Alterations in the Iron Homeostasis Network of Hepatocytes Caused by Hepatitis B Virus.

BACKGROUND: It is unclear whether hepatitis B virus (HBV) itself causes iron metabolism disorder in patients with chronic hepatitis B (CHB). In this study, we investigated the effect of HBV on iron metabolism at the clinical and cellular levels to determine the pathogenesis of CHB. MATERIALS: We enrolled 41 CHB patients and 20 healthy controls (HCs) in a retrospective study. Parameters of iron status included serum iron (SI), ferritin (SF), transferrin (TRF), soluble transferrin receptor (sTfR), transferrin saturation (TS), total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC), and hepcidin. Liver function indicators included serum alanine transaminase (ALT) and albumin. Furthermore, we investigated the correlations between iron markers and liver function indicators. Finally, the alterations in SF, TRF, transferrin receptor (TfR), and hepcidin expression were detected by RT-PCR, western blot, and cell immunofluorescence after HepG2 cells and Huh7 cells were transfected with the pSM2-HBV plasmid. We also measured these alterations between HepG2 cells and HepG2.215 cells. The significance of differences was analyzed by SPSS version 17.0. RESULTS: Compared with healthy controls, the CHB patients were more likely to have lower levels of serum hepcidin, TRF, sTfR, TIBC, and UIBC and higher levels of SI, SF, and TS (p < 0.05, all). In CHB patients, the levels of SI and SF correlated positively with ALT concentrations, and the serum hepcidin concentrations correlated positively with albumin concentrations (p < 0.05, all). The expression levels of ferritin, transferrin, and hepcidin mRNA and protein were significantly higher in HepG2.215 cells than in HepG2 cells, while expression levels of TfR were lower. The alterations in these iron markers in HepG2 and Huh7 cells that were transfected with pSM2-HBV plasmid were consistent with those in HepG2.215 cells. CONCLUSIONS: Serum iron markers tended to be abnormal in CHB patients. In hepatocytes, HBV promoted the expression of ferritin, transferrin, and hepcidin, while it inhibited the expression of TfR.

Prevalence of Small Intestinal Bacterial Overgrowth in Multiple Sclerosis: a Case-Control Study from China.

OBJECTIVE: It's hypothesized that gastrointestinal microbiota might play an important role in pathogenesis of multiple sclerosis (MS). The aim of the present study was to assess the prevalence of small intestinal bacterial overgrowth (SIBO) in MS patients compared with sex and age matched controls without MS. METHODS: The present study was a case-control type, it included 118 patients with definitive MS and 118 age-sex matched controls. Progression of disability was assessed using the Multiple Sclerosis Severity Score (MSSS). All patients and controls underwent the glucose breath test to assess SIBO. RESULTS: Forty-five of the 118 MS patients were SIBO positive (38.14%; 95%CI: 29.37%-46.90%) compared with 10 of 118 in the control group (8.47%; 95% confidence interval [CI]: 3.45%-13.50%); the difference was statistically significant (P<0.0001; Odds ratios (OR), 4.50; 95% CI, 2.38-8.50). In addition, 102 out of the 118 patients (86.4%) presented at least one GI symptom. Constipation (78.0%), Bloating (46.6%), and fecal incontinence (44.1%) were common. Multivariate analysis showed that expanded disability status scale (EDSS) score and MSSS were the only factors associated with the SIBO-positive status in MS patients (OR, 3.44; 95% CI, 1.56-6.99; and OR, 2.76; 95% CI, 1.42-4.94, respectively). CONCLUSION: SIBO is highly prevalent in Chinese patients with MS. Further analytical work is required to establish a causal association between SIBO and MS risk and progression.

A novel rodent model of posterior ischemic optic neuropathy.

OBJECTIVES: To develop a reliable, reproducible rat model of posterior ischemic optic neuropathy (PION) and study the cellular responses in the optic nerve and retina. METHODS: Posterior ischemic optic neuropathy was induced in adult rats by photochemically induced ischemia. Retinal and optic nerve vasculature was examined by fluorescein isothiocyanate­dextran extravasation. Tissue sectioning and immunohistochemistry were used to investigate the pathologic changes. Retinal ganglion cell survival at different times after PION induction, with or without neurotrophic application, was quantified by fluorogold retrograde labeling. RESULTS: Optic nerve injury was confirmed after PION induction, including local vascular leakage, optic nerve edema, and cavernous degeneration. Immunostaining data revealed microglial activation and focal loss of astrocytes, with adjacent astrocytic hypertrophy. Up to 23%, 50%, and 70% retinal ganglion cell loss was observed at 1 week, 2 weeks, and 3 weeks, respectively, after injury compared with a sham control group. Experimental treatment by brain-derived neurotrophic factor and ciliary neurotrophic factor remarkably prevented retinal ganglion cell loss in PION rats. At 3 weeks after injury, more than 40% of retinal ganglion cells were saved by the application of neurotrophic factors. CONCLUSIONS: Rat PION created by photochemically induced ischemia is a reproducible and reliable animal model for mimicking the key features of human PION. CLINICAL RELEVANCE: The correspondence between the features of this rat PION model to those of human PION makes it an ideal model to study the pathophysiologic course of the disease, most of which remains to be elucidated. Furthermore, it provides an optimal model for testing therapeutic approaches for optic neuropathies.

Foxn4 is required for retinal ganglion cell distal axon patterning.

The regulation of retinal ganglion cell (RGC) axon growth and patterning in vivo is thought to be largely dependent on interactions with visual pathway and target cells. Here we address the hypothesis that amacrine cells, RGCs' presynaptic partners, regulate RGC axon growth or targeting. We asked whether amacrine cells play a role in RGC axon growth in vivo using Foxn4(-/-) mice, which have fewer amacrine cells, but a normal complement of RGCs. We found that Foxn4(-/-) mice have a similar reduction in most subtypes of amacrine cells examined. Remarkably, spontaneous retinal waves were not affected by the reduction of amacrine cells in the Foxn4(-/-) mice. There was, however, a developmental delay in the distribution of RGC projections to the superior colliculus. Furthermore, RGC axons failed to penetrate into the retinorecipient layers in the Foxn4(-/-) mice. Foxn4 is not expressed by RGCs and was not detectable in the superior colliculus itself. These findings suggest that amacrine cells are critical for proper RGC axon growth in vivo, and support the hypothesis that the amacrine cell-RGC interaction may contribute to the regulation of distal projections and axon patterning.

A chemical screen identifies novel compounds that overcome glial-mediated inhibition of neuronal regeneration.

A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced four "hit compounds," which act with nanomolar potency on several different neuronal types and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR (epidermal growth factor receptor) activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.

ATP and NO dually control migration of microglia to nerve lesions.

Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 microM ATP caused maximal movement of microglia in leech nerve cords. The nucleotides ADP, UTP, and the nonhydrolyzable ATP analog AMP-PNP (adenyl-5'-yl imidodiphosphate) also caused movement, whereas AMP, cAMP, and adenosine were without effect. Both movement in ATP and migration after injury were slowed by 50 microM reactive blue 2 (RB2), an antagonist of purinergic receptors, without influencing the direction of movement. This contrasted with the effect of the NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide), which misdirected movement when applied at 1 mM. The cPTIO reduced cGMP immunoreactivity without changing the immunoreactivity of eNOS (endothelial nitric oxide synthase), which accompanies increased NOS activity after nerve cord injury, consistent with involvement of sGC. Moreover, the sGC-specific inhibitor LY83583 applied at 50 microM had a similar effect, in agreement with previous results with methylene blue. Taken together, the experiments support the hypothesis that ATP released directly or indirectly by injury activates microglia to move, whereas NO that activates sGC directs migration of microglia to CNS lesions.

In vivo three-dimensional high-resolution imaging of rodent retina with spectral-domain optical coherence tomography.

PURPOSE: To demonstrate the application of high-resolution spectral-domain optical coherence tomography (SD-OCT) for three-dimensional (3D) retinal imaging of small animals and quantitative retinal information extraction using 3D segmentation of the OCT images. METHODS: A high-resolution SD-OCT system was built for in vivo imaging of rodent retina. OCT fundus images similar to those acquired with a scanning laser ophthalmoscope (SLO) were constructed from the measured OCT data, which provided precise spatial registration of the OCT cross-sectional images on the fundus. A 3D segmentation algorithm was developed for calculation of the retinal thickness map. OCT images were compared by histologic examination. RESULTS: High-quality OCT images of the retinas of mice (B6/SJLF2 for normal retina, rhodopsin-deficient Rho(-/-) for photoreceptor degeneration, and LH(BETA)T(AG) for retinoblastoma) and rat (Wistar) were acquired. The OCT images compared well with histology. Not only was a 3D image of the tumor in a retinoblastoma mouse model successfully imaged in vivo but the tumor volume was extracted from the 3D image. Retinal thickness maps were calculated that enabled successful quantitative comparison of the retinal thickness distribution between the normal (202.3 +/- 9.3 microm) and the degenerative (102.7 +/- 12.6 microm) mouse retina. CONCLUSIONS: High-resolution spectral-domain OCT provides unprecedented high-quality 2D and 3D in vivo visualization of retinal structures of mouse and rat models of retinal diseases. With the capability of 3D quantitative information extraction and precise spatial registration, the OCT system made possible longitudinal study of ocular diseases that has been impossible to conduct.

Repair and regeneration of functional synaptic connections: cellular and molecular interactions in the leech.

A major problem for neuroscience has been to find a means to achieve reliable regeneration of synaptic connections following injury to the adult CNS. This problem has been solved by the leech, where identified neurons reconnect precisely with their usual targets following axotomy, re-establishing in the adult the connections formed during embryonic development. It cannot be assumed that once axons regenerate specific synapses, function will be restored. Recent work on the leech has shown following regeneration of the synapse between S-interneurons, which are required for sensitization of reflexive shortening, a form of non-associative learning, the capacity for sensitization is delayed. The steps in repair of synaptic connections in the leech are reviewed, with the aim of understanding general mechanisms that promote successful repair. New results are presented regarding the signals that regulate microglial migration to lesions, a first step in the repair process. In particular, microglia up to 900 microm from the lesion respond within minutes by moving rapidly toward the injury, controlled in part by nitric oxide (NO), which is generated immediately at the lesion and acts via a soluble guanylate cyclase (sGC). The cGMP produced remains elevated for hours after injury. The relationship of microglial migration to axon outgrowth is discussed.

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS.

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.