Ana Raït El Habib (1) ; Ana Raït El Habib (2) / Fadila Khetmi, chant [acc. voc. et instr.]

[Traditions. Afrique du Nord. Tunisie]

Biochemical characterization of a myelin fraction isolated from rat brain aggregating cell cultures.

Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15-day-old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A 'floating fraction' was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.

Minocycline promotes remyelination in aggregating rat brain cell cultures after interferon-γ plus lipopolysaccharide-induced demyelination.

Minocycline has been shown to inhibit microglia reactivity, and to decrease the severity and progression of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. It remained to be examined whether minocycline was also able to promote remyelination. In the present study, myelinating aggregating brain cell cultures were used as a model to study the effects of minocycline on microglial reactivity, demyelination, and remyelination. Cultures were treated simultaneously with two inflammatory agents, interferon-γ (IFN-γ) and lipopolysaccharide (LPS), which caused an inflammatory response accompanied by demyelination. The inflammatory response was characterized by microglial reactivity, upregulation of inflammatory cytokines and iNOS, and increased phophorylation of P38 and P44/42 mitogen activated protein (MAP) kinases. Minocycline inhibited microglial reactivity, and attenuated the increased phophorylation of P38 and P44/42 MAP kinases. Demyelination, determined by a decrease in myelin basic protein (MBP) content and immunoreactivity 48 h after the treatment with the inflammatory agents, was not prevented by minocycline. However, 1 week after demyelination was assessed, the MBP content was restored in presence of minocycline, indicating that remyelination was promoted. Concomitantly, in cultures treated with minocycline, the markers of oligodendrocyte precursors cells (OPCs) and immature oligodendrocytes NG2 and O4, respectively, were decreased compared to cultures treated with the inflammatory agents only. These results suggest that minocycline attenuates microglial reactivity and favors remyelination by enhancing the differentiation of OPCs and immature oligodendrocytes.

Does the physical disector method provide an accurate estimation of sensory neuron number in rat dorsal root ganglia?

The physical disector is a method of choice for estimating unbiased neuron numbers; nevertheless, calibration is needed to evaluate each counting method. The validity of this method can be assessed by comparing the estimated cell number with the true number determined by a direct counting method in serial sections. We reconstructed a 1/5 of rat lumbar dorsal root ganglia taken from two experimental conditions. From each ganglion, images of 200 adjacent semi-thin sections were used to reconstruct a volumetric dataset (stack of voxels). On these stacks the number of sensory neurons was estimated and counted respectively by physical disector and direct counting methods. Also, using the coordinates of nuclei from the direct counting, we simulate, by a Matlab program, disector pairs separated by increasing distances in a ganglion model. The comparison between the results of these approaches clearly demonstrates that the physical disector method provides a valid and reliable estimate of the number of sensory neurons only when the distance between the consecutive disector pairs is 60 microm or smaller. In these conditions the size of error between the results of physical disector and direct counting does not exceed 6%. In contrast when the distance between two pairs is larger than 60 microm (70-200 microm) the size of error increases rapidly to 27%. We conclude that the physical dissector method provides a reliable estimate of the number of rat sensory neurons only when the separating distance between the consecutive dissector pairs is no larger than 60 microm.

Building hippocampal circuits to learn and remember: insights into the development of human memory

The hippocampal formation is essential for the processing of episodic memories for autobiographical events that happen in unique spatiotemporal contexts. Interestingly, before 2 years of age, children are unable to form or store episodic memories for recall later in life, a phenomenon known as infantile amnesia. From 2 to 7 years of age, there are fewer memories than predicted based on a forgetting function alone, a phenomenon known as childhood amnesia. Here, we discuss the postnatal maturation of the primate hippocampal formation with the goal of characterizing the development of the neurobiological substrates thought to subserve the emergence of episodic memory. Distinct regions, layers and cells of the hippocampal formation exhibit different profiles of structural and molecular development during early postnatal life. The protracted period of neuronal addition and maturation in the dentate gyrus is accompanied by the late maturation of specific layers in different hippocampal regions that are located downstream from the dentate gyrus, particularly CA3. In contrast, distinct layers in several hippocampal regions, particularly CA1, which receive direct projections from the entorhinal cortex, exhibit an early maturation. In addition, hippocampal regions that are more highly interconnected with subcortical structures, including the subiculum, presubiculum, parasubiculum and CA2, mature even earlier. These findings, together with our studies of the development of human spatial memory, support the hypothesis that the differential maturation of distinct hippocampal circuits might underlie the differential emergence of specific "hippocampus-dependent" memory processes, culminating in the emergence of episodic memory concomitant with the maturation of all hippocampal circuits.

Coagulation Factor Xa Activates Thrombin and Signals Ischemic Neuronal Death Via Par-1 and JNK in Ischemic Neural Tissue

Background: The coagulation factor thrombin mediates ischemic neuronal deathand, at a low concentration, induces tolerance to ischemia.We investigated its modeof activation in ischemic neural tissue using an in vitro approach to distinguish therole of circulating coagulation factors from endogenous cerebral mechanisms. Wealso studied the signalling pathway downstream of thrombin in ischemia and afterthrombin preconditioning.Methods: Rat organotypic hippocampal slice cultures to 30 minute oxygen (5%)and glucose (1 mmol/L) deprivation (OGD).Results: Selective factor Xa (FXa) inhibition by fondaparinux during and afterOGD significantly reduced neuronal death in the CA1 after 48 hours. Thrombinactivity was increased in the medium 24 hours after OGD and this increasewas prevented by fondaparinux suggesting that FXa catalyzes the conversion ofprothrombin to thrombin in neural tissue after ischemia in vitro. Treatment withSCH79797, a selective antagonist of the thrombin receptor protease activatedreceptor-1 (PAR-1), significantly decreased neuronal cell death indicating thatthrombin signals ischemic damage via PAR-1. The JNK pathway plays an importantrole in cerebral ischemia and we observed activation of the JNK substrate,c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonistSCH79797, decreased the level of phospho-c-Jun Ser73. After thrombin preconditioningc-Jun was activated by phosphorylation in the nuclei of neurons of the CA1.Treatment with a synthetic thrombin receptor agonist resulted in the same c-Junactivation profile and protection against subsequent OGD indicating that thrombinalso signals via PAR-1 and c-Jun in cell protection.Conclusion: These results indicate that FXa activates thrombin in cerebral ischemia,leading via PAR-1 to the activation of the JNK pathway resulting in neuronal death.Thrombin induced tolerance also involves PAR-1 and JNK, revealing commonfeatures in cell death and survival signalling.

Induction of brain aquaporin 9 (AQP9) in catecholaminergic neurons in diabetic rats.

Aquaporin 9 facilitates the diffusion of water but also glycerol and monocarboxylates, known as brain energy substrates. AQP9 was recently observed in catecholaminergic neurons that are implicated in energy homeostasis and also possibly in neuroendocrine effects of diabetes. Recently it has been observed that the level of AQP9 expression in hepatocytes is sensitive to the blood concentration of insulin. Furthermore, insulin injection in the brain is known to be related to the energy homeostasis. Based on these observations, we investigated if the concentration of insulin affects the level of brain AQP9 expression and if so, in which cell types. This study has been carried out, in a model of the diabetic rat generated by streptozotocin injection and on brainstem slices. In diabetic rats showing a decrease in systemic insulin concentration, AQP9 is only increased in brain areas containing catecholaminergic neurons. In contrast, no significant change is detected in the cerebral cortex and the cerebellum. Using immunocytochemistry, we are able to show that the increase in AQP9 expression is specifically present in catecholaminergic neurons. In brainstem slice cultures, 2 microM insulin induces a significant decrease in AQP9 protein levels 6 h after application, suggesting that brain AQP9 is also regulated by the insulin. These results show that the level of expression of brain AQP9 is affected by variations of the concentration of insulin in a diabetic model and in vitro.

Macrophage migration inhibitory factor is critically involved in basal and fluoxetine-stimulated adult hippocampal cell proliferation and in anxiety, depression, and memory-related behaviors.

Intensive research is devoted to unravel the neurobiological mechanisms mediating adult hippocampal neurogenesis, its regulation by antidepressants, and its behavioral consequences. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed in the CNS, where its function is unknown. Here, we show, for the first time, the relevance of MIF expression for adult hippocampal neurogenesis. We identify MIF expression in neurogenic cells (in stem cells, cells undergoing proliferation, and in newly proliferated cells undergoing maturation) in the subgranular zone of the rodent dentate gyrus. A causal function for MIF in cell proliferation was shown using genetic (MIF gene deletion) and pharmacological (treatment with the MIF antagonist Iso-1) approaches. Behaviorally, genetic deletion of MIF resulted in increased anxiety- and depression-like behaviors, as well as of impaired hippocampus-dependent memory. Together, our studies provide evidence supporting a pivotal function for MIF in both basal and antidepressant-stimulated adult hippocampal cell proliferation. Moreover, loss of MIF results in a behavioral phenotype that, to a large extent, corresponds with alterations predicted to arise from reduced hippocampal neurogenesis. These findings underscore MIF as a potentially relevant molecular target for the development of treatments linked to deficits in neurogenesis, as well as to problems related to anxiety, depression, and cognition.

Influence of ethanol dose and pigmentation on the incorporation of ethyl glucuronide into rat hair.

Ethyl glucuronide (EtG) is a minor and specific metabolite of ethanol. It is incorporated into growing hair, allowing a retrospective detection of alcohol consumption. However, the suitability of quantitative EtG measurements in hair to determine the quantity of alcohol consumed has not clearly been demonstrated yet. The purpose of this study was to evaluate the influence of ethanol dose and hair pigmentation on the incorporation of EtG into rat hair. Ethanol and EtG kinetics in blood were investigated after a single administration of ethanol. Eighteen rats were divided into four groups receiving 0 (control group), 1, 2, or 3g ethanol/kg body weight. Ethanol was administered on 4 consecutive days per week for 3 weeks by intragastric route. Twenty-eight days after the initial ethanol administration, newly grown hair was shaved. Pigmented and nonpigmented hair were analyzed separately by gas chromatography coupled to tandem mass spectrometry. Blood samples were collected within 12h after the ethanol administration. EtG and ethanol blood levels were measured by liquid chromatography coupled to tandem mass spectrometry and headspace gas chromatography-flame ionization detector, respectively. No statistically significant difference was observed in EtG concentrations between pigmented and nonpigmented hair (Spearman's rho=0.95). Thus, EtG incorporation into rat hair was not affected by hair pigmentation. Higher doses of ethanol resulted in greater blood ethanol area under the curve of concentration versus time (AUC) and in greater blood EtG AUC. A positive correlation was found between blood ethanol AUC and blood EtG AUC (Spearman's rho=0.84). Increased ethanol administration was associated with an increased EtG concentration in hair. Blood ethanol AUC was correlated with EtG concentration in hair (Pearson's r=0.89). EtG concentration in rat hair appeared to reflect the EtG concentration in blood. Ethanol was metabolized at a median rate of 0.22 g/kg/h, and the median elimination half-life of EtG was 1.21 h. This study supports that the bloodstream is likely to display a major role in the hair EtG incorporation.

Thyroid hormones stimulate expression and modification of cytoskeletal protein during rat sciatic nerve regeneration.

Peripheral neurons can regenerate after axotomy; in this process, the role of cytoskeletal proteins is important because they contribute to formation and reorganization, growth, transport, stability and plasticity of axons. In the present study, we examined the effects of thyroid hormones (T3) on the expression of major cytoskeletal proteins during sciatic nerve regeneration. At various times after sciatic nerve transection and T3 local administration, segments of operated nerves from T3-treated rats and control rats were examined by Western blotting for the presence of neurofilament, tubulin and vimentin. Our results revealed that, during the first week after surgery, T3 treatment did not significantly alter the level of NF subunits and tubulin in the different segments of operated nerves compared to control nerves. Two or 4 weeks after operation, the concentration of NF-H and NF-M isoforms was clearly increased by T3 treatment. Moreover, under T3-treatment, NF proteins appeared more rapidly in the distal segment of operated nerves. Likewise, the levels of betaIII, and of acetylated and tyrosinated tubulin isotypes, were also up-regulated by T3-treatment during regeneration. However, only the tyrosinated tubulin form appeared earlier in the distal nerve segments. At this stage of regeneration, T3 had no effect on the level of vimentin expression. In conclusion, thyroid hormone improves and accelerates peripheral nerve regeneration and exerts a positive effect on cytoskeletal protein expression and transport involved in axonal regeneration. These results help us to understand partially the mechanism by which thyroid hormones enhance peripheral nerve regeneration. The stimulating effect of T3 on peripheral nerve regeneration may have considerable therapeutic potential.

Hippocampal atrophy predicts conversion to dementia after STN-DBS in Parkinson's disease.

BACKGROUND: Hippocampal atrophy (HA) is a known predictor of dementia in Alzheimer's disease. HA has been found in advanced Parkinson's disease (PD), but no predicting value has been demonstrated yet. The identification of such a predictor in candidates for subthalamic deep brain stimulation (STN-DBS) would be of value. Our objective was to compare preoperative hippocampal volumes (HV) between PD patients who subsequently converted to dementia (PDD) after STN-DBS and those who did not (PDnD). METHODS: From a cohort of 70 consecutive STN-DBS treated PD patients, 14 converted to dementia over 25.6+/-20.2 months (PDD). They were compared to 14 matched controls (PDnD) who did not convert to dementia after 43.9+/-11.7 months. On the preoperative 3D MPRAGE MRI images, HV and total brain volumes (TBV) were measured by a blinded investigator using manual and automatic segmentation respectively. RESULTS: PDD had smaller preoperative HV than PDnD (1.95+/-0.29 ml; 2.28+/-0.33 ml; p<0.01). This difference reinforced after normalization for TBV (3.28+/-0.48, 3.93+/-0.60; p<0.01). Every 0.1 ml decrease of HV increased the likelihood to develop dementia by 24.6%. A large overlap was found between PD and PDnD HVs, precluding the identification of a cut-off score. CONCLUSIONS: As in Alzheimer's disease, HA may be a predictor of the conversion to dementia in PD. This preoperative predictor suggests that the development of dementia after STN-DBS is related to the disease progression, rather then the procedure. Further studies are needed to define a cut-off score for HA, in order to affine its predictive value for an individual patient.

Differential regulation of Na-K-ATPase isoform gene expression by T3 during rat brain development.

A fetal rat telencephalon organotypic cell culture system was found to reproduce the developmental pattern of Na-K-adenosinetriphosphatase (ATPase) gene expression observed in vivo [Am. J. Physiol. 258 (Cell Physiol. 27): C1062-C1069, 1990]. We have used this culture system to study the effects of triiodothyronine (T3; 0.003-30 nM) on mRNA abundance and basal transcription rates of Na-K-ATPase isoforms. Steady-state mRNA levels were low at culture day 6 (corresponding to the day of birth) but distinct for each isoform alpha 3 much greater than beta 1 = beta 2 greater than alpha 2 greater than alpha 1. At culture day 6, T3 did not modify mRNA abundance of any isoform. At culture day 12 (corresponding to day 7 postnatal), T3 increased the mRNA level of alpha 2 (4- to 7-fold), beta 2 (4- to 5-fold), alpha 1 (3- to 6-fold), and beta 1 (1.5-fold), whereas alpha 3 mRNA levels remained unchanged. Interestingly, the basal transcription rate for each isoform differed strikingly (alpha 2 greater than alpha 1 much greater than beta 1 = beta 2 greater than alpha 3) but remained stable throughout 12 days of culture and was not regulated by T3. Thus we observed an inverse relationship between rate of transcription and rate of mRNA accumulation for each alpha-isoform, suggesting that alpha 1- and alpha 2-mRNA are turning over rapidly whereas alpha 3-mRNA is turning over slowly. Our data indicate that one of the mechanisms by which T3 selectively controls Na-K-ATPase gene expression during brain development in vitro occurs at the posttranscriptional level.

Evaluation of the new photosensitizer Stakel (WST-11) for photodynamic choroidal vessel occlusion in rabbit and rat eyes.

PURPOSE: To evaluate the photodynamic potential of a new hydrosoluble photosensitizer (WST-11, Stakel; Steba Biotech, Toussus-Le-Noble, France), for use in occlusion of normal choroidal vessels in the rabbit eye and CNV (choroidal neovascularization) in the rat eye. METHODS: Occlusive and nonocclusive parameters of Stakel and verteporfin photodynamic therapy (PDT) were investigated in pigmented rabbits. Eyes were followed by fluorescein angiography (FA) and histology at various intervals after PDT. RESULTS: When occlusive parameters (fluence of 50 J/cm(2), 5 mg/kg drug dose and DLI [distance to light illumination] of 1 minute) were used, Stakel PDT was efficient immediately after treatment without associated structural damage of the RPE and retina overlying the treated choroid in the rabbit eye. Two days later, total occlusion of the choriocapillaries was seen in 100% of the treated eyes, along with accompanying histologic structural changes in the overlying retina. When the occlusive parameters (fluence, 100 J/cm2; drug dose, 12 mg/m2; and DLI, 5 minutes) of verteporfin PDT were used, occlusion of the choriocapillaries was observed in 89% of the treated eyes. Histology performed immediately after treatment demonstrated structural damage of the overlying retina and RPE layer. Weaker, nonocclusive Stakel PDT parameters (25 J/cm2, 5 mg/kg, and DLI of 10 minutes) did not induce choriocapillary occlusion or retinal lesions on FA or histology. Weaker, nonocclusive verteporfin PDT parameters (10 J/cm2, 0.2 mg/kg, and DLI of 5 minutes) did not induce choriocapillary occlusion. However, histology of these eyes showed the presence of damage in the retinal and choroidal tissues. Moreover, preliminary results indicate that selective CNV occlusion can be achieved with Stakel PDT in the rat eye. CONCLUSIONS: Unlike verteporfin PDT, Stakel PDT does not cause direct damage to the RPE cell layer or retina. These observations indicate that Stakel PDT may have a high potential for beneficial therapeutic outcomes in treatment of AMD.

Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model.

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.