Solid Phase Synthesis of a Functionalized Bis-Peptide Using …
Published 5/15/2012
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In JoVE (5)
- Intravitreous Injection for Establishing Ocular Diseases Model
- Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region
- Laser-Induced Chronic Ocular Hypertension Model on SD Rats
- Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus
- Morphometric Analyses of Retinal Sections
Other Publications (10)
- Brain Research
- Experimental Neurology
- Cellular and Molecular Neurobiology
- Journal of Ocular Biology, Diseases, and Informatics
- Cellular & Molecular Immunology
- Journal of Ocular Biology, Diseases, and Informatics
- Cellular & Molecular Immunology
- Journal of Cellular Biochemistry
- Age (Dordrecht, Netherlands)
- Experimental Gerontology
Articles by Kin Chiu in JoVE
Intravitreous Injection for Establishing Ocular Diseases Model
The University of Hong Kong - HKU
Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.
Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region
Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong - HKU
Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.
Laser-Induced Chronic Ocular Hypertension Model on SD Rats
Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong - HKU
Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.
Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus
The University of Hong Kong - HKU
This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.
Morphometric Analyses of Retinal Sections
1Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, 2Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, 3State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong
This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.
Other articles by Kin Chiu on PubMed
Calpain and N-methyl-d-aspartate (NMDA)-induced Excitotoxicity in Rat Retinas
Calpain-mediated proteolysis has been implicated as a major process in neuronal cell death in both acute insults and the chronic neurodegenerative disorders in the central nerves system. However, activation of calpain also plays a protective function in the early phase of excitotoxic neuronal death. The exact role of calpains in neuronal death and recovery after exposure to N-methyl-D-aspartate (NMDA) is not clearly known. The purpose of present study was to examine the involvement of mu- and m-calpain in NMDA-induced excitotoxicity in the adult rat retina. Increased immunoreactivity of mu-calpain was noted in RGC layer cells and in the inner nuclear layer with maximal expression at 12 h after NMDA injection. This was further confirmed with Western blotting. TdT-mediated biotin-dUTP nick end labeling (TUNEL) positive cells in the inner retina co-localized with moderate or intense mu-calpain immunoreactivity. In contrast, there was no remarkable change in m-calpain immunoreactivity at any time point after NMDA injection. Simultaneous injection of 2 nmol of a calpain inhibitor (calpain inhibitor II) significantly reduced the number of TUNEL-positive cells in the inner retina at 18 h after NMDA injection and preserved RGC-like cells counted at 7 days after injection. The results of this study showed that mu-calpain may be involved in mediating NMDA-induced excitotoxicity in the rat retina and calpain inhibitors may play a therapeutic role in NMDA related disease.
Neuroprotective Effects of Lycium Barbarum Lynn on Protecting Retinal Ganglion Cells in an Ocular Hypertension Model of Glaucoma
Glaucoma is one of the major neurological disorders in eye leading to irreversible blindness in elderly. Increase in intraocular pressure (IOP) has been considered to be the major risk factor for the progressive loss of retinal ganglion cells (RGCs) in retina. While attenuation of IOP has been a major pharmaceutical target, reduction of IOP cannot prevent progressive loss of RGCs. In this regard, urgent need for alternative treatment has to be investigated. Anti-aging medicinal herb Lycium barbarum L. has been used for centuries in Eastern World to protect the eyes and maintain good health. Using an ocular hypertension (OH) model in rat by laser photocoagulation of episcleral and limbal veins, we attempted to investigate whether L. barbarum can promote RGCs survival against elevated IOP. Oral administration of L. barbarum in Sprague-Dawley rats (250-280 g) significantly reduced the loss of RGCs, although elevated IOP was not significantly altered. Rats fed with the 1 mg/kg extract could nearly totally escape from pressure-induced loss of RGCs. In conclusion, this is the first in vivo report showing the therapeutic function of L. barbarum against neurodegeneration in the retina of rat OH model. The results demonstrate that this extract may be a potential candidate for the development of neuroprotective drug against the loss of RGCs in glaucoma.
The Selective Vulnerability of Retinal Ganglion Cells in Rat Chronic Ocular Hypertension Model at Early Phase
Glutamate neurotoxicity has been postulated to play a prominent role in glaucoma. In this study the possible roles of two subunits of glutamate receptors during the early phase of retinal ganglion cell (RGC) loss in a rat chronic ocular hypertension (COH) model were investigated. COH was induced by applying argon laser to the episcleral and limbal veins of the right eye of rats, the observation times were at 4, 14 and 28 days after the first laser. RGCs were retrogradely labeled by putting Fluoro-Gold (FG) on the surface of both side superior colliculus. Immunohistochemical staining using specific antibodies against N-methyl-D-aspartate receptor 1 (NR1) or glutamate receptor 2/3 (GluR2/3) was performed on the retinal sections of normal and COH eyes. Fluorescent images were captured using confocal laser scanning microscope and the number of NR1 and GluR2/3 labeled cells were counted and cell size was measured using Stereo Investigator. During the observation period, the numbers of NR1 and GluR2/3 positive RGCs in the RGC layer were reduced parallel to the loss of RGC. The dramatic loss of GluR2/3 immunoreactive neurons occurred starting immediately after the first laser to 4 days while the dramatic loss of NR1 immunoreactive neurons occurred from 14 to 28 days after the first laser. Size difference was detected in NR1 immunoreactive RGCs, large ones were more sensitive to the high ocular pressure. These results suggest that both NR1 and GluR2/3 are involved in the mediation of RGC death in the early stage of COH.
Modulation of Microglia by Wolfberry on the Survival of Retinal Ganglion Cells in a Rat Ocular Hypertension Model
The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of lipopolysaccharide decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.
Modulation of Neuroimmune Responses on Glia in the Central Nervous System: Implication in Therapeutic Intervention Against Neuroinflammation
It has long been known that the brain is an immunologically privileged site in normal conditions. Although the cascade of immune responses can occur as long as there is a neuronal injury or a potent immune stimulation, how the brain keeps glial cells in a quiescent state is still unclear. Increasing efforts have been made by several laboratories to elucidate how repression of immune responses is achieved in the neuronal environment. The suppression factors include neurotransmitters, neurohormones, neurotrophic factors, anti-inflammatory factors, and cell-cell contact via adhesion molecules or CD200 receptor. This review discusses how these factors affect the cascade of cerebral immune responses because no single factor listed above can fully account for the immune suppression. While several factors contribute to the suppression of immune responses, activation of glial cells and their production of pro-inflammatory factors do occur as long as there is a neuronal injury, suggesting that some neuronal components facilitate immune responses. This review also discusses which signals initiate or augment cerebral immune responses so that stimulatory signals override the suppressive signals. Increasing lines of evidence have demonstrated that immune responses in the brain are not always detrimental to neurons. Attempt to simply clear off inflammatory factors in the CNS may not be appropriate for neurons in neurological disorders. Appropriate control of immune cells in the CNS may be beneficial to neurons or even neuroregeneration. Therefore, understanding the mechanisms underlying immune suppression may help us to reshape pharmacological interventions against inflammation in many neurological disorders.
Erratum: Modulation of Microglia by Wolfberry on the Survival of Retinal Ganglion Cells in a Rat Ocular Hypertension Model
The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of bacterial endotoxin lipopolysaccharide (LPS) decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.[This corrects the article on p. in vol. .].
Modulation of Morphological Changes of Microglia and Neuroprotection by Monocyte Chemoattractant Protein-1 in Experimental Glaucoma
Monocyte chemoattractant protein-1 (MCP-1)/CCL2 is a C-C chemokine involved in the activation and recruitment of monocytic cells to injury sites. MCP-1/CCL2 can induce either neuroprotection or neurodestruction in vitro, depending on the experimental model. We aim to use MCP-1/CCL2 as an experimental tool to investigate the morphological changes of microglia when loss of healthy retinal ganglion cells (RGCs) is exacerbated or attenuated in an experimental glaucoma model. While a high concentration (1000 ng) of MCP-1/CCL2 and lipopolysaccharide (LPS)-exacerbated RGC loss, 100 ng MCP-1/CCL2 provided neuroprotection towards RGC. Neuroprotective MCP-1/CCL2 (100 ng) also upregulated insulin-like growth factor-1 (IGF-1) immunoreactivity in the RGCs. The neuroprotective effect of MCP-1/CCL2 was not due to the massive infiltration of microglia/macrophages. Taken together, this is the first report showing that an appropriate amount of MCP-1/CCL2 can protect RGCs in experimental glaucoma.
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.
Neurodegeneration of the Retina in Mouse Models of Alzheimer's Disease: What Can We Learn from the Retina?
Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease commonly found among elderly. In addition to cognitive and behavioral deficits, vision abnormalities are prevalent in AD patients. Recent studies investigating retinal changes in AD double-transgenic mice have shown altered processing of amyloid precursor protein and accumulation of β-amyloid peptides in neurons of retinal ganglion cell layer (RGCL) and inner nuclear layer (INL). Apoptotic cells were also detected in the RGCL. Thus, the pathophysiological changes of retinas in AD patients are possibly resembled by AD transgenic models. The retina is a simple model of the brain in the sense that some pathological changes and therapeutic strategies from the retina may be observed or applicable to the brain. Furthermore, it is also possible to advance our understanding of pathological mechanisms in other retinal degenerative diseases. Therefore, studying AD-related retinal degeneration is a promising way for the investigation on (1) AD pathologies and therapies that would eventually benefit the brain and (2) cellular mechanisms in other retinal degenerations such as glaucoma and age-related macular degeneration. This review will highlight the efforts on retinal degenerative research using AD transgenic mouse models.
Decreased C-Jun Expression Correlates with Impaired Spinal Motoneuron Regeneration in Aged Mice Following Sciatic Nerve Crush
Post-injury nerve regeneration of the peripheral nervous system declines with age, but the mechanisms underlying the weakened axonal regeneration are not well understood. Increased synthesis and activity of the AP-1 transcription factor c-Jun have been implicated in efficient motor axonal regeneration. In the present study, we evaluated the hypothesis that the impaired regenerative capacity in the aged is associated with impaired induction of c-Jun. In non-manipulated young adult or aged mice, no c-Jun and its phosphorylated form were detected in the ventral horn of the spinal cord. Following nerve crush, significant c-Jun and phosphorylated c-Jun occurred in the injured motoneurons of young adult mice, but not in aged animals. In accord with the immunohistochemistry, Western blots also showed that sciatic nerve crush induced c-Jun and its phosphorylation expression in the ventral horn of young adult but not in aged mice. Changes in c-Jun mRNA level detected by in situ hybridization are congruent with that in c-Jun protein content, showing an increase at 5days after crush in young adult but not aged. Moreover, compared with young adult mice, aged mice showed impaired motor axonal regeneration. These results demonstrate that the impaired motor axonal regeneration seen in aged mice is correlated with impaired c-Jun expression and phosphorylation following injury. These data provide a neurobiological explanation for the poor outcome associated with nerve repair in the aged.