Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin.
Background
Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin.Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC 50  of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR) staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect.
Results
Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC 50  of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR) staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect.Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed neuron-astrocyte cultures from heme-mediated oxidative injury. Selective inhibition of neuronal HO-2 may have a beneficial effect after CNS hemorrhage.
Conclusions
These results suggest that HO-2 gene deletion protects neurons in mixed neuron-astrocyte cultures from heme-mediated oxidative injury. Selective inhibition of neuronal HO-2 may have a beneficial effect after CNS hemorrhage.

Background Hemin is a potent oxidant that accumulates in intracranial hematomas and may contribute to neural cell injury [ 1 , 2 ]. It is also the preferred substrate for heme oxygenase-2, the constitutively-expressed isoform that accounts for most CNS heme oxygenase (HO) under normal conditions [ 3 ]. In pathologic states, HO frequently has an antioxidant effect, putatively due to the protection provided by increased cellular bilirubin, decreased heme, and up-regulation of other antioxidants [ 4 - 7 ]. However, in models that are relevant to CNS hemorrhage, HO inhibitors have surprisingly been found to be protective [ 8 - 10 ]. All HO inhibitors that are currently available have numerous non-specific actions that may complicate the interpretation of experimental results, including inhibition of nitric oxide synthase and guanyl cyclase, and modification of voltage-gated calcium currents [ 11 - 14 ]. Some may also have a direct antioxidant effect that is unrelated to HO inhibition [ 15 ]. In order to investigate HO-2 in heme-mediated injury more specifically, we have cultured neurons and astrocytes derived from HO-2 knockout mice and genetically-similar wild type controls. In recent studies, we observed that astrocytes derived from mutant mice were more vulnerable to hemin [ 16 ]. Conversely, HO-2 gene deletion decreased the vulnerability of neurons to hemoglobin [ 17 ]. Neither wild type nor knockout astrocytes were injured by hemoglobin at the micromolar concentrations that are feasible in vitro. HO-2 gene deletion per se did not result in a compensatory increase in HO-1 in these cultures, and produced minimal or no change in other cellular antioxidants [ 16 , 17 ]. The disparate effects of HO-2 gene deletion on hemin toxicity to astrocytes and hemoglobin toxicity to neurons may reflect the inability of neurons to tolerate the products of heme metabolism, i.e. iron, carbon monoxide, and bilirubin. Alternatively, it may reflect the different oxidant properties of hemin and hemoglobin. Although the oxidant effect of hemoglobin may be due in part to hemin release to membrane lipids [ 18 ], other mechanisms may also contribute. Extracellular hemoglobin undergoes autoxidation, which produces superoxide [ 19 ]. In addition to being an oxidant, superoxide reacts with globin amino acids in a complex fashion to generate a variety of reactive species, including thiyl radicals, hydroxyl radicals, and hydrogen peroxide [ 20 , 21 ]. It is also noteworthy that hemoglobin is highly water soluble while hemin is quite lipophilic; their accumulation in separate cellular compartments may lead to a different pattern of site-specific oxidative damage [ 22 , 23 ]. The present study was designed to test the effect of HO-2 gene deletion on the oxidative neuronal injury produced by extracellular hemin. We specifically tested the hypothesis that targeted deletion of the HO-2 gene attenuated oxidative cell injury in a primary cell culture model of hemin toxicity.
Background
Hemin is a potent oxidant that accumulates in intracranial hematomas and may contribute to neural cell injury [ 1 , 2 ]. It is also the preferred substrate for heme oxygenase-2, the constitutively-expressed isoform that accounts for most CNS heme oxygenase (HO) under normal conditions [ 3 ]. In pathologic states, HO frequently has an antioxidant effect, putatively due to the protection provided by increased cellular bilirubin, decreased heme, and up-regulation of other antioxidants [ 4 - 7 ]. However, in models that are relevant to CNS hemorrhage, HO inhibitors have surprisingly been found to be protective [ 8 - 10 ].All HO inhibitors that are currently available have numerous non-specific actions that may complicate the interpretation of experimental results, including inhibition of nitric oxide synthase and guanyl cyclase, and modification of voltage-gated calcium currents [ 11 - 14 ]. Some may also have a direct antioxidant effect that is unrelated to HO inhibition [ 15 ]. In order to investigate HO-2 in heme-mediated injury more specifically, we have cultured neurons and astrocytes derived from HO-2 knockout mice and genetically-similar wild type controls. In recent studies, we observed that astrocytes derived from mutant mice were more vulnerable to hemin [ 16 ]. Conversely, HO-2 gene deletion decreased the vulnerability of neurons to hemoglobin [ 17 ]. Neither wild type nor knockout astrocytes were injured by hemoglobin at the micromolar concentrations that are feasible in vitro. HO-2 gene deletion per se did not result in a compensatory increase in HO-1 in these cultures, and produced minimal or no change in other cellular antioxidants [ 16 , 17 ].The disparate effects of HO-2 gene deletion on hemin toxicity to astrocytes and hemoglobin toxicity to neurons may reflect the inability of neurons to tolerate the products of heme metabolism, i.e. iron, carbon monoxide, and bilirubin. Alternatively, it may reflect the different oxidant properties of hemin and hemoglobin. Although the oxidant effect of hemoglobin may be due in part to hemin release to membrane lipids [ 18 ], other mechanisms may also contribute. Extracellular hemoglobin undergoes autoxidation, which produces superoxide [ 19 ]. In addition to being an oxidant, superoxide reacts with globin amino acids in a complex fashion to generate a variety of reactive species, including thiyl radicals, hydroxyl radicals, and hydrogen peroxide [ 20 , 21 ]. It is also noteworthy that hemoglobin is highly water soluble while hemin is quite lipophilic; their accumulation in separate cellular compartments may lead to a different pattern of site-specific oxidative damage [ 22 , 23 ].The present study was designed to test the effect of HO-2 gene deletion on the oxidative neuronal injury produced by extracellular hemin. We specifically tested the hypothesis that targeted deletion of the HO-2 gene attenuated oxidative cell injury in a primary cell culture model of hemin toxicity.Results Effect of HO-2 gene deletion on hemin neurotoxicity In preliminary experiments, we observed that overnight (14 h) exposure to low micromolar concentrations of hemin consistently produced morphologic evidence of neuronal injury in wild-type cultures (Fig.  1 ). This time interval was therefore used for cytotoxicity studies. Consistent with prior observations in pure astrocyte cultures [ 24 ], no morphologic evidence of injury was observed in the astrocyte monolayer at hemin concentrations up to 10 μM. In order to specifically assess neuronal injury in this study, the concentrations used were limited to this range. In wild-type cultures, cell injury as quantified by LDH release was observed at 1 μM hemin and then increased exponentially, to release of 69.7 ± 8.6% of neuronal LDH at 3 μM (Fig  2A ). The calculated EC 50  was 1.85 μM. LDH release was significantly reduced in knockout cultures subjected to the same treatment. At 3 μM hemin, only 12.6 ± 4.1% of LDH had been released at this time point. Control experiments demonstrated that these low hemin concentrations do not interfere with the LDH assay. Cell death was also quantified by analysis of fluorescence intensity after staining cultures with propidium iodide. Using this method, widespread neuronal death was also observed at 3–10 μM hemin in wild type cultures, and the calculated EC 50  was 1.05 μM. Propidium staining of nuclei was significantly reduced in cultures prepared from HO-2 knockout mice (Fig.  2B ). The maximal fluorescence was produced by exposure to 10 μM hemin, which was 37.0 ± 3.2% of that observed in control sister cultures treated with NMDA to kill all neurons. Effect of HO-2 gene deletion on markers of cell oxidation In order to assess reactive oxygen species formation after hemin exposure, cultures were stained with 20 μM dihydrorhodamine 123 after 4 hour hemin exposure. This time interval was used because it preceded cell lysis, and therefore allowed cell retention of the reduced fluorophore. A marked increase in fluorescence was observed in cultures prepared from wild type mice (Fig.  3 ). This signal was concentrated in neuronal cell bodies and processes. In contrast, fluorescence in cultures prepared from HO-2 knockout mice was minimal, and did not exceed that observed in cultures subjected to medium exchanges only. In order to further investigate the effect of HO-2 gene deletion on oxidative stress produced by hemin, cells were harvested after 4 hour hemin exposure. Protein carbonyl groups (i.e. aldehydes and ketones), which are markers of oxidation, were then derivatized and detected with a dinitrophenylhydrazone antibody. Increased immunoreactivity was apparent in lysates of wild type cultures treated with hemin (Figure  4 ). A prominent band was present at approximately 44 kDa, along with a higher molecular weight smear. At 10 μM hemin, the carbonyl signal intensity in wild type cultures was 2.3-fold higher than in cultures exposed to culture medium only, compared with only 1.3-fold higher in knockout cultures. Hemin neurotoxicity is attenuated by iron chelators Based on our prior observations in astrocytes and neuroblastoma cells [ 24 , 25 ], we hypothesized that the toxic product produced by hemin breakdown in primary murine neurons was iron. In order to test this hypothesis, the effect of iron chelators on hemin neurotoxicity in wild type cultures was assessed. Most cell death, as detected by both LDH release and PI staining, was prevented by concomitant treatment with phenanthroline, a lipid-soluble iron chelator (Fig.  5 ). Deferoxamine, which is water soluble, was less potent; its effect when applied at a concentration tenfold greater than that of hemin reached statistical significance only when injury was assessed by PI staining.
Results
Effect of HO-2 gene deletion on hemin neurotoxicity In preliminary experiments, we observed that overnight (14 h) exposure to low micromolar concentrations of hemin consistently produced morphologic evidence of neuronal injury in wild-type cultures (Fig.  1 ). This time interval was therefore used for cytotoxicity studies. Consistent with prior observations in pure astrocyte cultures [ 24 ], no morphologic evidence of injury was observed in the astrocyte monolayer at hemin concentrations up to 10 μM. In order to specifically assess neuronal injury in this study, the concentrations used were limited to this range. In wild-type cultures, cell injury as quantified by LDH release was observed at 1 μM hemin and then increased exponentially, to release of 69.7 ± 8.6% of neuronal LDH at 3 μM (Fig  2A ). The calculated EC 50  was 1.85 μM. LDH release was significantly reduced in knockout cultures subjected to the same treatment. At 3 μM hemin, only 12.6 ± 4.1% of LDH had been released at this time point. Control experiments demonstrated that these low hemin concentrations do not interfere with the LDH assay. Cell death was also quantified by analysis of fluorescence intensity after staining cultures with propidium iodide. Using this method, widespread neuronal death was also observed at 3–10 μM hemin in wild type cultures, and the calculated EC 50  was 1.05 μM. Propidium staining of nuclei was significantly reduced in cultures prepared from HO-2 knockout mice (Fig.  2B ). The maximal fluorescence was produced by exposure to 10 μM hemin, which was 37.0 ± 3.2% of that observed in control sister cultures treated with NMDA to kill all neurons.
Effect of HO-2 gene deletion on hemin neurotoxicity
In preliminary experiments, we observed that overnight (14 h) exposure to low micromolar concentrations of hemin consistently produced morphologic evidence of neuronal injury in wild-type cultures (Fig.  1 ). This time interval was therefore used for cytotoxicity studies. Consistent with prior observations in pure astrocyte cultures [ 24 ], no morphologic evidence of injury was observed in the astrocyte monolayer at hemin concentrations up to 10 μM. In order to specifically assess neuronal injury in this study, the concentrations used were limited to this range. In wild-type cultures, cell injury as quantified by LDH release was observed at 1 μM hemin and then increased exponentially, to release of 69.7 ± 8.6% of neuronal LDH at 3 μM (Fig  2A ). The calculated EC 50  was 1.85 μM. LDH release was significantly reduced in knockout cultures subjected to the same treatment. At 3 μM hemin, only 12.6 ± 4.1% of LDH had been released at this time point. Control experiments demonstrated that these low hemin concentrations do not interfere with the LDH assay.Cell death was also quantified by analysis of fluorescence intensity after staining cultures with propidium iodide. Using this method, widespread neuronal death was also observed at 3–10 μM hemin in wild type cultures, and the calculated EC 50  was 1.05 μM. Propidium staining of nuclei was significantly reduced in cultures prepared from HO-2 knockout mice (Fig.  2B ). The maximal fluorescence was produced by exposure to 10 μM hemin, which was 37.0 ± 3.2% of that observed in control sister cultures treated with NMDA to kill all neurons.Effect of HO-2 gene deletion on markers of cell oxidation In order to assess reactive oxygen species formation after hemin exposure, cultures were stained with 20 μM dihydrorhodamine 123 after 4 hour hemin exposure. This time interval was used because it preceded cell lysis, and therefore allowed cell retention of the reduced fluorophore. A marked increase in fluorescence was observed in cultures prepared from wild type mice (Fig.  3 ). This signal was concentrated in neuronal cell bodies and processes. In contrast, fluorescence in cultures prepared from HO-2 knockout mice was minimal, and did not exceed that observed in cultures subjected to medium exchanges only. In order to further investigate the effect of HO-2 gene deletion on oxidative stress produced by hemin, cells were harvested after 4 hour hemin exposure. Protein carbonyl groups (i.e. aldehydes and ketones), which are markers of oxidation, were then derivatized and detected with a dinitrophenylhydrazone antibody. Increased immunoreactivity was apparent in lysates of wild type cultures treated with hemin (Figure  4 ). A prominent band was present at approximately 44 kDa, along with a higher molecular weight smear. At 10 μM hemin, the carbonyl signal intensity in wild type cultures was 2.3-fold higher than in cultures exposed to culture medium only, compared with only 1.3-fold higher in knockout cultures.
Effect of HO-2 gene deletion on markers of cell oxidation
In order to assess reactive oxygen species formation after hemin exposure, cultures were stained with 20 μM dihydrorhodamine 123 after 4 hour hemin exposure. This time interval was used because it preceded cell lysis, and therefore allowed cell retention of the reduced fluorophore. A marked increase in fluorescence was observed in cultures prepared from wild type mice (Fig.  3 ). This signal was concentrated in neuronal cell bodies and processes. In contrast, fluorescence in cultures prepared from HO-2 knockout mice was minimal, and did not exceed that observed in cultures subjected to medium exchanges only.In order to further investigate the effect of HO-2 gene deletion on oxidative stress produced by hemin, cells were harvested after 4 hour hemin exposure. Protein carbonyl groups (i.e. aldehydes and ketones), which are markers of oxidation, were then derivatized and detected with a dinitrophenylhydrazone antibody. Increased immunoreactivity was apparent in lysates of wild type cultures treated with hemin (Figure  4 ). A prominent band was present at approximately 44 kDa, along with a higher molecular weight smear. At 10 μM hemin, the carbonyl signal intensity in wild type cultures was 2.3-fold higher than in cultures exposed to culture medium only, compared with only 1.3-fold higher in knockout cultures.Hemin neurotoxicity is attenuated by iron chelators Based on our prior observations in astrocytes and neuroblastoma cells [ 24 , 25 ], we hypothesized that the toxic product produced by hemin breakdown in primary murine neurons was iron. In order to test this hypothesis, the effect of iron chelators on hemin neurotoxicity in wild type cultures was assessed. Most cell death, as detected by both LDH release and PI staining, was prevented by concomitant treatment with phenanthroline, a lipid-soluble iron chelator (Fig.  5 ). Deferoxamine, which is water soluble, was less potent; its effect when applied at a concentration tenfold greater than that of hemin reached statistical significance only when injury was assessed by PI staining.
Hemin neurotoxicity is attenuated by iron chelators
Based on our prior observations in astrocytes and neuroblastoma cells [ 24 , 25 ], we hypothesized that the toxic product produced by hemin breakdown in primary murine neurons was iron. In order to test this hypothesis, the effect of iron chelators on hemin neurotoxicity in wild type cultures was assessed. Most cell death, as detected by both LDH release and PI staining, was prevented by concomitant treatment with phenanthroline, a lipid-soluble iron chelator (Fig.  5 ). Deferoxamine, which is water soluble, was less potent; its effect when applied at a concentration tenfold greater than that of hemin reached statistical significance only when injury was assessed by PI staining.Discussion In prior experiments, we demonstrated that targeted deletion of the HO-2 gene in primary neuron/astrocyte cultures did not alter expression of HO-1, and had little or no effect on other cellular antioxidants [ 17 ]. This simplified system therefore permits investigation of HO-2 without the confounding compensatory effects that have been observed in whole animal models [ 26 , 27 ]. We have previously reported that HO-2 gene deletion increased the vulnerability of astrocytes to hemin, the preferred substrate of HO, in cultures containing only astrocytes. The present study targeted neurons in mixed neuron/astrocyte cultures by using hemin concentrations that did not injure astrocytes [ 24 ]. In this model, the opposite was observed. HO-2 deletion attenuated hemin-induced ROS formation and reduced levels of oxidized proteins. Consistent with the oxidative nature of hemin toxicity, neuronal death was reduced in knockout cultures. Although hemin is a highly reactive pro-oxidant, its breakdown as catalyzed by the heme oxygenases generates biologically active and potentially toxic products. Prior in vitro studies suggest that neurons are particularly vulnerable to these, i.e. iron, carbon monoxide, and bilirubin [ 28 - 30 ]. The present results suggest that when neurons are provided with an excess of HO substrate, the toxicity of breakdown products outweighs any benefit provided by hemin removal. The protective effect of iron chelators suggests that this phenomenon is at least partly due to iron neurotoxicity. Inorganic iron is toxic to cultured cortical neurons, with an EC 50  of approximately 10 μM [ 17 ]. The lower EC 50  for hemin is not surprising, given its lipophilicity and accumulation in cell membranes [ 31 ]. The latter phenomenon likely accounts for the greater efficacy of phenanthroline, which unlike deferoxamine is lipophilic. Deferoxamine is quite effective against hemoglobin neurotoxicity in this culture system [ 32 ], suggesting that hemoglobin releases its iron either in the medium or in an aqueous cellular compartment. The present results are consistent with observations that heme oxygenase inhibitors are protective in models of CNS hemorrhage [ 2 , 8 , 9 ], in contrast to the beneficial effect of HO in ischemia [ 33 ]. In a recent study, Koeppen et al. [ 2 ] observed that repeated administration of tin mesoporphyrin protected thalamic neurons from the delayed degeneration that occurred in tissue adjacent to injected autologous blood. Similarly, Huang et al. [ 9 ] observed that tin protoporphyrin attenuated edema formation after stereotactic hemoglobin injection into the rat striatum. It is noteworthy that the number of astrocytes per neuron is significantly higher in the human brain than in rodents [ 34 ]. The deleterious effect of HO inhibition on heme mediated injury to astrocytes may therefore be less prominent in animal models than in clinical intracerebral hemorrhage [ 35 ]. The disparate effect of HO on neurons and astrocytes exposed to extracellular hemin suggests that it may be somewhat difficult to target it effectively after CNS hemorrhage. All currently available HO inhibitors inhibit both HO-2, which is predominantly neuronal in vivo [ 36 ], and HO-1, which is induced mainly in glial cells [ 37 ]. The protection that these non-selective agents provide to neurons may be negated by their deleterious effect on astrocytes. Further investigation is needed for the development of strategies that would permit the selective inhibition or down-regulation of HO-2 in neurons.
Discussion
In prior experiments, we demonstrated that targeted deletion of the HO-2 gene in primary neuron/astrocyte cultures did not alter expression of HO-1, and had little or no effect on other cellular antioxidants [ 17 ]. This simplified system therefore permits investigation of HO-2 without the confounding compensatory effects that have been observed in whole animal models [ 26 , 27 ]. We have previously reported that HO-2 gene deletion increased the vulnerability of astrocytes to hemin, the preferred substrate of HO, in cultures containing only astrocytes. The present study targeted neurons in mixed neuron/astrocyte cultures by using hemin concentrations that did not injure astrocytes [ 24 ]. In this model, the opposite was observed. HO-2 deletion attenuated hemin-induced ROS formation and reduced levels of oxidized proteins. Consistent with the oxidative nature of hemin toxicity, neuronal death was reduced in knockout cultures.Although hemin is a highly reactive pro-oxidant, its breakdown as catalyzed by the heme oxygenases generates biologically active and potentially toxic products. Prior in vitro studies suggest that neurons are particularly vulnerable to these, i.e. iron, carbon monoxide, and bilirubin [ 28 - 30 ]. The present results suggest that when neurons are provided with an excess of HO substrate, the toxicity of breakdown products outweighs any benefit provided by hemin removal. The protective effect of iron chelators suggests that this phenomenon is at least partly due to iron neurotoxicity. Inorganic iron is toxic to cultured cortical neurons, with an EC 50  of approximately 10 μM [ 17 ]. The lower EC 50  for hemin is not surprising, given its lipophilicity and accumulation in cell membranes [ 31 ]. The latter phenomenon likely accounts for the greater efficacy of phenanthroline, which unlike deferoxamine is lipophilic. Deferoxamine is quite effective against hemoglobin neurotoxicity in this culture system [ 32 ], suggesting that hemoglobin releases its iron either in the medium or in an aqueous cellular compartment.The present results are consistent with observations that heme oxygenase inhibitors are protective in models of CNS hemorrhage [ 2 , 8 , 9 ], in contrast to the beneficial effect of HO in ischemia [ 33 ]. In a recent study, Koeppen et al. [ 2 ] observed that repeated administration of tin mesoporphyrin protected thalamic neurons from the delayed degeneration that occurred in tissue adjacent to injected autologous blood. Similarly, Huang et al. [ 9 ] observed that tin protoporphyrin attenuated edema formation after stereotactic hemoglobin injection into the rat striatum. It is noteworthy that the number of astrocytes per neuron is significantly higher in the human brain than in rodents [ 34 ]. The deleterious effect of HO inhibition on heme mediated injury to astrocytes may therefore be less prominent in animal models than in clinical intracerebral hemorrhage [ 35 ].The disparate effect of HO on neurons and astrocytes exposed to extracellular hemin suggests that it may be somewhat difficult to target it effectively after CNS hemorrhage. All currently available HO inhibitors inhibit both HO-2, which is predominantly neuronal in vivo [ 36 ], and HO-1, which is induced mainly in glial cells [ 37 ]. The protection that these non-selective agents provide to neurons may be negated by their deleterious effect on astrocytes. Further investigation is needed for the development of strategies that would permit the selective inhibition or down-regulation of HO-2 in neurons.Conclusions Targeted deletion of the heme oxygenase-2 gene mitigates oxidative stress in cultured neurons exposed to hemin, and is cytoprotective. Selective inhibition of neuronal heme oxygenase may have a beneficial effect after CNS hemorrhage.
Conclusions
Targeted deletion of the heme oxygenase-2 gene mitigates oxidative stress in cultured neurons exposed to hemin, and is cytoprotective. Selective inhibition of neuronal heme oxygenase may have a beneficial effect after CNS hemorrhage.Methods Cell cultures The HO-2 knockout mice which were used in this study are descended from mutants produced by Poss et al. [ 38 ], and have a C57BL/6 X 129/Sv genetic background. All mice were obtained from our local breeding colony, and were provided with food and water ad libitum and a 12 hour light/dark cycle. All breeding mice were the offspring of heterozygotes. Genotype was determined by polymerase chain reaction (PCR) using genomic DNA isolated from tail clippings; primers were previously published [ 17 ]. Cortical cell cultures were prepared from fetal mice at gestational age 15–17 d as previously described [ 39 ]. Under a dissecting microscope, cortices were dissected free from other brain tissue, minced with forceps, and incubated in medium containing 0.075%-acetylated trypsin at 37°C for one hour. Tissue was then collected by low speed centrifugation, and was dissociated by trituration through a flamed Pasteur pipette in plating medium containing Eagle's minimal essential medium (MEM), 5% fetal bovine serum (Hyclone, Logan, UT), 5% heat inactivated equine serum (Hyclone), glutamine (2 mM), and glucose (23 mM). The cell suspension was diluted with additional plating medium, and cells were plated on confluent astrocyte cultures in 24-well plates (Primaria, Falcon) at a density of 3 hemispheres/plate. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air. Two-thirds of the culture medium was replaced at days 4 and 8 in vitro with MEM containing 10% equine serum, 2 mM glutamine, and 23 mM glucose. After ten days in vitro this feeding procedure was performed daily. Hemin exposure Hemin was freshly prepared as a 1 mM stock solution and was diluted to the desired concentration with minimal essential medium containing 10 mM glucose (MEM10). Experiments were conducted at 12–16 days in vitro. Serum was washed out of cultures with MEM10 (> 1000X dilution) prior to addition of hemin. Cultures were incubated at 37°C in a 5% CO2 atmosphere for the entire exposure interval. Detection of reactive oxygen species ROS formation was quantified by staining with dihydrorhodamine 123 (DHR, Molecular Probes, Eugene, OR), which is a cell-permeable, non-fluorescent compound that is oxidized by cellular peroxides to fluorescent rhodamine [ 40 ]. Fluorescence intensity is directly proportional to cellular oxidative stress. In order to prevent oxidation of DHR by hemin in the medium, cultures were washed free of hemin prior to DHR addition. After incubation with 20 μM DHR in MEM10 for 15 min, the medium was replaced, and cultures were imaged using a Nikon inverted microscope with epifluorescence attachment. Images were captured immediately after illumination (25 msec exposure). Photomicrographs of random 100X fields were analyzed with IPLab image analysis software (Scanalytics, Inc., Fairfax, VA). The low fluorescence in control cultures exposed to experimental medium only was subtracted from mean values to define the signal associated with hemin exposure. Detection of protein oxidation Protein oxidation was assessed using the Oxyblot™ kit (Chemicon, Inc., Temecula, CA). At the end of the hemin exposure interval, culture medium was aspirated, and cells were washed and then harvested in 100 μl of lysis buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1% sodium dodecyl sulfate. Carbonyl groups were derivatized to 2, 4-dintrophenylhydrazone (DNP-hydrazone) by reaction with 2, 4-dinitrophenylhydrazine, following the manufacturer's instructions. Proteins were then separated on a 12% polyacrylamide gel and were transferred onto a polyvinylidene difluoride Imobilon-P transfer membrane filter (Millipore, Billerica, MA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Carbonylated proteins were detected with rabbit anti-DNP (1:150) followed by goat anti-rabbit IgG (1:300). Immunoreactive proteins were visualized using Super Signal West Femto Reagent (Pierce Biotechnology, Rockford, IL) and Kodak ImageStation 400. Quantification of cell death After examination of cultures using phase contrast microscopy, cell death was quantified by measurement of LDH activity in the culture medium, as previously described [ 41 ]. To facilitate comparisons, values were scaled to the mean value in sister cultures exposed to NMDA 300 μM for 40 h. This treatment releases essentially all neuronal LDH in this system without injuring astrocytes [ 42 ]. Since the low micromolar concentrations of hemin that were used in this study do not injure cultured cortical astrocytes [ 24 ], the contribution of astrocyte LDH to the total signal is negligible. Cell death was also quantified by staining with propidium iodide (13 μg/ml for 15 min). When viewed with a rhodamine filter, the nuclei of cells with disrupted membranes stain red, while cells with intact membranes exclude propidium. Random 100X fields were captured with a Nikon Diaphot epifluorescence microscope and were analyzed with IPLab image analysis software. As with LDH data, fluorescence intensity was scaled to that in sister cultures treated with NMDA 300 μM for 40 h, which kills all neurons. Propidium iodide fluorescence was not observed in cells that had an astrocyte phenotype after treatment with NMDA or hemin at the concentrations used in this study.
Methods
Cell cultures The HO-2 knockout mice which were used in this study are descended from mutants produced by Poss et al. [ 38 ], and have a C57BL/6 X 129/Sv genetic background. All mice were obtained from our local breeding colony, and were provided with food and water ad libitum and a 12 hour light/dark cycle. All breeding mice were the offspring of heterozygotes. Genotype was determined by polymerase chain reaction (PCR) using genomic DNA isolated from tail clippings; primers were previously published [ 17 ]. Cortical cell cultures were prepared from fetal mice at gestational age 15–17 d as previously described [ 39 ]. Under a dissecting microscope, cortices were dissected free from other brain tissue, minced with forceps, and incubated in medium containing 0.075%-acetylated trypsin at 37°C for one hour. Tissue was then collected by low speed centrifugation, and was dissociated by trituration through a flamed Pasteur pipette in plating medium containing Eagle's minimal essential medium (MEM), 5% fetal bovine serum (Hyclone, Logan, UT), 5% heat inactivated equine serum (Hyclone), glutamine (2 mM), and glucose (23 mM). The cell suspension was diluted with additional plating medium, and cells were plated on confluent astrocyte cultures in 24-well plates (Primaria, Falcon) at a density of 3 hemispheres/plate. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air. Two-thirds of the culture medium was replaced at days 4 and 8 in vitro with MEM containing 10% equine serum, 2 mM glutamine, and 23 mM glucose. After ten days in vitro this feeding procedure was performed daily.
Cell cultures
The HO-2 knockout mice which were used in this study are descended from mutants produced by Poss et al. [ 38 ], and have a C57BL/6 X 129/Sv genetic background. All mice were obtained from our local breeding colony, and were provided with food and water ad libitum and a 12 hour light/dark cycle. All breeding mice were the offspring of heterozygotes. Genotype was determined by polymerase chain reaction (PCR) using genomic DNA isolated from tail clippings; primers were previously published [ 17 ].Cortical cell cultures were prepared from fetal mice at gestational age 15–17 d as previously described [ 39 ]. Under a dissecting microscope, cortices were dissected free from other brain tissue, minced with forceps, and incubated in medium containing 0.075%-acetylated trypsin at 37°C for one hour. Tissue was then collected by low speed centrifugation, and was dissociated by trituration through a flamed Pasteur pipette in plating medium containing Eagle's minimal essential medium (MEM), 5% fetal bovine serum (Hyclone, Logan, UT), 5% heat inactivated equine serum (Hyclone), glutamine (2 mM), and glucose (23 mM). The cell suspension was diluted with additional plating medium, and cells were plated on confluent astrocyte cultures in 24-well plates (Primaria, Falcon) at a density of 3 hemispheres/plate. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air. Two-thirds of the culture medium was replaced at days 4 and 8 in vitro with MEM containing 10% equine serum, 2 mM glutamine, and 23 mM glucose. After ten days in vitro this feeding procedure was performed daily.Hemin exposure Hemin was freshly prepared as a 1 mM stock solution and was diluted to the desired concentration with minimal essential medium containing 10 mM glucose (MEM10). Experiments were conducted at 12–16 days in vitro. Serum was washed out of cultures with MEM10 (> 1000X dilution) prior to addition of hemin. Cultures were incubated at 37°C in a 5% CO2 atmosphere for the entire exposure interval.
Hemin exposure
Hemin was freshly prepared as a 1 mM stock solution and was diluted to the desired concentration with minimal essential medium containing 10 mM glucose (MEM10). Experiments were conducted at 12–16 days in vitro. Serum was washed out of cultures with MEM10 (> 1000X dilution) prior to addition of hemin. Cultures were incubated at 37°C in a 5% CO2 atmosphere for the entire exposure interval.Detection of reactive oxygen species ROS formation was quantified by staining with dihydrorhodamine 123 (DHR, Molecular Probes, Eugene, OR), which is a cell-permeable, non-fluorescent compound that is oxidized by cellular peroxides to fluorescent rhodamine [ 40 ]. Fluorescence intensity is directly proportional to cellular oxidative stress. In order to prevent oxidation of DHR by hemin in the medium, cultures were washed free of hemin prior to DHR addition. After incubation with 20 μM DHR in MEM10 for 15 min, the medium was replaced, and cultures were imaged using a Nikon inverted microscope with epifluorescence attachment. Images were captured immediately after illumination (25 msec exposure). Photomicrographs of random 100X fields were analyzed with IPLab image analysis software (Scanalytics, Inc., Fairfax, VA). The low fluorescence in control cultures exposed to experimental medium only was subtracted from mean values to define the signal associated with hemin exposure.
Detection of reactive oxygen species
ROS formation was quantified by staining with dihydrorhodamine 123 (DHR, Molecular Probes, Eugene, OR), which is a cell-permeable, non-fluorescent compound that is oxidized by cellular peroxides to fluorescent rhodamine [ 40 ]. Fluorescence intensity is directly proportional to cellular oxidative stress. In order to prevent oxidation of DHR by hemin in the medium, cultures were washed free of hemin prior to DHR addition. After incubation with 20 μM DHR in MEM10 for 15 min, the medium was replaced, and cultures were imaged using a Nikon inverted microscope with epifluorescence attachment. Images were captured immediately after illumination (25 msec exposure). Photomicrographs of random 100X fields were analyzed with IPLab image analysis software (Scanalytics, Inc., Fairfax, VA). The low fluorescence in control cultures exposed to experimental medium only was subtracted from mean values to define the signal associated with hemin exposure.Detection of protein oxidation Protein oxidation was assessed using the Oxyblot™ kit (Chemicon, Inc., Temecula, CA). At the end of the hemin exposure interval, culture medium was aspirated, and cells were washed and then harvested in 100 μl of lysis buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1% sodium dodecyl sulfate. Carbonyl groups were derivatized to 2, 4-dintrophenylhydrazone (DNP-hydrazone) by reaction with 2, 4-dinitrophenylhydrazine, following the manufacturer's instructions. Proteins were then separated on a 12% polyacrylamide gel and were transferred onto a polyvinylidene difluoride Imobilon-P transfer membrane filter (Millipore, Billerica, MA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Carbonylated proteins were detected with rabbit anti-DNP (1:150) followed by goat anti-rabbit IgG (1:300). Immunoreactive proteins were visualized using Super Signal West Femto Reagent (Pierce Biotechnology, Rockford, IL) and Kodak ImageStation 400.
Detection of protein oxidation
Protein oxidation was assessed using the Oxyblot™ kit (Chemicon, Inc., Temecula, CA). At the end of the hemin exposure interval, culture medium was aspirated, and cells were washed and then harvested in 100 μl of lysis buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1% sodium dodecyl sulfate. Carbonyl groups were derivatized to 2, 4-dintrophenylhydrazone (DNP-hydrazone) by reaction with 2, 4-dinitrophenylhydrazine, following the manufacturer's instructions. Proteins were then separated on a 12% polyacrylamide gel and were transferred onto a polyvinylidene difluoride Imobilon-P transfer membrane filter (Millipore, Billerica, MA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Carbonylated proteins were detected with rabbit anti-DNP (1:150) followed by goat anti-rabbit IgG (1:300). Immunoreactive proteins were visualized using Super Signal West Femto Reagent (Pierce Biotechnology, Rockford, IL) and Kodak ImageStation 400.Quantification of cell death After examination of cultures using phase contrast microscopy, cell death was quantified by measurement of LDH activity in the culture medium, as previously described [ 41 ]. To facilitate comparisons, values were scaled to the mean value in sister cultures exposed to NMDA 300 μM for 40 h. This treatment releases essentially all neuronal LDH in this system without injuring astrocytes [ 42 ]. Since the low micromolar concentrations of hemin that were used in this study do not injure cultured cortical astrocytes [ 24 ], the contribution of astrocyte LDH to the total signal is negligible. Cell death was also quantified by staining with propidium iodide (13 μg/ml for 15 min). When viewed with a rhodamine filter, the nuclei of cells with disrupted membranes stain red, while cells with intact membranes exclude propidium. Random 100X fields were captured with a Nikon Diaphot epifluorescence microscope and were analyzed with IPLab image analysis software. As with LDH data, fluorescence intensity was scaled to that in sister cultures treated with NMDA 300 μM for 40 h, which kills all neurons. Propidium iodide fluorescence was not observed in cells that had an astrocyte phenotype after treatment with NMDA or hemin at the concentrations used in this study.
Quantification of cell death
After examination of cultures using phase contrast microscopy, cell death was quantified by measurement of LDH activity in the culture medium, as previously described [ 41 ]. To facilitate comparisons, values were scaled to the mean value in sister cultures exposed to NMDA 300 μM for 40 h. This treatment releases essentially all neuronal LDH in this system without injuring astrocytes [ 42 ]. Since the low micromolar concentrations of hemin that were used in this study do not injure cultured cortical astrocytes [ 24 ], the contribution of astrocyte LDH to the total signal is negligible.Cell death was also quantified by staining with propidium iodide (13 μg/ml for 15 min). When viewed with a rhodamine filter, the nuclei of cells with disrupted membranes stain red, while cells with intact membranes exclude propidium. Random 100X fields were captured with a Nikon Diaphot epifluorescence microscope and were analyzed with IPLab image analysis software. As with LDH data, fluorescence intensity was scaled to that in sister cultures treated with NMDA 300 μM for 40 h, which kills all neurons. Propidium iodide fluorescence was not observed in cells that had an astrocyte phenotype after treatment with NMDA or hemin at the concentrations used in this study.Abbreviations DHR: dihydrorhodamine; DNP: dinitrophenylhydrazone; HO: heme oxygenase; LDH: lactate dehydrogenase; MEM10: minimal essential medium containing 10 mM glucose; NMDA: N-methyl-D-aspartate; PI: propidium iodide; ROS: reactive oxygen species.
Abbreviations
DHR: dihydrorhodamine; DNP: dinitrophenylhydrazone; HO: heme oxygenase; LDH: lactate dehydrogenase; MEM10: minimal essential medium containing 10 mM glucose; NMDA: N-methyl-D-aspartate; PI: propidium iodide; ROS: reactive oxygen species.Authors' contributions RFR designed the study, collected and analyzed data, and wrote the manuscript. JC also participated in data collection and analysis, and edited the manuscript. LBZ participated in genotyping and edited the manuscript. All authors reviewed and approved the final manuscript.
Authors' contributions
RFR designed the study, collected and analyzed data, and wrote the manuscript. JC also participated in data collection and analysis, and edited the manuscript. LBZ participated in genotyping and edited the manuscript. All authors reviewed and approved the final manuscript.


Background A circadian rhythm of symptoms has been reported in allergic rhinitis and some studies have shown the dosing time of antihistamines to be of importance for optimizing symptom relief in this disease. The objective of this study was to examine the efficacy of morning vs. evening dosing of the antihistamine desloratadine at different time points during the day.
Background
A circadian rhythm of symptoms has been reported in allergic rhinitis and some studies have shown the dosing time of antihistamines to be of importance for optimizing symptom relief in this disease. The objective of this study was to examine the efficacy of morning vs. evening dosing of the antihistamine desloratadine at different time points during the day.Methods Patients ≥ 18 years, with seasonal allergic rhinitis received desloratadine 5 mg orally once daily in the morning (AM-group) or evening (PM-group) for two weeks. Rhinorrhea, nasal congestion, sneezing and eye symptoms were scored morning and evening. Wilcoxon rank sum and 2-way ANOVA test were used.
Methods
Patients ≥ 18 years, with seasonal allergic rhinitis received desloratadine 5 mg orally once daily in the morning (AM-group) or evening (PM-group) for two weeks. Rhinorrhea, nasal congestion, sneezing and eye symptoms were scored morning and evening. Wilcoxon rank sum and 2-way ANOVA test were used.Results Six-hundred and sixty-three patients were randomized; 336 in the AM-group; 327 in the PM-group. No statistically significant differences were seen between the AM and PM group at any time points. In the sub-groups with higher morning or evening total symptom score no difference in treatment efficacy was seen whether the dose was taken 12 or 24 hours before the higher score time. There was a circadian variation in baseline total symptom score; highest during daytime and lowest at night. The circadian variation in symptoms was reduced during treatment. This reduction was highest for daytime symptoms.
Results
Six-hundred and sixty-three patients were randomized; 336 in the AM-group; 327 in the PM-group. No statistically significant differences were seen between the AM and PM group at any time points. In the sub-groups with higher morning or evening total symptom score no difference in treatment efficacy was seen whether the dose was taken 12 or 24 hours before the higher score time. There was a circadian variation in baseline total symptom score; highest during daytime and lowest at night. The circadian variation in symptoms was reduced during treatment. This reduction was highest for daytime symptoms.Conclusions A circadian rhythm was seen for most symptoms being more pronounced during daytime. This was less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time.
Conclusions
A circadian rhythm was seen for most symptoms being more pronounced during daytime. This was less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time.

Background Allergic rhinitis is a common illness, which affects approximately 15 % of the population [ 1 ] and has a large impact on the quality of life of the patients. In some studies the symptoms of allergic rhinitis have shown a circadian rhythm with morning symptoms being most prominent in a majority of patients [ 1 - 6 ]. Antihistamines are important medications in the treatment of allergic rhinitis. One should expect that the effect of an antihistamine is best near or shortly after peak serum level is attained. If this also coincides with the peak in allergy symptoms, an optimal treatment effect should be expected. In one study evening dosing of the antihistamine mequitazine (half-life of 38–45 hours and time to peak serum level about 6 hours) gave better symptom relief than morning dosing on morning symptoms [ 4 , 7 ]. Desloratadine has as mequitazine a rather long half life of 27 hours, and the time to peak serum level at about 3 hours [ 8 ]. Evening dosing of this antihistamine may be expected to give better symptom relief than morning dosing on peak morning symptoms. Some studies have also confirmed a circadian variation in efficacy of some antihistamines on histamine induced skin reactions [ 9 , 10 ]. The aim of this study was to examine the efficacy of the antihistamine desloratadine at different time points during the day and to evaluate whether the time of dosing of desloratadine has any impact on the treatment efficacy in seasonal allergic rhinitis (SAR).
Background
Allergic rhinitis is a common illness, which affects approximately 15 % of the population [ 1 ] and has a large impact on the quality of life of the patients. In some studies the symptoms of allergic rhinitis have shown a circadian rhythm with morning symptoms being most prominent in a majority of patients [ 1 - 6 ].Antihistamines are important medications in the treatment of allergic rhinitis. One should expect that the effect of an antihistamine is best near or shortly after peak serum level is attained. If this also coincides with the peak in allergy symptoms, an optimal treatment effect should be expected. In one study evening dosing of the antihistamine mequitazine (half-life of 38–45 hours and time to peak serum level about 6 hours) gave better symptom relief than morning dosing on morning symptoms [ 4 , 7 ]. Desloratadine has as mequitazine a rather long half life of 27 hours, and the time to peak serum level at about 3 hours [ 8 ]. Evening dosing of this antihistamine may be expected to give better symptom relief than morning dosing on peak morning symptoms. Some studies have also confirmed a circadian variation in efficacy of some antihistamines on histamine induced skin reactions [ 9 , 10 ].The aim of this study was to examine the efficacy of the antihistamine desloratadine at different time points during the day and to evaluate whether the time of dosing of desloratadine has any impact on the treatment efficacy in seasonal allergic rhinitis (SAR).Methods This was a randomized, open label, parallel group, multicenter study of two weeks duration in patients with SAR during the birch or grass pollen season. Eighty one medical centers in the Nordic countries participated. The inclusion criteria were: patients 18 years or above with a minimum of two years history of SAR confirmed by either a positive skin prick test or a positive serologic allergen test to the relevant seasonal allergen; clinically symptomatic with SAR at baseline/inclusion with a minimum total nasal symptom score (rhinorrhea, congestion, itching and sneezing) of at least 6 and rhinorrhea being minimum 2 (moderate); willingness to adhere to dosing and visit schedule. Females of childbearing potential had to use medically accepted methods of birth control and written informed consent had to be obtained from all patients. The exclusion criteria were: pulmonary disease, perennial rhinitis, sinusitis, rhinitis medicamentosa, pollen desensitization during the last 6 months, respiratory tract infection within the last two weeks, structural nasal abnormalities (including polyps), use of oral, nasal, ocular decongestants, corticosteroids in any form (except mild dermatological group I corticosteroids allowed in only small areas), other antihistamines (oral or topical), any investigational drug during the last 30 days, pregnant or nursing females. The patients were randomized into one of two treatment groups with dosing of 5 mg desloratadine tablets either in the morning between 07 – 09 (AM-group) or evening between 19 – 21 (PM-group) in a 1:1 ratio. Randomizing was computer generated for the whole study population using SAS version 6.12 and performed in blocks of eight. Each subject unit (bottle with medication) was labelled with randomization number. Physicians in the different Nordic countries recruited the patients. They assigned the medication in consecutive order. The study was monitored by Schering-Plough. The following symptoms were assessed using a scale from 0 to 3 (0=none, 1=mild, 2=moderate, 3=severe): rhinorrhea, nasal congestion, sneezing, itching nose and eye symptoms (itching, burning, tearing, redness). These symptoms were recorded in a patient diary every morning (AM 12 hours reflective and AM last hour) and evening (PM 12 hours reflective and PM last hour) both at baseline and during the 2 weeks treatment period. Interference with sleep and interference with daily activity were also assessed by the patients every day using the same scale from 0 to 3. In addition, the number of hours spent outdoors was recorded. Visit 1 was at day 0 at the start of baseline, visit 2 after one week and visit 3 after two weeks. A wash-out period prior to Visit 1 was necessary if the patient had been on any drugs which could interfere with the study results (e.g. no other commonly used antihistamines allowed during the prior 10 days). Baseline symptoms were recorded in the evening at day 0 and the following morning (day 1) after which the patients started taking the study medication as randomized. A physical examination was performed at visit 1 and 3. All adverse events were recorded. The study period was from April 11 th  2001 to September 2 nd  2002. Pollen counts were not recorded. The primary objective was to evaluate the efficacy of 5 mg desloratadine taken orally once daily in the morning versus evening. The primary efficacy variable was the mean change from baseline for the AM last hour Total Symptom Score (TSS) over the 2 weeks treatment period. TSS is the sum of the individual symptom scores for the following symptoms that in prior studies [ 2 , 3 ] have shown a circadian rhythm: rhinorrhea, nasal stuffiness/congestion, sneezing and eye symptoms (maximum score 12). Since nasal itching had shown little circadian rhythm in these studies, this symptom was omitted from the TSS. AM last hour was chosen as primary time point since the symptoms had in the same studies shown to be worst in the morning. The study was designed to enrol 700 patients in order to have 600 evaluable patients. This sample size was chosen to detect with 90 % power and 5 % significance level, a difference between treatment groups of 0.6 units or more in mean change from baseline diary TSS, assuming a pooled SD of 2.25 units. In a study on morning vs. evening dosing of the antihistamine mequitazine the differences in dosing-time-related efficacy increased in the sub-group of patients having predominantly morning symptoms [ 4 ]. A sub-group analysis was therefore performed on patients with a higher TSS (≥ 1 point difference at baseline) in the morning (AM last hour) than in the evening (PM last hour) and patients with higher TSS in the evening than in the morning in this study. A comparison was then done on the treatment efficacy seen 12 hours and 24 hours after dosing (AM vs. PM dosing) in these patients. All patients receiving at least one dose of study drug and having at least one post dose registration were included in the efficacy analysis (intention-to-treat, ITT), and confirmatory analysis were based on evaluable patients with no protocol violations. Statistical analyses were made with 2-way ANOVA. For evaluation of response of therapy Wilcoxon rank sum test was used. Adverse events were tabulated. The study protocol and the patient informed consent form were approved by Ethics Committees and Health Authorities in each of the participating countries.
Methods
This was a randomized, open label, parallel group, multicenter study of two weeks duration in patients with SAR during the birch or grass pollen season. Eighty one medical centers in the Nordic countries participated. The inclusion criteria were: patients 18 years or above with a minimum of two years history of SAR confirmed by either a positive skin prick test or a positive serologic allergen test to the relevant seasonal allergen; clinically symptomatic with SAR at baseline/inclusion with a minimum total nasal symptom score (rhinorrhea, congestion, itching and sneezing) of at least 6 and rhinorrhea being minimum 2 (moderate); willingness to adhere to dosing and visit schedule. Females of childbearing potential had to use medically accepted methods of birth control and written informed consent had to be obtained from all patients.The exclusion criteria were: pulmonary disease, perennial rhinitis, sinusitis, rhinitis medicamentosa, pollen desensitization during the last 6 months, respiratory tract infection within the last two weeks, structural nasal abnormalities (including polyps), use of oral, nasal, ocular decongestants, corticosteroids in any form (except mild dermatological group I corticosteroids allowed in only small areas), other antihistamines (oral or topical), any investigational drug during the last 30 days, pregnant or nursing females.The patients were randomized into one of two treatment groups with dosing of 5 mg desloratadine tablets either in the morning between 07 – 09 (AM-group) or evening between 19 – 21 (PM-group) in a 1:1 ratio. Randomizing was computer generated for the whole study population using SAS version 6.12 and performed in blocks of eight. Each subject unit (bottle with medication) was labelled with randomization number. Physicians in the different Nordic countries recruited the patients. They assigned the medication in consecutive order. The study was monitored by Schering-Plough.The following symptoms were assessed using a scale from 0 to 3 (0=none, 1=mild, 2=moderate, 3=severe): rhinorrhea, nasal congestion, sneezing, itching nose and eye symptoms (itching, burning, tearing, redness). These symptoms were recorded in a patient diary every morning (AM 12 hours reflective and AM last hour) and evening (PM 12 hours reflective and PM last hour) both at baseline and during the 2 weeks treatment period. Interference with sleep and interference with daily activity were also assessed by the patients every day using the same scale from 0 to 3. In addition, the number of hours spent outdoors was recorded.Visit 1 was at day 0 at the start of baseline, visit 2 after one week and visit 3 after two weeks. A wash-out period prior to Visit 1 was necessary if the patient had been on any drugs which could interfere with the study results (e.g. no other commonly used antihistamines allowed during the prior 10 days). Baseline symptoms were recorded in the evening at day 0 and the following morning (day 1) after which the patients started taking the study medication as randomized. A physical examination was performed at visit 1 and 3. All adverse events were recorded. The study period was from April 11 th  2001 to September 2 nd  2002. Pollen counts were not recorded.The primary objective was to evaluate the efficacy of 5 mg desloratadine taken orally once daily in the morning versus evening. The primary efficacy variable was the mean change from baseline for the AM last hour Total Symptom Score (TSS) over the 2 weeks treatment period. TSS is the sum of the individual symptom scores for the following symptoms that in prior studies [ 2 , 3 ] have shown a circadian rhythm: rhinorrhea, nasal stuffiness/congestion, sneezing and eye symptoms (maximum score 12). Since nasal itching had shown little circadian rhythm in these studies, this symptom was omitted from the TSS. AM last hour was chosen as primary time point since the symptoms had in the same studies shown to be worst in the morning. The study was designed to enrol 700 patients in order to have 600 evaluable patients. This sample size was chosen to detect with 90 % power and 5 % significance level, a difference between treatment groups of 0.6 units or more in mean change from baseline diary TSS, assuming a pooled SD of 2.25 units.In a study on morning vs. evening dosing of the antihistamine mequitazine the differences in dosing-time-related efficacy increased in the sub-group of patients having predominantly morning symptoms [ 4 ]. A sub-group analysis was therefore performed on patients with a higher TSS (≥ 1 point difference at baseline) in the morning (AM last hour) than in the evening (PM last hour) and patients with higher TSS in the evening than in the morning in this study. A comparison was then done on the treatment efficacy seen 12 hours and 24 hours after dosing (AM vs. PM dosing) in these patients.All patients receiving at least one dose of study drug and having at least one post dose registration were included in the efficacy analysis (intention-to-treat, ITT), and confirmatory analysis were based on evaluable patients with no protocol violations. Statistical analyses were made with 2-way ANOVA. For evaluation of response of therapy Wilcoxon rank sum test was used. Adverse events were tabulated.The study protocol and the patient informed consent form were approved by Ethics Committees and Health Authorities in each of the participating countries.Results Patients Six hundred and sixty-three patients were randomized at baseline; 336 to the AM-group and 327 to the PM-group. The two groups were comparable with respect to demographics and baseline characteristics (Tab 1). To assess the primary parameter 310 in the AM and 294 in the PM group fulfilled the criteria for ITT. Of the AM group 259 and of the PM group 254 patients completed the study without any violation. Mean baseline TSS varied between 4.64 and 6.10, a difference of 31%; highest during daytime (PM 12 hours reflective) and lowest at night (AM 12 hours reflective). The circadian variation at baseline was more evident for sneezing (around 60% difference between night and day), rhinorrhea and eye symptoms, less so for nasal itching and hardly noticeable for nasal congestion. Fig.  1  shows total and individual symptom scores at baseline and during two weeks treatment. The circadian variation was much less apparent during treatment with desloratadine (Fig.  1 ). Figure 1 Symptom scores.  Total and individual symptom scores at baseline and over two weeks treatment period  Baseline:  ○ AM-group; Δ PM-group,  Treatment:  ● AM-group; ▲ PM-group  1  Max score = 12,  2  Max score = 3 Efficacy During the two weeks period the mean reduction in TSS ± SE for AM last hour (primary efficacy variable) was 1.63 ± 0.17 (30 %) for the AM-group and 1.80 ± 0.17 (35 %) for the PM-group. There was no statistically significant difference (ITT-analysis) between the groups at this time point (p = 0.456) or at any other time points. The reduction in TSS was highest (2.5 – 41%) for day time symptoms (PM previous 12 hours) and lowest at night. This was evident for all individual symptoms except for nasal congestion. In the subgroup analysis comparing TSS AM last hour and PM last hour at baseline, 32 % of the patients had more severe symptoms in the morning (≥ 1 point difference in TSS) than in the evening, and 37 % had more severe symptoms in the evening. Looking at these two sub-groups, no difference in treatment efficacy on TSS was seen 12 or 24 hours post dosing (Fig.  2 ). Figure 2 Sub-group Total Symptom Score.  These sub-groups consists of patients with higher (one or more score points) morning TSS (AM last hour) than evening TSS (PM last hour) at baseline and of patients with higher evening TSS (PM last hour) than morning TSS (AM last hour) at baseline. There was no statistical significant difference in the treatment efficacy between the AM-group and the PM-group. According to their diaries the patients spent in average more than 3.5 hours outdoors daily. The score for the interference of SAR on the patients' sleep and daily activity at baseline and throughout the study is shown in Fig.  3 . Figure 3 Sleep and daily activity.  The score for interference with sleep and daily activity at baseline and during treatment shows that there is a higher interference with daily activity at baseline and during treatment than with sleep. Safety The incidence of treatment related adverse events were comparable between the groups, 20 % in the AM-group and 18 % in the PM-group, headache being most frequent, 7 % and 4 % respectively.
Results
Patients Six hundred and sixty-three patients were randomized at baseline; 336 to the AM-group and 327 to the PM-group. The two groups were comparable with respect to demographics and baseline characteristics (Tab 1). To assess the primary parameter 310 in the AM and 294 in the PM group fulfilled the criteria for ITT. Of the AM group 259 and of the PM group 254 patients completed the study without any violation. Mean baseline TSS varied between 4.64 and 6.10, a difference of 31%; highest during daytime (PM 12 hours reflective) and lowest at night (AM 12 hours reflective). The circadian variation at baseline was more evident for sneezing (around 60% difference between night and day), rhinorrhea and eye symptoms, less so for nasal itching and hardly noticeable for nasal congestion. Fig.  1  shows total and individual symptom scores at baseline and during two weeks treatment. The circadian variation was much less apparent during treatment with desloratadine (Fig.  1 ). Figure 1 Symptom scores.  Total and individual symptom scores at baseline and over two weeks treatment period  Baseline:  ○ AM-group; Δ PM-group,  Treatment:  ● AM-group; ▲ PM-group  1  Max score = 12,  2  Max score = 3
Patients
Six hundred and sixty-three patients were randomized at baseline; 336 to the AM-group and 327 to the PM-group. The two groups were comparable with respect to demographics and baseline characteristics (Tab 1). To assess the primary parameter 310 in the AM and 294 in the PM group fulfilled the criteria for ITT. Of the AM group 259 and of the PM group 254 patients completed the study without any violation. Mean baseline TSS varied between 4.64 and 6.10, a difference of 31%; highest during daytime (PM 12 hours reflective) and lowest at night (AM 12 hours reflective). The circadian variation at baseline was more evident for sneezing (around 60% difference between night and day), rhinorrhea and eye symptoms, less so for nasal itching and hardly noticeable for nasal congestion. Fig.  1  shows total and individual symptom scores at baseline and during two weeks treatment. The circadian variation was much less apparent during treatment with desloratadine (Fig.  1 ).Efficacy During the two weeks period the mean reduction in TSS ± SE for AM last hour (primary efficacy variable) was 1.63 ± 0.17 (30 %) for the AM-group and 1.80 ± 0.17 (35 %) for the PM-group. There was no statistically significant difference (ITT-analysis) between the groups at this time point (p = 0.456) or at any other time points. The reduction in TSS was highest (2.5 – 41%) for day time symptoms (PM previous 12 hours) and lowest at night. This was evident for all individual symptoms except for nasal congestion. In the subgroup analysis comparing TSS AM last hour and PM last hour at baseline, 32 % of the patients had more severe symptoms in the morning (≥ 1 point difference in TSS) than in the evening, and 37 % had more severe symptoms in the evening. Looking at these two sub-groups, no difference in treatment efficacy on TSS was seen 12 or 24 hours post dosing (Fig.  2 ). Figure 2 Sub-group Total Symptom Score.  These sub-groups consists of patients with higher (one or more score points) morning TSS (AM last hour) than evening TSS (PM last hour) at baseline and of patients with higher evening TSS (PM last hour) than morning TSS (AM last hour) at baseline. There was no statistical significant difference in the treatment efficacy between the AM-group and the PM-group. According to their diaries the patients spent in average more than 3.5 hours outdoors daily. The score for the interference of SAR on the patients' sleep and daily activity at baseline and throughout the study is shown in Fig.  3 . Figure 3 Sleep and daily activity.  The score for interference with sleep and daily activity at baseline and during treatment shows that there is a higher interference with daily activity at baseline and during treatment than with sleep.
Efficacy
During the two weeks period the mean reduction in TSS ± SE for AM last hour (primary efficacy variable) was 1.63 ± 0.17 (30 %) for the AM-group and 1.80 ± 0.17 (35 %) for the PM-group. There was no statistically significant difference (ITT-analysis) between the groups at this time point (p = 0.456) or at any other time points. The reduction in TSS was highest (2.5 – 41%) for day time symptoms (PM previous 12 hours) and lowest at night. This was evident for all individual symptoms except for nasal congestion.In the subgroup analysis comparing TSS AM last hour and PM last hour at baseline, 32 % of the patients had more severe symptoms in the morning (≥ 1 point difference in TSS) than in the evening, and 37 % had more severe symptoms in the evening. Looking at these two sub-groups, no difference in treatment efficacy on TSS was seen 12 or 24 hours post dosing (Fig.  2 ).According to their diaries the patients spent in average more than 3.5 hours outdoors daily. The score for the interference of SAR on the patients' sleep and daily activity at baseline and throughout the study is shown in Fig.  3 .Safety The incidence of treatment related adverse events were comparable between the groups, 20 % in the AM-group and 18 % in the PM-group, headache being most frequent, 7 % and 4 % respectively.
Safety
The incidence of treatment related adverse events were comparable between the groups, 20 % in the AM-group and 18 % in the PM-group, headache being most frequent, 7 % and 4 % respectively.Discussion This study was randomized but without a placebo control. Since this study was a comparison between two different dosing times of the same medication, a placebo control was superfluous. The study was not blinded as there is no reason to believe that neither the patients nor the physicians should have a biased opinion as to the time of dosing. To blind such a study, the patients need to take study medication from different boxes in the morning and evening. However, this method was not used since this may complicate the study and impair patient compliance. A circadian rhythm has been found in many diseases, also in allergic rhinitis [ 1 - 6 ]. The effect of an antihistamine may be modulated [ 9 - 13 ] by variations in allergen exposure, hormonal activity, organ sensitivity and plasma concentration of the drug. In this study we have shown that desloratadine maintains its effect at different time points throughout the day and thus the effect appears unaffected by a modulating factor. The baseline period in this study lasted 24 hours which is the same as in the study on mequitazine [ 4 ] and other studies [ 16 , 17 ]. In some studies of the effect of antihistamines the baseline period has been longer [ 14 , 15 ]. It would have been difficult to keep patients in the Nordic countries off medication for more than one day in addition to any washout period during the pollen season. We do not believe that the duration of baseline influenced the results of this comparative study. The circadian rhythm at baseline found in this study with maximum symptoms during the day differs from some other studies [ 1 - 6 ] where more patients had the most severe symptoms in the morning. This difference may partly be due to patient selection. Patients with perennial rhinitis were excluded from our study. Thus indoor allergens do not influence symptom variation. The patients spent several hours outdoors during the day in the pollen season. It seems likely that this exposure would influence the symptoms. The circadian variation was not apparent during treatment, possibly because the suppression of symptoms by desloratadine is more observable when symptoms are most prominent. The best effect of mequitazine was obtained after evening dosing (12 hours before peak of symptoms) compared to morning dosing (24 hours before peak of symptoms). In our study no difference in treatment efficacy was seen 12 or 24 hours after dosing in the sub-group analysis of patients with higher baseline morning or evening TSS. Whatever the cause for this discrepancy between these two antihistamines, other antihistamines may show a variation in effect during the day not only on dermal symptoms [ 9 , 12 ] but also on nasal ones. Thus studies on the effect of other antihistamines in allergic rhinitis should be encouraged. The adverse events recorded were of a magnitude and nature as seen in other studies of desloratadine and other antihistamines [ 14 - 17 ]. Many patients have circadian variations in symptoms. The peak of symptoms can be at different time points from patient to patient. Individual dosing time of medication may improve symptom relief. Desloratadine, however, apparently shows no circadian variation in effect.
Discussion
This study was randomized but without a placebo control. Since this study was a comparison between two different dosing times of the same medication, a placebo control was superfluous. The study was not blinded as there is no reason to believe that neither the patients nor the physicians should have a biased opinion as to the time of dosing. To blind such a study, the patients need to take study medication from different boxes in the morning and evening. However, this method was not used since this may complicate the study and impair patient compliance.A circadian rhythm has been found in many diseases, also in allergic rhinitis [ 1 - 6 ]. The effect of an antihistamine may be modulated [ 9 - 13 ] by variations in allergen exposure, hormonal activity, organ sensitivity and plasma concentration of the drug. In this study we have shown that desloratadine maintains its effect at different time points throughout the day and thus the effect appears unaffected by a modulating factor.The baseline period in this study lasted 24 hours which is the same as in the study on mequitazine [ 4 ] and other studies [ 16 , 17 ]. In some studies of the effect of antihistamines the baseline period has been longer [ 14 , 15 ]. It would have been difficult to keep patients in the Nordic countries off medication for more than one day in addition to any washout period during the pollen season. We do not believe that the duration of baseline influenced the results of this comparative study.The circadian rhythm at baseline found in this study with maximum symptoms during the day differs from some other studies [ 1 - 6 ] where more patients had the most severe symptoms in the morning. This difference may partly be due to patient selection. Patients with perennial rhinitis were excluded from our study. Thus indoor allergens do not influence symptom variation. The patients spent several hours outdoors during the day in the pollen season. It seems likely that this exposure would influence the symptoms. The circadian variation was not apparent during treatment, possibly because the suppression of symptoms by desloratadine is more observable when symptoms are most prominent.The best effect of mequitazine was obtained after evening dosing (12 hours before peak of symptoms) compared to morning dosing (24 hours before peak of symptoms). In our study no difference in treatment efficacy was seen 12 or 24 hours after dosing in the sub-group analysis of patients with higher baseline morning or evening TSS. Whatever the cause for this discrepancy between these two antihistamines, other antihistamines may show a variation in effect during the day not only on dermal symptoms [ 9 , 12 ] but also on nasal ones. Thus studies on the effect of other antihistamines in allergic rhinitis should be encouraged.The adverse events recorded were of a magnitude and nature as seen in other studies of desloratadine and other antihistamines [ 14 - 17 ].Many patients have circadian variations in symptoms. The peak of symptoms can be at different time points from patient to patient. Individual dosing time of medication may improve symptom relief. Desloratadine, however, apparently shows no circadian variation in effect.Conclusions A circadian rhythm was seen for most SAR symptoms at baseline, being most distressing during daytime, possibly due to long outdoor exposure. This circadian variation is less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time.
Conclusions
A circadian rhythm was seen for most SAR symptoms at baseline, being most distressing during daytime, possibly due to long outdoor exposure. This circadian variation is less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time.Competing interests The study was funded by Schering-Plough in the Nordic countries. None of the authors will gain financially from the publication. There are no patents pending. There are no other competing interests.
Competing interests
The study was funded by Schering-Plough in the Nordic countries.None of the authors will gain financially from the publication. There are no patents pending. There are no other competing interests.Authors' contributions RH participated in the design of the protocol and is the main author of the article. KH participated in the design of the protocol and as investigator. OB participated as principal investigator in Sweden and enrolled most patients in the study. SF participated in the statistical analysis, drafted the tables and figures and participated in drafting the manuscript. TØ was project leader and participated in the design of the protocol, the statistical analysis and drafting of the manuscript. Table 1 Demographics Treatment groups Demographic Characteristics AM-group (n = 336) PM-group (n = 327) p-value Age (years)   Mean (SD) 35.4 (11.0) 36.5 (12.0) 0.232 a   Min-Max 18–69 18–75 Age Group (years)   18–29 115 (34.2 %) 113 (34.6 %)   30–39 113 (33.6 %) 95 (29.1 %)   40–49 70 (20.8 %) 66 (20.2 %)   ≥ 50 38 (11.3 %) 53 (16.2 %) Sex   Male (%) 152 (45) 163 (50) 0.235 b   Female (%) 184 (55) 164 (50) Duration of SAR History (years)   Mean (SD) 14.2 (9.2) 14.4 (10.3) 0.662 a   Min-Max 2–50 1–55 c a  t-test for differences between the means of the two treatment groups b  χ 2 -test for the distribution between the two groups c  One patient in the PM-group had only one year duration of SAR history although the inclusion criterion was 2 years
Authors' contributions
RH participated in the design of the protocol and is the main author of the article.KH participated in the design of the protocol and as investigator.OB participated as principal investigator in Sweden and enrolled most patients in the study.SF participated in the statistical analysis, drafted the tables and figures and participated in drafting the manuscript.TØ was project leader and participated in the design of the protocol, the statistical analysis and drafting of the manuscript.DemographicsTreatment groupsDemographic CharacteristicsAge (years)Age Group (years)SexDuration of SAR History (years)a  t-test for differences between the means of the two treatment groupsb  χ 2 -test for the distribution between the two groupsc  One patient in the PM-group had only one year duration of SAR history although the inclusion criterion was 2 years