Investigating degeneration of the retina in young and aged tau P301L mice.

AIMS: Tau is a microtubule-binding protein facilitating the stability of the cytoskeleton. It is important for neurons as several neurodegenerative diseases involve hyperphosphorylation and aggregation of tau. It is known that mutated tau P301L results in aggregation of tau proteins, leading to neuronal loss in the brain. The aim of this study was to investigate the effect of tau mutation on the retina using a transgenic tau P301L mouse model. MAIN METHODS: Morphometric analysis was utilized to quantify the neurodegenerative changes, including the thickness of the inner nuclear layer (INL), and the density and size of retinal ganglion cells (RGCs). Sections of retina tissue stained by hematoxylin and eosin (H&E) and immunohistochemistry were analyzed. Comparisons were made between the tau P301L mice and control mice, as well as between different age groups. KEY FINDINGS: A significant decrease in the thickness of the INL in tau P301L mice was found when compared with that of control mice. The effect was more pronounced in the peripheral area, and the effect increased with age. Regarding density of RGCs, tau P301L mice showed a similar age-related decline as in control mice. Furthermore, the RGCs from tau P301L mice increased in size with age, and the RGCs from control mice decreased in size with age. SIGNIFICANCE: Tau may be an age-independent factor of accelerated neurodegeneration, with effects differing by types of neurons and regions of the retina.

Morphometric analyses of retinal sections.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)-induced excitotoxicity(1), retinal ischemia-reperfusion injury(2) and glaucoma(3). Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity(4). Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure(3,5,6). Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies. The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes. This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience - MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

Up-regulation of crystallins is involved in the neuroprotective effect of wolfberry on survival of retinal ganglion cells in rat ocular hypertension model.

Wolfberry (fruit of Lycium barbarum Linn) has been known for balancing 'Yin' and 'Yang' in the body, nourishing the liver and kidney, improving visual acuity for more than 2,500 years in oriental countries. The active components in wolfberry include L. barbarum polysaccharide (LBP), zeaxanthine, betaine, cerebroside and trace amounts of zinc, iron, and copper. Each of them confers distinct beneficial effects and together they help to explain widespread use of wolfberry in the eastern world. Earlier study reported the neuroprotective effects of LBP on retinal ganglion cell (RGC) in an experimental model of glaucoma and the underlying in vivo cellular mechanisms of LBP neuroprotection deserve further exploration. In this study, we adopted proteomics, functional genomics, to evaluate pharmacological effects of LBP on the neuronal survival pathways. Among the significantly changed proteins induced by LBP feeding on ocular hypertension (OH) retinas, only proteins in crystallin family were focused in this study. The proteomic results were further confirmed using the Western blotting of the retinas and immunohistochemical staining of the retinal sections. We demonstrated that neuroprotective effect of-wolfberry extract-LBP on the survival of RGCs may be mediated via direct up-regulation of neuronal survival signal betaB2-crystallin.