Activity of docetaxel, carboplatin, and doxorubicin in patient-derived triple-negative breast cancer xenografts.

Triple-negative breast cancer (TNBC) is highly responsive to neoadjuvant polychemotherapy regimens including anthracyclines, taxanes, and, more recently, carboplatin. However, there is inadequate information on the individual contribution of each of these agents to the global activity of the combinations, and the use of combinations of up to four of these drugs is associated with relevant toxicity. Identifying single-drug activity in the clinical neoadjuvant setting is challenging. We developed patient-derived xenografts (PDXs) from several chemotherapy-naïve TNBC samples to assess the antitumor activity of single drugs and combinations of drugs. PDXs were established from chemotherapy-naïve TNBC samples. Nine TNBC PDX models (all of which corresponded to a basal-like phenotype according to the PAM50 classifier) were treated with carboplatin, docetaxel, and doxorubicin and the combination of docetaxel and carboplatin. Only one of nine PDX models showed sensitivity to doxorubicin, while eight of nine PDX models showed sensitivity to docetaxel and carboplatin as single agents. The 3 PDX models derived from patients with gBRCA-1 or gPALB2 mutations were very sensitive to carboplatin single agent. All 6 PDX models from patients without hereditary germ-line mutations showed increased sensitivity to the combination of docetaxel and carboplatin. In the present study, docetaxel and carboplatin single agents were active drugs against basal-like TNBC, while doxorubicin monotherapy showed low activity. The combination of docetaxel and carboplatin was more effective than the drugs used as single agents, except in the PDX from patients with gBRCA1/PALB2 mutations.

The Value of Mouse Models of Rare Diseases: A Spanish Experience.

Animal models are invaluable for biomedical research, especially in the context of rare diseases, which have a very low prevalence and are often complex. Concretely mouse models provide key information on rare disease mechanisms and therapeutic strategies that cannot be obtained by using only alternative methods, and greatly contribute to accelerate the development of new therapeutic options for rare diseases. Despite this, the use of experimental animals remains controversial. The combination of respectful management, ethical laws and transparency regarding animal experimentation contributes to improve society's opinion about biomedical research and positively impacts on research quality, which eventually also benefits patients. Here we present examples of current advances in preclinical research in rare diseases using mouse models, together with our perspective on future directions and challenges.

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage.

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke.

Estrogen Receptor-Alpha (ESR1) Governs the Lower Female Reproductive Tract Vulnerability to Candida albicans.

Estradiol-based therapies predispose women to vaginal infections. Moreover, it has long been known that neutrophils are absent from the vaginal lumen during the ovulatory phase (high estradiol). However, the mechanisms that regulate neutrophil influx to the vagina remain unknown. We investigated the neutrophil transepithelial migration (TEM) into the vaginal lumen. We revealed that estradiol reduces the CD44 and CD47 epithelial expression in the vaginal ectocervix and fornix, which retain neutrophils at the apical epithelium through the estradiol receptor-alpha. In contrast, luteal progesterone increases epithelial expression of CD44 and CD47 to promote neutrophil migration into the vaginal lumen and Candida albicans destruction. Distinctive to vaginal mucosa, neutrophil infiltration is contingent to sex hormones to prevent sperm from neutrophil attack; although it may compromise immunity during ovulation. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil TEM.

Fibrinogen nitrotyrosination after ischemic stroke impairs thrombolysis and promotes neuronal death.

Ischemic stroke is an acute vascular event that compromises neuronal viability, and identification of the pathophysiological mechanisms is critical for its correct management. Ischemia produces increased nitric oxide synthesis to recover blood flow but also induces a free radical burst. Nitric oxide and superoxide anion react to generate peroxynitrite that nitrates tyrosines. We found that fibrinogen nitrotyrosination was detected in plasma after the initiation of ischemic stroke in human patients. Electron microscopy and protein intrinsic fluorescence showed that in vitro nitrotyrosination of fibrinogen affected its structure. Thromboelastography showed that initially fibrinogen nitrotyrosination retarded clot formation but later made the clot more resistant to fibrinolysis. This result was independent of any effect on thrombin production. Immunofluorescence analysis of affected human brain areas also showed that both fibrinogen and nitrotyrosinated fibrinogen spread into the brain parenchyma after ischemic stroke. Therefore, we assayed the toxicity of fibrinogen and nitrotyrosinated fibrinogen in a human neuroblastoma cell line. For that purpose we measured the activity of caspase-3, a key enzyme in the apoptotic pathway, and cell survival. We found that nitrotyrosinated fibrinogen induced higher activation of caspase 3. Accordingly, cell survival assays showed a more neurotoxic effect of nitrotyrosinated fibrinogen at all concentrations tested. In summary, nitrotyrosinated fibrinogen would be of pathophysiological interest in ischemic stroke due to both its impact on hemostasis - it impairs thrombolysis, the main target in stroke treatments - and its neurotoxicity that would contribute to the death of the brain tissue surrounding the infarcted area.

Tonsils of the soft palate do not mediate the response of pigs to oral vaccination with heat-inactivated Mycobacterium bovis.

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-ß), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research.

Nitro-oxidative stress after neuronal ischemia induces protein nitrotyrosination and cell death.

Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.

Interleukin-10 regulates progenitor differentiation and modulates neurogenesis in adult brain.

The adult subventricular zone (SVZ) is the main neurogenic niche in the adult brain of mice and rats. The adult SVZ contains neural stem cells (NSCs) that primarily differentiate into committed neuroblasts. The newly generated neuroblasts accumulate in dorsal SVZ where they further differentiate and initiate a long migration pathway to their final destination, the olfactory bulb (OB). Here, we report a new role for Interleukin 10 (IL-10) that is different to its well-known anti-inflammatory properties. We show that the IL-10 receptor is expressed in Nestin-positive progenitors restricted to the dorsal SVZ in adult brain. Using IL-10 gain models, we observed that IL-10 maintains neural progenitors in an undifferentiated state by keeping progenitors in an active cycle where pro-neural gene markers (Nestin, Sox1, Sox2, Musashi, Mash1) are upregulated and neuronal gene expression (Numb, DCX, TUBB3) is downregulated. In addition, IL-10 reduces neuronal differentiation and ultimately impairs endogenous neurogenesis. Consistently, in the absence of IL-10, in vivo neuronal differentiation of SVZ progenitors is enhanced and the incorporation of new neurons in the adult OB is increased. Thus, our results provide the first evidence that IL-10 acts as a growth factor on SVZ progenitors and regulates neurogenesis in normal adult brain.

Antioxidant CR-6 protects against reperfusion injury after a transient episode of focal brain ischemia in rats.

Oxidative and nitrosative stress are targets for intervention after ischemia/reperfusion. The aim of this study was to explore the effect of CR-6, a vitamin-E analogue that is antioxidant and scavenger of nitrogen-reactive species. Sprague-Dawley rats had the middle cerebral artery (MCA) occluded either for 90 mins or permanently. Cortical perfusion was continuously monitored by laser-Doppler flowmetry. CR-6 (100 mg/kg) was administered orally either at 2 and 8 h after MCA occlusion, or at 2 h only. Infarct volume, neurological deficit, and signs of reperfusion injury were evaluated. CR-6 was detected in plasma and brain by HPLC. CR-6 reduced glutathione consumption in the ischemic brain and superoxide generation in the isolated MCA. CR-6 decreased infarct volume and attenuated the neurological deficit at 1 and 7 days after ischemia/reperfusion, but not after permanent ischemia. Immediately after reperfusion, cortical blood flow values returned to their baseline (+/-20%) in several animals, whereas others showed hyper-perfusion (>20% of baseline). Reactive hyperemia was associated with adverse events such as increased cortical BBB leakage, edema, protein nitrotyrosination, COX-2 expression, and neutrophil accumulation; and with a poorer outcome, and CR-6 attenuated these effects. In conclusion, oral CR-6 administration after transient ischemia protects the brain from reperfusion injury.

Participation of oxidative stress on rat middle cerebral artery changes induced by focal cerebral ischemia: beneficial effects of 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6).

Cerebral ischemia followed by reperfusion alters vessel properties of brain arteries in rats, inducing an inflammatory response and excessive generation of reactive oxygen species. This study investigated the participation of oxidative stress on vessel properties after ischemia/reperfusion and the beneficial effects of 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6). The right middle cerebral artery was occluded (90 min) and reperfused (24 h). Sham-operated animals were used as controls. Ischemic rats were treated either with CR-6 (100 mg/kg in 1 ml olive oil) or vehicle (1 ml olive oil) administered orally at 2 and 8 h after the onset of ischemia. The structural, mechanical, and myogenic properties of the middle cerebral artery (MCA) were assessed by pressure myography. Superoxide anion ( ) production was evaluated by ethidium fluorescence, and protein tyrosine nitrosylation was determined by immunofluorescence. Infarct volume was smaller in rats treated with CR-6. In MCA, ischemia/reperfusion increased wall thickness, cross-sectional area, wall/lumen, and decreased wall stress. CR-6 treatment prevented all of these changes induced by ischemia/reperfusion. However, impaired myogenic response and larger lumen diameter in active conditions observed after ischemia/reperfusion were not modified by CR-6. Treatment with CR-6 prevented the increase in production and partially prevented the enhanced protein tyrosine nitrosylation that occurred in response to ischemia/reperfusion. Our findings suggest that oxidative stress is involved in the alterations of MCA properties observed after ischemia/reperfusion and that CR-6 induces protection.

Transient benefits but lack of protection by sodium pyruvate after 2-hour middle cerebral artery occlusion in the rat.

Sodium pyruvate has shown protective effects in various experimental models of brain ischemia. The main holdup of this drug is that most of the benefits are reported with a very narrow time window for intervention. Here we investigated whether pyruvate could protect the brain against ischemic damage using a model of 2-hour middle cerebral artery occlusion in the rat. The time course of blood pyruvate after i.p. administration of sodium pyruvate (400 mg/kg) was studied. Animals were treated with the drug or with vehicle 45 min after reperfusion following 2-hour ischemia. Tissue ATP content was determined 5 and 10 h after ischemia onset, and infarct volume was measured at days 1 and 2. The neurological score was evaluated before and after treatment in the different experimental groups. Pyruvate prevented the drop of cortical ATP induced by ischemia in the ipsilateral cortex and ameliorated the neurological deficit at 5 h after the onset of ischemia, supporting some beneficial effects of the treatment. However, these effects were not sustained at 10 h. Furthermore, pyruvate failed to significantly reduce infarct volume and the neurological deficit at 24 and 48 h, in spite of some trend to smaller infarction after pyruvate administration. Therefore, under the present experimental conditions, systemic administration of sodium pyruvate at 3 h after the beginning of ischemia exerted only a transient benefit but not a persistent protection against brain damage.

Endothelial dysfunction in rat mesenteric resistance artery after transient middle cerebral artery occlusion.

Stroke triggers a local and systemic inflammatory response leading to the production of cytokines that can influence blood vessel reactivity. In this study, we aimed to assess whether cerebral ischemia/reperfusion could affect vasoconstriction and vasodilatation on mesenteric resistance arteries (MRA) from Wistar Kyoto rats. The right middle cerebral artery was occluded (90 min) and reperfused (24 h). Sham-operated animals were used as controls. Plasma levels of interleukin (IL)-6 and IL-1beta were measured at 24 h. Vasoconstrictor and vasodilator responses were recorded in a wire myograph. Protein expression was determined by Western blot and immunofluorescence, and superoxide anion (O(2)(.)) production was evaluated by ethidium fluorescence. In MRA, ischemia/reperfusion increased plasma levels of IL-6, O2. production, protein expression of cyclooxygenase-2, and protein tyrosine nitrosylation, but it impaired acetylcholine (ACh) vasodilatation without modifying the vasodilatations to sodium nitroprusside or the contractions to phenylephrine and KCl. Superoxide dismutase (SOD) and indomethacin reversed the impairment of ACh relaxation induced by ischemia/reperfusion. However, N(omega)-nitro-l-arginine methyl ester affected similarly ACh-induced vasodilatations in MRA of ischemic and sham-operated rats. Protein expression of endothelial and inducible nitric-oxide synthase, copper/zinc SOD, manganese SOD, and extracellular SOD was similar in both groups of rats. Our results show MRA endothelial dysfunction 24 h after brain ischemia/reperfusion. Excessive production of O2. in MRA mediates endothelial dysfunction, and the increase in plasma cytokine levels after brain ischemia/reperfusion might be involved in this effect.

Acute effects of three isoflavone class phytoestrogens and a mycoestrogen on cerebral microcirculation.

Phytoestrogens and mycoestrogens are naturally occurring plant and fungus secondary metabolites with estrogen-like structure and/or actions. We aimed to check the hypothesis that phytoestrogens and mycoestrogens, due to their ability to elicit cerebral vasodilation, can induce acute increases in brain blood perfusion. For this purpose, we continuously recorded cerebrocortical perfusion by laser-Doppler flowmetry in anesthetized rats receiving intracarotid infusions (1 mg/kg) of one of the following estrogenic compounds: biochanin A, daidzein, genistein or zearalanone. We have shown the ability of two isoflavone class phytoestrogens (daidzein and biochanin A) and the mycoestrogen zearalanone to induce acute increases in brain blood flow when locally infused into the cerebral circulation of anesthetized rats. The isoflavone genistein failed to induce a significant increase in brain perfusion. No concomitant changes in blood pressure were recorded during the cerebral effects of the estrogenic compounds. Therefore, these microcirculatory effects were due to direct actions of the estrogenic compounds on the cerebrovascular bed.

Chronic intracerebroventricular delivery of the secretory phospholipase A2 inhibitor, 12-epi-scalaradial, does not improve outcome after focal cerebral ischemia-reperfusion in rats.

Phospholipase A2s (PLA2s) seem to be involved in the pathophysiology of ischemic brain injury, but their specific role is far from being completely understood. The present study was carried out to ascertain how and to what extent secretory PLA2s (sPLA2s) activity influences outcome after cerebral ischemia-reperfusion, and to correlate this with the inflammatory response. To do this we used the potent and selective sPLA2 inhibitor, 12-epi-scalaradial. Male Wistar rats were separated into three groups: a control group receiving intracerebroventricular vehicle, and two groups receiving intracerebroventricular 0.005 or 0.5 microg/h 12-epi-scalaradial. Every animal was subjected to middle cerebral artery (MCA) occlusion (90 min, intraluminal thread technique) under continuous moni-torization of cerebrocortical perfusion (CP, laser-Doppler flowmetry), followed by reperfusion (3 days). Neurological status, infarct volume, and myeloperoxidase (MPO) activity were the main end points. Three days after the 90-min ischemia period, neurological examination did not reveal significant differences between the three groups of rats. Control rats showed a mean infarct volume of 145.9 +/- 24.7 mm3 (21 +/- 4.1% of the ipsilateral hemisphere volume), while mean infarct volume in rats treated with 0.005 or 0.5 microg/h 12-epi-scalaradial increased to 164.8 +/- 86.8 mm3 (22.0 +/- 10.9%) and 211.5 +/- 12.2 mm3 (28 +/- 3%, P < 0.05), respectively. Treatment with the highest dose of 12-epi-scalaradial (0.5 microg/h) increased MPO activity in the ipsilateral hemisphere by about 140% (from 0.59 +/- 0.59 to 1.42 +/- 1.03 units of activity/g of tissue in comparison with the control ischemic hemisphere, P < 0.05). Overall, our results point to a positive rather than a negative influence of sPLA2 activity during ischemia. This, along with its inability to decrease the inflammatory response, does not allow to propose the use of 12-epi-scalardial as a potential drug for stroke therapy.

Heat shock proteins, end effectors of myocardium ischemic preconditioning?

The purpose of this study was to investigate (1) whether ischemia-reperfusion increased the content of heat shock protein 72 (Hsp72) transcripts and (2) whether myocardial content of Hsp72 is increased by ischemic preconditioning so that they can be considered as end effectors of preconditioning. Twelve male minipigs (8 protocol, 4 sham) were used, with the following ischemic preconditioning protocol: 3 ischemia and reperfusion 5-minute alternative cycles and last reperfusion cycle of 3 hours. Initial and final transmural biopsies (both in healthy and ischemic areas) were taken in all animals. Heat shock protein 72 messenger ribonucleic acid (mRNA) expression was measured by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method using complementary DNA normalized against the housekeeping gene cyclophilin. The identification of heat shock protein 72 was performed by immunoblot. In our "classic" preconditioning model, we found no changes in mRNA hsp72 levels or heat shock protein 72 content in the myocardium after 3 hours of reperfusion. Our experimental model is valid and the experimental techniques are appropriate, but the induction of heat shock proteins 72 as end effectors of cardioprotection in ischemic preconditioning does not occur in the first hours after ischemia, but probably at least 24 hours after it, in the so-called "second protection window."

Dietary phytoestrogens improve stroke outcome after transient focal cerebral ischemia in rats.

As phytoestrogens are postulated as being neuroprotectants, we assessed the hypothesis that dietary isoflavone-type phytoestrogens are neuroprotective against ischemic stroke. Transient focal cerebral ischemia (90 min) was induced by middle cerebral artery occlusion (MCAO) following the intraluminal thread technique, both in rats fed with soy-based diet and in rats fed with isoflavone-free diet. Cerebro-cortical laser-Doppler flow (cortical perfusion, CP), arterial blood pressure, core temperature, PaO2, PaCO2, pH and glycemia were measured before, during and after MCAO. Neurological examination and infarct volume measurements were carried out 3 days after the ischemic insult. Dietary isoflavones (both glycosides and aglycones) were measured by high-performance liquid chromatography. Neither pre-ischemic, intra-ischemic nor post-ischemic CP values were significantly different between the soy-based diet and the isoflavone-free diet groups. Animals fed with the soy-based diet showed an infarct volume of 122 +/- 20.2 mm3 (19 +/- 3.3% of the whole ipsilateral hemisphere volume). In animals fed with the isoflavone-free diet the mean infarct volume was significantly higher, 191 +/- 26.7 mm3 (28 +/- 4.1%, P < 0.05). Neurological examination revealed significantly higher impairment in the isoflavone-free diet group compared with the soy-based diet group (3.3 +/- 0.5 vs. 1.9 +/- 0.5, P < 0.05). These results demonstrate that dietary isoflavones improve stroke outcome after transient focal cerebral ischemia in such a way that a higher dietary isoflavone content results in a lower infarct volume and a better neurological status.

Relaxant effect of sildenafil in the rabbit basilar artery.

We hypothesized that sildenafil, inhibitor of phosphodiesterase-5 (PDE-5), interacts with the nitric oxide (NO)-cGMP pathway in the cerebral arteries and shows vasoactive effects. To prove it in the isolated rabbit basilar artery, we compared the effects of sildenafil with other PDE-5 inhibitors, assessed the endothelial dependence of the vasoactive responses, and used modulators of the cGMP and cAMP signaling processes. Sildenafil (10 nM-0.1 mM) induced concentration-dependent relaxations of endothelin-1 (10 nM)-precontracted basilar artery, which were partially inhibited both in endothelium-denuded arteries and in arteries precontracted by depolarization with KCl (50 mM). Endothelin-1 (1 pM-30 nM) induced concentration-dependent contractions that were inhibited by sildenafil (0.1-100 microM). Zaprinast (10 nM-0.1 mM) and MBCQ (1 nM-0.1 mM), PDE-5 inhibitors, induced concentration-dependent relaxations with lower and higher potency than sildenafil, respectively. Sildenafil-induced relaxation was inhibited in arteries preincubated with the NO synthase inhibitor L-NAME (0.1 mM) or the soluble guanylyl cyclase inhibitor ODQ (10 microM). Preincubation with sildenafil (0.1 microM) enhanced the relaxations induced by acetylcholine (0.1 nM-0.1 mM) and the NO donor sodium nitroprusside (0.1 nM-0.1 mM), but not those induced by the cell-permeable cGMP analogue 8-Br-cGMP (1 nM-0.1 mM) and the adenylyl cyclase activator forskolin (0.1 nM-10 microM). These results show that sildenafil has vasoactive effects in isolated cerebral arteries. By enhancing the NO-cGMP signaling pathway in the cerebrovascular wall, sildenafil induces vasodilation, prevents vasoconstriction, and potentiates the effect of other NO-dependent vasodilators.

Inhibition of iNOS activity by 1400W decreases glutamate release and ameliorates stroke outcome after experimental ischemia.

BACKGROUND AND PURPOSE: It has been shown that the reversed operation of glutamate transporters when ATP levels fall accounts for most glutamate release induced by severe cerebral ischemia. Nitric oxide (NO) is formed after ischemia and causes ATP depletion. Our purpose is to test if NO release from inducible NO synthase (iNOS) after stroke may cause a delayed glutamate release due to ATP depletion that might underlie progression of the ischemic infarct. We have studied the effect of the highly selective inhibitor of iNOS activity 1400W on brain ATP levels, extracellular glutamate, and stroke outcome after transient focal cerebral ischemia in rats. METHODS: To induce focal ischemia, the middle cerebral artery (MCA) was occluded by using the intraluminal thread technique (tMCAO). 1400W was administered, after tMCAO, by using an Alzet osmotic pump to yield a drug delivery rate of 2.5 mg/kg/h. Results. Postischemic treatment with 1400W induced a reduction in the neurofunctional impairment and in the total volume of brain infarct. Western blot analysis showed ischemia-induced expression of iNOS. Treatment with 1400W partially prevented delayed ATP reduction and produced inhibition of the subsequent delayed increase in glutamate levels caused by the ischemic insult. CONCLUSIONS: Our data indicate that 1400W improves stroke outcome, an effect concomitant to the inhibition of both ischemia-induced decrease in brain ATP levels and increase in glutamate release. These results provide evidence indicating that the expression of iNOS induced by ischemia may contribute to the progression of the ischemic infarct and have important therapeutic implications for the management of stroke.

Single-dose ebselen does not afford sustained neuroprotection to rats subjected to severe focal cerebral ischemia.

Oxygen free radicals have been involved in the pathophysiology of cerebral ischemia, especially after spontaneous or thrombolytic reperfusion. In this study with rats, we have combined a severe focal ischemic insult (2 h) and a prolonged reperfusion time (7 days) to assess the possible sustained neuroprotective effect of ebselen (10 or 100 mg/kg), a small, lipophilic organoselenium compound which mimics glutathione peroxidase. Parietal cortical perfusion was measured by laser-Doppler flowmetry, and focal cerebral ischemia was carried out by the intraluminal thread method. We have measured plasma selenium levels, brain reduced glutathione levels, as a marker of oxidative stress, and infarct volume associated with cerebral ischemia. Focal ischemia did not alter reduced glutathione levels, while 60 min reperfusion following ischemia induced a significant (P < 0.05) decrease in reduced glutathione levels of the ipsilateral hemisphere. Pretreatment with ebselen, which induced significant (P < 0.05) increase in plasma selenium levels, did not significantly alter the decrease in reduced glutathione levels. The ischemic insult induced 30% mortality on average, with deaths always occurring within 12-48 h. Surviving rats suffered up to 25% body weight loss 1 week after the ischemic insult. Infarct volumes were 26.8 +/- 4.7% of the hemisphere in placebo-treated rats, 26.6 +/- 3.6% in 10 mg/kg ebselen-treated rats, and 25.6 +/- 6.4% in 100 mg/kg ebselen-treated rats (not significantly different). Single-dose administration of ebselen does not reduce the size of brain infarct resulting from severe focal cerebral ischemia in rats. In contrast to previous studies with relatively earlier endpoints, we have delayed the measurement of infarct volume to 1 week after the ischemic insult.

Pharmacological profile of phytoestrogens in cerebral vessels: in vitro study with rabbit basilar artery.

As a previous step to consider their use in the pharmacology for stroke, we investigated the effects of four phytoestrogens (i.e. genistein, daidzein, zearalanone and biochanin A) on cerebral vessels. Cerebral vascular responses were analyzed by conventional recording of isometric tension in rabbit basilar artery segments kept in organ bath under standard conditions. The four phytoestrogens elicited concentration-dependent relaxant responses of different potency in basilar artery segments previously contracted with either 5x10(-2) M KCl or 10(-4) M UTP. Neither endothelium removal, 10(-4) M N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric oxide (NO) synthase inhibitor), 10(-5) M1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, selective inhibitor of NO-sensitive guanylyl cyclase), 10(-5) M 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS2028, specific soluble guanylyl cyclase inhibitor), nor 10(-5) M indomethacin (prostaglandin biosynthesis inhibitor) modified the phytoestrogen-elicited vasorelaxant responses. On the other hand, Ca(2+)-elicited contractile responses were effectively inhibited in the presence of phytoestrogens. Phytoestrogens act as cerebrovascular relaxants by a mechanism which involves Ca(2+) entry blockade in the vascular smooth muscle rather than stimulation of vasorelaxant endothelium-related mechanisms such as NO/cGMP or prostaglandins.