300 confocal microscope and were continuously perfused with extracellular solution . The argon laser excitation of the dye was at 488 nm and the emissions were captured at wavelengths greater than 510 nm by Olympus Fluoview v4 .2 software at a frequency of 0.5 Hz for a continuous time period of 280 s. Agonist application was via a U-tube -LSB- 2 -RSB- . ADP stocks were treated with hexokinase to remove any ATP contamination . Agonist applications were at intervals of ∼ 10 min . Antagonists were perfused for 8 min before test solution application so as to allow complete equilibration . Cells displaying ` puff ' artefacts to just extracellular solution were discounted from analysis -LSB- 1 -RSB- . Baseline measurements of fluorescence were calculated as a mean of the 10 s of recording prior to agonist application . Peak changes in fluorescence for neuronal and glial cells were expressed as a ratio of the baseline -LRB- increases in fluorescence are expressed as a self-ratio , baseline value is 1 -RRB- . Spinal cord cells were cultured for 2 -- 5 days as described previously and agonists were applied to individual wells and incubated for 10 min . The cells were subsequently lysed in a solution -LRB- in mM ; 10 β-glycerophosphate , 1 EDTA , 1 EGTA , 50 Tris -- HCl , 1 benzamidine , 1 sodium orthovanadate , 0.2 PMSF , 50 sodium fluoride , 1 mg/ml pepstatin A , 1 mg/ml leupeptin , 0.1 % β-mercaptoethanol , 1 % Triton X-100 -RRB- . The samples were then mixed with sodium dodecyl sulphate sample buffer -LRB- containining β-mercaptoethanol -RRB- at a ratio of 1:1 and denatured for 5 min at 95 °C prior to loading on a 10 % sodium dodecyl sulphate polyacrylamide gel . The protein was then transferred onto a nitrocellulose membrane and subsequently blocked with blocking medium -LRB- 5 % non-fat milk powder and Tris-Tween 20 , buffered salts-TTBS -RRB- overnight at 4 °C . Nitrocellulose membranes were then incubated in 5 % blocking medium containing either anti-phospho-ERK antibody -LRB- Cell Signaling Technology , MA , USA -RRB- at a dilution of 1:1000 or anti-non-phosphorylated ERK antibody -LRB- Cell Signaling Technology , MA , USA -RRB- at a dilution 1:1000 . Immunoreactive bands were visualised by incubation of nitrocellulose membranes with anti rabbit peroxidase conjugate at 1:1500 for 2 h at room temperature and then detected with chemiluminiscence -LRB- ECL Plus , Amersham , UK -RRB- and recorded onto hyperfilm -LRB- Amersham , UK -RRB- . Western blots were analysed using Image J , NIH , USA -- http://rsb.info.nih.gov . Phospho ERK intensity was corrected for loading using non-phosphorylated ERK blotting and expressed as fold over basal . Total RNA was isolated from either intact spinal cord or spinal cord using the RNeasy Mini Kit -LRB- QIAGEN , Valencia , CA -RRB- . The RNA was treated with DNase I -LRB- Sigma -RRB- to remove any DNA contamination . Half of the purified RNA was reverse-transcribed to cDNA using SuperScript II-reverse transcriptase -LRB- Invitrogen , Carlsbad , CA -RRB- , and the remainder of the RNA was used as a negative control to ensure that there was no genomic contamination . The primers have been described previously -LSB- 1 -RSB- . PCR reactions were carried out with 2 μl of cDNA and a final concentration of 500 nM of each primer . Amplification took place in a Techne Genius PCR machine -LRB- Techne , Cambridge , UK -RRB- using the following protocol : denaturation at 94 °C for 5 min ; repeated cycles of denaturation at 94 °C , followed by annealing at 57 °C and extension at 72 °C , each for 30 s ; final extension at 72 °C for 10 min . Thirty-five cycles were used . The amplification products were examined by electrophoresis on a 1 % agarose gel and visualized using ethidium bromide under UV light . Statistical tests were preformed using an unpaired t-test with a p-value of < 0.05 considered as significant . Data are expressed as mean ± standard error of the mean . Spinal cord cells cultured from neonates for 3 -- 7 days contained both neuronal and glial cells that could be distinguished by their morphology and staining with glial fibrillary acidic protein ; approx 43 % of cells were glia and 57 % neuronal -LRB- n = 576 cells from 4 coverslips -RRB- . We have previously used calcium imaging to look at P2Y receptor signalling in mouse superior cervical ganglia -LSB- 1 -RSB- and similar protocols were used in this study . ADP -LRB- 10 μM -RRB- evoked robust increases in intracellular calcium for both neurons -LRB- 64 % cells responding with peak self-ratio amplitude of 2.20 ± 0.14 , n = 120 cells from 12 coverslips -RRB- and glial cells -LRB- 57 % of cells responded with a peak self-ratio amplitude of 2.17 ± 0.2 , n = 120 cells from 12 coverslips -RRB- . Responses were not sustained and generally decayed during the time-course of application -LRB- self-ratio at end of 30 s application 1.34 ± 0.08 and 1.27 ± 0.04 for neurons and glia , respectively , n = 20 for each -RRB- . The selective P2Y1 receptor antagonist MRS2179 reduced ADP evoked calcium rises in a concentration-dependent manner and these effects were reversed on antagonist washout -LRB- Fig. 1 -RRB- . ADP evoked calcium rises were essentially abolished in spinal cord cultures from P2Y1 receptor deficient mice with small residual responses in 11 % of neurons -LRB- self-ratio 1.14 ± 0.05 , n = 100 cells from 10 coverslips -RRB- and 17 % of glial cells -LRB- self-ratio 1.21 ± 0.05 , n = 115 cells from 11 coverslips -RRB- -LRB- Fig. 2 -RRB- . Responses to the P2Y2/4 receptor agonist UTP -LRB- 100 μM -RRB- in cultures from P2Y1 receptor deficient mice were the same as for WT for neurons -LRB- 45 and 48 % responding with amplitudes of 1.96 ± 0.09 and 1.79 ± 0.08 , n = 60 , 114 from 6 and 12 coverslips , respectively for WT and P2Y1 receptor deficient mice -RRB- and glial cells -LRB- 58 and 62 % responding with amplitudes of 1.88 ± 0.13 and 1.67 ± 0.04 , n = 60 , 119 from 6 to 12 coverslips , respectively for WT and P2Y1 receptor deficient mice -RRB- indicating that Gαq coupled receptor calcium signalling was not affected in the P2Y1 receptor deficient mouse -LRB- Fig. 2 -RRB- . Taken together these results define a role of the P2Y1 receptor in calcium signalling in spinal cord neurons and glial cells . These studies support the role of P2Y1 receptors in calcium signalling in the nervous system -LSB- 3,9,12 -RSB- and corroborate previous studies using sympathetic ganglion cultures from the P2Y1 receptor deficient mouse -LSB- 1 -RSB- . P2Y1 receptor stimulation has also been reported to mediate ERK phosphorylation in recombinant -LSB- 10 -RSB- and native systems -LSB- 8 -RSB- and this may contribute to changes in the spinal cord following injury . In spinal cord cultures ADP evoked a time-dependent phosphorylation of p44/42 ERK that was detected at 5 min , peaked at 10 min , and declined back to basal levels by 30 min -LRB- Fig. 3A -RRB- . When just media was added to the cultures there was no change in ERK phosphorylation -LRB- Fig. 3B -RRB- indicating that ERK stimulation does not result from a mechanical artefact . Adenosine -LRB- 10 μM -RRB- also had no effect demonstrating that adenosine receptors are not involved . We therefore compared ADP evoked ERK phosphorylation in spinal cord cultures from WT and P2Y1 receptor deficient mice . Surprisingly ADP -LRB- 10 μM -RRB- evoked similar levels of ERK phosphorylation -LRB- ∼ 3-fold increase over basal -RRB- in WT and P2Y1 receptor deficient cultures -LRB- Fig. 3 -RRB- demonstrating that a receptor other than the P2Y1 receptor mediates this ADP response . Of the currently eight identified P2Y receptor genes ADP is an agonist at Gαq coupled P2Y1 , and Gαi coupled P2Y12 and P2Y13 receptors -LSB- 11 -RSB- . ADP is also an agonist at the human P2Y11 receptor ; however a corresponding mouse homologue of the P2Y11 receptor has not been detected suggesting that mice do not express P2Y11-like receptors -LSB- 11 -RSB- . Transcripts for P2Y1 ,12 & 13receptors were detected in 3 -- 7 days old spinal cord cultures -LRB- data not shown -RRB- . Amplification of RNA for ADP sensitive P2Y12 & 13 receptors raised the possibility that these may contribute to the ERK phosphorylation . AR-C66931MX -LSB- 6 -RSB- is an antagonist of P2Y12 and P2Y13 receptors with a reported pA2 of 8.7 at P2Y12 receptors and 0.01 μM reduced ADP responses by ∼ 80 % at recombinant P2Y13 receptors -LRB- see review -LSB- 11 -RSB- -RRB- . AR-C66931MX -LRB- 10 μM a supra-maximal concentration at P2Y12 receptors -RRB- had no effect on ADP evoked ERK phosphorylation indicating that P2Y12 receptors did not mediate the ERK phosphorylation . AR-C66931MX may have complex actions at P2Y13 receptors , possibly dependent on cell type , and has been described as an activator of P2Y13 receptor dependent high density lipoprotein endocytosis in hepatocytes -LSB- 5 -RSB- questioning the use of this compound as a selective antagonist at the P2Y13 receptor . Thus it remains to be determined whether the P2Y13 receptor , or a novel ADP sensitive receptor , mediates the ADP evoked ERK phosphorylation in the spinal cord .

O O B O O B O O O B I O O O O O O B I O O O O B O B O O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O B O O B O O O O O O O B O O O O O B O O B I O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B I B O O O O O O O O O O O O B O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O B O O O O B O B O O O O O O O O O O O O O O O O O O B O B O O O O O B I O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O B O O B O O B I B O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O B I O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O B I O O O O B O O O O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O B O O O O O O O O O O B O O O O O O B O O O O B O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B O O B O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O B O O O O O B O O B O O B I O

p2y1
p2y12
p2y13
spinal cord
calcium
phosphorylation
neurons
glia
adp


Polymorphisms of Human Leukocyte Antigen Genes in Korean Children with Kawasaki Disease Background Kawasaki disease is a leading cause of acquired heart disease in children . The prevalence rate varies in different ethnic groups . Recently , with the clinical application of molecular genetic technology , human leukocyte antigen -LRB- HLA -RRB- polymorphisms associated with several diseases have been identified by DNA analysis . This study aimed to assess the association of HLA alleles with susceptibility and complications of Kawasaki disease in Korean children . Kawasaki disease is a systemic vasculitis that currently is a leading cause of acquired heart disease in children . The etiology of Kawasaki disease continues to be unknown , although it was described 4 decades ago in 1967 by Kawasaki -LSB- 15 -RSB- . Diagnosis continues to depend primarily on clinical manifestations . It appears that the pathogenesis of Kawasaki disease is related to infection of a susceptible host coupled with epidemiologic and ethnic factors . In a study examining the epidemiologic characteristics of Kawasaki disease in San Diego , CA , USA , Asians/Pacific Islanders were 2.7 times more likely and Hispanics one-third as likely to be hospitalized for Kawasaki disease compared with the children of other ethnic groups combined -LSB- 4 -RSB- . The sibling risk ratio is also known to be higher , than the risk of being affected with Kawasaki disease without sibling history , and cases of Kawasaki disease in parents and children have been reported -LSB- 10 , 21 -RSB- . For these reasons , many reports have suggested involvement of the human leukocyte antigen -LRB- HLA -RRB- system . The results of such studies vary depending on the ethnic group studied . Several reports show the association of HLA genotypes with Kawasaki disease including HLA-Bw22 -LRB- now referred to as HLA-B54 -RRB- in Japanese -LSB- 14 , 20 -RSB- , HLA-B51 in white populations -LSB- 13 , 16 , 18 , 19 -RSB- , and the major histocompatibility complex class I chain-related gene A -LRB- MICA -RRB- genes and others in southern Chinese population -LSB- 6 , 12 -RSB- . The major histocompatibility complex -LRB- MHC -RRB- class II genes in Kawasaki disease show variable correlations -LSB- 2 , 8 , 27 -RSB- . To date , there have been no reports supporting a consistent relationship between Kawasaki disease and HLA genes in Korean patients . Therefore , the current study aimed to assess the association of HLA-A , - B,-C , and - DRB1 alleles with Kawasaki disease in Korean children . Materials and Methods Subjects From March 2004 to December 2004 , 74 patients with Kawasaki disease -LRB- 44 boys and 30 girls -RRB- attending the pediatric outpatient clinic or admitted to St. Vincent 's Hospital in Korea were recruited for this study . The mean age of the patients at diagnosis was 2.7 ± 1.9 years . For comparison , genetically unrelated healthy Korean adults -LRB- n = 159 -RRB- who had no history of Kawasaki disease were studied as a healthy control group . Blood samples were obtained after informed consent in compliance with the institutional review board of St. Vincent 's hospital , College of Medicine , The Catholic University of Korea . The diagnosis of Kawasaki disease was established by clinical manifestations meeting the defined criteria according to the report of the Research Committee on Kawasaki Disease of the Japanese Ministry of Health and Welfare in addition to exclusion of any other possible illnesses -LSB- 25 -RSB- . Coronary complications included the following lesions detected by echocardiography : an internal diameter exceeding 3 mm in patients younger than 5 years and exceeding 4 mm in patients age 5 years or older , dilation of a segmental luminal diameter to at least 1.5 times that of an adjacent segment , or a definitely irregular internal lumen of the coronary arteries . The clinical characteristics of the patients are shown in Table 1 . Table 1Profiles of subjects with Kawasaki diseasen -LRB- % -RRB- Male/female44/30 -LRB- 60/40 -RRB- Typical KD/atypical KD65/9 -LRB- 88/12 -RRB- Family history of KD3 -LRB- 4.1 -RRB- Recurrence history of KD4 -LRB- 5.4 -RRB- Coronary complications21 -LRB- 28 -RRB- Other complications of KDGallbladder hydrops14 -LRB- 19 -RRB- Pyuria25 -LRB- 34 -RRB- Hepatopathy38 -LRB- 51 -RRB- Arthritis10 -LRB- 14 -RRB- KD , Kawasaki disease Genomic DNA was extracted by standard methods using the AccuPrep DNA extraction kit -LRB- Bioneer , Daejeon , Korea -RRB- from peripheral blood collected -LRB- 4 ml -RRB- with ethylenediaminetetraacetic acid -LRB- EDTA -RRB- and kept at − 20 °C . HLA-A , - B , and - C Genotyping The genotyping of HLA-A , - B , and - C was performed by the amplification refractory mutation system -LRB- ARMS -RRB- -- polymerase chain reaction -LRB- PCR -RRB- method . Each reaction contained a primer mix consisting of the allele - or group-specific primer pairs as well as internal control primers matching nonallelic sequences . Specific amplifications of the HLA-A , - B , and - C genes were performed using forward and reverse primers -LRB- 44 for HLA-A , 47 for HLA-B , and 33 for HLA-C -RRB- designed according to the published nucleotide sequences -LSB- 5 , 17 , 26 -RSB- . The PCR procedure was carried out in a reaction -LRB- 13 μl -RRB- , containing 100 to 200 ng genomic DNA , 0.8 X buffer -LRB- 40 mmol/l KCl , 1.2 mmol/l MgCl2 , 8 mmol/l Tris-HCl pH 8.8 , 0.08 % Triton X-100 -RRB- , 5 % dimethylsulphoxide -LRB- DMSO -RRB- , 200 μmol/l of each dNTP , 0.25 U Taq DNA polymerase -LRB- Boehringer , Mannheim , Germany -RRB- , 1 μmol/l of each sequence-specific primer , and 0.2 μmol/l of internal control primers . The amplifications were performed in a My Cycler thermocycler -LRB- Bio-Rad , Hercules , USA -RRB- . For amplification , 30 cycles were performed using the following steps : heating to 96 °C for 1 min to denature the DNA ; denaturation at 96 °C for 25 s and at 70 °C for 45 s ; annealing and extension at 72 °C for 30 s -LRB- for the first 5 cycles -RRB- , 96 °C for 25 s , 65 °C 45 s , 72 °C for 30 s -LRB- for the next 21 cycles -RRB- ; 96 °C for 25 s , 55 °C for 60 s , 72 °C for 120 s -LRB- for the last 4 cycles -RRB- ; and a final 1 min extension at 72 °C . The presence or absence of PCR products was determined after separation of the samples on a 1.5 % agarose gel containing 0.5 μg/ml of ethidium bromide . HLA-DRB1 Genotyping The PCR sequence-specific primer -LRB- SSP -RRB- method was used for HLA-DRB1 genotyping . Each reaction contained a primer mix consisting of the allele - or group-specific primer pairs as well as the internal control primers that matched the nonallelic sequences . Specific amplification of the HLA-DRB1 gene was performed using 47 forward and reverse primers for HLA-DRB1 designed on the basis of the published nucleotide sequences -LSB- 23 -RSB- . The PCR procedure was carried out in a reaction -LRB- 10 μl -RRB- containing 100 ng genomic DNA , 1 X buffer -LRB- 60 mmol/l Tris-HCl pH 9.0 , 15 mmol/l ammonium sulphate , 2.5 mmol/l MgCl2 -RRB- , 200 μmol/l of each dNTP , 0.25 U Taq DNA polymerase -LRB- Boehringer Mannheim , Germany -RRB- , 1 μmol/l of each sequence-specific primer , and 0.2 μmol/l of the internal control primers . The amplifications performed in a My Cycler thermocycler -LRB- Bio-Rad , Hercules , USA -RRB- . For amplification , 27 cycles were performed using the following steps : heating to 96 °C for 3 min to denature the DNA ; denaturation at 96 °C for 20 s ; annealing and extension at 66 °C for 60 s -LRB- for the first 10 cycles -RRB- , 62 °C for 80 s -LRB- for the next 10 cycles -RRB- , and 61 °C for 120 s -LRB- for the last 7 cycles -RRB- ; and a final 10 min extension at 72 °C . The presence or absence of PCR products was determined after separation of samples on a 1.5 % agarose gel containing 0.5 μg/ml of ethidium bromide . Statistical Analysis Statistical differences between Kawasaki disease patients and healthy control subjects and between subgroups of Kawasaki disease patients were tested with chi-square and Fisher 's exact test . The quantification of the relationship of the frequencies studied , in cases with Kawasaki disease , was performed by calculating the relative risks using the method of Woolf . Haldane 's modification was applied for cases in which the variables included zero . A p value less than 0.05 was considered to be significant . In this study , the data were not adjusted for multiple comparisons because the sample size was not large enough for multiple-comparison analysis correction . Results The Genotype Distribution and Allele Frequencies of HLA-A for the Patients With Kawasaki Disease and the Healthy Control Group In the analysis of the polymorphisms of HLA-A alleles , no statistical difference was found in the frequency of alleles between the patients with Kawasaki disease and the control group . However , the frequency of HLA-A26 alleles was significantly decreased in the Kawasaki disease patients without coronary complications -LRB- CC -RRB- compared with the healthy control group -LRB- p < 0.04 ; relative risk -LRB- RR -RRB- = 0.2 -RRB- . In a comparison between subgroups , with and without CC , no significant difference was identified -LRB- Table 2 -RRB- . Table 2The allele frequencies of human leukocyte antigen-A -LRB- HLA-A -RRB- in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls -LRB- n = 159 -RRB- n -LRB- % -RRB- KD patients -LRB- n = 74 -RRB- n -LRB- % -RRB- Coronary complicationsPresent -LRB- n = 21 -RRB- n -LRB- % -RRB- Absent -LRB- n = 53 n -LRB- % -RRB- HLA-A015 -LRB- 3.1 -RRB- 5 -LRB- 6.8 -RRB- 2 -LRB- 9.5 -RRB- 3 -LRB- 5.7 -RRB- 0285 -LRB- 53.5 -RRB- 43 -LRB- 58.1 -RRB- 12 -LRB- 57.1 -RRB- 31 -LRB- 58.5 -RRB- 035 -LRB- 3.1 -RRB- 3 -LRB- 4.1 -RRB- 0 -LRB- 0.0 -RRB- 3 -LRB- 5.7 -RRB- 1131 -LRB- 19.5 -RRB- 21 -LRB- 28.4 -RRB- 6 -LRB- 28.6 -RRB- 15 -LRB- 28.3 -RRB- 2457 -LRB- 35.8 -RRB- 33 -LRB- 44.6 -RRB- 12 -LRB- 57.1 -RRB- 21 -LRB- 39.6 -RRB- 2625 -LRB- 15.7 -RRB- 6 -LRB- 8.1 -RRB- 4 -LRB- 19.0 -RRB- 2 -LRB- 3.8 -RRB- a3020 -LRB- 12.6 -RRB- 4 -LRB- 5.4 -RRB- 1 -LRB- 4.8 -RRB- 3 -LRB- 5.7 -RRB- 3114 -LRB- 8.8 -RRB- 6 -LRB- 8.1 -RRB- 0 -LRB- 0.0 -RRB- 6 -LRB- 11.3 -RRB- 323 -LRB- 1.9 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 3338 -LRB- 23.9 -RRB- 15 -LRB- 20.3 -RRB- 2 -LRB- 9.5 -RRB- 13 -LRB- 24.5 -RRB- 681 -LRB- 0.6 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- KD , Kawasaki disease ; RR , relative riskap < 0.04 ; RR = 0.2 -LRB- 95 % confidence interval -LSB- CI -RSB- , 0.05 -- 0.90 -RRB- vs healthy control subjects The Genotype Distribution and Allele Frequencies of HLA-B for the Patients With Kawasaki Disease and the Healthy Control Group In analysis of the polymorphisms of HLA-B alleles , there was a significant increase in the frequency of HLA-B35 and - B75 alleles in the patients with Kawasaki disease compared with the control group -LRB- p < 0.006 ; RR = 3.1 vs p < 0.02 ; RR = 8.2 -RRB- . When the patients with Kawasaki disease were divided into two subgroups , with or without CC , the Kawasaki disease patients without CC showed a significantly increased frequency of HLA-B35 and - B75 alleles compared with the healthy control group -LRB- p < 0.02 ; RR = 3.1 vs p < 0.003 ; RR = 11 -RRB- -LRB- Table 3 -RRB- . Although the frequency of the HLA-B35 alleles were similar in the groups with and without CC -LRB- 19 % vs 18.9 % -RRB- , the HLA-B75 allele was found only in the Kawasaki disease patients without CC -LRB- 0.0 % vs. 13.2 % -RRB- . Therefore , the HLA-B35 allele was implicated in susceptibility to Kawasaki disease and the HLA-B75 allele in susceptibility to Kawasaki disease without CC . In a comparison between the subgroups , with and without CC , there was no overall significant difference identified between the two groups . Table 3The allele frequencies of human leukocyte antigen-B -LRB- HLA-B -RRB- in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls -LRB- n = 159 -RRB- n -LRB- % -RRB- KD patients -LRB- n = 74 -RRB- n -LRB- % -RRB- Coronary complicationsPresent -LRB- n = 21 -RRB- n -LRB- % -RRB- Absent -LRB- n = 53 -RRB- n -LRB- % -RRB- HLA-B0719 -LRB- 11.9 -RRB- 9 -LRB- 12.2 -RRB- 3 -LRB- 14.3 -RRB- 6 -LRB- 11.3 -RRB- 082 -LRB- 1.3 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 1320 -LRB- 12.6 -RRB- 5 -LRB- 6.8 -RRB- 2 -LRB- 9.5 -RRB- 3 -LRB- 5.7 -RRB- 146 -LRB- 3.8 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 2711 -LRB- 6.9 -RRB- 3 -LRB- 4.1 -RRB- 1 -LRB- 4.8 -RRB- 2 -LRB- 3.8 -RRB- 3511 -LRB- 6.9 -RRB- 14 -LRB- 18.9 -RRB- a4 -LRB- 19.0 -RRB- 10 -LRB- 18.9 -RRB- b375 -LRB- 3.1 -RRB- 4 -LRB- 5.4 -RRB- 2 -LRB- 9.5 -RRB- 2 -LRB- 3.8 -RRB- 387 -LRB- 4.4 -RRB- 3 -LRB- 4.1 -RRB- 0 -LRB- 0.0 -RRB- 3 -LRB- 5.7 -RRB- 392 -LRB- 1.3 -RRB- 3 -LRB- 4.1 -RRB- 2 -LRB- 9.5 -RRB- 1 -LRB- 1.9 -RRB- 4429 -LRB- 18.2 -RRB- 10 -LRB- 13.5 -RRB- 3 -LRB- 14.3 -RRB- 7 -LRB- 13.2 -RRB- 4618 -LRB- 11.3 -RRB- 7 -LRB- 9.5 -RRB- 2 -LRB- 9.5 -RRB- 5 -LRB- 9.4 -RRB- 470 -LRB- 0.0 -RRB- 1 -LRB- 1.4 -RRB- 0 -LRB- 0.0 -RRB- 1 -LRB- 1.9 -RRB- 4813 -LRB- 8.2 -RRB- 2 -LRB- 2.7 -RRB- 0 -LRB- 0.0 -RRB- 2 -LRB- 3.8 -RRB- 5129 -LRB- 18.2 -RRB- 13 -LRB- 17.6 -RRB- 5 -LRB- 23.8 -RRB- 8 -LRB- 15.1 -RRB- 529 -LRB- 5.7 -RRB- 3 -LRB- 4.1 -RRB- 2 -LRB- 9.5 -RRB- 1 -LRB- 1.9 -RRB- 5423 -LRB- 14.5 -RRB- 10 -LRB- 13.5 -RRB- 3 -LRB- 14.3 -RRB- 7 -LRB- 13.2 -RRB- 553 -LRB- 1.9 -RRB- 4 -LRB- 5.4 -RRB- 1 -LRB- 4.8 -RRB- 3 -LRB- 5.7 -RRB- 562 -LRB- 1.3 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 0 -LRB- 0.0 -RRB- 572 -LRB- 1.3 -RRB- 1 -LRB- 1.4 -RRB- 0 -LRB- 0.0 -RRB- 1 -LRB- 1.9 -RRB- 5815 -LRB- 9.4 -RRB- 5 -LRB- 6.8 -RRB- 0 -LRB- 0.0 -RRB- 5 -LRB- 9.4 -RRB- 594 -LRB- 2.5 -RRB- 3 -LRB- 4.1 -RRB- 0 -LRB- 0.0 -RRB- 3 -LRB- 5.7 -RRB- 6011 -LRB- 6.9 -RRB- 7 -LRB- 9.5 -RRB- 2 -LRB- 9.5 -RRB- 5 -LRB- 9.4 -RRB- 6123 -LRB- 14.5 -RRB- 13 -LRB- 17.6 -RRB- 6 -LRB- 28.6 -RRB- 7 -LRB- 13.2 -RRB- 6229 -LRB- 18.2 -RRB- 14 -LRB- 18.9 -RRB- 1 -LRB- 4.8 -RRB- 13 -LRB- 24.5 -RRB- 674 -LRB- 2.5 -RRB- 2 -LRB- 2.7 -RRB- 1 -LRB- 4.8 -RRB- 1 -LRB- 1.9 -RRB- 717 -LRB- 4.4 -RRB- 1 -LRB- 1.4 -RRB- 0 -LRB- 0.0 -RRB- 1 -LRB- 1.9 -RRB- 752 -LRB- 1.3 -RRB- 7 -LRB- 9.5 -RRB- c0 -LRB- 0.0 -RRB- 7 -LRB- 13.2 -RRB- dKD , Kawasaki disease ; RR , relative riskap < 0.006 ; RR = 3.1 -LRB- 95 % confidence interval -LSB- CI -RSB- , 1.29 -- 7.62 -RRB- vs healthy control subjectsbp < 0.02 ; RR = 3.1 -LRB- 95 % CI , 1.16 -- 8.43 -RRB- vs healthy control subjectscp < 0.02 ; RR = 8.2 -LRB- 95 % CI , 1.74 -- 38.68 -RRB- vs healthy control subjectsdp < 0.003 ; RR = 11 -LRB- 95 % CI , 2.79 -- 51.13 -RRB- vs healthy control subjects The Genotype Distribution and Allele Frequency of HLA-C for the Patients With Kawasaki Disease and the Healthy Control Group In the analysis of the polymorphisms of HLA-C alleles , there was a significant increase in the frequency of the HLA-Cw09 allele in the patients with Kawasaki disease compared with the healthy control group -LRB- p < 0.04 ; RR = 2.0 -RRB- . When the Kawasaki disease patients were divided into two subgroups , with or without CC , the Kawasaki disease patients without CC showed a significantly increased frequency of the HLA-Cw09 allele compared with the healthy control group -LRB- p < 0.05 ; RR = 2.1 -RRB- . However , no significant difference was found between the subgroups with and without CC -LRB- Table 4 -RRB- . Table 4The allele frequencies of human leukocyte antigen-C -LRB- HLA-C -RRB- in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls -LRB- n = 159 -RRB- n -LRB- % -RRB- KD patients -LRB- n = 74 -RRB- n -LRB- % -RRB- Coronary complicationsPresent -LRB- n = 21 -RRB- n -LRB- % -RRB- Absent -LRB- n = 53 -RRB- n -LRB- % -RRB- HLA-Cw0159 -LRB- 37.1 -RRB- 22 -LRB- 29.7 -RRB- 6 -LRB- 28.6 -RRB- 16 -LRB- 30.2 -RRB- 020 -LRB- 0.0 -RRB- 1 -LRB- 1.4 -RRB- 0 -LRB- 0.0 -RRB- 1 -LRB- 1.9 -RRB- 0421 -LRB- 13.2 -RRB- 11 -LRB- 14.9 -RRB- 2 -LRB- 9.5 -RRB- 9 -LRB- 17.0 -RRB- 056 -LRB- 3.8 -RRB- 1 -LRB- 1.4 -RRB- 0 -LRB- 0.0 -RRB- 1 -LRB- 1.9 -RRB- 0625 -LRB- 15.7 -RRB- 10 -LRB- 13.5 -RRB- 5 -LRB- 23.8 -RRB- 5 -LRB- 9.4 -RRB- 0744 -LRB- 27.7 -RRB- 20 -LRB- 27.0 -RRB- 6 -LRB- 28.6 -RRB- 14 -LRB- 26.4 -RRB- 0829 -LRB- 18.2 -RRB- 10 -LRB- 13.5 -RRB- 3 -LRB- 14.3 -RRB- 7 -LRB- 13.2 -RRB- 0923 -LRB- 14.5 -RRB- 19 -LRB- 25.7 -RRB- a5 -LRB- 23.8 -RRB- 14 -LRB- 26.4 -RRB- b1039 -LRB- 24.5 -RRB- 24 -LRB- 32.4 -RRB- 5 -LRB- 23.8 -RRB- 19 -LRB- 35.8 -RRB- 1211 -LRB- 6.9 -RRB- 4 -LRB- 5.4 -RRB- 2 -LRB- 9.5 -RRB- 2 -LRB- 3.8 -RRB- 1434 -LRB- 21.4 -RRB- 18 -LRB- 24.3 -RRB- 7 -LRB- 33.3 -RRB- 11 -LRB- 20.8 -RRB- 158 -LRB- 5.0 -RRB- 1 -LRB- 1.4 -RRB- 1 -LRB- 4.8 -RRB- 0 -LRB- 0.0 -RRB- KD , Kawasaki disease ; RR , relative riskap < 0.04 ; RR = 2.0 -LRB- 95 % confidence interval -LSB- CI -RSB- , 0.97 -- 4.29 -RRB- vs healthy control subjectsbp < 0.05 ; RR = 2.1 -LRB- 95 % CI , 0.92 -- 4.87 -RRB- vs healthy control subjects The Genotype Distribution and the Allele Frequencies of HLA-DRB1 for the Patients With Kawasaki Disease and the Healthy Control Group Among the HLA-DRB1 alleles , there was no increase in the frequency of alleles in the Kawasaki disease patients compared with the healthy control group . However , after subgrouping of the Kawasaki disease patients , with or without CC , the frequency of HLA-DRB1 * 11 was significantly increased in the Kawasaki disease patients with CC compared with the healthy control group -LRB- p < 0.04 ; RR = 4.7 -RRB- . In a comparison between the subgroups , with and without CC , the frequency of the HLA-DRB1 * 09 allele was increased in the Kawasaki disease patients with CC compared with the Kawasaki disease patients without CC -LRB- 33.3 % vs 9.4 % ; p < 0.04 -RRB- . However , there was no significant difference in the frequency of these alleles when each was compared with the healthy control group . The frequency of the HLA-DRB1 * 04 allele was slightly increased in the Kawasaki disease patients without CC compared with the healthy control group -LRB- p < 0.05 ; RR = 1.9 -RRB- -LRB- Table 5 -RRB- . Table 5The allele frequencies of human leukocyte antigen-DRB1 -LRB- HLA-DRB1 -RRB- in the patients with Kawasaki disease and the control subjectsHLA allelesHealthy controls -LRB- n = 159 -RRB- n -LRB- % -RRB- KD patients -LRB- n = 74 -RRB- n -LRB- % -RRB- Coronary complicationsPresent -LRB- n = 21 -RRB- n -LRB- % -RRB- Absent -LRB- n = 53 -RRB- n -LRB- % -RRB- HLA-DRB10129 -LRB- 18.2 -RRB- 8 -LRB- 10.8 -RRB- 1 -LRB- 4.8 -RRB- 7 -LRB- 13.2 -RRB- 037 -LRB- 4.4 -RRB- 1 -LRB- 1.4 -RRB- 0 -LRB- 0.0 -RRB- 1 -LRB- 1.9 -RRB- 0448 -LRB- 30.2 -RRB- 30 -LRB- 40.5 -RRB- 6 -LRB- 28.6 -RRB- 24 -LRB- 45.3 -RRB- a0726 -LRB- 16.4 -RRB- 8 -LRB- 10.8 -RRB- 1 -LRB- 4.8 -RRB- 7 -LRB- 13.2 -RRB- 0832 -LRB- 20.1 -RRB- 14 -LRB- 18.9 -RRB- 3 -LRB- 14.3 -RRB- 11 -LRB- 20.8 -RRB- 0928 -LRB- 17.6 -RRB- 12 -LRB- 16.2 -RRB- 7 -LRB- 33.3 -RRB- b5 -LRB- 9.4 -RRB- 104 -LRB- 2.5 -RRB- 4 -LRB- 5.4 -RRB- 2 -LRB- 9.5 -RRB- 2 -LRB- 3.8 -RRB- 1110 -LRB- 6.3 -RRB- 8 -LRB- 10.8 -RRB- 5 -LRB- 23.8 -RRB- c3 -LRB- 5.7 -RRB- 1218 -LRB- 11.3 -RRB- 11 -LRB- 14.9 -RRB- 2 -LRB- 9.5 -RRB- 9 -LRB- 17.0 -RRB- 1332 -LRB- 20.1 -RRB- 10 -LRB- 13.5 -RRB- 3 -LRB- 14.3 -RRB- 7 -LRB- 13.2 -RRB- 1421 -LRB- 13.2 -RRB- 10 -LRB- 13.5 -RRB- 3 -LRB- 14.3 -RRB- 7 -LRB- 13.2 -RRB- 1535 -LRB- 22.0 -RRB- 20 -LRB- 27.0 -RRB- 5 -LRB- 23.8 -RRB- 15 -LRB- 28.3 -RRB- 164 -LRB- 2.5 -RRB- 2 -LRB- 2.7 -RRB- 1 -LRB- 4.8 -RRB- 1 -LRB- 1.9 -RRB- KD , Kawasaki disease ; RR , relative riskap < 0.05 ; RR = 1.9 -LRB- 95 % confidence interval -LSB- CI -RSB- , 0.96 -- 3.82 -RRB- vs healthy control subjectsbp < 0.04 between KD patients with and without coronary complicationcp < 0.04 ; RR = 4.7 -LRB- 95 % CI , 1.26 -- 17.17 -RRB- vs healthy control subjects Discussion The prevalence of CC among the patients with Kawasaki disease in this study was higher than previously reported -LRB- 28.4 % vs 5 % -RRB- -LSB- 22 -RSB- . This is likely because the patients included had a previous diagnosis of Kawasaki disease and were attending our clinic for this reason . Patients with more severe Kawasaki disease , including those with CC , present for medical care more frequently than patients without CC , and this may explain the higher proportion of CC in our study . Three patients -LRB- 4 % -RRB- had a positive family history , and four patients -LRB- 5.4 % -RRB- had a history of Kawasaki disease recurrence . Prior studies reported the familial occurrence and recurrence rates for Kawasaki disease to be 1 % and 3 % , respectively -LSB- 11 , 24 , 28 -RSB- . Our data were confined to Korean children and focused on patients who had Kawasaki disease with CC . Therefore , further research on the effect of the family history and recurrence of Kawasaki disease in association with HLA polymorphisms is needed . Studies have associated HLA alleles with many different human diseases , such as HLA-B27 with ankylosing spondylitis and Reiter syndrome , HLA-B35 with subacute thyroiditis , and HLA-DR8 with juvenile rheumatoid arthritis . Most of the early studies on the genetics of Kawasaki disease were conducted using serologic testing that measured HLA antigens . The association of the HLA-Bw22 allele with Kawasaki disease in Japanese studies was evaluated with the microcytotoxicity test used for tissue typing of 205 patients with Kawasaki disease and 500 control samples in the late 1970s -LSB- 14 -RSB- . On basis of these studies , a gene was thought to control susceptibility to Kawasaki disease and was linked to the Japanese-specific HLA antigen . Since the clinical application of genetic technology became available in the 1980s , polymorphisms at many HLA loci have been identified . In addition , there were changes in nomenclature , and the alleles were reclassified . For example , the previously labeled HLA-Bw22 allele was changed to the HLA B54 allele -LSB- 1 , 3 -RSB- . Our study on the polymorphisms of HLA genes was conducted using the PCR-ARMS method for the HLA class I genes and the PCR-SSP method for the HLA class II genes . There was a significant increase in the frequencies of HLA-B35 , - B75 , and - Cw09 alleles in the patients with Kawasaki disease compared with the healthy control group . As previously mentioned , this study showed that the HLA-B35 allele was related to susceptibility to Kawasaki disease and the HLA-B75 allele to Kawasaki disease without CC in Korean children . These results suggest that polymorphisms -LRB- in some of the B and C loci -RRB- in HLA class I genes are associated with Kawasaki disease in Korean children ; consistent with prior reports on other ethnic groups -LSB- 12 , 14 , 16 , 19 -RSB- . Review of the medical literature on Kawasaki disease and HLA shows that there is a trend toward an association between the HLA-B loci and Kawasaki disease . However , to date , there is no confirmed relationship to a particular locus . It is likely that the HLA-B locus is not the only locus associated with Kawasaki disease . This can be the case because of its functional variation or the potential effects from other related genes around the HLA-B locus . The HLA-B locus and other associated genes might be useful genetic markers for Kawasaki disease . The HLA-B35 gene is known to play a major role in a variety of infections . For example , in cases with HIV infection , the patients with an increased frequency of HLA-B35 show rapid progression of HIV -LSB- 9 -RSB- . Infection has been implicated in the etiology of Kawasaki disease . The finding in our study that the frequency of the HLA-B35 allele was increased in Korean children with Kawasaki disease suggests a role for infection in Kawasaki disease . The HLA-DRB1 alleles showed no increased frequency in the Kawasaki disease patients compared with the healthy control group . However , when the patients were compared by subgroups , with and without CC , the frequency of HLA-DRB1 * 11 was significantly increased in the Kawasaki disease patients with CC -LRB- p < 0.04 ; RR = 4.7 -RRB- , and HLA-DRB1 * 04 was increased in the Kawasaki disease patients without CC -LRB- p < 0.05 ; RR = 1.9 -RRB- compared with the healthy control group . Comparison of the subgroups with and without CC showed that the frequency of the HLA-DRB1 * 09 allele was increased in Kawasaki disease patients with CC compared with the Kawasaki disease patients who had no CC -LRB- 33.3 % vs 9.4 % ; p < 0.04 -RRB- , and that there was no significant difference in comparison with the healthy control group . For the HLA-DRB1 * 11 allele , the finding of a higher allele frequency compared with the healthy control group was amplified by the subgrouping of Kawasaki disease patients with and without CC . Future studies with a larger sample size are needed to confirm these findings . Several reports describe the presence of HLA-DRB1 and the progression of infections -LSB- 7 -RSB- . However , most of the reports on the association of HLA-DRB1 with Kawasaki disease concluded that despite a regional association , their data failed to support a consistent role for HLA class II alleles in Kawasaki disease . If there was any role , it was predicted to be minor -LSB- 2 , 8 -RSB- . Interpopulation discrepancies of the frequencies of HLA alleles make generalization of results difficult . Matched population profiles are needed for disease association studies because ethnic differences can provide altered disease associations . In this study , we compared HLA alleles of Kawasaki disease patients with known polymorphic loci of HLA genes in healthy Korean adults . The results we report on the polymorphisms of HLA genes in Kawasaki disease patients may be limited to the Korean population . However , this information may be helpful in future studies on HLA genetic polymorphisms in Kawasaki disease . Further studies with a larger sample size are needed for confirmation of our findings .

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