Wei-Yi Ong

A light and electron microscopic study of NG2 chondroitin sulfate proteoglycan-positive oligodendrocyte precursor cells in the normal and kainate-lesioned rat hippocampus

The adult brain contains a large population of oligodendrocyte precursor cells that can be identified using antibodies against the NG2 chondroitin sulfate proteoglycan. The functions of this newly recognized class of glial cells in the normal or pathological brain are not well understood. To begin to elucidate these functions, we have examined the morphology and distribution of oligodendrocyte precursor cells in the hippocampus and neocortex of normal and kainate-lesioned rats by anti-NG2 immunocytochemistry using light and electron microscopy. Large numbers of oligodendrocyte precursor cells were present in all layers of the neocortex and hippocampus. These cells differed in their morphology from astrocytes, oligodendrocytes and microglia. The processes of these cells often surrounded unlabeled areas of clear cytoplasm. At the electron microscopic level, some of the profiles that were enclosed by oligodendrocyte precursor cell processes contained synaptic vesicles. Other enclosed profiles were dendrites or dendritic spines. NG2-immunopositive processes were also observed to interpose between axon terminals containing round vesicles and dendrites with thick postsynaptic densities. After kainate injection, the NG2-positive oligodendrocyte precursor cells in the hippocampus displayed reactive changes characterized by swollen cell bodies, an increased number of small, filopodial-like processes, and higher levels of immunodetectable NG2. Both viable and degenerating oligodendrocyte precursor cells were observed with electron microscopy. These observations emphasize the dynamic nature of the oligodendrocyte precursor cell and suggest that, in addition to participating in the glial reactions to excitotoxic damage, oligodendrocyte precursor cells may regulate the stability, structure and function of synapses in the normal central nervous system.

A light and electron microscopic study of the CB1 cannabinoid receptor in primate brain

The immunohistochemical distribution and subcellular localization of the cannabinoid CB1 receptor was determined in the adult monkey using a polyclonal antiserum raised against the amino terminus of the rat CB1 receptor. At the level of light microscopy, our results generally parallel earlier studies investigating CB1 distribution in rodent brain with a few differences. In particular, high levels of receptor were found in the cortex, hippocampus, amygdala, cerebellum. However significant differences were also noted. The most striking differences were high levels of CB1 receptor in the monkey substantia nigra pars compacta, cerebellar Purkinje cells, and the principal cells of the hippocampus, while few receptors were found in the globus pallidus or substantia nigra pars reticulata. In contrast, in a previous study investigating the rat, using the same antibody, the opposite staining pattern was observed. At the electron microscopic level CB1 receptor was restricted to neurons. Here it was found both pre- and postsynaptically, particularly on dendritic spines and axon terminals. The CB1 receptor is widely distributed in higher brain regions in the monkey. While its distribution is similar to that in the rat, there are major differences, some of which may be significant when extrapolating the behavioral effects of cannabinoids observed in rodents to primates (e.g., humans). The ultrastructural localization of the CB1 receptor suggests that it modulates neuronal excitability by both pre- and postsynaptic mechanisms.

Characterization, purification, and stability of gold nanoparticles

Impurities in the synthesized gold nanoparticle (AuNP) solution are systematically identified followed by determining an optimal purification process and evaluating the stability as well as oxidation state of the purified 20-nm AuNPs. Quantified non-AuNP components and a newly speciated byproduct (acetate) complete the stoichiometric equation of AuNP synthesis through the citrate reduction method. Among the five tested centrifugation forces (3000-11,000g) and durations (10-60 min), optimal purification of AuNPs was achieved by centrifugation operating at 7000 g for 20 min which satisfactorily recovers ∼80% of AuNPs without detectable impurities. Storage in the dark at 4 °C prolongs the stability of the purified AuNP suspensions up to 20 days. AuNPs employed in this study persist in their atomic status without being oxidized, even after they were aerosolized in air or heated at 500 °C. This work demonstrates how impurities are identified and removed, and the purified AuNPs can be a reference material to evaluate toxicity or reactivity of other engineered nanomaterials.

Translocation and effects of gold nanoparticles after inhalation exposure in rats

This study was carried out to test the hypothesis that nanogold particles can accumulate in the olfactory bulb, and translocate from the lung to other organs after inhalation exposure. Gold nanoparticles were aerosolized and introduced through an exposure chamber. The number concentration of airborne nano-sized particles was 2×106 #NSPs/cm3 with >75% of particulates between 30 and 110 nm. Exposure for 5 days resulted in significant increase of Au in the lung and olfactory bulb as detected by ICP-MS, but after 15 days, significant accumulation of gold was detected in the lung, esophagus, tongue, kidney, aorta, spleen, septum, heart and blood. Microarray analysis showed downregulation of many genes related to muscle in the nanogold-exposed lung. Lipidomic analysis of the lung showed a specific decrease in phosphatidylserine 36:1 species. We conclude that nanogold is able to translocate from the lung to other organs with time, and causes significant effects in exposed tissues.

Differential expression of apolipoprotein D and apolipoprotein E in the kainic acid-lesioned rat hippocampus.

Expression of apolipoprotein D, a member of the lipocalin superfamily of transporter proteins, was investigated in the kainic acid-lesioned rat hippocampus. Using an anti-rat apolipoprotein D antibody and biotin avidin-enhanced immunocytochemistry, in the normal rat hippocampus there was little apolipoprotein D expression, that was restricted mainly to scattered astrocytes. By contrast, kainic acid-injected rats showed apolipoprotein D immunoreactivity in the pyramidal neurons of the affected CA fields 24-48 h after injection of the excitotoxin, at a time when there was no histological evidence of cell death. Apolipoprotein D immunoreactivity peaked by day 3, coincident with neuronal cell death, and declined thereafter, reaching very low levels by day 7. Besides pyramidal neurons, apolipoprotein D immunoreactivity was also observed in a small number of reactive glial cells in the affected CA fields, but not in the vascular compartments at any time-point. In contrast to the neuronal expression of apolipoprotein D, apolipoprotein E immunoreactivity was observed predominantly in degenerating astrocytes. In conclusion, following excitotoxic injury with kainic acid, apolipoprotein D is expressed in hippocampal pyramidal neurons destined for subsequent cell death.

Distribution of hydroxynonenal-modified proteins in the kainate-lesioned rat hippocampus: evidence that hydroxynonenal formation precedes neuronal cell death

Decomposition of lipid peroxides gives rise to a wide range of aldehydes. 4-Hydroxyalkenals and in particular 4-hydroxynonenal (HNE) are often the most toxic products. Frequently, it is unclear at which stage in the tissue injury process HNE is formed, i.e., is it a late stage or an early stage in which HNE contributes to subsequent cell death? The present study was carried out using an antibody to HNE-modified proteins to elucidate the time course and distribution of HNE in the lesioned hippocampus after kainate injections. HNE was absent from normal neurons, but dense staining to HNE was observed in degenerating neurons after kainate injection. The increase in HNE staining occurred as early as 1 d postinjection, at a time when there was no histological evidence of cell death. HNE immunoreactivity was observed in the degenerating CA1 and CA3 fields at 3 d and 1 week postinjection, but was confined to a cluster of neurons at the edge of the degenerating CA fields, at 2 and 3 weeks postinjection. These observations suggest that HNE formation is an early event after this tissue injury, and may contribute to later cell death.

A Nuclear Microscopic Study of Elemental Changes in the Rat Hippocampus After Kainate-Induced Neuronal Injury

Abstract: The effect of intracerebroventricular kainate injection on the elemental composition of the hippocampus was studied in adult Wistar rats, at 1 day and 1, 2, 3, and 4 weeks postinjection, using a nuclear microscope. An increase in calcium concentration was observed on the injected side from 1 day postinjection. The increase peaked at 3 weeks postinjection, reaching a concentration of 18 times normal. Large numbers of glial cells but no neurons were observed in the lesioned CA fields at this time, suggesting that an increased calcium level was present in glial cells. This was confirmed by high‐resolution elemental maps of the lesioned areas, which showed very high intracellular calcium concentrations in almost all glial cells. It is possible that the high intracellular calcium level could activate calcium‐dependent enzymes, including calpain II and cytosolic phospholipase A2, shown to be expressed in reactive glial cells after kainate injections. In addition to calcium, an increase in iron content was also observed at the periphery of the glial scar at 4 weeks postinjection. Because free iron could catalyze the formation of free radicals, the late increase in iron content may be related to oxygen radical formation during neurodegeneration.

Neuronal localization of sterol regulatory element binding protein-1 in the rodent and primate brain: a light and electron microscopic immunocytochemical study.

Sterol regulatory element binding proteins are membrane-bound transcription factors that activate expression of several genes controlling cellular cholesterol and fatty acid homeostasis. The present study aimed to investigate the in vivo expression of sterol regulatory element binding protein-1 in the normal rodent and primate brain, and in the brain in Niemann-Pick type C disease mice. These mutant animals have lysosomal cholesterol accumulation and progressive neurodegeneration caused by an inactivating mutation of the NPC1 gene whose protein product functions in vesicular lipid trafficking. Western blot analysis of rat hippocampal homogenates with an affinity purified rabbit polyclonal antibody directed against an internal epitope of sterol regulatory element binding protein-1 identified a major 68,000 mol. wt protein consistent with the amino-terminal, transcriptionally active fragment of sterol regulatory element binding proteins-1. Immunocytochemically, this antibody revealed dense sterol regulatory element binding protein-1 staining of nuclei and light staining of the cytoplasm of cells in the neocortex and hippocampus in the rat, mouse and monkey brain. By electron microscopy of immunogold-labeled brain sections, these densely labeled cells were found to be neurons. In contrast, normal glial cells had little or no sterol regulatory element binding protein-1 immunoreactivity even at a developmental stage (postnatal day 9) which coincides with active myelination in the rat brain. Also, in contrast to the normal mouse brain, Niemann-Pick type C mice showed reduced staining of cortical and hippocampal neuronal nuclei. Since sterol regulatory element binding protein-1 has been shown to be a transcriptional regulator of fatty acid synthesis in vivo, the current findings of a predominantly neuronal nuclear expression of the 68,000 mol. wt transcriptionally active fragment of sterol regulatory element binding protein-1 highlights the established role of phospholipid metabolites and other fatty-acid containing lipids in neuronal signal transduction and other neuronal functions. Reduced sterol regulatory element binding protein-1 expression in neurons in Niemann-Pick type C may reflect a deficiency in fatty acid synthesis that could contribute to the neuronal dysfunction in this disorder.

Apolipoprotein D gene expression in the rat brain and light and electron microscopic immunocytochemistry of apolipoprotein D expression in the cerebellum of neonatal, immature and adult rats.

Apolipoprotein D gene and protein expression were investigated in the rat brain and cerebellum, respectively, during development. Apolipoprotein D gene expression was first observed in embryonic day 12 rat brain, with a moderate increase in apolipoprotein D messenger RNA levels towards the later part (embryonic days 15-17) of gestation. In the postnatal rat brain, a marked induction of apolipoprotein D messenger RNA occurred at postnatal day 10, with progressively higher levels of apolipoprotein D messenger RNA observed up to postnatal day 20. Somewhat lower, but none the less high, levels of apolipoprotein D messenger RNA continued to be present in brains of adult animals. In the immature cerebellum (day 3 up to one- to two-week-old rats), there were many densely labeled apolipoprotein D-immunoreactive cells that had features of oligodendrocyte precursors. Purkinje neurons showed apolipoprotein D immunoreactivity in one- to two-week-old animals, after which there appeared to be some decrease in staining. Oligodendrocytes in the cerebella of two-week-old animals were strongly apolipoprotein D positive, with immunoreactivity declining in older animals. These results reveal a maturation-associated induction of apolipoprotein D gene expression in the rat brain, and expression of apolipoprotein D in glial (immature oligodendrocyte) cells in the immature cerebellum, followed by specific expression of apolipoprotein D in Purkinje neurons.

A nuclear microscopic and histochemical study of iron concentrations and distribution in the midbrain of two age groups of monkeys unilaterally injected with MPTP

The present study was carried out to elucidate the concentration and distribution of iron in the substantia nigra of two age groups of monkeys after experimental hemi-Parkinsonism induced by unilateral internal carotid injections of MPTP. Iron levels and distribution were detected using the nuclear microscope, which is able to provide structural and quantitative elemental analysis of biological tissue down to the parts per million (ppm) level of analytical sensitivity. Five weeks after unilateral lesioning with MPTP, we observed a 30-65% loss of neurons in the injected substantia nigra of each monkey, compared with the contralateral control non-lesioned side. In monkeys less than 7 years of age, the iron was distributed fairly uniformly and showed little evidence of focal deposits. In monkeys greater than 7 years of age, we observed many dense focal deposits of iron in the substantia nigra. A comparison between iron distributions in nuclear microscopic scans and cell distributions in the same sections stained by the Nissl technique showed that areas containing high iron concentrations were present not where large-diameter neurons with abundant Nissl substance (presumed dopaminergic neurons) were located but in a region ventral to these cell bodies, i.e., in the substantia nigra pars reticulata. These distributions were present on the control side as well as the MPTP-injected side. Since a previous study has shown that unilateral MPTP injection results in lesions of the substantia nigra of the same side but negligible injury to the opposite side, this implies that the iron deposits existed in the older monkeys before MPTP injections (i.e. they occurred normally). The accumulation of iron in the substantia nigra with age suggests the possibility of localised damage to neurons through the catalysis of free radicals.