Amoeboid Microglial Cells and not Astrocytes Synthesize TNF-alpha in Swiss Mouse Brain Cell Cultures

The role of tumour necrosis factor (TNF-alpha) in brain physiology and pathology has been the focus of several studies. However, the source of this lymphokine in the central nervous system and the regulation of its synthesis is still poorly understood. We have therefore used purified astrocytes and brain macrophages in culture to compare the abilities of these two cell types to synthesize TNF-alpha and its mRNA. We find that, in the Swiss mouse, no significant TNF activity or TNF-alpha mRNA are produced by astrocytes, even following activation with lipopolysaccharides (LPS). On the other hand, purified microglial cells express a cytotoxic activity able to kill TNF-sensitive LM cells. Part of this activity is released into the culture medium and part remains bound to the membrane after mild paraformaldehyde treatment, demonstrating the existence in the culture of the soluble and membrane-bound forms of TNF activity. The fact that amoeboid microglial cells, and not astrocytes, are the actual source of TNF in brain cultures was further demonstrated by Northern blot analysis and in situ hybridization using a TNF-alpha specific oligonucleotide probe. The definition of the cell type which, in the CNS, is responsible for TNF synthesis will allow the regulation of this lymphokine to be analysed and opens the way for a better understanding of the interactions between amoeboid microglial cells and the other cell types which make up the nervous system.     

IL-6 Expression in Neurons of Transgenic Mice Causes Reactive Astrocytosis and Increase in Ramified Microglial Cells but no Neuronal Damage

Growing evidence suggests that aberrant production of inflammatory cytokines within the central nervous system (CNS) contributes to the development of pathological conditions. To test the cause—effect relationship between the overproduction of interleukin-6 (IL-6) in the CNS and the onset of neuropathological changes, we have generated transgenic mice in which human IL-6 expression has been targeted to the neurons by using the rat neuron-specific enolase promoter. These mice develop reactive astrocytosis and an increase in ramified microglial cells but do not show histological or behavioural signs of neuron damage at the light microscope level. We thus conclude that a constant release of human IL-6 by neuronal subpopulations in mice is sufficient to activate cells potentially capable of modulating the local immune response, but at the same time is compatible with normal neuron functions.     

Effect of Memantine on the Levels of Neuropeptides and Microglial Cells in the Brain Regions of Rats with Neuropathic Pain

Neuropathic pain induced by sciatic nerve injury not only causes peripheral dysfunctions but also affects the cortical and subcortical regions of the brain. It is still unknown whether neuropathic pain could relate to behavioral and neurochemical alterations in the central nervous system. This paper deals with the effect of peripheral neuropathic pain on mechanical allodynia, neuropeptide levels, neuropeptide-degrading enzyme activities, and microglial cells in the brain regions of rats by applying chronic constriction injury, a partial sciatic nerve injury. We examined the possible protection effect on the allodynia and changes in levels of neuropeptides and microglial activation in chronic constriction injury of the rat brain by memantine. On 4 days after chronic constriction injury, the induction of mechanical allodynia was suppressed by memantine treatment. Reductions in the substance P in the hypothalamus and somatostatin in the periaqueductal gray of chronic constriction injury rat brain were reversed by memantine. This suggests the role of these neuropeptides in pain information processing in the brain. Immunohistochemical experiments revealed that the expression of CD11b, a marker protein of microglia, was increased in the hypothalamus and periaqueductal gray in the chronic constriction injury rat brain as compared with the controls, and memantine treatment could suppress the activation of microglia, suggesting the involvement of microglia in pain mechanism. The present behavioral, biochemical, and immunohistochemical studies demonstrated that peripheral neuropathic pain affects the neuropeptide levels and microglial activation in the brain regions, and these events described above may play an important role in neuropathic pain pathogenesis.     

Analysis of Molecular Expression in Adipose Tissue-Derived Mesenchymal Stem Cells : Prospects for Use in the Treatment of Intervertebral Disc Degeneration

Objective

Recent studies have shown encouraging progress toward the use of autogenic and allogenic mesenchymal stem cells (MSCs) to arrest, or even lead to partial regeneration in, intervertebral disc (IVD) degeneration. However, this technology is still in its infancy, and further development is required. The aim of this study was to analyze whether rat adipose-derived mesenchymal stem cells (ADMSC) can differentiate towards IVD-like cells after treatment with transforming growth factor β3 (TGF-β3) in vitro. We also performed quantitative analysis of gene expression for ADMSC only, ADMSCs treated with TGF-β3, and co-cultured ADMSCs treated with TGF-β3.

Methods

ADMSCs were sub-cultured to homogeneity and used in fluorocytometry assays for CD11, CD45, and CD90/Thy1. ADMSCs were differentiated in spheroid culture towards the chondrogenic lineage by the presence of TGF-β3, dexamethasone, and ascorbate. We also co-cultured pure ADMSCs and nucleus pulposus cells in 24-well plates, and performed immunohistochemical staining, western blotting, and RT-PCR for quantitativeanalysis of gene expression.

Results

Results of fluorocytometry were positive for CD90/Thy1 and negative for CD11 and CD45. TGF-β3-mediated induction of ADMSCs led to the expression of the differentiation markers of intervertebral disc-like cells, such as aggrecan, collagen II, and sox-9. Co-cultured ADMSCs treated with TGF-β3 showed higher expression of differentiation markers and greater extracellular matrix production compared with ADMSCs treated with TGF-β3 alone.

Conclusion

ADMSC treated with TGF-β3 may be an attractive source for regeneration therapy in degenerative IVD. These findings may also help elucidate the pathologic mechanism of MSC therapy in the degeneration of IVD in vivo.     

A Collection of cDNA Clones with Specific Expression Patterns in Mouse Brain

A total of 950 cDNA clones were randomly selected from mouse cerebellar cDNA libraries, and of these, about 130 clones were found to correspond to mRNAs which were expressed unequally between the cerebellum and other parts of mouse brain. Their distribution patterns in adult mouse brain were analysed by in situ hybridization, and eight clones were found restricted to specific regions of the brain, including four clones specific to cerebellar granule cells and one clone specific to Purkinje cells. Another 27 clones were preferentially expressed in a diverse, but distinctive subpopulation of brain cells. Among them seven clones were especially abundant in specific nuclei, and three in specific fibre bundles. These clones will be useful in defining new subpopulations of brain cells characterized by different gene expression.     

The Relationship of Microglial Cells to Dying Neurons During Natural Neuronal Cell Death and Axotomy-induced Degeneration of the Rat Retina

The interactions between dying neurons and phagocytotic cells within the developing and injured retina remain controversial. The present work explored the role of microglia and investigated whether so-called resident microglial cells are permanently responsible for removing cell debris whenever it is produced. As a first goal, I characterized some quantitative and morphometric features of the small ipsilateral retinocollicular projections and analysed the permanent function of phagocytosing microglia with these projections as a paradigm. To achieve this, I combined the fluorescent dyes Dil and 4Di-10ASP, both of which persist in the labelled ganglion cells after injection into the superior colliculus (SC), and retrograde labelling. After neuronal degradation, the dyes accompany the degradation products, become interiorized and then persist within the phagocytosing microglia. Consequently, early labelling of microglial cells can be assessed by injecting one dye into the SC during the first postnatal day of life, that is, prior to advanced natural neuronal cell death. Labelling of the remaining ipsilaterally projecting neurons with the second dye following intraorbital axotomy in adulthood and during subsequent neuronal death would therefore result in double labelling of some microglial cells, if these were involved in phagocytosis during both the natural and the induced phases of neuronal degradation. The ganglion cells which survived natural neuronal cell death remained fluorescent for 3 months after labelling with either dye on the day of the animal's birth, indicating that both fluorescent probes persisted within neurons. Quantitatively, 1770+/-220 ganglion cells/mm2 were labelled within the contralateral retina and a total population of 1442+/-120 cells/retina were observed within the periphery of the inferior/temporal quadrant of the ipsilateral retina. A smaller, ipsilateral projection of 150+/-24 cells/retina was uniformly scattered throughout the rest of the retinal surface. Transient projections of ganglion cells to either the contralateral or the ipsilateral colliculi and death of labelled ganglion cells during the first postnatal days resulted in labelling of 210+/-36 microglial cells/mm2 within the contralateral retina and a total number of 800+/-120 cells/retina within the inferior/temporal and 200+/-22 cells/retina within the rest of the retina. These labelled microglial cells were observed in adulthood and indicated that after taking away the neuronal cell debris they persisted within the retinal tissue. The small number of prelabelled ganglion cells which formed persistent ipsilateral projections until adulthood were axotomized by transecting the optic nerve, and resulted in additional labelling of microglial cells with the second fluorescent dye as well. Double-labelled microglia were observed within the inferior/temporal quadrant (3500+/-240 cells/retina) and to a lesser extent (340+/-40 cells/retina) scattered over the entire retinal surface. The chronotopological sequence of microglial labelling paralleled that of ganglion cell degeneration. Injection of protease inhibitors into the vitreous body during optic nerve transection retarded retrograde glial cell degeneration, probably by blocking microglial proteases. The results directly proved that the same microglial cells which remove neuronal cell debris in the postnatal retina were reactivated later in life to proteolytically degrade and then phagocytose neurons which had altered because of the axotomy.     

Transplantation of Placenta-Derived Mesenchymal Stem Cells in the EAE Mouse Model of MS

Stem cell-based regenerative medicine raises great hope for the treatment of multiple sclerosis (MS). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are being tested in clinical trials. Bone marrow is the traditional source of human MSCs, but human term placenta appears to be an excellent alternative because of its availability, without ethical issues. In this study, the therapeutic effect of human placental MSCs (PL-MSCs) was evaluated in experimental autoimmune encephalomyelitis (EAE), the mice model of MS. EAE mice were transplanted intra-cerebrally with PL-MSCs or with the vehicle saline 5 or 10 days after first MOG injection. The mice were monitored for a month after therapy. A daily EAE score revealed a decrease in disease severity in the transplanted animals when compared to saline. Survival was significantly higher in the transplanted animals. In vitro experiments demonstrated that conditioned media from LPS-activated astrocytes stimulated PL-MSCs to express the gene TNF-α-stimulated gene/protein 6 (TSG-6). The same mRNA expression was obtained when PL-MSCs were exposed to TNF-α or IL1-β. These results demonstrate that PL-MSCs have a therapeutic effect in the EAE mice model. We assume that this effect is caused by reduction of the anti-inflammatory protein, TSG-6, of the inflammatory damage.     

Quantitative Comparison of Primary Cilia Marker Expression and Length in the Mouse Brain

Primary cilia are small, special cellular organelles that provide important sensory and signaling functions during the development of mammalian organs and coordination of postnatal cellular processes. Dysfunction of primary cilia are thought to be the main cause of ciliopathies, a group of genetic disorders characterized by overlapping developmental defects and prominent neurodevelopmental features. Although, disrupted cilia-linked signaling pathways have been implicated in the regulation of numerous neuronal functions, the precise role of primary cilia in the brain are still unknown. Importantly, studies of recent years have highlighted that different functions of primary cilia are reflected by their diverse morphology and unique signaling components localized in the ciliary membrane. In the present study, we conducted a comparative analysis of the expression pattern, distribution and length of adenylyl cyclase 3, somatostatin receptor 3, and ADP-ribosylation factor-like protein 13B expressing primary cilia in the mouse brain. We show that cilia of neurons and astrocytes display a well characterized distribution and ciliary marker arrangements. Moreover, quantitative comparison of their length, density and occurrence rate revealed that primary cilia exhibit region-specific alternations. In summary, our study provides a comprehensive overview of the cellular organization and morphological traits of primary cilia in regions of the physiological adult mouse brain.     

Malfunctioning DNA Damage Response (DDR) Leads to the Degeneration of Nigro-Striatal Pathway in Mouse Brain

Pronounced neuropathology is a feature of ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS), which are both genomic instability syndromes. The Nbs1 protein, which is defective in NBS, is a component of the Mre11/RAD50/NBS1 (MRN) complex. This complex plays a major role in the early phase of the cellular response to double strand breaks (DSBs) in the DNA. Among others, MRN is required for timely activation of the protein kinase ATM (A-T mutated), which is disrupted in patients with A-T. Earlier reports show that Atm-deficient mice exhibit severe degeneration of tyrosine hydroxylase (TH)-positive dopaminergic nigro-striatal neurons and their terminals in the striatum. This cell loss is accompanied by a large reduction in immunoreactivity for the dopamine transporter protein (DAT) in the striatum. To test whether Nbs1 inactivation also affects the integrity of the nigro-striatal pathway, we examined this pathway in a murine model with conditional inactivation of the Nbs1 gene in central nervous system (Nbs1-CNS-Δ). We report that this model has a reduction in TH-positive cells in the substantia nigra. This phenomenon was seen at very early age, while Atm−/− mice showed a progressive age-dependent reduction. Furthermore, we observed an age-dependent increase in the level of TH in the striatum of Atm−/− and Nbs1-CNS-Δ mice. In addition to the altered expression of TH, we also found a reduction of DAT in the striatum of both Atm−/− and Nbs1-CNS-Δ mice at 60 days of age. Finally, microglial recruitment and alterations in the levels of various neurotrophic factors were also observed. These results indicate that malfunctioning DNA damage response severely affects the integrity of the nigro-striatal pathway and suggest a new neurodegenerative pathway in Parkinsonian syndromes.     

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies

The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmd ^mdx , SJL/J, Large ^myd and Lama2 ^dy2J /J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-β1 and Pro-collagen 1α2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-β1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.     

TNF-αⅠ型受体对小鼠脑血管内皮细胞自由基产生和MnSOD表达的影响

目的探讨TNF-αⅠ型受体(TNFR1)对小鼠脑血管内皮细胞(BVEC)内氧自由基产生及锰超氧化物歧化酶(MnSOD)基因表达的影响。方法体外培养TNFR1基因敲除小鼠脑血管内皮细胞(BVEC/RI)和野生型小鼠脑血管内皮细胞,分别给予5ng/mLTNF-α刺激24h,用荧光显微镜观察细胞内氧自由基的产生,荧光定量PCR法及Western-blot法测定MnSOD基因mRNA和蛋白表达。结果给予TNF-α刺激后,细胞内活性氧(ROS)、锰超氧化物歧化酶基因mRNA及蛋白表达仅在BVEC内明显增高,而在BVEC/RI无明显变化。结论TNF-α可能通过作用于脑血管内皮细胞TNFR1增加细胞内氧自由基产生以及MnSOD基因及蛋白表达,从而介导了细胞氧化应激。     

Ischemia-Induced Increase in Microvascular Phosphodiesterase 4D Expression in Rat Hippocampus Associated with Blood Brain Barrier Permeability: Effect of Age

Phosphodiesterase 4D (PDE4D) is one of 16 PDEs expressed in cerebral microvessels, and may be involved in regulating blood-brain barrier (BBB) permeability. To assess the possible role of PDE4D in stroke-related injury in young versus aged rats, we measured microvascular PDE4D expression, parenchymal albumin immunoreactivity, and changes in the inside bore of the brain microvasculature. Ischemia caused severe hippocampal CA1 damage, associated with significant increases in vascular PDE4D and parenchymal albumin immunoreactivities. This effect was greater in the younger animals, which also had a greater increase in PDE4D expression. Ischemia significantly decreased tissue density in the perimicrovascular space in both young and aged animals. In addition, internal bore circumference and cross-sectional area of the hippocampal microvessels increased dramatically following ischemia. Increased PDE4D expression following cerebral ischemia may play a role in changing BBB permeability, which could secondarily affect ischemic outcome.     

Baclofen Modulates the Expression and Release of Neurotrophins in Schwann-Like Adipose Stem Cells

Neurotrophins are a group of polypeptides that specifically influence neuronal activity during development and adult life in the central and peripheral nervous system (PNS). In particular, Schwann cells (SC) in the PNS exert a neurotrophic role following up-regulation of several growth factors, including nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). Also SC-like cells derived from adipose tissue (dASC), which have molecular and functional properties similar to SC, can produce and secrete NGF and BDNF. Interestingly, gamma-aminobutyric acid (GABA) and its receptors have been also suggested as modulators of development and myelination in PNS. Therefore, it was interesting to investigate whether the stimulation of the GABA-B receptor may regulate the expression of neurotrophins in SC and dASC. Our findings demonstrated that the specific GABA-B receptors agonist baclofen influences the expression and the secretion NGF and BDNF. In particular, 2 and 24 h of baclofen exposure lead to increased neurotrophins expression in both SC and dASC, as measured by western blot. Moreover, enzyme-linked immunosorbent assay showed that also the levels of released neurotrophins were modified after baclofen treatments. The possibility to modulate the neurotrophic potential of adult stem cell, acting on functional GABAergic receptors, could represent a novel pharmacological approach to improve nerve regeneration.     

Differential Expression of Syntrophins and Analysis of Alternatively Spliced Dystrophin Transcripts in the Mouse Brain

Expression of syntrophin genes, encoding members of the dystrophin-associated protein complex, was studied in the mouse brain. In the hippocampal formation there is distinctive co-localization of specific syntrophins with certain dystrophin isoforms in neurons, e.g. α₁,-syntrophin with the C-dystrophin in CA regions and β₂-syntrophin with the G-dystrophin in the dentate gyrus. Expression of the al-syntrophin is predominant in CA regions and the olfactory bulb and it is also present in the cerebral cortex and the dentate gyrus. The β₂-syntrophin mRNA is most abundant in the dentate gyrus and is also evident in the pituitary, the cerebral cortex and in Ammon's horn and in traces in the caudate putamen. The choroid plexus was labelled by both α₁ and β₂-syntrophin-specific probes. The expression of syntrophins in the brain correlates with expression of dystrophins and dystroglycan. There are brain areas such as the cerebral cortex where several different syntrophins and dystrophins are expressed together. Syntrophin expression co-localizes with utrophin in the choroid plexus and caudate putamen. Finally, no syntrophin was detected in the cerebellar Purkinje cells where the specific dystrophin isoform (P-type) is present. This specific distribution of syntrophins in the brain is particularly interesting, as muscle syntrophin interacts with neuronal nitric oxide synthase. This may suggest that the dystrophin-associated protein complex may be involved in synaptic organisation and signal transduction machinery in both muscle and neurons. The dystrophin isoform, with exons 71–74 spliced out and hence lacking syntrophin binding sites, had been believed to be predominant in the brain, but our analyses using in situ hybridization, S1 nuclease protection and the semi-quantitative polymerase chain reaction revealed that this alternatively spliced mRNA is a minor, low abundance form in the brain.     

红景天苷和脑源性神经营养因子与神经干细胞共移植对致鼠神经干细胞定向分化的研究

目的：探讨红景天苷和脑源性神经营养因子（BDNF）、神经干细胞（NSCs）共移植对致鼠NSCs定向分化影响。方法：将戊四氮致大鼠分为模型组、NSCs组、NSCs＋BDNF组和NSCs＋BDNF＋红景天苷组。取新生大鼠海马组织,将培养的NSCs与BDNF＋红景天苷＋BDNF和基础培养基分别移植至致鼠海马组织中,苏木精-伊红染色及免疫组化检测不同时间点5-溴脱氧尿嘧啶核苷（BrdU）、谷氨酸脱羧酶（GAD65）阳性细胞数,并观察大鼠行为学改变。结果：NSCs＋BDNF＋红景天苷共移植组与其他组比较,各时间点BrdU、GAD65阳性细胞数均增多（P〈0.05）。第3周开始,大鼠癫发作次数最少（P〈0.05）。结论：BDNF与红景天苷联合有利于神经干细胞向γ-氨基丁酸能神经元分化。两者联合移植至致鼠后能减少大鼠的癫发作次数。     

Suppression of c-Fos Protein and mRNA Expression in Pentylenetetrazole-Induced Kindled Mouse Brain by Isoxylitones

An early immediate gene c-fos has been proposed as the gene responsible for turning on molecular events that might underlie the long-term neural changes occurring during kindling. We have evaluated the effects of novel anticonvulsant isomeric compounds isoxylitones [(E/Z)-2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine] on the c-Fos protein and mRNA expression in the brain samples of kindled mice and compared it with the normal and untreated kindled groups. Kindling was induced in male NMRI mice by repeated administration of sub-convulsive dose (50 mg/kg) of pentylenetetrazole (PTZ) until a seizure score of 4-5 was achieved. The c-Fos expression was quantified by combination of immunohistochemistry and RT-PCR protocols. Both the immunohistochemical and RT-PCR analysis revealed a marked increase in the expression of c-fos mRNA and protein in the brain regions tested in case of PTZ-kindled control group compared to normal control. In contrast, the isoxylitone (30 mg/kg)-treated group demonstrated significant reduction of c-Fos expression compared to PTZ-kindled control animals. However, low expression of c-fos mRNA was only detected in the thalamus of the isoxylitone-treated brain samples. Based on these observations, we suggest that isoxylitones may have the capacity to control the seizure pattern by mechanism such as the suppression of c-Fos protein and mRNA levels in different regions of the brain. Further investigations to explore the mechanism of action of these compounds are under process.     

Sexually Dimorphic Expression of Reelin in the Brain of a Mouse Model of Alzheimer Disease

Recent evidence highlights the protective role of reelin against amyloid β (Aβ)-induced synaptic dysfunction and cognitive impairment in Alzheimer disease (AD). In this study, exploiting TgCRND8 mice that overexpress a mutant form of amyloid β precursor protein (AβPP) and display an early onset of AD neuropathological signs, we addressed the question whether changes of reelin expression eventually precede the appearance of Aβ-plaques in a sex-dependent manner. We show that sex-associated and brain region-specific differences in reelin expression appear long before Aβ-plaque formation. However, in spite of a downregulation of reelin expression compared to males, TgCRND8 females display fewer Aβ-plaques, suggesting that additional factors, other than sex and reelin level, influence amyloidosis in this mouse model.