Susanne Müller

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.

Candesartan but not ramipril pretreatment improves outcome after stroke and stimulates neurotrophin BNDF/TrkB system in rats

Objectives Drugs interfering with the renin–angiotensin system (RAS) have been shown to reduce the incidence of stroke in patients at risk and to afford neuroprotection in experimental brain ischemia. This study aimed to compare potential neuroprotective effects of systemic pretreatment with the angiotensin receptor blocker, candesartan, and the angiotensin-converting enzyme (ACE)-inhibitor, ramipril, in normotensive Wistar rats after focal cerebral ischemia, with special emphasis on the regulation of neurotrophins. Methods Equipotent subcutaneous doses of candesartan and ramipril were determined via inhibition of pressor responses to intravenously injected angiotensin II (Ang II) or angiotensin I (Ang I), respectively. Accordingly, animals were treated with candesartan (0.1 mg/kg body weight, twice daily), ramipril (0.01 and 0.1 mg/kg body weight, twice daily) or vehicle (0.9% saline, twice daily), respectively, 5 days prior to middle cerebral artery occlusion (MCAO) with reperfusion. Severity of stroke was estimated via infarct size [magnetic resonance imaging (MRI) 48 h after MCAO] and neurological outcome (24 h, 48 h after MCAO). Measurements of neurotrophins/receptors in brain tissue were performed 48 h after MCAO. Results Pretreatment with candesartan and ramipril (low dose) did not reduce blood pressure during MCAO, whereas ramipril high dose did. Candesartan, but not ramipril at any dose, significantly reduced stroke volume and improved neurological outcome. Poststroke mRNA and protein of the neurotrophin receptor, TrkB, were significantly elevated in animals treated with candesartan, but not ramipril. Conclusions Systemic pretreatment with a sub-hypotensive, RAS-blocking dose of candesartan affords neuroprotection after focal ischemia, associated with increased activity of the neurotrophin BDNF/TrkB system. Ramipril at sub-hypotensive and hypotensive, RAS-blocking doses showed no significant neuroprotective effects.

Resident microglia, and not peripheral macrophages, are the main source of brain tumor mononuclear cells.

Gliomas consist of multiple cell types, including an abundant number of microglia and macrophages, whereby their impact on tumor progression is controversially discussed. To understand their unique functions and consequently manipulate either microglia or macrophages in therapeutic approaches, it is essential to discriminate between both cell populations. Because of the lack of specific markers, generally total body irradiated chimeras with labeled bone marrow cells were used to identify infiltrated cells within the brain. However, total body irradiation (TBI) affects the blood–brain barrier integrity, which in turn potentially facilitates immune cell infiltration. In this study, changes on the blood–brain barrier were avoided using head‐protected irradiation (HPI). Head protection and total body irradiated chimeras exhibited similar reconstitution levels of the myeloid cell lineage in the blood, enabling the comparable analyses of brain infiltrates. We demonstrate that the HPI model impeded a massive unspecific influx of donor‐derived myeloid cells into naive as well as tumor‐bearing brains. Moreover, experimental artifacts such as an enlarged distribution of infiltrated cells and fourfold increased tumor volumes are prevented in head‐protected chimeras. In addition, our data evidenced for the first time that microglia are able to up‐regulate CD45 and represent an inherent part of the CD45high population in the tumor context. All in all, HPI allowed for the unequivocal distinction between microglia and macrophages without alterations of tumor biology and consequently permits a detailed and realistic description of the myeloid cell composition in gliomas.

Comparison between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain.

Angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have become well established drugs for the treatment of major risk factors of stroke. Since several studies provided evidence that ARBs and TZDs also have additional anti-inflammatory effects, we hypothesized that a combined treatment with the ARB, candesartan, and the TZD, pioglitazone, ameliorates ischemia-induced brain injury and inflammation by synergistic anti-inflammatory actions. Normotensive Wistar rats were pre-treated for 5 days with vehicle (0.9% NaCl), 0.2 mg/kg/day candesartan (s.c.), and/or 2 and/or 20 mg/kg/day pioglitazone (p.o.), respectively and underwent 90 min of middle cerebral artery occlusion (MCAO) with successive reperfusion. Neurological deficits and infarct size were determined 24 h and 48 h after MCAO, respectively, followed by tissue sampling. Animals treated with candesartan, pioglitazone, and the combination of candesartan and pioglitazone had reduced neurological deficits 24 h and 48 h after MCAO, respectively (P<0.05-0.01). Infarct size was reduced by treatment of candesartan, pioglitazone, and their respective combination (each P<0.05) 48 h after stroke compared to vehicle. Treatment with candesartan, pioglitazone, and their combination resulted in significantly reduced mRNA expression of the inflammatory markers CXCL1 and TNFalpha in vivo (P<0.01). The combination of candesartan plus pioglitazone is equally effective compared to their single applications concerning neuroprotection and attenuation of inflammation after MCAO. Therefore, we conclude that a direct synergistic neuroprotective action of parallel ARB and TZD treatment is unlikely.

MR elastography in a murine stroke model reveals correlation of macroscopic viscoelastic properties of the brain with neuronal density.

The aim of this study was to investigate the influence of neuronal density on viscoelastic parameters of living brain tissue after ischemic infarction in the mouse using MR elastography (MRE). Transient middle cerebral artery occlusion (MCAO) in the left hemisphere was induced in 20 mice. In vivo 7‐T MRE at a vibration frequency of 900 Hz was performed on days 3, 7, 14 and 28 (n = 5 per group) after MCAO, followed by the analysis of histological markers, such as neuron counts (NeuN). MCAO led to a significant reduction in the storage modulus in the left hemisphere relative to contralateral values (p = 0.03) without changes over time. A correlation between storage modulus and NeuN in both hemispheres was observed, with correlation coefficients of R = 0.648 (p = 0.002, left) and R = 0.622 (p = 0.003, right). The loss modulus was less sensitive to MCAO, but correlated with NeuN in the left hemisphere (R = 0.764, p = 0.0001). In agreement with the literature, these results suggest that the shear modulus in the brain is reduced after transient ischemic insult. Furthermore, our study provides evidence that the in vivo shear modulus of brain tissue correlates with neuronal density. In diagnostic applications, MRE may thus have diagnostic potential as a tool for image‐based quantification of neurodegenerative processes. Copyright

Determination of the brain-blood partition coefficient for water in mice using MRI

Cerebral blood flow (CBF) quantification is a valuable tool in stroke research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides repetitive, noninvasive CBF quantification. Many MRI techniques require the knowledge of the brain–blood partition coefficient (BBPC) for water. Adopting an MRI protocol described by Roberts et al (1996) in humans, we determined the BBPC for water in 129S6/SvEv mice from proton density measurements of brain and blood, calibrated with deuterium oxide/water phantoms. The average BBPC for water was 0.89±0.03 mL/g, with little regional variation within the mouse brain.

The possible impact of noise-induced Ca2+-dependent activity in the central auditory pathway: A manganese-enhanced MRI study

Noise exposure at high intensities leads to a temporary shift of hearing thresholds (TTS) and is followed by a permanent threshold shift (PTS). Permanent threshold shift is not only associated with cochlear damage as the primary site-of-lesion, but also with subsequent structural and functional changes within the central auditory pathway. The aim of the present study was to monitor neuronal activity within central auditory structures in mice after noise exposure at different time intervals using manganese-enhanced magnetic resonance imaging (MEMRI). The results demonstrate for the first time that calcium-dependent activity patterns are modified in several structures of the central auditory system as the result of a noise-induced hearing loss (NIHL). The MEMRI data demonstrate that temporary threshold shift is correlated with an activity increase in hierarchically lower structures of the auditory pathway. This seems to be indicative of a direct noise impact at the first stage of central auditory processing. However, noise-dependent changes of higher auditory structures were found as well in the phase of PTS. Repeated noise exposure was found to induce an additional elevation of calcium-dependent activity in all investigated auditory structures - without a significant shift in auditory thresholds. Sustained manganese accumulation was present in the auditory brainstem after moderate acoustic stimulation as well without PTS induction. The long-lasting enhancement of MEMRI signals suggests a noise-induced activity increase of various calcium-dependent processes of different origin (such as neuroprotective mechanisms). The present findings could be helpful to better understand the time-course of different symptoms in NIHL and the individual susceptibility to noise.

Adolescent hyperactivity and impaired coordination after neonatal hyperoxia.

In preterm infants, the risk to develop attention-deficit/hyperactivity disorder is 3 to 4-fold higher than in term infants. Moreover, preterm infants exhibit deficits in motor coordination and balance. Based on clinical data, higher oxygen levels in preterm infants lead to worse neurological outcome, and experimental hyperoxia causes wide-ranging cerebral changes in neonatal rodents. We hypothesize that hyperoxia in the immature brain may affect motor activity in preterm infants. We subjected newborn mice from P6 to P8 to 48 h of hyperoxia (80% O(2)) and tested motor activity in running wheels starting at adolescent age P30. Subsequently, from P44 to P53, regular wheels were replaced by complex wheels with variable crossbar positions to assess motor coordination deficits. MRI with diffusion tensor imaging was performed in the corpus callosum to determine white matter diffusivity in mice after hyperoxia at ages P30 and P53 in comparison to control animals. Adolescent mice after neonatal hyperoxia revealed significantly higher values for maximum velocity and mean velocity in regular wheels than controls (P<0.05). In the complex running wheels, however, maximum velocity was decreased in animals after hyperoxia, as compared to controls (P<0.05). Decreased fractional anisotropy and increased radial diffusion coefficient were observed in the corpus callosum of P30 and P53 mice after neonatal hyperoxia compared to control mice. Hyperoxia in the immature brain causes hyperactivity, motor coordination deficits, and impaired white matter diffusivity in adolescent and young adult mice.

MRI heralds secondary nigral lesion after brain ischemia in mice: a secondary time window for neuroprotection

Cerebral ischemia in the territory of the middle cerebral artery (MCA) can induce delayed neuronal cell death in the ipsilateral substantia nigra (SN) remote from the primary ischemic lesion. This exofocal postischemic neuronal degeneration (EPND) may worsen stroke outcomes. However, the mechanisms leading to EPND are poorly understood. Here, we studied the time course of EPND via sequential magnetic resonance imaging (MRI) and immunohistochemistry for up to 28 days after 30 minutes occlusion of the MCA (MCAo) and reperfusion in the mouse. Furthermore, the effects of delayed treatment with FK506 and MK-801 on the development of EPND were investigated. Secondary neuronal degeneration in the SN occurred within the first week after MCAo and was characterized by a marked neuronal cell loss on histology. Sequential neuroimaging examinations revealed transient MRI changes, which were detectable as early as day 4 after MCAo and thus heralding histologic evidence of EPND. Treatment with MK-801, an established anti-excitotoxic agent, conferred protection against EPND even when initiated days after the initial ischemic event, which was not evident with FK506. Our findings define a secondary time window for delayed neuroprotection after stroke, which may provide a promising target for the development of novel therapies.

Mortality and morbidity in different immunization protocols for experimental autoimmune myocarditis in rats

We aimed to investigate the histological and clinical presentations of experimental autoimmune myocarditis (EAM) induced by different immunization schemes.