Treatment with thyroxine restores myelination and clinical recovery after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) remains a major cause of white matter injury in preterm infants with no viable therapeutic strategy to restore myelination. Maturation of oligodendrocytes and myelination is influenced by thyroid hormone (TH) signaling, which is mediated by TH receptor α (TRα) and TRß. In the brain, cellular levels of TH are regulated by deiodinases, with deiodinase-2 mediating TH activation and deiodinase-3 TH inactivation. Therefore, we hypothesized that IVH would decrease TH signaling via changes in the expression of deiodinases and/or TRs, and normalization of TH signaling would enhance maturation of oligodendrocytes and myelination in preterm infants with IVH. These hypotheses were tested using both autopsy materials from human preterm infants and a rabbit model of IVH. We found that deiodinase-2 levels were reduced, whereas deiodinase-3 levels were increased in brain samples of both humans and rabbits with IVH compared with controls without IVH. TRα expression was also increased in human infants with IVH. Importantly, treatment with TH accelerated the proliferation and maturation of oligodendrocytes, increased transcription of Olig2 and Sox10 genes, augmented myelination, and restored neurological function in pups with IVH. Consistent with these findings, the density of myelinating oligodendrocytes was almost doubled in TH-treated human preterm infants compared with controls. Thus, in infants with IVH the combined elevation in deiodinase-3 and reduction in deiodinase-2 decreases TH signaling that can be worsened by an increase in unliganded TRα. Given that TH promotes neurological recovery in IVH, TH treatment might improve the neurodevelopmental outcome of preterm infants with IVH.

Bone morphogenetic protein inhibition promotes neurological recovery after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) results in neural cell death and white matter injury in premature infants. No therapeutic strategy is currently available against this disorder. Bone morphogenetic protein (BMP) signaling suppresses oligodendrocyte development through basic-helix-loop-helix (bHLH) transcription factors and promotes astrocytosis. Therefore, we hypothesized that IVH in premature newborns initiates degeneration and maturation arrest of oligodendrocyte lineage and that BMP inhibition alleviates hypomyelination, gliosis, and motor impairment in the survivors of IVH. To test the hypotheses, a rabbit model of IVH was used in which premature rabbit pups (E29) are treated with intraperitoneal glycerol at 2 h of age to induce IVH; and the pups with IVH exhibit hypomyelination and gliosis at 2 weeks of postnatal age. Maturation of oligodendrocyte lineage was evaluated by specific markers, and the expression of bHLH transcription factors was assessed. BMP levels were measured in both premature rabbit pups and autopsy materials from premature infants. Recombinant human noggin was used to suppress BMP action; and neurobehavioral performance, myelination and gliosis were assessed in noggin-treated pups compared with untreated controls. We found that IVH resulted in apoptosis and reduced proliferation of oligodendrocyte progenitors, as well as arrested maturation of preoligodendrocytes in rabbits. BMP4 levels were significantly elevated in both rabbit pups and human premature infants with IVH compared with controls. Importantly, BMP inhibition by recombinant human noggin restored the levels of phospho-Smad1/5/8, Olig2 transcription factor, oligodendrocyte maturation, myelination, astrocyte morphology, and motor function in premature pups with IVH. Hence, BMP inhibition might enhance neurological recovery in premature infants with IVH.

Hypoxia-ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter.

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia-ischemia. This relatively brief period of hypoxia-ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13+/-0.25 counts/microm(2)), and declined significantly during hypoxia-ischemia (0.93+/-0.15 counts/microm(2): P<0.001). Astrocytes had lower glutamate levels (0.7+/-0.07 counts/microm(2)), and showed a relatively small decline during hypoxia-ischemia. Axonal regions contained high levels of glutamate (1.84+/-0.20 counts/microm(2)), much of which was lost during hypoxia-ischemia (0.72+/-0.20 counts/microm(2): P>0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia-ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90+/-0.20 counts/microm(2)), and ependymal cells (2.20+/-0.28 counts/microm(2)), and hypoxia-ischemia produced a large fall in ependymal glutamate (0.97+/-0.08 counts/microm(2): P>0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia.

Multicenter study of acetaminophen hepatotoxicity reveals the importance of biological endpoints in genomic analyses.

Gene expression profiling is a widely used technique with data from the majority of published microarray studies being publicly available. These data are being used for meta-analyses and in silico discovery; however, the comparability of toxicogenomic data generated in multiple laboratories has not been critically evaluated. Using the power of prospective multilaboratory investigations, seven centers individually conducted a common toxicogenomics experiment designed to advance understanding of molecular pathways perturbed in liver by an acute toxic dose of N-acetyl-p-aminophenol (APAP) and to uncover reproducible genomic signatures of APAP-induced toxicity. The nonhepatotoxic APAP isomer N-acetyl-m-aminophenol was used to identify gene expression changes unique to APAP. Our data show that c-Myc is induced by APAP and that c-Myc-centered interactomes are the most significant networks of proteins associated with liver injury. Furthermore, sources of error and data variability among Centers and methods to accommodate this variability were identified by coupling gene expression with extensive toxicological evaluation of the toxic responses. We show that phenotypic anchoring of gene expression data is required for biologically meaningful analysis of toxicogenomic experiments.

Quantitative analysis of immunogold labeling for basic fibroblast growth factor according to the activational stages of mast cells.

OBJECTIVE: To analyze whether immunogold labeling density for basic fibroblastic growth factor in granules is compatible with the activation stage of mast cells. STUDY DESIGN: Cytoplasmic features and granules of 46 mast cells were examined at the ultrastructural level. The cells were classified according to their activation stage, namely, whether resting, initially activated, fully degranulated or piecemeal degranulated. Granules were classified as electron lucent, moderate or dense granules. Gold particles per secretory granules in the cells were counted. Recently described quantitative analysis techniques were used for evaluation. RESULTS: There is a statistically meaningful relationship between the activation stage of mast cells and their immunogold labeling density. The number of different types of granules encountered in a cell depends on the type of the cell. The distribution of gold particles among the secretory granules depends upon the cell. The type of granule does not have an individual effect on the number of particles, as indicated by an overall statistical analysis of granules, cells and their interaction effects. CONCLUSION: A count of gold particles in the cells can be used as a strong biological indicator. Therefore the number of gold particles might be very useful for comparative studies related to the secretion of this growth factor under different conditions.

Ultrastructural immunolocalization of basic fibroblast growth factor in endothelial cells: morphologic evidence for unconventional secretion of a novel protein.

Basic fibroblast growth factor (bFGF) is one of the most potent angiogenic factors. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion. Recent studies suggest that there is an unconventional secretory pathway for this growth factor. The aim of this study was to identify the specific location of bFGF in endothelial cells and to find morphologic evidences concerning its synthesis, storage and release from endothelial cells. The capillaries in hippocampus, adrenal gland, kidney, peripheral nerves as well as the vessels in connective tissues were analysed by using immunogold labeling techniques at electron microscope level. Results show that endogenous bFGF is mainly located in the nuclei of endothelial cells. Slight immunoreactivity is found in the cytoplasm. Immunolabeling is notably absent in pinocytotic vesicles, Golgi complexes, endoplasmic reticulum, nuclear membrane and intercellular junctions. These results provide morphologic evidence suggesting that endothelial cells might export bFGF via unique cellular pathways that are clearly distinct from classical signal peptide mediated secretion and/or release of this protein could be directly through mechanically induced disruptions of these cells. The current study support the recent hypothesis related with unconventional secretory pathway for bFGF as some other "cargo" proteins.

On the biomarkers and mechanisms of konzo, a distinct upper motor neuron disease associated with food (cassava) cyanogenic exposure.

Konzo is a self-limiting central motor-system disease associated with food dependency on cassava and low dietary intake of sulfur amino acids (SAA). Under conditions of SAA-deficiency, ingested cassava cyanogens yield metabolites that include thiocyanate and cyanate, a protein-carbamoylating agent. We studied the physical and biochemical modifications of rat serum and spinal cord proteins arising from intoxication of young adult rats with 50-200mg/kg linamarin, or 200mg/kg sodium cyanate (NaOCN), or vehicle (saline) and fed either a normal amino acid- or SAA-deficient diet for up to 2 weeks. Animals under SAA-deficient diet and treatment with linamarin or NaOCN developed hind limb tremors or motor weakness, respectively. LC/MS-MS analysis revealed differential albumin carbamoylation in animals treated with NaOCN, vs. linamarin/SAA-deficient diet, or vehicle. 2D-DIGE and MALDI-TOF/MS-MS analysis of the spinal cord proteome showed differential expression of proteins involved in oxidative mechanisms (e.g. peroxiredoxin 6), endocytic vesicular trafficking (e.g. dynamin 1), protein folding (e.g. protein disulfide isomerase), and maintenance of the cytoskeleton integrity (e.g. α-spectrin). Studies are needed to elucidate the role of the aformentioned modifications in the pathogenesis of cassava-associated motor-system disease.

Monocyclic and dicyclic hydrocarbons: structural requirements for proximal giant axonopathy.

The chromogenic and neurotoxic gamma-diketone 1,2-diacetylbenzene (1,2-DAB), but not its isomer 1,3-DAB, induces blue discoloration of tissues and urine, clustering of axonal microtubules and proximal neurofilament-filled axonal swellings in rodents. The remarkable chromogenic property of 1,2-DAB, a monocyclic aromatic hydrocarbon, arises from reaction with lysine residues of proteins and formation of dimeric and polymeric derivatives. Tetralin, a dicyclic solvent structurally related to acetyl ethyl tetramethyl tetralin, a chromogenic and neurotoxic agent, reportedly induces excretion of green urine, and causes neurological disturbances in humans. Monocyclic aromatic 1,2,4-triethylbenzene (1,2,4-TEB), but not its isomer 1,3,5-TEB, is also reportedly chromogenic and induces neurophysiological deficits in rodents consistent with axonal neuropathy, but without neuropathological confirmation. We treated 12-week-old C57Bl/6 mice by gavage with 300, 600, or 900 mg/kg/day 1,2,4-TEB, or equivalent doses of 1,3,5-TEB, 3 days/week, for up to 12 weeks, or intraperitoneally with 400 mg/kg/day tetralin, or 50 or 100 mg/kg/day of its alpha-tetralol analogue, 5 days/week, for up to 5 weeks. Animals treated with 1,2,4-TEB, but not 1,3,5-TEB, tetralin or alpha-tetralol, developed hind limb weakness, excreted greenish urine, and showed 1,2-DAB-like neuropathology. These findings support the hypothesis that 1,2-spaced ethyl (or acetyl) moieties on a benzene ring of hydrocarbons are required for hydrocarbons to induce chromogenic changes and proximal giant neurofilamentous axonopathy. Key molecular targets of these compounds likely reside in the axon where they serve to maintain normal cytoskeletal organization.

Age related optic nerve axonal loss in adult Brown Norway rats.

The effect of age on the number and morphology of optic nerve axons in adult Brown Norway rats (5-31 months old) (n=29) was examined using transmission electron microscopy (TEM). By manually counting every axon in areas representing 60% of the optic nerve cross-section, we found a significant negative correlation between age and axon count (R(2)=0.18, P<0.05). However, when the oldest animals were omitted, the relationship was no longer statistically significant. Simultaneously, the proportion of spontaneously degenerating axons increased at an exponential rate (R(2)=0.79, P<0.05), with significantly more degeneration in the 31-month group than in 5-month-old animals (ANOVA, P<0.05). This study demonstrates, using quantitative TEM methods, that optic nerve axonal numbers are relatively constant throughout the majority of the adult life of the Brown Norway rat, an increasingly popular strain for glaucoma research. Total axonal loss with aging is substantially less than that reported for other strains. The reduction in axonal numbers and the rate of axonal degeneration do not appear significantly altered until the last few months of life, failing to support some studies that have concluded that optic nerve axon loss in adult rats is linear. However, they do agree with other studies in the rat, and a similar study performed in non-human primate eyes, that concluded that aging changes in the optic nerve and retina follow a complex pattern. Therefore, the impact of animal age must be considered when modeling the course and pathophysiology of experimental glaucomatous optic nerve damage in rats.