Journal of Neuroscience Research

We have systematically optimized the concentrations of 20 components of a previously published serumfree medium (Brewer and Cotman, Brain Res 494: 65‐74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum‐freemedium Supplement, B27, produced neuron survival above 60% independent of plating density above 160 plated cells/mm² For isolated cells (<100 cells/mm²), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm² by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrnous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/ Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron‐specific enolase. Excellent long‐term viability is achieved after 4 weeks in culture with greater than 90%viability for cells plated at 640/mm² and greater than 50 viability for cells plated at 160/mm² Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones. © 1993 Wiley‐Liss, Inc.

Việc ức chế tích tụ peptide amyloid β (Aβ) và sự hình thành các sợi fibril β-amyloid (fAβ) từ Aβ, cũng như sự bất ổn của những fAβ đã hình thành trước đó trong hệ thần kinh trung ương, sẽ là những đích điều trị hấp dẫn để điều trị bệnh Alzheimer (AD). Chúng tôi đã báo cáo trước đây rằng axit nordihydroguaiaretic (NDGA) và các polyphenol liên quan đến rượu vang ức chế sự hình thành fAβ từ Aβ(1–40) và Aβ(1–42) và làm mất ổn định fAβ(1–40) và fAβ(1–42) đã hình thành trước đó tỷ lệ theo liều lượng trong thí nghiệm in vitro. Sử dụng phân tích quang phổ huỳnh quang với thioflavin T và nghiên cứu kính hiển vi điện tử, chúng tôi đã kiểm tra tác động của curcumin (Cur) và axit rosmarinic (RA) lên sự hình thành, mở rộng, và phá bỏ ổn định của fAβ(1–40) và fAβ(1–42) ở pH 7.5 và 37°C trong thí nghiệm in vitro. Sau đó, chúng tôi so sánh hoạt động kháng tạo amyloid của Cur và RA với NDGA. Cur và RA ức chế sự hình thành fAβ từ Aβ(1–40) và Aβ(1–42) cũng như sự mở rộng của chúng theo liều lượng. Ngoài ra, chúng cũng làm mất ổn định những fAβ đã hình thành theo liều lượng. Tổng thể, hoạt động của Cur, RA và NDGA là giống nhau. Các nồng độ hiệu quả (EC₅₀) của Cur, RA và NDGA cho sự hình thành, mở rộng và phá bỏ ổn định của fAβ là từ 0.1–1 μM. Mặc dù cơ chế mà Cur và RA ức chế sự hình thành fAβ từ Aβ và phá vỡ sự ổn định của fAβ trong thí nghiệm in vitro vẫn chưa rõ ràng, chúng có thể là một phân tử quan trọng cho sự phát triển của các phương pháp điều trị cho AD. © 2004 Wiley‐Liss, Inc.

The entry of T‐lymphocytes into the parenchyma of the central nervous system is a critical early feature in the pathogenesis of many experimental and spontaneously occurring immune‐mediated illnesses. The physiological mechanisms controlling this entry have not been elucidated. This study reports that T‐cell entry into the rat CNS appears to be primarily dependent upon the activation state of the lymphocytes; T‐lymphoblasts enter the CNS (and all other tissues examined) in an apparently random manner while T cells not in blast phase are excluded. Antigen specificity, MHC compatibility, T‐cell phenotypce and T‐cell receptor gene usage do not appear related to the ability of cells to enter. This study demonstrates that when T‐lymphoblasts are introduced into the circulation they rapidly appear in the CNS tissue. Their concentration in the CNS reaches a peak between 9 and 12 hr, and lymphocytes which have entered, exit within 1 to 2 days. Cells capable of reacting with a CNS antigen remain in the tissue or cyclically reenter to initiate inflammation if they are able to recognize their antigen in the correct MHC context. This observation also appears to pertain to the entry of activated T cells into many other tissues, although their concentrations in these non‐CNS sites was not quantitatied.

Microglia, one of three glial cell types in the central nervous system (CNS), play an important role as resident immunocompetent and phagocytic cells in the CNS in the event of injury and disease. It was del Rio Hortega in 1927 who determined that microglia belong a distinct glial cell type apart from astrocytes and oligodendrocytes, and since 1970s there has been wide recognition that microglia are immune effectors in the CNS that respond to pathological conditions and participate in initiation and progression of neurological disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and acquired immune deficiency syndrome dementia complex by releasing potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins. There is also evidence to suggest that microglia are capable of secreting neurotrophic or neuron survival factors upon activation via inflammation or injury. It is thus timely to review current status of knowledge on biology and immunology of microglia, and consider new directions of investigation on microglia in health and disease. © 2005 Wiley‐Liss, Inc.

The present study investigates the induction of neurogenesis, reduction of apoptosis, and promotion of basic fibroblast growth factor (bFGF) expression as possible mechanisms by which treatment of stroke with bone marrow stromal cells (MSCs) improves neurological functional recovery. Additionally, for the first time, we treated cerebral ischemia in female rats with intraveneous administration of MSCs. Female rats were subjected to 2 hr of middle cerebral artery occlusion (MCAo), followed by an injection of 3 × 10⁶ male (for Y chromosome labeling) rat MSCs or phosphate‐buffered saline (PBS) into the tail vein 24 hr after MCAo. All animals received daily injection of bromodeoxyuridine (BrdU; 50 mg/kg, i.p.) for 13 days after treatment for identification of newly synthesized DNA. Animals were sacrificed at 14 days after MCAo. Behavioral tests (rotarod and adhesive‐removal tests) were performed. In situ hybridization, immunohistochemistry, and terminal deoxynucleotidyltransferase (TdT)‐mediated dUTP‐biotin nick‐end labeling (TUNEL) were performed to identify transplanted MSCs (Y chromosome), BrdU, bFGF, and apoptotic cells in the brain. Significant recovery of behavior was found in MSC‐treated rats at 7 days in the somatosensory test and at 14 days in the motor test after MCAo compared with control, PBS‐treated animals (P < .05). MSCs were found to survive and preferentially localize to the ipsilateral ischemic hemisphere. Significantly more BrdU‐positive cells were located in the subventricular zone (P < .05), and significantly fewer apoptotic cells and more bFGF immunoreactive cell were found in the ischemic boundary area (P < .05) of MSC‐treated rats than in PBS‐treated animals. Here we demonstrate that intravenously administered male MSCs increase bFGF expression, reduce apoptosis, promote endogenous cellular proliferation, and improve functional recovery after stroke in female rats. © 2003 Wiley‐Liss, Inc.

Two fundamental questions about neuron cell culture were addressed. Can one serum‐free medium that was developed for optimum growth of hippocampal neurons support the growth of neurons from other regions of the brain? Is the region specific state of differentiation maintained in culture? To answer these questions, we isolated neurons from six other rat brain regions, placed them in culture in B27/Neurobasal^TM defined medium, and analyzed their morphology and growth dependence on cell density after 4 days in culture. Neuronal identity was confirmed by immunostaining with antibodies to neurofilament 200. Neurons from each brain region maintained distinctive morphologies in culture in the virtual absence of glia. Cells isolated from embryonic day 18 cerebral cortex by digestion with papain showed the same high survival as hippocampal neurons, e.g., 70% survival for cells plated at 160/mm². At this age and density, neurons from the septum showed slightly lower survival, 45%. Survival of dentate granule neurons from postnatal day four brains was 30‐40%, significantly lower, and relatively independent of plating density. This suggests an absence of dependence on trophic factors or contact for dentate granule neurons. Growth of cerebellar granule neurons isolated from postnatal day 7, 8, or 9 brains in B27/Neurobasal was compared to growth in BME/10% serum. Viability in serum‐free medium at 4 days was much better than that in serum, did not require KCI elevated to 25 mM, and occurred without substantial growth of glia. Cerebellar granule neurons plated at 1,280 cells/mm² were maintained in culture for three weeks with 17 of the original cell density surviving. Survival of cells isolated from embryonic day 18 substantia nigra was 50% at 160 cells/mm² after 4 days, similar to that of striatum, but slightly less than hippocampal neuron survival. The dopaminergic phenotype of the substantia nigral neurons was maintained over 2 weeks in culture as judged by immunoreactivity with antibodies to tyrosine hydroxylase. During this time, immunoreactivity was found in the processes as they grew out from the soma. Together, these studies suggest that B27/Neurobasal will be a useful medium for maintaining the differentiated growth of neurons from many brain regions. Potential applications of a common growth medium for different neurons are discussed. © 1995 Wiley‐Liss, Inc.

The perfusion of rat brain with ¹²⁵I‐transferrin resulted in a receptor‐mediated uptake of transferrin into the endothelium of the blood‐brain barrier followed by its detecation in the brain. During a pulsechase experiment, ¹²⁵I‐transferrin accumulated in the endothelial cells during the pulse, with a decrease of this intraendothelial radioactivity during the chase associated a concomitant increase in the nonvascular elements of the brain. The receptor‐mediated movement of transferrin across the blood‐brain barrier suggests that the brain may derive its iron through the transcytosis of iron‐loaded transferrin across the brain microvasculature. We discuss the likelihood that aluminum and other potentially toxic heavy metals, which also bind tightly to transferrin, may enter the brain by this pathway. We also discuss the possibility that other large molecules including neuroactive peptides and neurotrophic viruses may enter the brain through a similar receptor‐mediated, vesicular transcytotic route.

Alterations in transcription, RNA editing, translation, protein processing, and clearance are a consistent feature of Alzheimer's disease (AD) brain. To extend our initial study (Alzheimer Reports [2000] 3:161‐167), RNA samples isolated from control and AD hippocampal cornu ammonis 1 (CA1) were analyzed for 12633 gene and expressed sequence tag (EST) expression levels using DNA microarrays (HG‐U95Av2 Genechips; Affymetrix, Santa Clara, CA). Hippocampal CA1 tissues were carefully selected from several hundred potential specimens obtained from domestic and international brain banks. To minimize the effects of individual differences in gene expression, RNA of high spectral quality (A_260/280 ≥ 1.9) was pooled from CA1 of six control or six AD subjects. Results were compared as a group; individual gene expression patterns for the most‐changed RNA message levels were also profiled. There were no significant differences in age, postmortem interval (mean ≤ 2.1 hr) nor tissue pH (range 6.6–6.9) between the two brain groups. AD tissues were derived from subjects clinically classified as CDR 2‐3 (CERAD/NIA). Expression data were analyzed using GeneSpring (Silicon Genetics, Redwood City, CA) and Microarray Data Mining Tool (Affymetrix) software. Compared to controls and 354 background/alignment markers, AD brain showed a generalized depression in brain gene transcription, including decreases in RNA encoding transcription factors (TFs), neurotrophic factors, signaling elements involved in synaptic plasticity such as synaptophysin, metallothionein III, and metal regulatory factor‐1. Three‐ or morefold increases in RNAs encoding DAXX, cPLA₂, CDP5, NF‐κBp52/p100, FAS, βAPP, DPP1, NFIL6, IL precursor, B94, HB15, COX‐2, and CEX‐1 signals were strikingly apparent. These data support the hypothesis of widespread transcriptional alterations, misregulation of RNAs involved in metal ion homeostasis, TF signaling deficits, decreases in neurotrophic support and activated apoptotic and neuroinflammatory signaling in moderately affected AD hippocampal CA1. © 2002 Wiley‐Liss, Inc.

The widespread expression of circular RNAs (circRNAs) is regarded as a feature of gene expression in highly diverged eukaryotes. Recent studies have shown that circRNAs can act as a miRNA sponge to repress miRNA function, participate in splicing of target genes, translate genes into protein and interact with RNA binding proteins (RBPs). RBPs are a broad class of proteins involved in gene transcription and translation, and interaction with RBPs is considered an important part of circRNA function, which can serve as an essential element underlying the functions of circRNAs, including genesis, translation, transcriptional regulation of target genes, and extracellular transport. In this mini‐review, we attempt to explore in detail the relationship between circRNAs and RBPs, and the interactions between the two factors. The goal of this review is to investigate the emerging studies of RBPs and circRNAs to better understand how their interaction alters cellular function.

Peroxides are generated continuously in cells that consume oxygen. Among the different peroxides, hydrogen peroxide is the molecule that is formed in highest quantities. In addition, organic hydroperoxides are synthesized as products of cellular metabolism. Generation and disposal of peroxides is a very important process in the human brain, because cells of this organ consume 20% of the oxygen used by the body. To prevent cellular accumulation of peroxides and damage generated by peroxide‐derived radicals, brain cells contain efficient antioxidative defense mechanisms that dispose of peroxides and protect against oxidative damage. Cultured brain cells have been used frequently to investigate peroxide metabolism of neural cells. Efficient disposal of exogenous hydrogen peroxide was found for cultured astrocytes, oligodendrocytes, microglial cells, and neurons. Comparison of specific peroxide clearance rates revealed that cultured oligodendrocytes dispose of the peroxide quicker than the other neural cell cultures. Both catalase and the glutathione system contribute to the clearance of hydrogen peroxide by brain cells. For efficient glutathione‐dependent reduction of peroxides, neural cells contain glutathione in high concentration and have substantial activity of glutathione peroxidase, glutathione reductase, and enzymes that supply the NADPH required for the glutathione reductase reaction. This article gives an overview on the mechanisms involved in peroxide detoxification in brain cells and on the capacity of the different types of neural cells to dispose of peroxides. © 2004 Wiley‐Liss, Inc.