Gideon Ho

Effect of low-level laser treatment of tissue-engineered skin substitutes: contraction of collagen lattices

Fibroblast-populated collagen lattices (FPCL) are widely used in tissue-engineered artificial skin substitutes, but their main drawback is that interaction of fibroblasts and matrix causes contraction of the lattice, reducing it to about 20% of its original area. The effect of low-level laser treatment (LLLT) on the behavior of 3T3 fibroblasts seeded in collagen lattices containing 20% chondroitin-6-sulphate was investigated to determine whether LLLT could control the contraction of FPCL. A He-Ne laser was used at 632.8 nm to deliver a 5-mW continuous wave with fluences from 1 to 4 J/cm(2). Laser treatment at 3 J/cm(2) increased contraction of collagen lattices in the absence of cells but decreased contraction of cell seeded lattices over a 7-day period. The effect was energy dependent and was not observed at 1, 2, or 4 J/cm(2). There was no alteration in fibroblast viability, morphology, or mitochondrial membrane potential after any laser treatments, but the distribution of actin fibers within the cells and collagen fibers in the matrices was disturbed at 3 J/cm(2). These effects contribute to the decrease in contraction observed. LLLT may offer a means to control contraction of FPCL used as artificial skin substitutes.

Molecular imaging of retinal gliosis in transgenic mice induced by kainic acid neurotoxicity.

PURPOSE Gliosis is a universal response of the central nervous system to diverse insults. Here the authors aimed to develop a noninvasive fluorescence system to monitor and quantify retinal gliosis in real time. METHODS Transgenic mice expressing green fluorescent protein (GFP) under the control of the glial fibrillary acidic protein promoter were treated with excitatory neurotoxicant kainic acid (KA) through a single intraperitoneal injection to induce gliosis in the brain and the retina. The expression of the GFAP-GFP transgene as a surrogate reporter for gliosis was noninvasively and longitudinally imaged with a confocal scanning laser ophthalmoscope for 2 weeks to monitor the progression of gliosis. RESULTS The authors demonstrated that KA-induced gliosis (an elevation in GFP fluorescence intensity [FI]) could be noninvasively detected starting on day 3 and that it peaked on day 7, as quantified for the optic disc astrocytes. A significant increase in the FI in retinal glial cells was also visible on the processed images. Immunohistochemistry in defined regions of the brain (hippocampal CA1, CA3, dentate gyrus) known to be affected by KA neurotoxicity showed that severe gliosis in these regions occurred at day 7, when retinal gliosis peaked. CONCLUSIONS The current real-time fluorescent imaging method described here is a powerful preclinical tool to directly monitor retinal gliosis caused by various retinopathies. In addition, this molecular imaging method should be useful in assessing retinal neurotoxicity and in therapeutic development in a preclinical setting.

Imidazolium Salt (DBZIM) Reduces Gliosis in Mice Treated with Neurotoxicant 2′-CH3-MPTP

We have recently identified a class of imidazolium salts (IMSs) with antioxidative property and can function as scavengers for radical oxygen species (ROS) [ 18 ]. Here, we investigate one of the IMSs, 1,3‐bisbenzylimidazolium bromide (DBZIM), for its possible role in attenuating neurotoxicity and gliosis in the retina and the brain induced by a Parkinsonian neurtoxicant, methyl‐4(2′‐methylphenyl)‐1,2,3,6‐tetrahydropyridine (2′‐CH3‐MPTP), which is a free radical generating agent. In this study, we employ a molecular retinal imaging method, which we recently developed in a transgenic mouse model expressing green fluorescent protein (GFP) under the control of glial fibrillary acidic protein (GFAP) promoter [ 14 ], to assess the efficacy of DBZIM, since currently no in vitro system with a sufficient complexity is available for accurately assessing a compounds efficacy. The longitudinal imaging results showed DBZIM can effectively suppress the neurotoxicant‐induced retinal gliosis. Immunohistochemistry performed on the postmodern mouse brain confirmed that DBZIM also reduced striatal gliosis, and concomitantly attenuated the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). These findings suggest that DBZIM could be a useful small molecular compound for studying neurotoxicity and neuroprotection in the retina and the brain.

In Vivo Imaging and Quantification of Retinal Gliosis in Transgenic Mice

We propose an in vivo retinal imaging platform for quantifying gliosis in response to neurotoxicity of certain compounds. Gliosis, in the form of retinal GFAP-GFP expression, was non-invasively and longitudinally imaged using a confocal scanning laser ophthalmoscope (SLO) and its progression was monitored over a period of two weeks. However, the retinal images have very poor signal-to-noise ratio (SNR) and cellular resolution such that the analysis of GFAP-GFP expressing retinal cells from these images can be a very challenging task. We report an image averaging method based on a pixel rank matching criterion which significantly enhances both these image attributes. We then compute retinal gliosis by quantifying the fluorescence intensity (FI) around the optic disc at different time points. We demonstrated that the kainic acid (KA)-induced gliosis can be non-invasively detected starting on day 3, and peaked at day 7 (p les 0.05). The proposed method provides great potential for longitudinal studies on various retinopathies and for therapeutic development in a pre-clinical setting.

Molecular imaging reveals a correlation between 2'-CH3-MPTP-induced neonatal neurotoxicity and dopaminergic neurodegeneration in adult transgenic mice

We previously reported that a single subcutaneous (s.c.) injection of the neurotoxicant, 1‐methyl‐4‐(2′‐methylphenyl)‐1,2,3,6‐tetrahydropyridine or 2′‐CH3‐MPTP, to postnatal day 4 (PD4) mice caused acute and transient gliosis in the brain, which can be noninvasively monitored during a course of 8 h immediately after the dosing [Ho, G., Zhang, C.Y., Zhuo, L., 2007. Non‐invasive fluorescent imaging of gliosis in transgenic mice for profiling developmental neurotoxicity. Toxicol. Appl. Pharmacol. 221, 76–85]. In the current study, we examined the consequence of PD4 mice receiving multiple injections (4 × 8 mg/kg, s.c. in 2 h intervals) of the same neurotoxicant 24–72 h after the last injection. Here we showed that the multiple dosing scheme (with a higher cumulative dose) triggered a severe gliosis not only in the striatum and substantia nigra pars compacta (SNpc), but also in hippocampus and cerebellum when examined by noninvasive in vivo imaging and by immunohistochemistry (IHC), respectively, in the PD5 to PD7 mice. When neonates treated with the neurotoxicant at PD4 were allowed to develop to 10 weeks of age and examined with IHC, a majority of the dopaminergic (DA) neurons were found to be permanently depleted from the adult SNpc. Our findings suggest that neurotoxicant‐elicited neonatal gliosis can be used as an early molecular signature to predict the permanent loss of DA neurons in the developed brain. Since 2′‐CH3‐MPTP is an inducer of Parkinsonism in mice, the molecular imaging method described here is a relatively simple and powerful tool for longitudinally studying the developmental aspect of Parkinsonism.

The effect of low level laser therapy on the contraction of fibroblast populated collagen-GAG lattices in vitro

Paper presented at the Second Meeting of the European Tissue Engineering Society focusing on cell-seeding and proliferation on the lumina of PLLA tubules in a Dynamic Couette-Taylor Bioreactor.Abstract of paper from the Second Meeting of the European Tissue Engineering Society focusing on differential cellular responses to poly (Σ-caprolactone) films, and the effect of solvent-induced variations in surface microstructure.Abstract of paper from the Second Meeting of the European Tissue Engineering Society (ETES), Genoa, Italy, September 3-6, 2003. Describes the effect of low level laser therapy on the contraction of fibroblast populated collagen-GAG lattices in vitro.

Low level laser therapy on artificial skin substitutes : no significant effect on the proliferation rate of 3T3 mouse fibroblast cells

Summary of paper describing how low level laser therapy on artificial skin substitutes cause no significant effect on the proliferation rate of 3T3 mouse fibroblast cells.