The purpose of this investigation was to explore the potentiality of a novel animal model to be used for the in vivo evaluation of the ability of a drug delivery system to promote the passage through ...the blood–brain barrier (BBB) and/or to improve the brain localization of a bioactive compound. A Tween 80®-coated poly-l-lactid acid nanoparticles was used as a model of colloidal drug delivery system, able to trespass the BBB. Tacrine, administered in LiCl pre-treated rats, induces electrocorticographic seizures and delayed hippocampal damage. The toxic effects of tacrine-loaded poly-l-lactid acid nanoparticles (5mg/kg), a saline solution of tacrine (5mg/kg) and an empty colloidal nanoparticle suspension were compared following i.p. administration in LiCl-pre-treated Wistar rats. All the animals treated with tacrine-loaded nanoparticles showed an earlier outcome of CNS adverse symptoms, i.e. epileptic onset, with respect to those animals treated with the free compound (10min vs. 22min respectively). In addition, tacrine-loaded nanoparticles administration induced damage of neuronal cells in CA1 field of the hippocampus in all treated animals, while the saline solution of tacrine only in 60% of animals. Empty nanoparticles provided similar results to control (saline-treated) group of animals. In conclusion, the evaluation of time-to-onset of symptoms and the severity of neurodegenerative processes induced by the tacrine–lithium model of epilepsy in the rat, could be used to evaluate preliminarily the capability of a drug delivery system to trespass (or not) the BBB in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
BACKGROUND AND PURPOSE Tianeptine is an antidepressant affecting the glutamatergic system. In spite of its proven clinical efficacy, molecular effects of tianeptine are not entirely clear. Tianeptine ...modulates cytokine expression in the CNS and protects the hippocampus from chronic stress effects. HIV infection is associated with inflammation and neuronal loss, causing HIV-associated dementia (HAD). The human immunodeficiency virus type-1 glycoprotein gp120 has been proposed as a likely aetiological agent of HAD. In this study, we determined whether tianeptine protects astroglial cells from the neurodegenerative effects of gp120. EXPERIMENTAL APPROACH Human astroglial cells were treated with gp120 and tianeptine, and viability and apoptosis was monitored by TUNEL, annexin V, and activated caspase-3 staining and flow cytometry. Protein levels of glutamine synthase (GS), inducible and constitutive nitric oxide synthases (iNOS, cNOS) and nuclear factor kappaB (NF-kappaB) pathway were determined by Western blot analysis. The respective activities were assessed indirectly by measuring glutamine and nitrite concentrations or by luciferase reporter assays. KEY RESULTS Tianeptine showed an anti-apoptotic effect and prevented caspase-3 activation by gp120. The mechanism of tianeptine's action involved GS and cNOS stabilization and iNOS suppression. Moreover, tianeptine increased IkappaB-alpha levels in the absence of gp120 and blocked its degradation in response to gp120. This correlated with the suppression of basal and gp120-induced NF-kappaB transcriptional activity. CONCLUSIONS AND IMPLICATIONS Tianeptine clearly exerts neuroprotective effects in vitro by suppressing the molecular pro-inflammatory effects of gp120. Studies in animal models should be performed to evaluate the potential of tianeptine as a treatment for HAD. PUBLICATION ABSTRACT
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
It is well-established that 17β-estradiol (17β-E
2) confers neuroprotection to male and female rats exposed to focal cerebral ischemia, while less is known about the effects of the hormone under ...conditions of transient global ischemia. Since translocation of cytochrome
c from the mitochondria to the cytosol is a critical step in apoptotic cell death after cerebral ischemia, we have investigated whether 17β-E
2 interferes with such mechanism to exert neuroprotection. Global ischemia, induced in male Wistar rats by 5-min 4 vessel occlusion (4VO), resulted in a significant increase of cytosolic cytochrome
c (cyt-
c) levels as detected by Western blotting at 6
h after reperfusion. 17β-E
2 (0.2
mg/kg, i.p.) given 1
h before ischemia minimized cytochrome
c translocation and the latter effect was partially reversed by tamoxifen (0.25
mg/kg, i.p.). Bilateral cell counting revealed that delayed hippocampal damage typically caused by 4VO was abolished by 17β-E
2 and this was partially reversed by tamoxifen in the CA3 subregion, but not in CA1/CA2 or CA4. These findings provide the original observation that 17β-E
2 reduces delayed hippocampal damage caused by 4VO in male rats and blocks cytochrome
c translocation during the early stages of neuronal death, thus providing an important mechanism involved in estrogen-mediated neuroprotection.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Herbicides, including paraquat, may produce neurodegenerative effect when given both peripherally and into the brain though the pathophysiological mechanism is still unknown. Microinfusion of ...paraquat into the Substantia Nigra (50 microg) produced increased motor activity, jumping and circling opposite to the injection site, associated with ECoG desynchronization, high voltage epileptogenic spikes, and with neuropathological effects. These effects were accompanied by increase of malondialdehyde (MDA) levels in the Substantia Nigra, suggesting that paraquat was able to induce oxidative stress when injected directly into the rat brain. Pre-treatment of rats with M40401, a non peptidyl superoxide dismutase (SOD) mimetic given directly into the Substantia Nigra or i.p. prevented both behavioural, electrocorticogram and neuropathological effects and MDA elevation. Taken together, these results demonstrate that paraquat produces brain damage via abnormal formation of oxygen free radicals and that this effect may be counteracted by novel SOD mimetics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The role of the
L
‐arginine‐nitric oxide (NO) pathway on the formation of prostaglandin E
2
(PGE
2
) by human cultured astroglial cells incubated with interleukin‐1β (IL‐1β) and tumour necrosis ...factor‐α (TNF‐α) was investigated.
Incubation of T 67 astroglial cell line with IL‐β (10 ng ml
−1
) and TNF‐α (500 u ml
−1
) produced a significant (
P
<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE
2
levels in cell supernatants. N
ω
‐nitro‐
L
‐arginine methyl ester (
L
‐NAME; 20–300 μ
M
), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine‐treated astroglial cells and reduced the release of PGE
2
. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL‐1β and TNF‐α was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 μ
M
), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 μ
M
).
The inhibition elicited by
L
‐NAME on PGE
2
‐release by cytokine‐treated astroglial cells was reversed by adding AA (40 μ
M
), showing that the effect of NO on cytokine‐dependent PGE
2
release occurred at the cyclo‐oxygenase (COX) level. Furthermore, the release of PGE
2
in cytokine‐treated astroglial cells was inhibited by indomethacin (10 μ
M
), a COX inhibitor as well as by preincubating cells with dexamethasone (20 μ
M
), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX‐2) was involved.
On the other hand, pretreating astroglial cells with methylene blue (MB; 10 μ
M
), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE
2
release in cytokine‐treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites.
The present experiments demonstrated that the release of PGE
2
by astroglial cells pretreated with IL‐1β and TNF‐α is due to enhanced COX‐2 activity via activation of the
L
‐arginine‐NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract The removal of pathologically generated free radicals produced during ischemia, reperfusion and intracranical hemorrhage seems to be a viable approach to neuroprotection. However, at ...present, no neuroprotective agent has proven effective in focal ischemic stroke phase III trials, despite the encouraging data in animal models. This study aimed to explore the effect of the brain penetrant low molecular weight radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) in neurological damage subsequent to ischemia–reperfusion injury in Mongolian gerbils. We examined the intraperitoneal effects of IAC on temporary bilateral common carotid artery occlusion (BCCO) by means of morphological and histological analysis of the hippocampus. Significant dose-dependent protective effects of IAC (1 to 10 mg/kg b.w. ) against neuropathological and morphological brain changes were seen when administered i.p. 1 h before temporary BCCO in Mongolian gerbils. When administered up to 6 h after BCCO, IAC actually reverses the neurodegenerative processes (e.g. hippocampal cell viability) induced by ischemia in a dose-dependent fashion. Data show that IAC is highly effective in protecting and preventing oxidative brain damage associated with cerebral flow disturbances. It is also effective even in late treatment of the insult, emphasizing its potential role for the management of ischemic stroke patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The role of the L‐arginine‐nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin‐1β (IL‐1β) and tumour necrosis factor‐α ...(TNF‐α) was investigated.
Incubation of T 67 astroglial cell line with IL‐β (10 ng ml−1) and TNF‐α (500 u ml−1) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. Nω‐nitro‐L‐arginine methyl ester (L‐NAME; 20–300 μM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine‐treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL‐1β and TNF‐α was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 μM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 μM).
The inhibition elicited by L‐NAME on PGE2‐release by cytokine‐treated astroglial cells was reversed by adding AA (40 μM), showing that the effect of NO on cytokine‐dependent PGE2 release occurred at the cyclo‐oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine‐treated astroglial cells was inhibited by indomethacin (10 μM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 μM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX‐2) was involved.
On the other hand, pretreating astroglial cells with methylene blue (MB; 10 μM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine‐treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites.
The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL‐1β and TNF‐α is due to enhanced COX‐2 activity via activation of the L‐arginine‐NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Bartonella quintana, an emerging gram-negative pathogen, may cause trench fever, endocarditis, cerebral abscess and bacillary angiomatosis usually with the absence of septic shock in humans.
B. ...quintana lipopolysaccharide (LPS), a deep rough endotoxin with strong reactivity in the limulus amebocyte lysate (LAL)-assay, was studied in human whole blood and in a rat model. A significant (
P<0.05) increase of interleukin-8 (IL-8) concentration, comparable to the level induced by enterobacterial LPS, was stimulated in the human whole blood by
B. quintana LPS. Isolated human neutrophils delayed their apoptotic behavior in the presence of
B. quintana LPS. In the rat,
B. quintana LPS induced a significant (
P<0.001) increase in white blood cell count, both 30 and 60 min after intravenous injection. Such leukocytosis was inhibited by pretreatment with prazosin, an α-adrenergic antagonist.
B. quintana LPS did not significantly change heart rate (HR), hematocrit (HCT) and platelet count in the above reported in vivo model, and regarding mean blood pressure (MAP) only a very early (5 min after LPS) and mild (yet significant) hypotension was observed. In contrast, a long-lasting decrease of MAP was found in
Salmonella minnesota R595 LPS-treated animals. Blood TNFα levels did not change significantly from the baseline in rats injected with either saline or with
B. quintana LPS, on the contrary
S. minnesota R595 LPS-injected animals showed substantial increase of TNFα levels up to 2924 pg/ml at 60 min after LPS injection.
B. quintana LPS as well as
Salmonella LPS-injected rats exhibited an increase of the blood levels of GRO/CINC-1, particularly at 240 min after LPS administration. Apical part of rat gut villi showed several TUNEL-positive cells in tissue sections from
B. quintana LPS-treated animals. Taken together, our data demonstrates that
B. quintana LPS is able to selectively stimulate some inflammatory mediators.
B. quintana LPS-induced leukocytosis appears mediated by an α-adrenergic receptor. The delayed apoptotic process of leukocytes and the chemokine increase may explain the apoptotic cells found in the rat gut and the inflammatory reactions in some human
Bartonella diseases. This peculiar inflammatory pattern induced by
B. quintana LPS, may partially account for the lack of severe septic shock, observed in human
B. quintana infections.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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