- Research Article
- Open Access
The Interleukin-8 (IL-8/CXCL8) Receptor Inhibitor Reparixin Improves Neurological Deficits and Reduces Long-term Inflammation in Permanent and Transient Cerebral Ischemia in Rats
© Feinstein Institute for Medical Research 2007
- Received: 1 February 2007
- Accepted: 5 February 2007
- Published: 1 March 2007
Leukocyte infiltration is viewed as a pharmacological target in cerebral ischemia. We previously reported that reparixin, a CXCL8 receptor blocker that inhibits neutrophil infiltration, and related molecules can reduce infarct size in a rat model of transient middle cerebral artery occlusion (MCAO). The study aims were to compare the effects of reparixin in transient and permanent MCAO using varied treatment schedules and therapeutic windows to evaluate effects on long-term neurological deficits and late inflammatory response. Reparixin, administered for 1 to 3 days, 3.5 to 6 h after MCAO, ameliorates neurological function recovery and inhibits long-term inflammation. The infarct size reduction at 24 h, evaluated by TTC staining, is more pronounced in transient MCAO. MRI analysis identified a decrease in the progression of infarct size by reparixin that was more evident at 48 h in permanent MCAO, and was associated with a significantly improved recovery from long-term neurological deficits.
Blockade of inflammation is considered a possible approach to the therapy of cerebral ischemia. Leukocytic infiltration, particularly of polymorphonuclear neutrophils (PMN) is a key aspect of the deleterious aspects of inflammation in stroke (1–3), and CXCL8 or related chemokines are induced in stroke in animal models (4) as well as in patients (5,6). Recently, we described reparixin (formerly termed repertaxin), a small molecular weight inhibitor of CXCR1 and CXCR2, the receptors for the CXCL8 family of chemokines implicated in the recruitment of PMN active in vivo (7), and the drug is now undergoing clinical trials for other indications. A preliminary study of reparixin in two models of cerebral ischemia in the rat indicated that it was more effective against transient ischemia than in permanent ischemia, where there was only a trend for reduction in infarct size (8), consistent with the hypothesis that PMN are mediators mainly in the reperfusion injury.
To better characterize the effect of reparixin in the two models of cerebral ischemia, and hence the role of CXCR1/2 ligands in neuroinflammation, we undertook a series of experiments aiming at investigating not only its effect on infarct size but also on long-term neurological outcome. In fact, infarct size only partially correlates with functional outcome in patients, and it is suggested it should only be used as a surrogate marker in clinical trials (9).
Transient cerebral ischemia was induced in rats by 1.5 h middle cerebral artery (MCA) occlusion (MCAO). In some experiments, we used a permanent ischemia model, often termed three-vessel occlusion, where the permanent occlusion of the right MCA and of the ipsilateral carotid and the temporary (1 h) occlusion of the contralateral carotid induce a damage with a penumbra surrounding the fixed lesions in the MCA territory (10,11). In these animals we measured the infarct volume 24 h after surgery, using triphenyltetrazolium hydrochloride (TTC) staining, quantified PMN infiltrate by measuring brain myeloperoxidase (MPO) or by immunochemistry, and performed behavioral testing including sensorimotor tests (De Ryck’s (12), Bederson’s (13), and foot-fault tests (14)) for up to 1 month to evaluate neurological deficits. As the results on reduction of infarct size in the permanent ischemia model were not conclusive, we used MRI to follow up infarct size progression in these rats.
These experiments used treatment schedules chosen according to previous studies with reparixin in various models of ischemia (7,8,15). However, in this study, we also characterized the drug in terms of therapeutic window and compared different injection schedules, either bolus or continuous infusion to gain information useful for future clinical trials.
Finally, because we show elsewhere (16) that the neuroprotective action of erythropoietin induces long-term functional improvement associated with a decrease in the late inflammatory response, we also evaluated the effect of reparixin on late inflammation in the ischemic brain by evaluating immunohistochemical markers of astroglial activation one month after ischemia.
The results indicate that reparixin reduces not only short-term PMN infiltration and infarct size, but also decreases long-term inflammation and improves long-term neurological outcome in both transient and permanent ischemia models.
Male Crl:CD (SD)BR rats (Charles River, Calco, Italy) were used. Procedures involving animals and their care conformed to institutional guidelines that are in compliance with national (D.L. n.116, G.U. suppl. 40; February 18, 1992) and international laws and policies (EEC Council Directive 86/609, OJ L 358,1; December 12,1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996).
Reparixin (as L-lysine salt) was from Dompé pha.r.ma. s.p.a., L’Aquila, Italy. The drug was dissolved in saline and administered as described in the text.
Transient Cerebral Ischemia
We used an intraluminal occlusion method with subsequent reperfusion (17). Overnight fasted rats (300–330 g) were anesthetized with 2–3% isoflurane in N2O/O2 (70%:30%) and a Stren nylon filament suture, blunted at the tip by heat to 0.35 mm diameter, was advanced through the right common carotid artery (CA) and the internal CA up to 19 mm from the bifurcation of the common CA and the external CA. Heparin (30U) was administered intravenously (i.v.) before insertion of the filament. Reperfusion began 90 min after MCA occlusion. The same surgery was performed in sham-operated rats but no ischemia was performed. Rectal temperature was monitored during ischemia and reperfusion period and, when it started rising above 37°C, the animals were placed in a cold room (10°C) and 70% alcohol was applied if there was a sudden rise (18). Adequate MCA occlusion was judged from neurological behavior, shown by gait disturbances with circling to the left (17).
Permanent Cerebral Ischemia
Fed rats (250–280 g) were anesthetized with chloral hydrate (400 mg/kg). The common CAs were visualized and the right one was occluded. A hole adjacent and rostral to the right orbit allowed visualization of the MCA, which was cauterized distal to the rhinal artery. To produce a penumbra around this fixed MCA lesion, the contralateral common CA was occluded for 1 h using traction with fine forceps (10). Body core temperature was held thermostatically at 37°C. In the present study we selected 24 h for evaluation of the injury because this time provides maximum infarction for both permanent and transient MCA occlusion (19).
Quantification of Ischemic Volume
To evaluate the extent of injury the rats were killed 24 h after ischemia and the brains were removed, transferred to cold saline and twelve serial 1-mm thick sections were cut through the entire brain. Six alternate sections were stained with triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO, USA) as previously described (8,20). The extent of injury was quantified in six sections using a computerized image analysis system (AIS version 3.0 software, Imaging Research, St. Catherine’s, ON, Canada). The other six sections were frozen on dry ice and stored at −80°C until MPO was measured.
Immunohistochemical and Histochemical Analyses
For immunohistochemical analyses, rats were perfused with paraformaledhyde and immunohistochemistry was performed on 30 free-floating sections using anti-CD11b (MRC OX-42) mouse monoclonal antibody (1:50; Serotec, UK) or anti-GFAP mouse monoclonal antibody (1:250; Immunological Sciences, Rome) as previously described (21). To quantitate the extent glial reaction, the area of GFAP and CD11b immunoreactivity in the ischemic hemisphere was measured using a digital image analyzer (Olympus DP Software). Glial spreading was calculated as the percentage of the GFAP- or CD11b-positive area over the total area of the ischemic hemisphere.
Histochemical determination of PMN was done on 5 µm paraffin sections by the naphthol AS-D chloroacetate technique for esterase that stains PMN in red (7,22). To quantitatively assess PMN infiltration, MPO activity in the tissue homogenate from the two hemispheres was quantified in six alternate sections for each animal as previously described (8). MPO activity is expressed as ΔA/min/mg protein and is the difference between the ipsilateral and the contralateral hemisphere.
MRI measurements were taken 2, 24, and 48 h after ischemic insult using a 4.7T, vertical superwide bore magnet and a Bruker Advance II spectrometer with microimaging accessory. The ischemic volume was determined by trace of apparent diffusion coefficient maps (Tr(D)) computation as previously described (23,24). The progression of the ischemic damage was assessed over time using ANOVA for repeated measurements with lesion volume (evaluated by MRI) as dependent variable and the treatment as independent variable.
Neurological deficits were evaluated using the foot fault (14), Bederson’s (13), and De Ryck’s (12) tests. In the postural reflex test of Bederson rats were scored as follows: grade 5, normal; grade 4, moderate (forelimb flexion and no other abnormality); grade 3, severe (reduced resistance to lateral push toward the paretic side, and forelimb flexion); grade 2, severe (same behavior as grade 3, with circling toward the paretic side when pulling the tail on the table); grade 1, severe vere (same behavior as grade 2, with spontaneous circling); grade 0, no activity. The limb-placing test developed by De Ryck examines sensorimotor integration in limb placing responses to visual, vibrissae, tactile, and proprioceptive stimuli. For each test, limb placing scores were 0, no placing; 1 incomplete and/or delayed (> 2 seconds); or 2, immediate and complete placing. For each body side the maximum limb placing score was 16.
The foot-fault test measures the ability of the animal to integrate motor responses. The rats were placed on a grid with 2 cm spaces between 0.3 cm diameter metal rods and were observed for 2 min. With each weight-bearing step, the paw may fall or slip between the wires and this is recorded as foot fault. The number of foot-faults for the paws contralateral and ipsilateral to the infarction was recorded with the number of successful steps and the foot-fault index was calculated as the percentage of contralateral limb foot-faults per limb step minus the percentage of ipsilateral limb footfaults per limb step.
Effect of Reparixin on Ischemic Damage in Transient and Permanent Ischemia
Effect of reparixin on cortical vs. subcortical damage 24 h after transient MCAO
Saline (n = 6)
273 ± 24
181 ± 24
89 ± 9
Reparixin (n = 5)
68 ±8 **
12 ± 4 **
56 ± 9 *
Saline (n = 8)
271 ± 25
193 ± 19
78 ± 8
Reparixin (n = 7)
143 ± 23 *
64 ± 18 **
79 ± 8
To further confirm these results, we also evaluated by non-invasive MRI the evolution of cerebral infarct in permanent MCAO with or without reparixin (15 mg/kg, i.v. on the first day 1 h after MCAO followed by 3 s.c. doses at 2 h intervals).
Effect of Reparixin on Long-term Neurological Deficits
We next investigated the effect of reparixin on long-term (up to 1 month) neurological recovery using the treatment schedules described above.
Also in permanent ischemia, reparixin significantly improved neurological functions in rats as early as 1 day after MCAO (Figure 3, panels D–F).
Comparison of Different Treatment Schedules and Therapeutic Window in Transient MCAO
To better define the therapeutic window, we also tested reparixin administered 6 h after ischemia (4.5 h after reperfusion). In these experiments, reparixin (15 mg/kg) was given i.v. 6 h after MCAO followed by s.c. infusion for 48 h at a rate of 10mg/h/kg, as shown in Figure 4, panels G–I. This led to a significant improvement of 24-h neurological function as evaluated by De Ryck’s and Bederson’s tests, while there was only a trend for the foot fault test.
Reparixin Reduces PMN Infiltrate and Long-term Inflammation in Ischemic Brain
In transient ischemia, GFAP staining shows that, in contrast to coronal sections from control animals (A), ischemic rats present a stronger immunoreactivity (B) that is reduced and confined to a small subcortical region in reparixin-treated rats (C). The same anti-inflammatory effect of reparixin is detectable using anti-CD11b (D–F): while non-ischemic rats show little immunoreactivity (D), diffuse staining is present in the ipsilateral side of ischemic rats (E) and greatly diminished by reparixin either in terms of intensity or spreading (F).
Staining with GFAP in rats undergoing permanent ischemia show that, compared with control rats (G), ischemic rats show increased staining in the whole cortical and subcortical area corresponding to the side where the necrotic area is (H), while in reparixin-treated rats the staining is very low and limited to ischemic core (I). Of note, although permanent ischemia led to marked destruction of the tissue close to the focal zone, the preserved region showed GFAP immunoreactivity (H), and reparixin both reduced tissue loss and GFAP staining (I). As in the case of transient ischemia, CD11b immunoreactivity was strongly reduced in brains from rats undergoing permanent ischemia treated with reparixin (J-L).
Because loss of integrity close to the area of injury observed in ischemic brains can be considered indirect parameters of tissue damage (26), it is of interest to note that, in contrast to untreated ischemic rats, the sections obtained from reparixin-treated animals did not show any macroscopic feature of sufferance close to the injured area. Reparixin almost completely reduced long-term inflammation in the cortex, in agreement with the reduction in the infarct area where the cortical region is preferentially protected.
We also analyzed the morphology of the GFAP- and CD11b-positive cells in these samples. Analysis of higher magnification pictures (not shown) indicated that, in both models of MCAO, the morphology of GFAP-stained activated astrocytes does not seem different between the two groups. CD11b-positive cells show the typical morphology of activated microglia and, as described for GFAP, no difference in microglial morphology, at the single cell level, was detected by comparing the area of neuroinflammation of untreated and reparixin-treated mice.
Quantification of glial reaction in transient MCAO
46.5 ± 3.0
17.6 ± 1.6**
22.7 ± 1.0**
The protective action of reparixin demonstrates the pathogenic role of inflammation induced by chemokines, particularly PMN chemoattractants acting on CXCR1/2, in cerebral ischemia (27,28). This is in agreement with the notion that PMN infiltration is important in post-ischemic damage in the brain as suggested by several studies using anti-leukocyte strategies in experimental stroke (2).
We characterized the effect of reparixin, using different routes of administration, some of which, such as infusion, may better reproduce the clinical setting, in reducing infarct size in the model of transient ischemia and could show for the first time that reduction in infarct size achieved by an anti-CXCR1/2 agent is accompanied by an improved long-term neurological recovery.
This observation strengthens the concept that inflammatory pathways may be important pharmacological targets in cerebral ischemia even looking at long-term effects that may better reflect the balance between neuronal injury and neuronal repair. While it has been postulated that inflammatory responses in the CNS may be beneficial in some contexts as potential sources for trophic factors (29), others have pointed out that inflammation can actually inhibit neurogenesis and the latter may be improved by anti-inflammatory drugs (30–33). The recently published observation that CXCL1, by acting on CXCR2, is an anti-apoptotic factor for rat astrocytes (34) is in agreement with ours showing that CXCR1/2 inhibition decreases astrocytosis, and supports the concept of a pathogenic, rather than protective, role of astrocytosis in our models. These findings indicate that inhibition of inflammation, or at least its CXCR1/2-mediated component, does not hamper long-term recovery from stroke.
The improvement of neurological functions reported here in the model of permanent MCAO deserves further discussion. In fact, in an early report, showing that reparixin reduced PMN infiltration but did not reduce infarct size significantly, we concluded that reparixin was not protective in permanent ischemia (8), but meta-analysis of several experiments clearly shows a protective effect of reparixin in this model as well. A protective effect was evident also when evaluating damage by either MRI or neurological tests. Clearly, protection by reparixin is less marked in permanent MCAO than in transient MCAO, and this could easily be explained by the fact that PMN infiltration, and thus its pathogenic contribution, is greater when reperfusion takes place. Furthermore, reperfusion might allow production of toxic oxygen species by the infiltrating leukocytes.
Our observation that reparixin is more effective in reducing cortical infarction than subcortical infarction (Table 1), is compatible with the model of transient cerebral ischemia, where the MCA is occluded at the origin (at the junction of the anterior and middle cerebral arteries), producing an ischemic area in striatum and overlying cortex. During the ischemic time, the local cerebral blood flow of both areas is similarly reduced, but during reperfusion the cerebral blood flow remains depressed in the ischemic striatum and gradually recovers to its control value in the ischemic cortex (Takagi et al 1995). Consequently, as discussed in a recent review (Carmichael 2005), the striatum is the ischemic core zone and the infarction in this area is mostly necrotic and more resistant to most neuroprotective agents while cortical infarction is a region of delayed, progressive neuronal death or an ischemic penumbra more susceptible of neuroprotection. Interestingly, a similar pattern was reported with IL-1 receptor antagonist, another anti-inflammatory agent (35), and in a study showing that striatal injury by MCAO is not ameliorated by PMN depletion (36). Also the reduction of ischemic damage by erythropoietin, a neuroprotective agent that has a marked anti-inflammatory action in this context as well (16), is more pronounced in the cortical area than in the subcortical one (37). Of note, preliminary clinical trials with these two agents have indicated that they may be beneficial in the therapy of stroke (38,39).
In conclusion, the data from this study support the idea that CXCL8-mediated inflammation plays an important role in ischemic damage and provides a validation of the approach taking advantage of CXCR1/2 inhibitors.
R Bertini, B Cavalieri, and R Di Bitondo are employees of Dompé pha.r.ma. s.p.a., which produces reparixin; P Ghezzi and L Sironi received contract money from Dompé.
- Barone FC, Feuerstein GZ. (1999) Inflammatory mediators and stroke: new opportunities for novel therapeutics. J. Cereb. Blood Flow. Metab. 19:819–34.View ArticleGoogle Scholar
- Emerich DF, Dean RL, 3rd, Bartus RT. (2002) The role of leukocytes following cerebral ischemia: pathogenic variable or bystander reaction to emerging infarct? Exp. Neurol. 173:168–81.View ArticleGoogle Scholar
- Fagan SC, Hess DC, Hohnadel EJ, Pollock DM, Ergul A. (2004) Targets for vascular protection after acute ischemic stroke. Stroke. 35:2220–5.View ArticleGoogle Scholar
- Yamasaki Y et al. (1995) Transient increase of cytokine-induced neutrophil chemoattractant, a member of the interleukin-8 family, in ischemic brain areas after focal ischemia in rats. Stroke. 26:318–22; discussion 322–3.View ArticleGoogle Scholar
- Kostulas N, Pelidou SH, Kivisakk P, Kostulas V, Link H. (1999) Increased IL-1beta, IL-8, and IL-17 mRNA expression in blood mononuclear cells observed in a prospective ischemic stroke study. Stroke. 30:2174–9.View ArticleGoogle Scholar
- Tarkowski E, et al. (1997) Intrathecal release of pro- and anti-inflammatory cytokines during stroke. Clin. Exp. Immunol. 110:492–9.View ArticleGoogle Scholar
- Bertini R, et al. (2004) Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: prevention of reperfusion injury. Proc. Natl. Acad. Sci. U. S. A. 101:11791–6.View ArticleGoogle Scholar
- Garau A, et al. (2005) Neuroprotection with the CXCL8 inhibitor repertaxin in transient brain ischemia. Cytokine. 30:125–31.View ArticleGoogle Scholar
- Saver JL, et al. (1999) Infarct volume as a surrogate or auxiliary outcome measure in ischemic stroke clinical trials. The RANTTAS Investigators. Stroke. 30:293–8.View ArticleGoogle Scholar
- Brines ML, et al. (2000) Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc. Natl. Acad. Sci. U. S. A. 13397:10526–31.View ArticleGoogle Scholar
- Zimmerman GA, et al. (1995) Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine. Proc. Natl. Acad. Sci. U. S. A. 92:3744–8.View ArticleGoogle Scholar
- De Ryck M, Van Reempts J, Borgers M, Wauquier A, Janssen PA. (1989) Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke. 20:1383–90.View ArticleGoogle Scholar
- Bederson JB et al. (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 17:472–6.View ArticleGoogle Scholar
- Hernandez TD, Schallert T. (1988) Seizures and recovery from experimental brain damage. Exp. Neurol. 102:318–24.View ArticleGoogle Scholar
- Cavalieri B, et al. (2005) Neutrophil recruitment in the reperfused-injured rat liver was effectively attenuated by repertaxin, a novel allosteric non-competitive inhibitor of CXCl8 receptors: a therapeutic approach for the treatment of post-ischemic hepatic syndromes. Int. J. Immunopathol. Pharmacol. 18 475–86.View ArticleGoogle Scholar
- Villa P, et al. (2007) Reduced functional deficits, neuroinflammation, and secondary tissue damage after treatment of stroke by nonerythropoietic erythropoietin derivatives. J. Cereb. Blood Flow Metab. 27:552–63.View ArticleGoogle Scholar
- Memezawa H, Minamisawa H, Smith ML, Siesjo BK. (1992) Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. Exp. Brain. Res. 89:67–78.View ArticleGoogle Scholar
- Reglodi D, Somogyvari-Vigh A, Maderdrut JL, Vigh S, Arimura A. (2000) Postischemic spontaneous hyperthermia and its effects in middle cerebral artery occlusion in the rat. Exp. Neurol. 163:399–407.View ArticleGoogle Scholar
- Barone FC, et al. (1992) Reperfusion increases neutrophils and leukotriene B4 receptor binding in rat focal ischemia. Stroke. 23:1337–47; discussion 1347–8.View ArticleGoogle Scholar
- Bederson JB et al. (1986) Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke. 17:1304–8.View ArticleGoogle Scholar
- Savino C, et al. (2006) Delayed administration of erythropoietin and its non-erythropoietic derivatives ameliorates chronic murine autoimmune encephalomyelitis. J. Neuroimmunol. 172:27–37.View ArticleGoogle Scholar
- Moloney WC, McPherson K, Fliegelman L. (1960) Esterase activity in leukocytes demonstrated by the use of naphthol AS-D chloroacetate substrate. J. Histochem. Cytochem. 8:200–7.View ArticleGoogle Scholar
- Guerrini U, et al. (2002) New insights into brain damage in stroke-prone rats: a nuclear magnetic imaging study. Stroke. 33:825–30.View ArticleGoogle Scholar
- Sironi L, et al. (2003) Treatment with statins after induction of focal ischemia in rats reduces the extent of brain damage. Arterioscler. Thromb. Vasc. Biol. 23:322–7.View ArticleGoogle Scholar
- Macleod MR, O’Collins T, Howells DW, Donnan GA. (2004) Pooling of animal experimental data reveals influence of study design and publication bias. Stroke. 35:1203–8.View ArticleGoogle Scholar
- Eichenbaum KD, et al. (2005) Minimally invasive method for murine brain fixation. Biotechniques. 39:487–8, 490.View ArticleGoogle Scholar
- Beech JS et al. (2001) Neuroprotection in ischemia-reperfusion injury: an antiinflammatory approach using a novel broad-spectrum chemokine inhibitor. J. Cereb. Blood Flow Metab. 21:683–9.View ArticleGoogle Scholar
- Glabinski AR, Ransohoff RM. (1999) Chemokines and chemokine receptors in CNS pathology. J. Neurovirol. 5:3–12.View ArticleGoogle Scholar
- Schwartz M, Moalem G, Leibowitz-Amit R, Cohen IR. (1999) Innate and adaptive immune responses can be beneficial for CNS repair. Trends Neurosci. 22:295–9.View ArticleGoogle Scholar
- Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O. (2003) Inflammation is detrimental for neurogenesis in adult brain. Proc. Natl. Acad. Sci. U. S. A. 100:13632–7.View ArticleGoogle Scholar
- Monje ML, Toda H, Palmer TD. (2003) Inflammatory blockade restores adult hippocampal neurogenesis. Science. 302:1760–5.View ArticleGoogle Scholar
- Hoehn BD, Palmer TD, Steinberg GK. (2005) Neurogenesis in rats after focal cerebral ischemia is enhanced by indomethacin. Stroke. 36:2718–24.View ArticleGoogle Scholar
- Kluska MM, Witte OW, Bolz J, Redecker C. (2005) Neurogenesis in the adult dentate gyrus after cortical infarcts: effects of infarct location, N-methyl-D-aspartate receptor blockade and anti-inflammatory treatment. Neuroscience. 135:723–35.View ArticleGoogle Scholar
- Wang Y, Luo W, Stricker R, Reiser G. (2006) Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1. J. Neurochem. 98:1046–60.View ArticleGoogle Scholar
- Mulcahy NJ, Ross J, Rothwell NJ, Loddick SA. (2003) Delayed administration of interleukin-1 receptor antagonist protects against transient cerebral ischaemia in the rat. Br. J. Pharmacol. 140:471–6.View ArticleGoogle Scholar
- Beray-Berthat V, Croci N, Plotkine M, Margaill I. (2003) Polymorphonuclear neutrophils contribute to infarction and oxidative stress in the cortex but not in the striatum after ischemia-reperfusion in rats. Brain Res. 987:32–8.View ArticleGoogle Scholar
- Belayev L, et al. (2005) Neuroprotective effect of darbepoetin alfa, a novel recombinant erythropoietic protein, in focal cerebral ischemia in rats. Stroke. 36:1071–6.View ArticleGoogle Scholar
- Emsley HC, et al. (2005) A randomised phase II study of interleukin-1 receptor antagonist in acute stroke patients. J. Neurol. Neurosurg. Psychiatry. 76:1366–72.View ArticleGoogle Scholar
- Ehrenreich H, et al. (2002) Erythropoietin therapy for acute stroke is both safe and beneficial. Mol. Med. 8:495–505.View ArticleGoogle Scholar