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There are mixed reports on the neuroprotective properties of erythropoietin (EPO) in animal models of birth asphyxia. We investigated the effect of EPO on short-and long-term outcome after neonatal hypoxic-ischemic (HI) brain injury in mice and compared the effect of two different dose regimens of EPO. Nine-day-old mice were subjected to HI, and EPO was injected i.p. at 0, 24, and 48 h after HI in a dose of either 5 or 20 kU/kg. Paw preference in the cylinder rearing test (CRT) was used as a measure of sensorimotor function. Only in female mice, administration of EPO at 5 kU/kg but not 20 kU/kg improved sensorimotor function, reduced striatum atrophy and hippocampal lesion volume, and enhanced myelin basic protein (MBP) staining as determined at 4 and 9 wk after HI. In addition, at 72 h after HI, more Ki67 cells were found in the subventricular zone and dentate gyrus after EPO 5 kU/kg treatment, indicating an increase in progenitor cell proliferation. In conclusion, EPO improves sensorimotor function after neonatal HI and protects against striatum atrophy, hippocampus injury, and white matter loss. per 1000 live-born infants and is an important cause of cerebral palsy, epilepsy, and adverse developmental outcome (1,2). Experimental studies in newborn animals with HI showed that antioxidative, anti-inflammatory, and neurotrophic agents are neuroprotective (3,4). However, in clinical studies with newborns with HI encephalopathy, only mild hypothermia showed a modest neuroprotective effect if started within 6 h after birth in asphyxiated term newborns with moderate encephalopathy (5,6).Erythropoietin (EPO), a glycoprotein primarily recognized as a mediator promoting maturation and proliferation of erythroid progenitor cells (7,8), is an attractive drug for this purpose. EPO has been proven to cross the blood-brain barrier after systemic administration at a high dose and to reduce free radical formation and proinflammatory and apoptotic activity in models of brain damage (8 -11). EPO has also been shown to stimulate formation of new neurons because of its actions as a neurotrophic factor (12,13). The effects of EPO are mediated by binding of EPO to its receptor (EPOR), which is present in the brain at relatively high levels in regions known to be sensitive to hypoxia (14,15). Most studies in neonatal animals treated with EPO after HI indeed showed an improved histological outcome (8,16), although our study in rats with neonatal HI brain damage showed no histological improvement (17). However, Spandou et al. (18) reported that in a similar rat model, using a shorter duration of hypoxia, EPO induced effective regeneration of brain tissue.In human studies, no adverse effects of EPO treatment have been reported indicating that EPO is a safe drug for neonates (19). EPO has been increasingly used in preterm infants to treat neonatal anemia or to improve neurodevelopmental outcome, at a low (0.4 kU/kg) or a relatively high dose (3 kU/kg) (19 -21). Furthermore, EPO treatment (0.3-0.5 kU/kg) of perinatally asphyxi...

Background: Both hypothermia and erythropoietin (ePO) are reported to have neuroprotective effects after perinatal hypoxia-ischemia (hI). We investigated a possible additive effect of the use of a combination of hypothermia-ePO in a rat model of neonatal hI. Methods: at postnatal day 7, rats were subjected to hI and then randomized to 3 h of hypothermia, ePO, or both. sensorimotor function was assessed by the cylinder-rearing test (cRT) at 2 and 5 wk after hI. Brain lesion volume and white matter loss were determined by hematoxylin-eosin and luxol fast blue staining, respectively. results: Multivariable analysis using general linear modeling showed that hypothermia, ePO, and the interaction hypothermia × gender were determinants of sensorimotor function, both at 2 and 5 wk after hI. Neuroprotective effects of hypothermia at 5 wk were more pronounced in females, showing 52% improvement in the cRT. Maximal improvement in males was 26% after combined treatment with hypothermia and ePO. histological outcome was improved by hypothermia only with no additional effect of ePO or gender. conclusion: hypothermia after hI improved sensorimotor function in females more than in males. There was a borderline additive effect of ePO when combined with hypothermia. histology of brain lesion volume and white matter damage was improved only by hypothermia.P erinatal hypoxia-ischemia (HI) is an important cause of neonatal brain injury and is associated with long-term neurological sequelae such as cognitive dysfunction, developmental delay, seizures, and sensory and/or motor impairment (1,2). Several studies in newborn rodents indicate that reduction of brain temperature by 2-5 °C even for 3 h, when started within 6 h after HI, provides neuroprotection and improved behavioral outcome (3-6).The neuroprotective effects of hypothermia of mildly asphyxiated newborns have been demonstrated, but in clinical practice, morbidity and mortality remain considerable (30-45%) (7,8). The combination of hypothermia and other pharmacologic strategies after birth asphyxia may improve long-term neurodevelopment (9). Erythropoietin (EPO) has been shown to reduce brain lesion volume in an experimental setting of neonatal HI (10-13). In addition, EPO treatment of perinatally asphyxiated human term neonates has also proven to be beneficial (14). In recent studies with a rodent model, females appear to benefit more from neuroprotective interventions after HI, such as hypothermia, EPO,.The aim of the present study was to test whether the addition of EPO to hypothermia has additive neuroprotective effects as compared with a single treatment and to examine possible gender effects. Results HypothermiaSham-treated rats did not show a paw preference in the cylinder-rearing test (CRT) (paw preference < ±5%, data not shown). A significant paw preference of the unimpaired forepaw in the normothermia groups was observed at both 2 and 5 wk after HI (Figure 1). Differences between males and females in the normothermia group were not statistically significant. In fema...

Perinatal hypoxia-ischemia (HI) is an important cause of neonatal brain injury. Recent progress in the search for neuroprotective compounds has provided us with several promising drugs to reduce perinatal HI-induced brain injury. In the early stage (first 6 hours after birth) therapies are concentrated on prevention of the production of reactive oxygen species or free radicals (xanthine-oxidase-, nitric oxide synthase-, and prostaglandin inhibition), anti-inflammatory effects (erythropoietin, melatonin, Xenon) and anti-apoptotic interventions (nuclear factor kappa B- and c-jun N-terminal kinase inhibition); in a later stage stimulation of neurotrophic properties in the neonatal brain (erythropoietin, growth factors) can be targeted to promote neuronal and oligodendrocyte regeneration. Combination of pharmacological means of treatment with moderate hypothermia, which is accepted now as a meaningful therapy, is probably the next step in clinical treatment to fight post-asphyxial brain damage. Further studies should be directed at a more rational use of therapies by determining the optimal time and dose to inhibit the different potentially destructive molecular pathways or to enhance endogenous repair while at the same time avoiding adverse effects of the drugs used.

P erinatal hypoxia-ischemia (HI) is a significant cause of neonatal brain injury. Neonatal animal models of HI show that excessive production of nitric oxide (NO), mediated by nitric oxide synthases (NOS), play an important role in the pathogenesis of neuronal injury after HI in the neonate.1,2 Three isoforms of NOS exist: the constitutively expressed neuronal NOS, endothelial NOS, and the inducible NOS. In vitro studies have shown that selective inhibition of neuronal NOS and inducible NOS can be achieved by the NOS inhibitor 2-iminobiotin (2-IB). 3To transition treatment to the human term neonate, it is important to know the dose-response effect of 2-IB to identify the optimal dose to be given after perinatal HI. The aim of this study was to determine the dose-response characteristics of 2-IB in a preclinical animal model of perinatal HI and to establish the most effective dose (range) for future clinical trials. This study was performed in our piglet model of inhalational HI, which is clinically, electrophysiologically, and neuropathologically comparable with the term born human neonate. The HI insult was performed in term neonatal piglets (n=47) as previously described 5 ; 6 animals served as sham-operated controls. HI piglets were randomly assigned to blinded treatment with vehicle or 2-IB at 0.1, 0.2, or 1 mg/kg/dose i.v. immediately post-HI and dosing repeated every 4 h until 20 h (6 doses in total). aEEG background pattern, presence of epileptic activity, and neurobehavior were scored. 6At 48 h postinsult animals were euthanized and tissue analyzed for caspase-3 activity, tyrosine nitration, and histology (see the onlineonly Data Supplement). ResultsIn total, 47 piglets were subjected to HI; 16 piglets were only mildly affected (continuous normal voltage at 30 min post-HI; see the online-only Data Supplement for examples) and excluded from further analysis. Of the remaining 31 piglets, 10 were vehicle-treated, 7 received 0.1 mg/kg/dose, 9 received 0.2 mg/kg/dose, and 5 received 1.0 mg/kg/dose. There was no difference in birth weight, postnatal age, pH, arterial BE, PO 2 , PCO 2, duration of hypotension, heart rate, or temperature between treatment groups (see the online-only Data Supplement).Background and Purpose-To determine the optimal dose of 2-iminobiotin (2-IB) for the treatment of moderate to severe asphyxia in a neonatal piglet model of hypoxia-ischemia. Methods-Newborn piglets were subjected to a 30-minute hypoxia-ischemia insult and randomly treated with vehicle or 2-IB (0.1 mg/kg, 0.2 mg/kg, or 1.0 mg/kg). aEEG background and seizure activity were scored after hypoxia-ischemia every 4 h until 24 h and at 48 h and neurobehavioral scores were obtained. Brain tissue was collected and processed for analysis of caspase-3 activity, histology, and tyrosine nitration. Results-A dose range of 0.1 to 1.0 mg/kg/dose of 2-IB improved short-term outcome as demonstrated by an increased survival with a normal aEEG and decreased nitrotyrosine staining in the 2-IB-treated animals, indicating decreased cellular...

Recent progress has provided us with several promising neuroprotective compounds to reduce perinatal hypoxic-ischemic (HI) brain injury. In the early post HI phase, therapies can be concentrated on ion channel blockage (Xenon), anti-oxidation (allopurinol, 2-iminobiotin, and indomethacin), anti-inflammation (erythropoietin [EPO], melatonin), and anti-apoptosis (nuclear factor kappa B [NF-κB]and c-jun N-terminal kinase [JNK] inhibitors); in the later phase, therapies should be targeted to promote neuronal regeneration by stimulation of neurotrophic properties of the neonatal brain (EPO, growth factors, stem cells transplantation). Combination of pharmacological interventions with moderate hypothermia, which is the only established therapy for post HI brain injury, is probably the next step to fight HI brain damage in the clinical setting. Further studies should be concentrated on more rational pharmacological strategies by determining the optimal time and dose to inhibit the various potentially destructive molecular pathways and/or to enhance endogenous repair meanwhile avoiding the adverse effects.

For centuries, zeolites have been used for their utility in binding metals, and they feature in a multitude of agricultural and industrial applications in which the honeycombed zeolite structures form ideal ion exchangers, catalysts and binding agents. Zeolites are currently in a transition period, moving towards implementation in human ailments and diseases. Here, we postulated that zeolites may be able to counter the effects of excess iron and conducted a mouse model trial to gauge the utility of this notion. We used the transgenic mouse strain Mex TA g299 for a thirty‐week pilot trial in which iron polymaltose and/or the zeolite clinoptilolite was injected into the peritoneum twice weekly. Mice were sacrificed at the end of the trial period and examined by postmortem and histology for significant physiological differences between mouse subgroups. In this study, we demonstrated that a common zeolite, clinoptilolite, is able to maintain the general health and well‐being of mice and prevent iron‐induced deleterious effects following iron overload. When zeolites are given with iron biweekly as intraperitoneal injections, mice showed far less macroscopic visual organ discoloration, along with near normal histology, under iron overload conditions when compared to mice injected with iron only. The purpose of the present pilot study was to examine potential alternatives to current iron chelation treatments, and the results indicate an advantage to using zeolites in conditions of iron excess. Zeolites may have translational potential for use in cases of human iron overload.

Malignant mesothelioma (MM) is an almost invariably fatal cancer caused by asbestos exposure. The toxicity of asbestos fibers is related to their physicochemical properties and the generation of free radicals. We set up a pilot study to investigate the potential of the zeolite clinoptilolite to counteract the asbestos carcinogenesis by preventing the generation of reactive nitrogen and oxygen radicals. In cell culture experiments, clinoptilolite prevented asbestos‐induced cell death, reactive oxygen species production, DNA degradation, and overexpression of genes known to be up‐regulated by asbestos. In an asbestos‐induced transgenic mouse model of MM, mice were injected intraperitoneal injections with blue asbestos, with or without clinoptilolite, and monitored for 30 weeks. By the end of the trial all 13 mice injected with asbestos alone had reached humane end points, whereas only 7 of 29 mice receiving crocidolite and clinoptilolite reached a similar stage of disease. Post‐mortem examination revealed pinpoint mesothelioma‐like tumors in affected mice, and the absence of tumor formation in surviving mice. Interestingly, the macrophage clearance system, which was largely suppressed in asbestos‐treated mice, exhibited evidence of increased phagocytosis in mice treated with asbestos and clinoptilolite. Our study suggests that inhibiting the asbestos‐induced generation of reactive oxygen species and stimulating the macrophage system may represent a pathway to amelioration of asbestos‐induced toxicity. Additional studies are warranted to explore the underlying mechanisms responsible for our observations.