Brains of fetal rats (day 14.5 of gestation) were dissociated into single cells with collagenase and suspended in Dulbecco-modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum. The cell suspension (5 x 10' cells/2 ml medium) was cultured at 37切 ina 20 ml flask on a gyratory shaker at 70 rpm. Cells bound to each other randomly and formed small sized lumps in 20 min. Cell lumps adhered and became large sized aggregates. The size of cell aggregates increased up to about 2 mm in diameter within 24 h of cultivation. Cells, arranged randomly in 24 h-cultured aggregates, began to sort themselves out and reconstructed a tissue configuration similar to the brain primordium of day-16.5 fetal rat til 48 h of cultivation. The presence of heparin (100μg/ mI) in the culture medium resulted in the formation of only small sized aggregates (100-200 pm in diameter), and any histotypic pattern was not observed in these small sized aggregates. Therefore, one can consider that the adhesion of cell lumps was mediated by heparin-related molecules such as heparan sulfate proteoglycans and these molecules may be involved in the brain morphogenesis.

The proteoglycan content of the i1iac crest cartilage from a patient with osteogenesis imperfecta type 111 (for cIassification, see Ref. 2) was higher than normal controls, but no significant abnormalities were observed in the structure of the cartilage proteoglycan which was extracted with 4 M guanidine HCI containing protease inhibitors. However, the cartilage-type proteoglycan (proteoglycan-H), isolated from fracture caIluses, had a relatively high content of unsulfated chondroitin and chondroitin 6-sulfate. Another proteoglycan (proteoglycan-L), from the cartiIaginous area and gIycosaminoglycans from the osseous area of the fracture calluses, had a relatively normaI composition of isomeric chondroitin sulfates. This apparent abnormaIity in the glycosaminoglycan composition of proteoglycan-H may be related to the rapidly differentiating aspect of fracture caIluses, rather than an altered process of fracture healing in osteogenesis imperfecta.

Dexamethasone (DEX), a synthetic glucocorticoid, has been widely used to prevent the development of a variety of poor health conditions in premature infants including chronic lung disease, inflammation, circulatory failure, and shock. Although there are some reports of neurologic complications related to DEX exposure, its full effects on the premature brain have not been examined in detail. To investigate the effects of DEX on neural development, we first administered low doses (0.2 mg/kg bodyweight or less) of the glucocorticoid to neonatal rats on a daily basis during the first postnatal week and examined subsequent behavioral alterations at the juvenile stage. DEX-treated rats exhibited not only a significant reduction in both somatic and brain weights but also learning disabilities as revealed in the shuttle avoidance test. The hippocampi of DEX-treated rats displayed a high apoptotic and a low mitotic cell density compared to control rats on day 7 after birth. In a subsequent experiment, neural stem/progenitor cells were cultured in the presence of DEX for 6 days. The glucocorticoid inhibited cell growth without an increase in cell death. These results suggest that administration of DEX to premature infants induces neurological dysfunction via inhibition of the proliferation of neural stem/progenitor cells.

A highly sulfated chondroitin sulfate, CS-E, prevents excitatory amino acid-induced neuronal cell death by an as yet unknown mechanism. To reveal this mechanism, we pretreated neurons in culture with various inhibitors, and examined whether N-methyl-D-aspartic acid (NMDA)-induced neuronal cell death was reduced in the presence of CS-E. The inhibitors of protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) ameliorated NMDA-induced neuronal cell death, but did not affect the neuroprotective activity of CS-E. Among the growth factors with which CS-E can interact, high concentration of BDNF protected against the NMDA-induced neuronal cell death and strengthened neuroprotection by CS-E. CS-E, but neither CS-A nor CS-C, adsorbed to a subclass of neurons with nuclear condensation, namely pyknosis. Contactin-1 (CNTN-1), a putative receptor for neuritogenic activity of CS-E, was present in cortical neurons, but a neutralizing antibody to CNTN-1 did not block neuroprotective activity of CS-E. The results suggest that CS-E may prevent the progression of cell death at the early stages of excitotoxicity through a signaling pathway different from CNTN-1.

Chondroitin sulfate/dermatan sulfate (CS/DS) polysaccharides have been reported to play a crucial role in the proliferation and maintenance of neural stem cells (NSCs). However, little is known about the structural changes and functional role of CS/DS chains in the differentiation of NSCs. Western blots of NSCs, neurons and astrocytes in culture, with three CS-polysaccharide antibodies of different specificities, revealed marked differences in CS structure among the three cell types. To confirm this finding, we measured gene expression levels of CS sulfotransferases and C5-epimerase in these cell types, as these are responsible for producing the high structural diversity of CS/DS. Expressions of chondroitin 4-O-sulfotransferase, chondroitin 6-O-sulfotransferase, and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase mRNAs were low in cultures of differentiated neural cells, such as neurons and astrocytes, in comparison to NSCs. In contrast, expressions of uronyl 2-O-sulfotransferase and C5-epimerase mRNAs were higher in the differentiated neural cells than NSCs. Thus, we first provide evidence to support the hypothesis that CS/DS undergoes structural changes during NSC differentiation. The structural changes in CS/DS may be implicated in the regulation of NSC differentiation through interactions with growth/neurotrophic factors and cytokines.

The hypothalamo-neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS.

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that promotes neurite outgrowth. To identify the ligand of NGC, we applied a detergent-solubilized membrane fraction of fetal rat brains to an NGC-immobilized affinity column. Several proteins were eluted from the column including an 18 kDa-band protein recognized by an anti-pleiotrophin antibody. The binding of pleiotrophin (PTN) to NGC was confirmed by a quartz crystal microbalance method and had a Kd of 8.7 nM. PTN bound to the acidic amino acid cluster of the NGC extracellular domain. In addition, PTN bound to both chondroitin sulfate-bearing NGC and chondroitinase-treated NGC prepared from the neonatal rat brain. These results suggest that NGC interacts with PTN.

Chondroitin sulfate (CS), a polysaccharide moiety of proteoglycans, is one of the major components of the extracellular matrix in the central nervous system and is involved in various cellular events in the formation and maintenance of the neural network. In the developing brain, CS in the milieu of neural stem/progenitor cells (NSPCs) is believed to participate in the regulation of their functions such as proliferation and differentiation. NSPCs are expected to act as a potent cell type in cell replacement therapy for neurodegeneration in various neurological diseases. Recently, it has been shown that transplantation of NSPCs combined with removal of extracellular CS from the host nervous tissues gives a satisfactory outcome in some animal models of nervous tissue injuries including neonatal hypoxic-ischemic injury and adult spinal cord injury. The combination of cell transplantation with modification of the extracellular matrix of the host tissue could be a novel strategy for the treatment of incurable neurodegenerative diseases.

Chondroitin sulfate proteoglycans (CSPGs) consist of chondroitin sulfate (CS) glycosaminoglycans (GAGs) and core protein and regulate the migration, axonal outgrowth, and synaptogenesis in mammalian brains. In the present study, we investigated the localization of CSPGs, the effects of sensory deprivation on the density of perineuronal nets (PNNs), and the effects of chondroitinase ABC (Chase) on the formation of barrel structures in the posterior medial barrel subfield (PMBSF). In developing mouse and rat brains, the immunoreactivity of chondroitin-6-sulfate containing proteoglycan (CS-6-PG), phosphacan, and neurocan was stronger at barrel septa as compared with barrel hollows and surrounding cortex, while the labeling of Wisteria floribunda agglutinin (WFA) was observed at barrel hollows. In adult brains, CS-6-PG-immunoreactive and WFA-labeled PNNs were observed mainly at barrel hollows of mouse, but they were seen chiefly at barrel septa of rats. Sensory deprivation of facial vibrissae reduced the number of WFA-labeled PNNs at barrel hollows but not at barrel septa. Intracerebral injection of Chase did not affect the formation of barrel structures in the PMBSF. These data indicate species-dependent heterogeneity of CSPG expression and activity-dependent formation of PNNs in the PMBSF, but CS GAGs have no crucial function in constructing the barrel structures during early postnatal development.

Fetal growth retardation (FGR) is a critical problem in the neonatal period, because a substantial population of infants born with FGR go on to develop various developmental disorders. In the present study, we produced FGR model rats by continuous administration of a synthetic thromboxane A2 analogue (STA2) to pregnant rats. The FGR pups exhibited a significant delay in postnatal neurological development. Moreover, behavioral analyses revealed the presence of a learning disability in juvenile FGR male rats. To investigate the mechanism underlying the neurological disorders, histological and biochemical analyses of the brain of FGR rats were performed. The density of neurons in the cortical plate of an FGR brain was low compared with the brains of a similarly aged, healthy rat. Consistent with this finding, the density of TUNEL-positive cells was higher in the cortical plate of FGR brains. Western blot analyses showed that the levels of three brain-specific chondroitin sulfate proteoglycans (CSPGs), neurocan, phosphacan, and neuroglycan C, were all significantly reduced in the brain of neonatal FGR rats compared with those of the control. The reduction of CSPG-levels and morphological changes in the brain may be relevant to neurological dysfunction in FGR.

This chapter focuses on the changes in the expression of the axon growth-inhibitory chondroitin sulphate proteoglycans (CSPGs). CSPGs represent a varied class of complex extracellular matrix macromolecules. They share a general molecular structure comprising a central core protein with a number of covalently attached carbohydrate chains known as “glycosaminoglycans” (GAG). The experimental data indicates that the expression of a number of different CSPGs is increased following central nervous system (CNS) injury. The hyalectans neurocan—versican and brevican—plus NG2 and phosphacan are upregulated following injury and all have been shown to exhibit inhibitory effects on neurite outgrowth in vitro.

Perinatal hypoxia-ischemia (HI) remains a critical issue. Cell transplantation therapy could be a potent treatment for many neurodegenerative diseases, but limited works on this kind of therapy have been reported for perinatal HI. In this study, the therapeutic effect of transplantation with neural stem/ progenitor cells (NSPCs) and chondrotinase ABC (ChABC) in a neonatal HI rat model is evaluated. Histological studies showed that the unaffected area of the brain in animals treated with NSPCs together with ChABC was significantly larger than that in the animals treated with vehicle or NSPCs alone. The wet weight of the brain that received the combined treatment was also significantly higher than those of the vehicle and their individual treatments. These results indicate that intracerebroventricular injection of NSPCs with ChABC reduces brain injury in a rat neonatal HI model.

Research on proteoglycans originally advanced using connective tissues such as cartilage as a source. In the 1990s, it was found that multiple species of proteoglycans, both heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs), existed in the central nervous system (CNS). Of these proteoglycans, some such as neurocan, phosphacan/RPTPβ, and neuroglycan C are predominantly expressed in the CNS. Since the CNS contains a very small amount of fibrous collagen, if any, proteoglycans are the major glycoconjugate in the microenvironment of neural cells.

Bone morphogenetic protein (BMP) signaling is involved in differentiation of neural precursor cells into astrocytes, but its contribution to angiogenesis is not well characterized. This study examines the role of BMP signaling through BMP type IA receptor (BMPRIA) in early neural development using a conditional knockout mouse model, in which Bmpr1a is selectively disrupted in telencephalic neural stem cells. The conditional mutant mice show a significant increase in the number of cerebral blood vessels and the level of vascular endothelial growth factor (VEGF) is significantly upregulated in the mutant astrocytes. The mutant mice also show leakage of immunoglobulin around cerebral microvessels in neonatal mice, suggesting a defect in formation of the blood-brain-barrier. In addition, astrocytic endfeet fail to encircle cortical blood vessels in the mutant mice. These results suggest that BMPRIA signaling in astrocytes regulates the expression of VEGF for proper cerebrovascular angiogenesis and has a role on in the formation of the blood-brain-barrier.

Purpose: An increased mRNA expression of the genes coding for the extracellular matrix proteins neuroglycan C (NGC), interphotoreceptor matrix proteoglycan 2 (IMPG2), and CD44 antigen (CD44) has been observed during retinal degeneration in mice with a targeted disruption of the Rpe65 gene (Rpe65(-/-) mouse). To validate these data, we analyzed this differential expression in more detail by characterizing retinal NGC mRNA isoform and protein expression during disease progression.Methods: Retinas from C57/B16 wild-type and Rpe65(-/-) mice, ranging 2 to 18 months of age, were used. NGC, IMPG2, and CD44 mRNA expression was assessed by oligonucleotide microarray, quantitative PCR, and in situ hybridization. Retinal NGC protein expression was analyzed by western blot and immunohistochemistry.Results: As measured by quantitative PCR, mRNA expression of NGC and CD44 was induced by about 2 fold to 3 fold at all time points in Rpe65(-/-) retinas, whereas initially 4 fold elevated IMPG2 mRNA levels progressively declined. NGC and IMPG2 mRNAs were expressed in the ganglion cell layer, the inner nuclear layer, and at the outer limiting membrane. NGC mRNA was also detected in retinal pigment epithelium cells (RPE), where its mRNA expression was not induced during retinal degeneration. NGC-I was the major isoform detected in the retina and the RPE, whereas NGC-III was barely detected and NGC-II could not be assessed. NGC protein expression was at its highest levels on the apical membrane of the RPE. NGC protein levels were induced in retinas from 2- and 4-month-old Rpe65(-/-) mice, and an increased amount of the activity-cleaved NGC ectodomain containing an epidermal growth factor (EGF)-like domain was detected.Conclusions: During retinal degeneration in Rpe65(-/-) mice, NGC expression is induced in the neural retina, but not in the RPE, where NGC is expressed at highest levels.