Kristin Lucht

The beta-lactam antibiotic, ceftriaxone, dramatically improves survival, increases glutamate uptake and induces neurotrophins in stroke.

Objective Ceftriaxone has been reported to reduce neuronal damage in amyotrophic lateral sclerosis and in an in-vitro model of neuronal ischaemia through increased expression and activity of the glutamate transporter, GLT1. We tested the effects of ceftriaxone on mortality, neurological outcome, and infarct size in experimental stroke in rats and looked for underlying mechanisms. Methods Male normotensive Wistar rats received ceftriaxone (200 mg/kg intraperitoneal) as a single injection 90 min after middle cerebral artery occlusion (90 min with reperfusion). Forty-eight hours after middle cerebral artery occlusion, infarct size (MRI) and neurological deficits were estimated. GLT1 expression was determined by real time RT-PCR, immunoblotting and promoter reporter assay, astrocyte GLT1 activity by measuring glutamate uptake. Bacterial load in various organs was measured by real time RT-PCR, neurotrophins and IL-6 by immunoblotting. Results Ceftriaxone dramatically reduced early (24-h) mortality from 34.5% (vehicle treatment, n = 29) to 0% (P < 0.01, n = 19). In a subgroup, followed up for 4 weeks, mortality persisted at 0%. Ceftriaxone strongly tended to reduce infarct size, it significantly improved neuronal survival within the penumbra, reduced neurological deficits (P < 0.001) and led to an upregulation of neurotrophins (P < 0.01) in the peri-infarct zone. Ceftriaxone did not increase GLT1 expression, but increased GLT1 activity (P < 0.05). Conclusion Ceftriaxone causes a significant reduction in acute stroke mortality in a poststroke treatment regimen in animal studies. Improved neurological performance and survival may be due to neuroprotection by activation of GLT1 and a stimulation of neurotrophins resulting in an increased number of surviving neurons in the penumbra.

Direct Angiotensin II Type 2 Receptor Stimulation in Nω-Nitro-l-Arginine-Methyl Ester–Induced Hypertension: The Effect on Pulse Wave Velocity and Aortic Remodeling

Pulse wave velocity (PWV), a direct marker of arterial stiffness, is an independent cardiovascular risk factor. Although the angiotensin II type 1 receptor blockade belongs to major antihypertensive and cardioprotective therapies, less is known about the effects of long-term stimulation of the angiotensin II type 2 receptor. Previously, compound 21, a selective nonpeptide angiotensin II type 2 receptor agonist improved the outcome of myocardial infarction in rats along with anti-inflammatory properties. We investigated whether compound 21 alone or in combination with angiotensin II type 1 receptor blockade by olmesartan medoxomil could prevent PWV increase and aortic remodeling in N&ohgr;-nitro-L-arginine-methyl ester (L-NAME)–induced hypertension. Male adult Wistar rats (n=65) were randomly assigned to control, L-NAME, L-NAME+compound-21, L-NAME+olmesartan, and L-NAME+olmesartan+compound-21 groups and treated for 6 weeks. We observed that L-NAME hypertension was accompanied by enhanced PWV, increased wall thickness, and stiffness of the aorta, along with elevated hydroxyproline concentration. Olmesartan completely prevented hypertension, PWV and wall thickness increase, and the increase of aortic stiffness and partly prevented hydroxyproline accumulation. Compound 21 partly prevented all of these alterations, yet without concomitant prevention of blood pressure rise. Although the combination therapy with olmesartan and compound 21 led to blood pressure levels, PWV, and wall thickness comparable to olmesartan-alone–treated rats, only in the combination group was complete prevention of increased hydroxyproline deposition achieved, resulting in even more pronounced stiffness reduction. We conclude that chronic angiotensin II type 2 receptor stimulation prevented aortic stiffening and collagen accumulation without preventing hypertension in rats with inhibited NO synthase. These effects were additive to angiotensin II type 1 receptor blockade, yet without additional blood pressure–lowering effect, and they seem to be NO and blood pressure independent.

AT2-receptor stimulation enhances axonal plasticity after spinal cord injury by upregulating BDNF expression

It is widely accepted that the angiotensin AT2-receptor (AT2R) has neuroprotective features. In the present study we tested pharmacological AT2R-stimulation as a therapeutic approach in a model of spinal cord compression injury (SCI) in mice using the novel non-peptide AT2R-agonist, Compound 21 (C21). Complementary experiments in primary neurons and organotypic cultures served to identify underlying mechanisms. Functional recovery and plasticity of corticospinal tract (CST) fibers following SCI were monitored after application of C21 (0.3mg/kg/dayi.p.) or vehicle for 4 weeks. Organotypic co-culture of GFP-positive entorhinal cortices with hippocampal target tissue served to evaluate the impact of C21 on reinnervation. Neuronal differentiation, apoptosis and expression of neurotrophins were investigated in primary murine astrocytes and neuronal cells. C21 significantly improved functional recovery after SCI compared to controls, and this significantly correlated with the increased number of CST fibers caudal to the lesion site. In vitro, C21 significantly promoted reinnervation in organotypic brain slice co-cultures (+50%) and neurite outgrowth of primary neurons (+25%). C21-induced neurite outgrowth was absent in neurons derived from AT2R-KO mice. In primary neurons, treatment with C21 further induced RNA expression of anti-apoptotic Bcl-2 (+75.7%), brain-derived neurotrophic factor (BDNF) (+53.7%), the neurotrophin receptors TrkA (+57.4%) and TrkB (+67.9%) and a marker for neurite growth, GAP43 (+103%), but not TrkC. Our data suggest that selective AT2R-stimulation improves functional recovery in experimental spinal cord injury through promotion of axonal plasticity and through neuroprotective and anti-apoptotic mechanisms. Thus, AT2R-stimulation may be considered for the development of a novel therapeutic approach for the treatment of spinal cord injury.

The Promyelocytic Leukemia Zinc Finger (PLZF) Protein Exerts Neuroprotective Effects in Neuronal Cells and is Dysregulated in Experimental Stroke

Stroke is one of the major medical burdens in industrialized countries. Animal experiments indicate that blockade of the angiotensin AT1 receptor (AT1R) improves neurological outcome after cerebral ischemia. These protective effects are partially mediated by the angiotensin AT2 receptor (AT2R). The transcription factor promyelocytic leukemia zinc finger (PLZF) was identified as a direct adapter protein of the AT2R. Furthermore, our group was able to demonstrate that PLZF also directly binds and mediates the effects of the human (pro)renin receptor [(P)RR] which is involved in brain development. Therefore, we hypothesized that PLZF is involved in neuroprotection.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin–angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1–7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that—at least in astrocytes—both receptors functionally depend on each other.

Novel therapy approach in primary stroke prevention: Simultaneous inhibition of endothelin converting enzyme and neutral endopeptidase in spontaneously hypertensive, stroke-prone rats improves survival

Abstract Objectives: Stroke, frequently a consequence of hypertension, is one of the leading causes of death and neurological disabilities worldwide. In the ischemic brain, levels of endothelin-1, one of the most potent vasoconstrictors, are raised. Anti-inflammatory and neuroprotective effects of endothelin antagonists after stroke have been described in literature. Based on these findings, we investigated the protective effect of the endothelin converting enzyme/neutral endopeptidase blocker, SLV 338, in salt-loaded, stroke-prone, spontaneously hypertensive rats. Methods: Male, 8-week-old spontaneously hypertensive stroke-prone rats were put on a high salt diet and treated with either 30 mg/kg or 100 mg/kg SLV 338 or vehicle for 27 weeks. Blood pressure, neurological outcome, body weight, and mortality were investigated throughout treatment. In weeks 1 and 9, animals were housed in metabolic cages for collection of urinary and blood samples and assessment of salt water and food intake. In weeks 22 and 27, additional blood samples were taken. At the end of the study, all brains were analyzed using magnetic resonance imaging. Results: SLV 338 was well tolerated in all animals. Neurological outcome and infarct size were similar in all groups. Albuminuria was considerably delayed and the incidence of stroke significantly lowered in treated animals. In spontaneously hypertensive stroke-prone rats, treatment with SLV 338 significantly (P = 0·01) improved survival in comparison to the vehicle treated group in a blood pressure-independent manner. Discussion: Our data in spontaneously hypertensive stroke-prone rats demonstrate that combined endothelin converting enzyme/neutral endopeptidase inhibition could offer a new therapeutic approach for primary stroke prevention and improvement of mortality. The mechanism seems to be blood pressure-independent.

931 INHIBITION OF MICROGLIA ACTIVATION AND MIGRATION BY DIRECT AT2-RECEPTOR STIMULATION

Objectives: Previously we have shown that stimulation of the AT2-receptor reduces synthesis of proinflammatory cytokines in vitro and in vivo in various cell types. In the CNS, microglia represent the major population of immune cells playing a detrimental role in stroke, multiple sclerosis and spinal cord injury. We hypothesized that AT2R stimulation with Compound 21 (C21) will modify microglial activation and migration. Methods: Microglia were isolated from C57BL/6 mice, stimulated with LPS (10 &mgr;g/ml) plus INF&ggr; (100U/ml) for 24hrs and co-treated either with C21 (1 &mgr;M) or vehicle. Microglial activation was analyzed by measurement of nictric oxide release and by a migration assay using xCelligence System with ADP (50 &mgr;M) as a chemoattractant. Immune cell infiltration in vivo was studied in an EAE -animal model of multiple sclerosis in C57BL/6 mice treated daily either with C21 (0.3 mg/kgBW) or vehicle for 28 days. The infiltration was analyzed in spinal cord tissue by immunohistology and by FACS. Results: Stimulation with LPS/INF&ggr; induced NO release (39.4 vs. 2.7 &mgr;M, p<0.001) that was reduced by C21 (30.6 &mgr;M, p<0.05). ADP-mediated microglial migration (increase by 53% vs. control) was almost completely abolished by C21 (p<0.01). EAE induced massive infiltration of microglia into the spinal cord (p<0.05 for histology; p<0.01 for FACS). C21-treatment tended to reduce the infiltration score in histological sections (p>0.05) and significantly lowered the number of microglia in lumbar spinal cord as estimated by FACS (5.215 vs. 11.83 for vehicle, p<0.05). Conclusions: AT2R-stimulation may be considered a novel therapeutic approach to reduce inflammation in the CNS.

1033 DIRECT AT2-RECEPTOR STIMULATION IMPROVES NEUROLOGICAL OUTCOME IN A MOUSE MODEL OF MULTIPLE SCLEROSIS

Objectives: The angiotensin AT2-receptor (AT2R) acts neuroprotective in models of neuronal injury. Here we identify the impact of direct AT2R stimulation by the selective non-peptide agonist C21 in myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmune encephalomyelitis (MOG-EAE), a mouse model mimicking many aspects of multiple sclerosis. Furthermore, we examined the effects of C21 on remyelination using an in vitro model of neuroinflammation. Design and methods: C57BL/6 mice were immunized with the MOG35–55 fragment and treated from day 3 before immunization until day 28 after immunization with C21 (0.3 mg/kg bw i.p.) or vehicle. Neurological deficits were evaluated daily. Spinal cord tissue was extracted on day 28 post-immunization and stained by fluoromyelin for myelin quantification. Furthermore, to test whether AT2R-stimulation can promote remyelination, aggregating brain cell cultures consisting of neurons, astrocytes, oligodendrocytes and microglia were prepared from whole embryonic rat brain and treated for 7 days with C21 (1 &mgr;M) or vehicle after IFN-&ggr;/LPS-induced demyelination. The degree of myelination was determined by immunofluorescence for MOG. Staining was quantified by ImageJ. Results: Preventive application of AT2R agonist C21 resulted in a significantly ameliorated course of MOG-EAE and prevented demyelination in spinal cord tissue. Additionally, AT2R stimulation led to a significant increase in MOG-positive area after IFN-&ggr;/LPS-induced demyelination in vitro (93.02% vs 78.13%, p < 0.01) when compared to vehicle treated aggregates. In vitro effects of C21 were inhibited by the specific AT2R antagonist PD 123319. Conclusions: Direct AT2R stimulation improves neurological outcome, protects against demyelination and promotes remyelination. Our findings identify AT2R stimulation as a potential new therapeutic target for demyelinating diseases.

1029 AT2 receptor stimulation and vascular remodeling in L-NAME-induced Hypertension

Background: We investigated, whether AT2 receptor (AT2R) stimulation by compound 21 alone or combined with AT1 receptor (AT1R) blockade prevented aortic stiffening in L-NAME-induced hypertension. Design and methods: Male adult Wistar rats (n = 65) were randomized into 6 groups: control, L-NAME and L-NAME + either compound 21, olmesartan or compound 21 + olmesartan. Blood pressure (BP) was measured each week. After 6-week treatment, aortic hydroxyproline content, PWV, wall thickness (WT) and inner diameter were determined and aortic stiffness (elasticity modulus) was estimated. Results: L-NAME led to increased BP, PWV, WT, aortic stiffness and hydroxyproline concentration. Olmesartan completely prevented BP, PWV, WT and stiffness increase and partly prevented hydroxyproline accumulation. Compound 21 partly prevented all these alterations, yet without concomitant prevention of BP rise. Although the combination therapy with olmesartan and compound 21 led to BP levels, PWV and WT comparable to olmesartan-alone-treated rats, only in the combination group complete prevention of increased hydroxyproline deposition was achieved, resulting in even more pronounced stiffness reduction. Conclusions: In rats with inhibited NO-synthase, the BP-independent effect on aortic stiffening and collagen accumulation by AT2R stimulation was additive to AT1R blockade.