Marie-Françoise Ritz

Effect of 17β-estradiol on functional outcome, release of cytokines, astrocyte reactivity and inflammatory spreading after spinal cord injury in male rats

The effect of 17beta-estradiol on the secondary damage following spinal cord injury (SCI) was examined in male rats subjected to moderate compression. Two doses of 17beta-estradiol (0.1 or 4 mg/kg) were injected i.p. immediately after spinal cord compression. Functional outcome was observed during 4 weeks following injury with two different tests. Release of cytokines (IL-1alpha, IL-1beta and IL-6) was assessed 6 h, 3 days and 1 week post-injury. Reactive astrocytes expressing the glial fibrillary acidic protein GFAP and vimentin, and diffusion of CD68-positive inflammatory cells were examined from 3 days to 4 weeks following SCI. Treatment with 17beta-estradiol significantly increased locomotor function from the first week until 4 weeks post-SCI. The injured spinal cord of 17beta-estradiol-treated rats expressed more IL-1alpha, IL-1beta and IL-6 than controls 6 h after injury. Moreover, 17beta-estradiol-treated rats showed reactive astrocytes as soon as 3 days following SCI, with increased GFAP expression, smaller lesion areas and more limited diffusion of CD68-positive cells after 1 week post-injury compared to controls. The number of CD68-positive cells was also reduced in 17beta-estradiol-treated rats one week post-SCI. However, these differences between 17beta-estradiol-treated and control rats disappeared after 4 weeks. These results suggest that 17beta-estradiol protects the spinal cord by stimulating early cytokines release and astroglial responses. These stimulations may prevent the area of damage from expanding and inflammatory cells to spread in the surrounding tissue during the critical first week following SCI. Although transient, these effects improved the locomotor recovery that was sustained over 4 weeks after injury.

Acute treatment with red wine polyphenols protects from ischemia-induced excitotoxicity, energy failure and oxidative stress in rats

Red wine polyphenolic compounds (RWPC) possess numerous neuroprotective activities that may be beneficial for treating cerebral ischemia. To investigate the in vivo effects of an acute treatment with RWPC during stroke, male Wistar rats were subjected to transient ischemia for 90 min and immediately treated with RWPC. The extracellular concentrations of excitatory amino acids, free radical scavengers and energy metabolites during occlusion and reperfusion were monitored using microdialysis. The brain lesions were measured 24 h after reperfusion using immunohistological staining. We found that acute treatment with RWPC significantly reduced the burst of amino acids glutamate, aspartate and taurine in response to ischemia and increased the levels of free radical scavengers ascorbic and uric acids during occlusion or at early reperfusion, respectively. The concentration of glucose was improved during occlusion whereas the level of lactate strongly decreased during reperfusion in RWPC treated animals, suggesting an increased use of this substrate by surviving neurons. RWPC also significantly improved blood flow during reperfusion and brain tissue preservation as observed 24 h after MCAO in treated animals. These findings strongly suggest that RWPC are agents able to fight against the excitotoxic, oxidative pathways and metabolic dysfunction induced by cerebral ischemia.

Acute effects of 17β-estradiol on the extracellular concentration of excitatory amino acids and energy metabolites during transient cerebral ischemia in male rats

Elevation of extracellular levels of amino acids has been implicated in the pathogenesis of stroke. The failure of brain energy metabolism due to the lack of oxygen and glucose contributes also to cell loss. Estrogen has been shown to protect brain cells against ischemia by a still unclear mechanism. We used intracerebral microdialysis to monitor the effects of acute 17beta-estradiol treatment on the release of glutamate and aspartate and on the levels of the energy metabolites glucose and lactate. In male rats subjected to 90 min of transient middle cerebral artery occlusion followed by 24-h reperfusion, acute treatment with 17beta-estradiol (0.8 mg/kg, i.v.) at the time of occlusion reduced the ischemic infarct by about 50%. In these treated rats, the ischemia-induced increases of extracellular levels of glutamate and aspartate were significantly and rapidly reduced. The reduction of glucose level during occlusion was not affected by 17beta-estradiol treatment; however, the increase of extracellular lactate was reduced during occlusion and reperfusion, probably due to the reduced glutamate-driven astrocytic glycolysis. These data suggest that acute treatment with 17beta-estradiol at the onset of occlusion significantly reduces the ischemia-induced excitotoxicity in the cortex, a mechanism that may participate in the neuroprotective effect on cellular survival.

17β-Estradiol Effect on the Extracellular Concentration of Amino Acids in the Glutamate Excitotoxicity Model in the Rat

Estrogen has demonstrated a neuroprotective role in a rat model of glutamate excitotoxicity and other neurodegenerative disorders. We studied the effect of 17β-estradiol on glutamate-induced increases in amino acids levels (aspartate, histidine, taurine and GABA) in the rat cortex. Local perfusion of glutamate produced a transient increase of aspartate, histidine, taurine and GABA in the extracellular fluid. Pretreatment with 17β-estradiol significantly reduced the increases of taurine and moderately attenuated that of histidine, whereas aspartate and GABA releases were not modified. The effect of 17β-estradiol on histidine release was reversed by the antiestrogen tamoxifen, suggesting a receptor-dependent mechanism. Good correlations between the volumes of the glutamate-induced lesions and the extracellular concentrations of taurine and aspartate were observed. These findings suggest that the attenuation of the glutamate-induced release of taurine by 17β-estradiol may participate in the neuroprotective effects of 17β-estradiol and that increased levels of aspartate and taurine are markers for the severity of the glutamate-induced cortical lesions.

Traumatic Spinal Cord Injury Alters Angiogenic Factors and TGF-Beta1 that may Affect Vascular Recovery

Traumatic spinal cord injury (SCI) disrupts the blood-spinal cord barrier and reduces the blood supply caused by microvascular changes. Vessel regression and neovascularization have been observed in the course of secondary injury contributing to microvascular remodeling after trauma. Spatio-temporal distribution of blood vessels and modulation of gene expression of several angiogenic factors have been investigated in rats after spinal cord compression injury. Rarefaction of vessels was detectable at the injury site 2 days after SCI before they disappeared in the developing cavity after 2 and 4 weeks, whereas no changes were observed in the penumbra. Investigation of the temporal expression of angiogenic genes using quantitative RT-PCR disclosed a constant down-regulation of the vascular endothelial growth factor (VEGF), and transient decreases of angiopoietin-1 (Ang-1), platelet-derived growth factor-BB (PDGF-BB), as well as placental growth factor (PlGF), with the lowest values obtained 3 days after injury, when compared to the expression levels obtained in sham-operated rats. Hepatocyte growth factor (HGF) was the only angiogenic factor with a constant increased gene expression when compared with controls, starting at day 3 post-SCI. mRNA levels of transforming growth factor-beta 1 (TGF-β1) were elevated at every time point following SCI, whereas those encoding for the cysteine-rich protein CCN1/CYR61 were upregulated after 2 h, 6 h, and 1 week only. Our data provide an overview of the temporal modulated expression of the major angiogenic factors, hampering revascularization in the lesion during the phase of secondary injury. These findings should be considered in order to improve therapeutic interventions.

Necessity for re-vascularization after spinal cord injury and the search for potential therapeutic options.

Disruption of the blood-spinal cord barrier (BSCB) and microvascular changes leading to reduction of blood supply represent hallmarks of spinal cord secondary injury causing further deterioration of the traumatized patient. Injury to the blood vessels starts with prominent hemorrhage and generation of inflammation. Furthermore, spinal cord ischemia and extravasation of blood components contribute to edema formation resulting in death of neural cells. Endogenous attempts of re-vascularization have been observed although these newly formed vessels display morphological and functional abnormalities. The unfavorable regulation of angiogenic and counterregulatory anti-angiogenic factors during the complicated course of vessel remodeling after SCI is suspected to participate in the failure of re-vascularization and vessel stabilization. Repression of the expression of angiogenic factors such as vascular endothelial growth factor-A (VEGF-A), placental growth factor (PlGF), angiopoietin-1 (Ang1), and platelet-derived growth factor-BB (PDGF-BB) contributes to vessel regression. Therefore, therapeutic applications of angiogenic factors following SCI are promising strategies to restore blood flow in the lesion.

EFFECTS OF ISOFLURANE ON GLUTAMATE AND TAURINE RELEASES, BRAIN SWELLING AND INJURY DURING TRANSIENT ISCHEMIA AND REPERFUSION

The volatile anesthetic agent isoflurane was thought to provide neuroprotection against ischemic damage; however, this effect remains controversial. Using the middle cerebral artery occlusion model and intracerebral microdialysis, the authors monitored the variations of glutamate and taurine concentrations in the extra-cellular space in male rats anesthetized with pentobarbital or isoflurane. Brain injury and edema were evaluated 24 h after ischemia. Isoflurane prevented the ischemia-induced efflux of glutamate and reduced the release of taurine. No difference in the size of the brain lesions was observed with both anesthetics, and isoflurane induced the formation of a bigger brain edema and reduced taurine release. These results suggest that inhibiting glutamate release during ischemia may not be sufficient to improve brain outcome after transient ischemia.

Gene expression suggests spontaneously hypertensive rats may have altered metabolism and reduced hypoxic tolerance.

Cerebral small vessel disease (SVD) is an important cause of stroke, cognitive decline and vascular dementia (VaD). It is associated with diffuse white matter abnormalities and small deep cerebral ischemic infarcts. The molecular mechanisms involved in the development and progression of SVD are unclear. As hypertension is a major risk factor for developing SVD, Spontaneously Hypertensive Rats (SHR) are considered an appropriate experimental model for SVD. Prior work suggested an imbalance between the number of blood microvessels and astrocytes at the level of the neurovascular unit in 2-month-old SHR, leading to neuronal hypoxia in the brain of 9-month-old animals. To identify genes and pathways involved in the development of SVD, we compared the gene expression profile in the cortex of 2 and 9-month-old of SHR with age-matched normotensive Wistar Kyoto (WKY) rats using microarray-based technology. The results revealed significant differences in expression of genes involved in energy and lipid metabolisms, mitochondrial functions, oxidative stress and ischemic responses between both groups. These results strongly suggest that SHR suffer from chronic hypoxia, and therefore are unable to tolerate ischemia-like conditions, and are more vulnerable to high-energy needs than WKY. This molecular analysis gives new insights about pathways accounting for the development of SVD.

CD133 Expressing Pericytes and Relationship to SDF-1 and CXCR4 in Spinal Cord Injury

Compression injury to the spinal cord (SC) results in vascular changes affecting the severity of the primary damage of the spinal cord. The recruitment of bone marrow (BM)-derived cells contribute to revascularization and tissue regeneration in a wide range of ischemic pathologies. Involvement of these cells in the vascular repair process has been investigated in an animal model of spinal cord injury (SCI). Temporal gene and protein expression of the BM-derived stem cell markers CD133 and CD34, of the mobilization factor SDF-1 and its receptor CXCR4 were determined following SC compression injury in rats. CD133 was expressed in uninjured tissue by cells surrounding arterioles identified as pericytes by co-expression of alpha-SMA. These cells mostly disappeared 2 days after injury but repopulated the tissue after 2 weeks. CD34 was expressed by endothelial cells and CD11b+ macrophages/microglia invading the injured tissue as observed 2 weeks following injury. SDF-1 was induced in reactive astrocytes and endothelial cells not until 2 weeks post-SCI. Comparison of the variation between CD34, CD133, CXCR4, and SDF-1 revealed a corresponding trend of CD133 with the SDF-1 expression. This study showed that resident microvascular CD133+ pericytes with presumptive stem cell potential are sensitive to SCI. Their decline following SCI and the delayed induction of SDF-1 may contribute to vessel destabilisation and inefficient revascularization. In addition, none of the analyzed markers could be assigned clearly to BM-derived cells. Together, our findings suggest that effective recruitment of pericytes may serve as a therapeutic option to improve microcirculation after SCI.

Identification of Inflammatory, Metabolic, and Cell Survival Pathways Contributing to Cerebral Small Vessel Disease by Postmortem Gene Expression Microarray

Cerebral small-vessel disease (SVD) is characterized by periventricular white matter (WM) changes and general brain atrophy. SVD is prevalent in elderly individuals and is frequently associated with the development of vascular dementia (VaD). Studies of the molecular basis of SVD are sparse. We have to gain further insight into the pathogenic mechanisms of SVD. Therefore, we compared gene expression patterns in the brains of SVD and control patients, in order to identify cellular pathways changed in diseased brains. We compared the expression of mRNA transcripts in postmortem, macroscopically normal-appearing human brain tissues isolated from frontal, temporal and occipital cortical and subcortical regions in 5 SVD and 5 non-SVD control patients. Significant expression changes were determined by fold change F>1.2 in either direction, and p<0.05. We identified 228 genes differentially expressed in cortex (89 up-, 139 down-regulated) and 555 genes in WM (223 up-, 332 down-regulated) in SVD patients. Pathway analyses revealed that upregulated genes were associated with inflammation and apoptosis in WM, suggesting active cell death. Downregulated genes were associated with coagulation and fatty and amino acids metabolisms. In the cortex, down-regulated genes were principally associated with neuronal functions. Our data revealed widespread changes in the transcriptome profiles in the cortex and WM of human SVD brains, with a predominance of changes in WM. We provide for the first time a comprehensive view of the molecular alterations in human SVD brains that seem to contribute to the neuropathogenesis of SVD.