Leping Zeng

Differential neuronal expression of receptor interacting protein 3 in rat retina: involvement in ischemic stress response

BackgroundReceptor-interacting protein 3 (RIP3), a member of RIP family proteins, has been shown to participate in programmed necrosis or necroptosis in cell biology studies. Evidence suggests that necroptosis may be a mode of neuronal death in the retina.ResultsIn the present study we determined the expression of RIP3 in normal rat retina and its changes following acute high intraocular pressure (aHIOP). RIP3 immunoreactivity (IR) was largely present in the inner retinal layers, localized to subsets of cells expressing neuron-specific nuclear antigen (NeuN), parvalbumin and calbindin in the ganglion cell layer (GCL) and inner nuclear layer (INL). No double labeling was detected for RIP3 with PKC-α or rhodopsin. RIP3 immunoreactivity was increased in the GCL at 6 hr and 12 hr, but reduced at 24 hr in the retina, without apparent alteration in laminar or cellular distribution pattern. Western blot analysis confirmed the above time-dependent alteration in RIP3 protein expression. RIP3 expressing cells frequently co-localized with propidium iodide (PI). A few co-localized cells were observed between RIP3 and Bax or cleaved caspase-3 in the GCL in 12 hr following aHIOP.ConclusionsThe results indicate that RIP3 is expressed differentially in retinal neurons in adult rats, including subsets of ganglion cells, amacrine and horizontal cells. RIP3 protein levels are elevated rapidly following aHIOP. RIP3 labeling co-localized with PI, Bax or cleaved caspase-3 among cells in the ganglion cell layer following aHIOP, which suggest its involvement of RIP3 in neuronal responses to acute ischemic insults.

BACE1 in the retina: a sensitive biomarker for monitoring early pathological changes in Alzheimer's disease.

Because of a lack of sensitive biomarkers, the diagnosis of Alzheimer′s disease (AD) cannot be made prior to symptom manifestation. Therefore, it is crucial to identify novel biomarkers for the presymptomatic diagnosis of AD. While brain lesions are a major feature of AD, retinal pathological changes also occur in patients. In this study, we investigated the temporal changes in β-site APP-cleaving enzyme 1 (BACE1) expression in the retina and brain to determine whether it could serve as a suitable biomarker for early monitoring of AD. APP/PS-1 transgenic mice, 3, 6 and 8 months of age, were used as an experimental group, and age-matched C57/BL6 wild-type mice served as the control group. In the Morris water maze test, there were no significant differences in escape latency or in the number of crossings in the target area among mice of different ages. Compared with wild-type mice, no changes in learning or memory abilities were detected in transgenic mice at 3 months of age. However, compared with wild-type mice, the escape latency was significantly increased in transgenic mice at 6 months, starting on day 3, and at 8 months, starting on day 2, during Morris water maze training. In addition, the number of crossings of the target area was significantly decreased in transgenic mice. The learning and memory abilities of transgenic mice were further worsened at 8 months of age. Immunohistochemical staining revealed no BACE1 plaques in wild-type mice at 3, 6 or 8 months or in transgenic mice at 3 months, but they were clearly found in the entorhinal cortex, hippocampus and prefrontal cortex of transgenic mice at 6 and 8 months. BACE1 expression was not detected in the retina of wild-type mice at 3 months, but weak BACE1 expression was detected in the ganglion cell layer, inner plexiform layer and outer plexiform layer at 6 and 8 months. In transgenic mice, BACE1 expression in the ganglion cell layer was increased at 3 months, and BACE1 expression in the ganglion cell layer, inner plexiform layer and outer plexiform layer was significantly increased at 6 and 8 months, compared with age-matched wild-type mice. Taken together, these results indicate that changes in BACE1 expression appear earlier in the retina than in the brain and precede behavioral deficits. Our findings suggest that abnormal expression of BACE1 in the retina is an early pathological change in APP/PS-1 transgenic mice, and that BACE1 might have potential as a biomarker for the early diagnosis of AD in humans.

Arctigenin, a Natural Lignan Compound, Induces Apoptotic Death of Hepatocellular Carcinoma Cells via Suppression of PI3‐K/Akt Signaling

In this study, we explored the cytotoxic effects of arctigenin, a natural lignan compound, on human hepatocellular carcinoma (HCC) cells and check the involvement of phosphatidylinositol 3‐kinase (PI3‐K)/Akt signaling. HCC cells were treated with different concentrations of arctigenin and cell viability and apoptosis were assessed. Manipulating Akt signaling was used to determine its role in the action of arctigenin. Arctigenin significantly inhibited the viability of HCC cells in a concentration‐dependent manner. Arctigenin induced apoptosis and activation of caspase‐9 and ‐3. Overexpression of a constitutively active Akt mutant blocked arctigenin‐induced apoptosis. Combinational treatment with arctigenin and the PI3‐K inhibitor LY294002 significantly enhanced apoptosis. Arctigenin reduced the expression of Bcl‐xL, Mcl‐1, and survivin and the phosphorylation of mTOR and S6K, which were significantly reversed by overexpression of constitutively active Akt. This is the first report about the anticancer activity of arctigenin in HCC cells, which is mediated by inactivation of PI3‐K/Akt signaling.

Distribution of thrombospondins and their neuronal receptor α2δ1 in the rat retina

The role of the extracellular matrix protein thrombospondins (TSPs) in promoting synaptogenesis is gaining more and more attention. The binding of TSP1 and TSP2 to their neuronal receptor α2δ1 stimulates excitatory synaptogenesis in the development and injury of the central nervous system; however, the specific cellular localization and expression of TSP1/2 and α2δ1 in healthy and damaged retinas is unknown. This, to a certain extent, has restricted the progress of research on the molecular mechanisms triggering synaptic plasticity after retinal injury. Here, the cellular localization and expression of TSP1/2 and their receptor α2δ1 was studied in healthy and damaged adult retina induced by elevated intraocular pressure (IOP) using double immunofluorescence labeling and confocal scanning microscopy. We showed the apparent differential distribution of TSP1 and TSP2 in the adult rat retina. TSP1 was confined to the ganglion cell layer and inner nuclear layer, in which it was preferentially expressed by ganglion cells, bipolar cells and horizontal cells but rarely expressed by glial cells. TSP2 staining was diffusely distributed in GFAP- and GS-immunopositive glial cells and processes in the inner retina. In rat retinas, α2δ1 staining was present in ganglion cells, bipolar cells, partial horizontal cells and amacrine cells and the presynaptic terminals. Müller cells and a minority of astrocytes also expressed α2δ1. On the seventh day of elevated IOP, TSP2 immunoreactivity was greatly increased, and immunopositive processes extended throughout the retinal layer and co-localized with GFAP- and GS-positive glial cells. TSP1 distribution in the retina, however, did not change distinctly. α2δ1-immunopositive processes were also increased on the seventh day after elevated IOP. Our study suggested that in the adult rat retina, TSP2, but not TSP1, secreted by glial cells may be involved in the synaptic plastic process after retinal injury through binding to its neuronal receptor α2δ1.

Differential Changes of Local Blood Supply in Rat Retinae Are Involved in the Selective Loss of Retinal Ganglion Cells Following the Acute High Intraocular Pressure

Purpose: To detect changes in local blood supply to central, middle, and peripheral retina following acute high intraocular pressure and to investigate the effects of changes in local retinal blood supply on the selective loss of retinal ganglion cells. Methods: The acute high intraocular pressure model of Sprague-Dawley rats was established by increasing the anterior chamber pressure to 110 mmHg via a normal saline perfusion system. Blood supply to the central, middle, and peripheral retina at 3, 6, and 12 h, and 1, 3, 7, and 14 d following induction of acute high intraocular pressure was detected by using gelatin-ink perfusion and fluorescent microsphere injection. Retinal ganglion cell loss following acute high intraocular pressure was detected by fluorogold retrograde labeling. Finally, the relationship between changes in local retinal blood supply and loss of retinal ganglion cells was investigated. Results: The increased ratio of blood supply of peripheral retina was less than that of the central and middle retina at 3 h to 14 d following acute high intraocular pressure. The percent of retinal ganglion cell loss in the peripheral retina was clearly greater than that in the central and middle retina during the first 3 d following induction of acute high intraocular pressure (p < 0.05). Using either the gelatin-ink infusion method or the microsphere injection method (p < 0.05), a significantly negative correlation between the percent of retinal ganglion cell loss and the corresponding increased local blood supply after induction of acute high intraocular pressure (r = -0.57 or -0.72) was found. Moreover, a significant negative correlation in the peripheral retina (r = -0.80 or -0.91; p < 0.05) was observed. Conclusion: A disparity exists between changes in local blood supply to the central and middle retina, and the peripheral retina following induction of acute high intraocular pressure in rats. This inequality of changes in local blood supply in rat retina is related to the selective loss of retinal ganglion cells.

Degenerative alterations in noradrenergic neurons of the locus coeruleus in Alzheimer's disease.

Mice carrying mutant amyloid-β precursor protein and presenilin-1 genes (APP/PS1 double transgenic mice) have frequently been used in studies of Alzheimers disease; however, such studies have focused mainly on hippocampal and cortical changes. The severity of Alzheimers disease is known to correlate with the amount of amyloid-β protein deposition and the number of dead neurons in the locus coeruleus. In the present study, we assigned APP/PS1 double transgenic mice to two groups according to age: young mice (5–6 months old) and aged mice (16–17 months old). Age-matched wild-type mice were used as controls. Immunohistochemistry for tyrosine hydroxylase (a marker of catecholaminergic neurons in the locus coeruleus) revealed that APP/PS1 mice had 23% fewer cells in the locus coeruleus compared with aged wild-type mice. APP/PS1 mice also had increased numbers of cell bodies of neurons positive for tyrosine hydroxylase, but fewer tyrosine hydroxylase-positive fibers, which were also short, thick and broken. Quantitative analysis using unbiased stereology showed a significant age-related increase in the mean volume of tyrosine droxylase-positive neurons in aged APP/PS1 mice compared with young APP/PS1 mice. Moreover, the mean volume of tyrosine hydroxylase-positive neurons was positively correlated with the total volume of the locus coeruleus. These findings indicate that noradrenergic neurons and fibers in the locus coeruleus are predisposed to degenerative alterations in APP/PS1 double transgenic mice.

An effective method of DNA extraction for bioleaching bacteria from acid mine drainage

An effective and versatile method for microorganism lysis and direct extraction of DNA from bioleaching bacteria was developed using pure cultures and an acid mine drainage (AMD) sediment sample. In the described method, microorganisms are treated at three different incubation temperatures: boiling water incubation for 6–10min, followed by 60 ± 5°C for 30min, then 72°C for 30min. The extracted DNA is purified using a phenol/chloroform/alcohol mixture and precipitated in absolute alcohol. The 16S ribosomal RNA (rRNA) and gyrB genes of the pure cultures were amplified using the polymerase chain reaction (PCR) and differentiated using repetitive intergenic DNA sequences amplification (Rep-PCR). For the AMD sediment sample, the 16S rRNA and gyrB genes of the amplicons were digested with Hin6I and MspI, and the restriction fragment length polymorphism analysis patterns were used as a fingerprint to discern community diversity. The results indicated that this method is a versatile, reproducible, effective, and rapid technique for routine DNA extraction from bioleaching bacteria. The low cost of this method also makes it attractive for large-scale studies.

Regulatory effects of inhibiting the activation of glial cells on retinal synaptic plasticity

Various retinal injuries induced by ocular hypertension have been shown to induce plastic changes in retinal synapses, but the potential regulatory mechanism of synaptic plasticity after retinal injury was still unclear. A rat model of acute ocular hypertension was established by injecting saline intravitreally for an hour, and elevating the intraocular pressure to 14.63 kPa (110 mmHg). Western blot assay and immunofluorescence results showed that synaptophysin expression had a distinct spatiotemporal change that increased in the inner plexiform layer within 1 day and spread across the outer plexiform layer after 3 days. Glial fibrillary acidic protein expression in retinae was greatly increased after 3 days, and reached a peak at 7 days, which was also consistent with the peak time of synaptophysin expression in the outer plexiform layer following the increased intraocular pressure. Fluorocitrate, a glial metabolic inhibitor, was intravitreally injected to inhibit glial cell activation following high intraocular pressure. This significantly inhibited the enhanced glial fibrillary acidic protein expression induced by high intraocular pressure injury. Synaptophysin expression also decreased in the inner plexiform layer within a day and the widened distribution in the outer plexiform layer had disappeared by 3 days. The results suggested that retinal glial cell activation might play an important role in the process of retinal synaptic plasticity induced by acute high intraocular pressure through affecting the expression and distribution of synaptic functional proteins, such as synaptophysin.

The correlation between rat retinal nerve fiber layer thickness around optic disc by using optical coherence tomography and histological measurements.

AIM To explore the correlation between the retinal nerve fiber layer (RNFL) thickness by using optical coherence tomography (OCT) and by histological measurements in normal adult rats and optic nerve transected rats. METHODS The RNFL thickness of 36 rats was scanned in a circle 3.46mm far from the optic disc by OCT. The two experimental groups were the normal group (n=20 rats) and the optic nerve transected group (n=16 rats). The latter group included 4 groups (n=4/group) surviving for 1 day, 3, 5 and 7 days. Then the RNFL thickness of the same retina area was also measured by NF-200 immunohistochemical staining method. Linear regression was used to analyze the correlation between the data obtained from these two methods. RESULTS The RNFL thickness of normal right eyes around optic disc by OCT was 72.35±5.71µm and that of the left eyes was 72.65±5.88µm (P=0.074). The RNFL thickness of the corresponding histological section by immunohistochemistry was 37.54±4.05µm (right eyes) and 37.38±4.23µm (left eyes) (P=0.059). There was a good correlation between the RNFL thickness measured by OCT and that measured by histology (R(2)=0.8131). After optic nerve transection, the trend of the RNFL thickness was thinner with the prolonged survival time. The correlation of the thickness detected by the above two methods was approximately (R(2)=0.8265). Value of the RNFL thickness in rats around optic disc measured by OCT was obviously higher than that measured by common histological measurement in normal adult rats and optic nerve transected rats. CONCLUSION The RNFL thickness measured by OCT has a strong correlation with that measured by histological method. Through OCT scanning, we found that the thickness of RNFL gradually becomes thinner in a time-dependent manner.

Expression of Glutamate and GABA during the Process of Rat Retinal Synaptic Plasticity Induced by Acute High Intraocular Pressure.

Acute high intraocular pressure (HIOP) can induce plastic changes of retinal synapses during which the expression of the presynaptic marker synaptophysin (SYN) has a distinct spatiotemporal pattern from the inner plexiform layer to the outer plexiform layer. We identified the types of neurotransmitters in the retina that participated in this process and determined the response of these neurotransmitters to HIOP induction. The model of acute HIOP was established by injecting normal saline into the anterior chamber of the rat eye. We found that the number of glutamate-positive cells increased successively from the inner part to the outer part of the retina (from the ganglion cell layer to the inner nuclear layer to the outer nuclear layer) after HIOP, which was similar to the spatiotemporal pattern of SYN expression (internally to externally) following HIOP. However, the distribution and intensity of GABA immunoreactivity in the retina did not change significantly at different survival time post injury and had no direct correlation with SYN expression. Our results suggested that the excitatory neurotransmitter glutamate might participate in the plastic process of retinal synapses following acute HIOP, but no evidence was found for the role of the inhibitory neurotransmitter GABA.