Thalidomide protects against ischemic neuronal damage induced by focal cerebral ischemia in mice

We aimed to examine whether thalidomide might inhibit the neuronal damage resulting from focal cerebral ischemia, and if so to explore the neuroprotective mechanism. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) in mice, and thalidomide was intraperitoneally administered a total of three times (at 10 min before, just before, and 1 h after MCAO). Thalidomide significantly reduced (a) the infarct area and volume at 24 and 72 h after MCAO and (b) the neurological score at 72 h after MCAO. Brains were also histochemically assessed for apoptosis and lipid peroxidation using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and an antibody recognizing 8-hydroxy-2′-deoxyguanosine (8-OHdG), respectively. Thalidomide reduced both the number of TUNEL-positive cells and the oxidative damage. However, post-treatment of thalidomide [20 mg/kg, three times (at just after, 1 h after, 3 h after MCAO)] did not reduce the infarct volume. In an in vitro study, we examined the effects of thalidomide on lipid peroxidation in mouse brain homogenates and on the production of various radical species. Thalidomide inhibited both the lipid peroxidation and the production of H₂O₂ and O₂ · ⁻ (but not HO⁻) radicals. We also measured the brain concentration of TNF-α by ELISA. The TNF-α level in the brain was significantly increased at 9–24 h after MCAO. However, thalidomide did not reduce the elevated TNF-α level at either 12 or 24 h after MCAO. These findings indicate that thalidomide has neuroprotective effects against ischemic neuronal damage in mice, and that an inhibitory action of thalidomide against oxidative stress may be partly responsible for these neuroprotective effects.     

Lipocalin-prostaglandin D synthase is a critical beneficial factor in transient and permanent focal cerebral ischemia

Prostaglandin D₂ (PGD₂) is the most abundant prostaglandin produced in the brain. It is a metabolite of arachidonic acid and synthesized by prostaglandin D₂ synthases (PGDS) via the cyclooxygenase pathway. Two distinct types of PGDS have been identified: hematopoietic prostaglandin D synthase (H-PGDS) and lipocalin-type prostaglandin D synthase (L-PGDS). Because relatively little is known about the role of L-PGDS in the CNS, here we examined the outcomes in L-PGDS knockout and wild-type (WT) mice after two different cerebral ischemia models, transient middle cerebral artery (MCA) occlusion (tMCAO) and permanent distal middle cerebral artery occlusion (pMCAO). In the tMCAO model, the MCA was occluded with a monofilament for 90 min and then reperfused for 4 days. In the pMCAO model, the distal part of the MCA was permanently occluded and the mice were sacrificed after 7 days. Percent corrected infarct volume and neurological score were determined after 4 and 7 days, respectively. L-PGDS knockout mice had significantly greater infarct volume and brain edema than did WT mice after tMCAO (P<0.01). Similarly, L-PGDS knockout mice showed greater infarct volume and neurological deficits as compared to their WT counterparts after pMCAO (P<0.01). Using the two models enabled us to study the role of L-PGDS in both early (tMCAO) and delayed (pMCAO) ischemic processes. Our findings suggest that L-PGDS is beneficial for protecting the brain against transient and permanent cerebral ischemia. These results provide a better understanding of the role played by the enzymes that control eicosanoid synthesis and how they can be utilized as potential targets to prevent damage following either acute or potentially chronic neurological disorders.     

Role of endothelial nitric oxide synthetase in arteriogenesis after stroke in mice

Arteriogenesis supports restored perfusion in the ischemic brain and improves long-term functional outcome after stroke. We investigate the role of endothelial nitric oxide synthetase (eNOS) and a nitric oxide (NO) donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate), in promoting arteriogenesis after stroke. Adult wild-type (WT, n=18) and eNOS-knockout (eNOS^−/−, n=36) mice were subjected to transient (2.5 h) right middle cerebral artery occlusion (MCAo) and were treated with or without DETA-NONOate (0.4 mg/kg) 24 h after MCAo. Functional evaluation was performed. Animals were sacrificed 3 days after MCAo for arterial cell culture studies, or 14 days for immunohistochemical analysis. Consistent with previous studies, eNOS^−/− mice exhibited a higher mortality rate (P<0.05, n=18/group) and more severe neurological functional deficit after MCAo than WT mice (P<0.05, n=12/group). Decreased arteriogenesis, was evident in eNOS^−/− mice compared with WT mice, as demonstrated by reduced vascular smooth muscle cell (VSMC) proliferation, arterial density and diameter in the ischemic brain. eNOS^−/− mice treated with DETA-NONOate had a significantly decreased mortality rate and improved functional recovery, and exhibited enhanced arteriogenesis identified by increased VSMC proliferation, and upregulated arterial density and diameter compared to eNOS^−/− mice after stroke (P<0.05, n=12/group). To elucidate the mechanisms underlying eNOS/NO mediated arteriogenesis, VSMC migration was measured in vitro. Arterial cell migration significantly decreased in the cultured common carotid artery (CCA) derived from eNOS^−/− mice 3 days after MCAo compared to WT arterial cells. DETA-NONOate-treatment significantly attenuated eNOS^−/−-induced decrease of arterial cell migration compared to eNOS^−/− control artery (P<0.05; n=6/group). Using VSMC culture, DETA-NONOate significantly increased VSMC migration, while inhibition of NOS significantly decreased VSMC migration (P<0.05; n=6/group). Our data indicated that eNOS not only promotes vascular dilation but also increases VSMC proliferation and migration, and thereby enhances arteriogenesis after stroke. Therefore, increase eNOS may play an important role in regulating of arteriogenesis after stroke.     

Calcium responses mediated by type 2 IP3-receptors are required for osmotic volume regulation of retinal glial cells in mice

Prevention of osmotic swelling of retinal glial (Müller) cells is required to avoid detrimental decreases in the extracellular space volume during intense neuronal activity. Here, we show that glial cells in slices of the wildtype mouse retina maintain the volume of their somata constant up to ∼4 min of perfusion with a hypoosmolar solution. However, calcium chelation with BAPTA/AM induced a rapid swelling of glial cell bodies. In glial cells of retinas from inositol-1,4,5-trisphosphate-receptor type 2-deficient (IP₃R2^−/−) mice, hypotonic conditions caused swelling of the cell bodies without delay. Exogenous ATP (acting at P2Y₁ receptors) prevented the swelling of glial cells in retinal slices from wildtype but not from IP₃R2^−/− mice. Müller cells from IP₃R2^−/− mice displayed a strongly reduced amplitude of the ATP-evoked calcium responses as compared to cells from wildtype mice. It is concluded that endogenous calcium signaling mediated by IP₃R2 is required for the osmotic volume regulation of retinal glial cells.     

Aggravated chronic brain injury after focal cerebral ischemia in aquaporin-4-deficient mice

The water channel aquaporin-4 (AQP4) is important in brain water homeostasis, and is also involved in astrocyte growth and glial scar formation. It has been reported that AQP4 deficiency attenuates acute ischemic brain injury as a result of reducing cytotoxic edema. Here, we determined whether AQP4 deficiency influences chronic brain injury after focal cerebral ischemia induced by 30 min of middle cerebral artery occlusion (MCAO). AQP4^−/− mice exhibited a lower survival rate and less body weight gain than wild-type mice, but their neurological deficits were similar to wild-type mice during 35 days after MCAO. At 35 days after MCAO, AQP4^−/− mice showed more severe brain atrophy and cavity formation in the ischemic hemisphere as well as more neuronal loss in the hippocampus. Furthermore, astrocyte proliferation and glial scar formation were impaired in AQP4^−/− mice. Therefore, AQP4 deficiency complicated by astrocyte dysfunction aggravates chronic brain injury after focal cerebral ischemia, suggesting that AQP4 may be important in the chronic phase of the post-ischemic recovery process.     

Risk factors,types and outcomes of arterial ischemic stroke in Polish pediatric patients: a retrospective single-center study

IntroductionVarious neurological complications may occur as a consequence of arterial ischemic stroke (AIS) and have an impact on daily activity of the patients, costs of their medical care and rehabilitation. The aim of this study was to analyze risk factors, stroke symptoms and post-stroke consequences in Polish pediatric patients depending on stroke subtype.Material and methodsWe retrospectively reviewed 77 children under the age of 18 years following their first AIS. Patients were white, Polish Caucasians, recruited in the Department of Pediatric Neurology at the Medical University of Silesia in Katowice (Poland). Statistical analysis was performed using Statistica 12.0.ResultsGender differed significantly between stroke subgroups (p = 0.030). The presence of focal cerebral arteriopathy (FCA) and chronic diseases was associated with type of AIS (p = 0.003 and p = 0.050, respectively). An outcome without neurological deterioration (normal outcome) was observed in 43% of children with lacunar anterior circulation infarct (LACI). Hemiparesis was present in almost all children with total anterior circulation infarct (TACI), in two thirds of children with partial anterior circulation infarct (PACI) and in almost 50% of children with LACI or posterior circulation infarct (POCI). In every child with hemiplegia the stroke symptom evolved into hemiparesis at follow-up. Additionally, patients with a normal outcome were older at the time of AIS than those with at least one neurological consequence (OR = 0.894, p = 0.034).ConclusionsThe presence and number of neurological outcomes depend on stroke subtypes. A relation between the presence of post-stroke deficits and age at onset was observed. The odds of deficit after ischemic stroke decreases by an average of 10.6% if the child is 1 year older at the time of AIS.     

Nylon filament coated with paraffin for intraluminal permanent middle cerebral artery occlusion in rats

A variety of intraluminal nylon filament has been used in rat middle cerebral artery occlusion (MCAO) models. However the lesion extent and its reproducibility vary among laboratories. The properties of nylon filament play a part of reasons for these variations. In the present study, we used paraffin-coated nylon filament for rat MCAO model, tested the effects and advanced improvement for making the rat MCAO. Forty male Sprague-Dawley (SD) rats were randomized into two groups, MCAO with traditional uncoated nylon filament (uMCAO) and MCAO with paraffin-coated nylon filament (cMCAO), three rats as normal group and sham group respectively. Assessment included mortality rates, model success rates, neurological deficit evaluation, and infarct volume. The study showed two rats died in uMCAO group, no rat died in cMCAO group within the 12 h. The model success rate of uMCAO was 100%, while the uMCAO group was 55% (n = 20, two died within 12 h, seven rats were excluded as the brain slices showed no TTC staining due to subarachanoid hemorrhage). Neurological evaluation demonstrated group cMCAO had more worse neurological outcomes than group uMCAO, and the difference was statistically signification (p < 0.05). TTC staining cMCAO group had significantly larger infarct volumes than uMCAO group, and also showed statistically significant difference (p < 0.05). The result demonstrated that the paraffin-coated nylon filament intraluminal occlusion provide better occlusion of middle cerebral artery than the uncoated nylon filament, improve the consistent of model, and raise the success rate to reduce the number of experimental animals. These positive results are much encouraging and interesting.     

Characterization of spontaneous inhibitory postsynaptic currents in cultured rat retinal amacrine cells

Spontaneous postsynaptic current is a reflection of spontaneous neurotransmitter release that plays multiple roles in a variety of neurobiological activities. In the present study, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) by patch-clamp techniques in cultured rat retinal GABAergic amacrine cells (ACs), which provide inhibitory inputs to both bipolar and ganglion cells in the inner retina, and examined if and how Ca²⁺ was involved in the induction of spontaneous GABA release from the terminals of these cells. sIPSCs were completely blocked by application of either 10 μM bicuculline or 10 μM gabazine, and the reversal potential of sIPSCs was close to E_Cl−, indicating that these events were exclusively mediated by GABA_A receptors. Increase of external Ca²⁺ concentrations from 2 to 5 mM significantly enhanced the frequency, but did not change the amplitude of sIPSCs. In contrast, perfusion of Ca²⁺-free external solution greatly reduced the events of sIPSCs and decreased the amplitude of sIPSCs. Consistently, the non-selective voltage-gated calcium channel blocker CdCl₂ (200 μM) considerably suppressed both the frequency and the amplitude of sIPSCs. Furthermore, the ryanodine receptor (RyR) antagonist dantrolene (10 μM) failed to affect sIPSCs, while the inositol 1,4,5-trisphosphate (IP₃) receptor antagonists 2-aminoethyl diphenylborinate (2-APB, 20 μM) and xestospongin C (XeC, 1 μM) significantly decreased the frequency of sIPSCs. In the presence of SKF96365 (10 μM), a non-specific transient receptor potential channel (TRP) blocker, 2-APB persisted to show its effect on sIPSCs. These results suggest that spontaneous GABA release from the terminals of GABAergic ACs is Ca²⁺-dependent, and both extracellular calcium influx through presynaptic calcium channels and Ca²⁺ release through activation of the IP₃-sensitive pathway, but not the ryanodine-sensitive one, from intracellular stores are responsible for the generation of sIPSCs under our experimental conditions.     

Hyperbaric oxygen preconditioning reduces ischemia–reperfusion injury by inhibition of apoptosis via mitochondrial pathway in rat brain

This study examined the hypothesis that apoptotic inhibition via mitochondrial pathway was involved in hyperbaric oxygen preconditioning (HBO-PC)–induced neuroprotection on ischemia–reperfusion injury in rat brain. Male Sprague–Dawley rats (250∼280 g, n=144) were divided into control, middle cerebral artery occlusion (MCAO) for 90 min, and HBO-PC plus MCAO groups. HBO-PC was conducted four times by giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at 12 h intervals for 2 days. At 24 h after the last HBO-PC, MCAO was performed and at 24 h after MCAO, neurological function, brain water content, infarct volume, and cell death were evaluated. Enzymatic activity of capase-3 and −9, and expression of cytochrome c, Bcl-2 and Bax proteins were performed in the samples from hippocampus, ischemic penumbra and core of the brain cortex, respectively. HBO-PC reduced brain edema, decreased infarction volume, and improved neurological recovery. HBO-PC reduced cytoplasm cytochrome c levels, decreased caspase enzyme activity, upregulated the ratio of Bcl-2 and Bax expression, and abated the apoptosis of ischemic tissue. HBO-PC protects brain tissues from ischemia–reperfusion injury by suppressing mitochondrial apoptotic pathways.     

Prostacyclin aerosol and inhaled nitric oxide fail to reverse pulmonary vasoconstriction induced by thromboxane analogue in dogs

Inhalation of either prostacyclin (PGI₂) as an aerosol or nitric oxide (NO) has been shown to elicit selective pulmonary vasodilation during hypoxic pulmonary vasoconstriction in dogs. Hypoxia may produce cardiovascular changes confounding interpretation of drug effects. Therefore, we investigated the effects of PGI₂-aerosol and inhaled NO (50 p.p.m.) on pulmonary pressure-flow relationships (P/Q_ plots) during thromboxane analogue (U46619) induced pulmonary vasoconstriction. In eight anaesthetized dogs infusion of U46619 (0.33 ± 0.18 μg kg_¹ min^-1) increased the slope (3.5 ± 1.1 to 8.4 ± 1.7 mmHg L^-1 min^-1, P < 0.001) and the intercept (4.4±2.3 to 10.2 ± 4.6 mmHg, P < 0.01) of P/Q plots indicating pulmonary vasoconstriction. Inhalation of both aerosolized PGI₂ solution (10μgmL^-1) and NO (50 p.p.m.) reduced neither the slope nor the intercept of the P/Q_ plots. Increasing the concentration of the aerosolized PGI₂ solution to 50 μg mL^-1: (n= 3) did not enhance the effect on pulmonary circulation but systemic vascular resistance fell by 23%. Oxygenation and intrapulmonary shunt remained unchanged during both PGI₂-aerosol and inhaled NO. The failure of PGI₂-aerosol to induce pulmonary vasodilation indicates that during aerosolization PGI₂-concentrations at receptor sites on pulmonary vessels were insufficient to surmount U46619 induced vasoconstriction; this notion is supported by unchanged arterial plasma concentrations of the PGI₂ degradation product 6-keto-PGF_lα. Considering that NO inhaled at comparable concentrations in sheep reversed U46619 induced pulmonary vasoconstriction, species differences may account for the failure of both PGI₂-aerosol and NO to dilate pulmonary vessels in dogs.     

Effect of anesthesia and cerebral blood flow on neuronal injury in a rat middle cerebral artery occlusion (MCAO) model

Middle cerebral artery occlusion (MCAO) models have become well established as the most suitable way to simulate stroke in experimental studies. The high variability in the size of the resulting infarct due to filament composition, rodent strain and vessel anatomy makes the setup of such models very complex. Beside controllable variables of homeostasis, the choice of anesthetics and the grade of ischemia and reperfusion played a major role for extent of neurological injury. Transient MCAO was induced during either isoflurane or ketamine/xylazine (ket/xyl) anesthesia with simultaneously measurement of cerebral blood flow (CBF) in 60 male Wistar rats (380–420 g). Neurological injury was quantified after 24 h. Isoflurane compared with ket/xyl improved mortality 24 h after MCAO (10 vs. 50 %, p = 0.037) and predominantly led to striatal infarcts (78 vs. 18 %, p = 0.009) without involvement of the neocortex and medial caudoputamen. Independent of anesthesia type, cortical infarcts could be predicted with a sensitivity of 67 % and a specificity of 100 % if CBF did not exceed 35 % of the baseline value during ischemia. In all other cases, cortical infarcts developed if the reperfusion values remained below 50 %. Hyperemia during reperfusion significantly increased infarct and edema volumes. The cause of frequent striatal infarcts after isoflurane anesthesia might be attributed to an improved CBF during ischemia (46 ± 15 % vs. 35 ± 19 %, p = 0.04). S-100β release, edema volume and upregulation of IL-6 and IL-1β expression were impeded by isoflurane. Thus, anesthetic management as well as the grade of ischemia and reperfusion after transient MCAO demonstrated important effects on neurological injury.     

Developmental regulation and neuroprotective effects of striatal tonic GABAA currents

Striatal neurons are known to express GABA_A receptor subunits that underlie both phasic and tonic inhibition. Striatal projection neurons, or medium spiny neurons (MSNs), are divided into two classes: MSNs containing the dopamine D1 receptor (D1-MSNs) form the direct pathway to the substantia nigra and facilitate movement while MSNs expressing the dopamine D2 receptor (D2-MSNs) form the pallidal pathway that inhibits movement. Consequently, modulating inhibition in distinct classes of MSNs will differentially impact downstream network activity and motor behavior. Given the powerful role of extrasynaptic inhibition in controlling neuronal excitability, we examined the nature of striatal tonic inhibition and its potential role in preventing excitotoxicity. Consistent with earlier studies in young (P16–P25) mice, tonic GABA currents in D2-MSNs were larger than in D1-MSNs. However, with age (>P30 mice) the tonic GABA currents increased in D1-MSNs but decreased in D2-MSNs. These data demonstrate a developmental switch in the MSN subtype expressing larger tonic GABA currents. Compared to wild-type, MSNs from adult mice lacking the GABA_AR δ subunit (Gabrd^−/− mice) had both decreased tonic GABA currents and reduced survival following an in vitro excitotoxic challenge with quinolinic acid. Furthermore, muscimol-induced tonic GABA currents were accompanied by reduced acute swelling of striatal neurons after exposure to NMDA in WT mice but not in Gabrd^−/− mice. Our data are consistent with a role for tonic inhibition mediated by GABA_AR δ subunits in neuroprotection against excitotoxic insults in the adult striatum.     

Hydrogen gas reduced acute hyperglycemia-enhanced hemorrhagic transformation in a focal ischemia rat model

Hyperglycemia is one of the major factors for hemorrhagic transformation after ischemic stroke. In this study, we tested the effect of hydrogen gas on hemorrhagic transformation in a rat focal cerebral ischemia model. Sprague–Dawley rats (n=72) were divided into the following groups: sham; sham treated with hydrogen gas (H₂); Middle Cerebral Artery Occlusion (MCAO); and MCAO treated with H₂ (MCAO+H₂). All rats received an injection of 50% dextrose (6 ml/kg i.p.) and underwent MCAO 15 min later. Following a 90 min ischemic period, hydrogen was inhaled for 2 h during reperfusion. We measured the level of blood glucose at 0 h, 0.5 h, 4 h, and 6 h after dextrose injection. Infarct and hemorrhagic volumes, neurologic score, oxidative stress (evaluated by measuring the level of 8 Hydroxyguanosine (8OHG), 4-Hydroxy-2-Nonenal (HNE) and nitrotyrosine), and matrix metalloproteinase (MMP)-2/MMP-9 activity were measured at 24 h after ischemia. We found that hydrogen inhalation for 2 h reduced infarct and hemorrhagic volumes and improved neurological functions. This effect of hydrogen was accompanied by a reduction of the expression of 8OHG, HNE, and nitrotyrosine and the activity of MMP-9. Furthermore, a reduction of the blood glucose level from 500±32.51 to 366±68.22 mg/dl at 4 h after dextrose injection was observed in hydrogen treated animals. However, the treatment had no significant effect on the expression of ZO-1, occludin, collagen IV or aquaporin4 (AQP4). In conclusion, hydrogen gas reduced brain infarction, hemorrhagic transformation, and improved neurological function in rats. The potential mechanisms of decreased oxidative stress and glucose levels after hydrogen treatment warrant further investigation.     

Functional outcome is impaired following traumatic brain injury in aging Nogo-A/B-deficient mice

Increasing age is associated with a poor prognosis following traumatic brain injury (TBI). CNS axons may recover poorly following TBI due to expression of myelin-derived inhibitors to axonal outgrowth such as Nogo-A. To study the role of Nogo-A/B in the pathophysiological response of the elderly to TBI, 1-year-old mice deficient in Nogo-A/B (Nogo-A/B homozygous^−/− mice), Nogo-A/B heterozygous^−/+ mice, and age-matched wild-type (WT) littermate controls were subjected to a controlled cortical impact (CCI) TBI. Sham-injured WT mice (7 months old) and 12 month old naïve Nogo-A/B^−/− and Nogo-A/B^−/+ served as controls. Neurological motor function was evaluated up to 3 weeks, and cognitive function, hemispheric tissue loss, myelin staining and hippocampal β-amyloid (Aβ) immunohistochemistry were evaluated at 4 weeks post-injury. In WT littermates, TBI significantly impaired learning ability at 4 weeks and neurological motor function up to 2 weeks post-injury and caused a significant loss of hemispheric tissue. Following TBI, Nogo-A/B^−/− mice showed significantly less recovery from neurological motor and cognitive deficits compared to brain-injured WT mice. Naïve Nogo-A/B^−/− and Nogo-A/B^−/+ mice quickly learned the MWM task in contrast to brain-injured Nogo-A/B^−/− mice who failed to learn the MWM task at 4 weeks post-injury. Hemispheric tissue loss and cortical lesion volume were similar among the brain-injured genotypes. Neither TBI nor the absence of NogoA/B caused an increased Aβ expression. Myelin staining showed a reduced area and density in the corpus callosum in brain-injured Nogo-A/B^−/− animals compared to their littermate controls. These novel and unexpected behavioral results demonstrate that the absence of Nogo-A/B may negatively influence outcome, possibly related to hypomyelination, following TBI in mice and suggest a complex role for this myelin-associated axonal growth inhibitor following TBI.     

LP-211 is a brain penetrant selective agonist for the serotonin 5-HT7 receptor

We have determined the pharmacological profile of the new serotonin 5-HT₇ receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211). Radioligand binding assays were performed on a panel of 5-HT receptor subtypes. The compound was also evaluated in vivo by examining its effect on body temperature regulation in mice lacking the 5-HT₇ receptor (5-HT₇^−/−) and their 5-HT₇^+/+ sibling controls. Disposition studies were performed in mice of both genotypes. It was found that LP-211 was brain penetrant and underwent metabolic degradation to 1-(2-diphenyl)piperazine (RA-7). In vitro binding assays revealed that RA-7 possessed higher 5-HT₇ receptor affinity than LP-211 and a better selectivity profile over a panel of 5-HT receptor subtypes. In vivo it was demonstrated that LP-211, and to a lesser degree RA-7, induced hypothermia in 5-HT₇^+/+ but not in 5-HT₇^−/− mice. Our results suggest that LP-211 can be used as a 5-HT₇ receptor agonist in vivo.     

Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in mice

The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS^−/− mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS^−/− mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS^−/− mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved.     

A role for macrophage migration inhibitory factor in protective immunity against Aspergillus fumigatus

Inflammation plays an important role in protective immunity against fungi, including the opportunistic pathogen, Aspergillus fumigatus. The balance between pro-inflammatory and anti-inflammatory cytokines is a key determinant of infection outcome. Since macrophage migration inhibitory factor (MIF) is an upstream regulator of many cytokines, we analyzed herein the role of endogenous MIF in the host control of hematogenously disseminated aspergillosis using MIF^−/− mice. As revealed by their mortality rate, MIF^−/− mice were more susceptible to disseminated infection than WT mice. Moreover, pharmacologic inhibition of MIF with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester, (ISO-1) increased the susceptibility of WT mice to lethal infection. The higher tissue fungal burden early in sublethal infection indicated increased susceptibility of MIF^−/− mice to sublethal infection as well. Substantial down-regulation of innate and acquired antifungal responses, characterized by decreased production of IL-1β, IL-6, TNF-α, IFN-γ and IL-17 in the spleen was noted in sublethally infected MIF^−/− mice. In contrast, IL-4 was higher in MIF^−/− than in WT mice. Taken together, our findings show that MIF contributes to host resistance against progressive invasive A. fumigatus infection by controlling downstream pro-inflammatory versus anti-inflammatory cytokine production thus determining the outcome of infection.     

The effect of heliox treatment in a rat model of focal transient cerebral ischemia

Manipulation of inhaled gases during ischemia/reperfusion is a potential novel therapy for acute stroke. We previously found that treatment with a mixture of 70%/30% helium/oxygen (heliox) or 100% oxygen protects the brain against acute focal ischemia–reperfusion injury. This study evaluates the potential neuro-protective effects of delayed heliox treatment and its dose response effects in a rat transient focal cerebral ischemia model. Adult male rats were subjected to 2-h middle cerebral artery occlusion and then assigned to 1 of 4 inhaled gas exposure groups: I: 70%/30% nitrogen/oxygen (control); II: 70%/30% helium/oxygen administered immediately after occlusion; III: 70%/30% helium/oxygen administered after a 30–60 min delay; or, IV: 40%/30%/30% nitrogen/helium/oxygen administered immediately after occlusion. Outcome measurements included infarct size and neurological deficit score. Mean infarct sizes from groups I to IV were 228, 35, 109, and 124 mm³ respectively (p = 0.012). Only group II had significantly smaller infarct size compared to the control group (p = 0.008). In addition, only Group II had a significantly lower neurological deficit score at 24 h post ischemia when compared to the control group (p < 0.001). Since heliox reduced infarct size and improved neurological deficit scores if initiated immediately after onset of ischemia, it may be a useful adjuvant to other stroke therapies.     

Contribution of glutamate transporter GLT-1 to removal of synaptically released glutamate at climbing fiber-Purkinje cell synapses

Rapid removal of synaptically released glutamate from the extracellular space ensures a high signal-to-noise ratio in excitatory neurotransmission. In the cerebellum, glial glutamate transporters, GLAST and GLT-1, are co-localized in the processes of Bergmann glia wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, the decay kinetics of climbing fiber-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(−/−) mice are not different from those in wild-type (WT) mice. This raises a possibility that GLT-1 plays a significant role in clearing glutamate at CF-PC synapses despite its smaller amount of expression. Here, we studied the functions of GLT-1 and GLAST in the clearance of glutamate using GLAST(−/−) mice and GLT-1(−/−) mice. In the presence of cyclothiazide (CTZ) that attenuates the desensitization of AMPA receptors, the decay time constant of CF-EPSCs (τ_w) in GLT-1(−/−) mice was slower than that in WT mice. However, the degree of this prolongation of τ_w was less prominent compared to that in GLAST(−/−) mice. The values of τ_w in GLT-1(−/−) mice and GLAST(−/−) mice were comparable to those estimated in WT mice in the presence of a potent blocker of glial glutamate transporters (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA) at 10 and 100 nM, which reduced the amplitudes of glutamate transporter currents elicited by CF stimulation in Bergmann glia to ∼81 and ∼28%, respectively. We conclude that GLT-1 plays a minor role compared to GLAST in clearing synaptically released glutamate at CF-PC synapses.