Assessment of renal fibrosis and anti-fibrotic agents using a novel diagnostic and stain-free second-harmonic generation platform.

Current histological measurement techniques for interstitial collagen, the basis of interstitial fibrosis, are semi-quantitative at best and only provide a ratio of collagen levels within tissues. The Genesis200 imaging system and supplemental image analysis software, FibroIndex from HistoIndex, is a novel, automated platform that uses second-harmonic generation (SHG) for imaging and characterization of interstitial collagen deposition and additional characteristics, in the absence of any staining. However, its ability to quantify renal fibrosis requires investigation. This study compared SHG imaging of renal fibrosis in mice with unilateral ureteric obstruction (UUO), to that of Masson's trichrome staining (MTS) and immunohistochemistry (IHC) of collagen I. Additionally, the platform generated data on collagen morphology and distribution patterns. While all three methods determined that UUO-injured mice underwent significantly increased renal fibrosis after 7 days, the HistoIndex platform additionally determined that UUO-injured mice had a significantly increased collagen-to-tissue cross reticulation ratio (all P < .001 vs sham group). Furthermore, in UUO-injured mice treated with the relaxin family peptide receptor-1 agonists, relaxin (0.5 mg/kg/day) or B7-33 (0.25 mg/kg/day), or angiotensin converting enzyme-inhibitor, perindopril (1 mg/kg/day) over the 7-day period, only the HistoIndex platform determined that the drug-induced prevention of renal fibrosis correlated with significantly reduced collagen fiber thickness and collagen-to-tissue cross reticulation ratio, but increased collagen fiber counts. Relaxin or B7-33 treatment also increased renal matrix metalloproteinase-2 and reduced tissue inhibitor of metalloproteinase-1 levels (all P < .01 vs UUO alone). This study demonstrated the diagnostic value of the HistoIndex platform over currently used staining techniques.

Imidazolium salt (DBZIM) reduces gliosis in mice treated with neurotoxicant 2'-CH(3) -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'-CH(3) -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 compound's 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.

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.

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'-CH(3)-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 x 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'-CH(3)-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.

Non-invasive fluorescent imaging of gliosis in transgenic mice for profiling developmental neurotoxicity.

Gliosis is a universal response of the brain to almost all types of neural insults, including neurotoxicity, neurodegeneration, viral infection, and stroke. A hallmark of gliotic reaction is the up-regulation of the astrocytic biomarker GFAP (glial fibrillary acidic protein), which often precedes the anatomically apparent damages in the brain. In this study, neonatal transgenic mice at postnatal day (PD) 4 expressing GFP (green fluorescent protein) under the control of a widely used 2.2-kb human GFAP promoter in the brain are treated with two model neurotoxicants, 1-methyl-4(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH(3)-MPTP), and kainic acid (KA), respectively, to induce gliosis. Here we show that the neurotoxicant-induced acute gliosis can be non-invasively imaged and quantified in the brain of conscious (un-anesthetized) mice in real-time, at 0, 2, 4, 6, and 8 h post-toxicant dosing. Therefore the current methodology could be a useful tool for studying the developmental aspects of neuropathies and neurotoxicity.