Research Proposal on "Connexin 43 Expression Following Retinal Ischemia"

Research Proposal 19 pages (4785 words) Sources: 50

[EXCERPT] . . . .

Connexin43 Expression Following Retinal Ischemia

Ischemia is a condition that occurs when there is an inadequate supply of blood delivered to the tissues generally resulting from a problem in the blood vessel. Retinal ischemia is stated by Renwick, et al. (2006) to result in the "loss of vision in a number of ocular diseases including acute glaucoma, diabetic retinopathy, hypertensive retinopathy and retinal vascular occlusion." (Renwick, et al., 2006) It is additionally reported by Renwick, et al. (2006) that recent studies have shown that most of the neuronal death that leads to loss of vision results from apoptosis." (Ibid)

Retinal Ischemia is reported in the work of Osborn, et al. (2004) to be a common cause of "...visual impairment and blindness. At the cellular level, ischemic retinal injury consists of a self-reinforcing destructive cascade involving neuronal depolarization, calcium influx and oxidative stress initiated by energy failure and increased glutamatergic stimulation. There is a cell-specific sensitivity to ischemic injury which may reflect variability in the balance of excitatory and inhibitory neurotransmitter receptors on a given cell." (Osborn, et al., 2004)

It is reported that several animal models and techniques for analysis have been utilized in the study of retina ischemia. Furthermore, Osborn et al. reports that "an increasing number of treatments have been shown to interrupt the ischemic cascade and attenuate the detrimental effects of fretinal ischemia." (2004) However, laboratory success has not been applied to the clinic and the reasons include: (1) difficulties with the route of administration; (2) dosage; and (3) adverse effects rendering some experimental treatments unusable in the clinic. (Osborne, et al., 2004)

I. TISSUE FIXATION (PARAFORMALDEHYDE FIXATION)

Confocal microscopy tissue fixation is the beginning of the process in order to preserve morphology. The chosen fixation method is one that must carefully balance two characteristics:

(1) 'Good' preservation of cellular 3-D structure; and (2) Adequate access to antigenic sites. (Department of Bioengineering University of Pennsylvania, 2000)

The goal is the preservation of "sufficient cellular organization to allow identifying the features of interest but yet not destroy the antigencity of the target. Fixation is also frequently combined with permeabilization to allow the staining solutions used in later steps access to the cytoplasm." (Department of Bioengineering University of Pennsylvania, 2000)

Stated to be a commonly used histological method for fixation and permeabilization is the treating the cells or tissues with solvents (alcohol or acetone). These methods are quick acting and good permeabilizing agents with one significant negative consequence and that being cellular shrinkage, although the degree of shrinkage "...may be almost insignificant for monolayers of cells, but will distort tissue samples dramatically." (Department of Bioengineering University of Pennsylvania, 2000)

Stated as a good compromise for use of Glutaraldehyde is that of paraformaldehyde (PF) as a fixative. Paraformaldehyde of the formalin solution which is commercially available (PF plus added methanol) is stated to preserve most structure "resolvable by at the confocal microscope level and generally "will not obscure the epitopes of interest." (Department of Bioengineering University of Pennsylvania, 2000)

The use of paraformaldehyde fixation makes a requirement of permeabilization with Triton X-100 or other detergent..." (Department of Bioengineering University of Pennsylvania, 2000) It is reported that should a component of the cytoplasm or nucleus needs to be labeled then the "plasma membrane must be made permeable to staining solutions." (Department of Bioengineering University of Pennsylvania, 2000)

It is reported that there are several methods that can be used to accomplish this of which are dependent partially on the chosen fixation method. Specifically stated is that there is no need to additionally permeabilize cells fixed with solvents as the "solvent has already extracted enough of the membrane therefore solvent fixation is twice as efficient. Cells however that are fixed with crosslinking aldehydes will need to have the integrity of the membrane breached through use of chemical agents.

Commonly used are DMSO and detergents such as Triton X-100, saponin or deosycholate. There should be careful adjustment to the detergent concentration so as to remove plasma membrane constituent selectively and thus allow access to the cytoplasm without alteration of the "antigenicity or morphology of the sample." (Department of Bioengineering University of Pennsylvania, 2000) Two labeling techniques are reported and those are:

(1) Direct labeling which consists of the use of a "...fluorescently labeled primary antibody or chemical legend to cause the structure of interest to become fluorescent. Advantages of this method include speed and ease of application. A potential disadvantage is lack of sensitivity; and (2) Indirect labeling which involves "...binding a primary antibody to the epitope of interest, followed by a fluorescently labeled secondary antibody. The primary advantage of using this technique is the great amplification of signal possible through an antibody cascade. Disadvantages include increased complexity, more time consuming, and often problems with non-specific antibody reactions. (Department of Bioengineering University of Pennsylvania, 2000)

Both of these labeling methods are stated to be suitable for confocal microscopy. The choice of the label is dependent on the equipment available and the availability of "certain fluors conjugated to required antibodies for use in multiple labeling schemes. In general, the laser lines available dictate which fluorophores may be used. Recent advances in biochemistry have created new families of fluorophores with very favorable signal-to-noise and quantum efficiency (QE) properties. In particular, the Cy dyes and the Alexa dyes are particularly useful. Both families have high QEs, are very resistant to photobleaching, and are available in a variety of excitation/emission wavelengths." (Department of Bioengineering University of Pennsylvania, 2000)

Fluorescence detection is stated to be only one way to use a confocal and stated to be a specifically "powerful technique" for the illustration of the cell layer details is the combination of the emitted fluorescence and transmitted Nomarski. (Department of Bioengineering University of Pennsylvania, 2000) It is reported that another non-fluorescence-based technique is reflection-mode confocal microscopy. Light reflected from the point of focus is collected and used as the source of signal for generating the image. Common samples used for reflection mode confocal microscopy are silver-enhanced gold-conjugated antibodies, or materials science samples." (Department of Bioengineering University of Pennsylvania, 2000)

II. HISTOLOGY USING HEMATOXYLIN AND EOSIN STAIN

The work of Fontaine (2002) entitled: "Neurogenerative and Neuroprotective Effects or Tumor Necrosis Factor (TNF) in Retinal Ischemia: Opposite Roles of TNF Receptor 1 and TNF Receptor 2" states that tumor necrosis factor (TNF) is a critical factor in various "acute and neurodegenerative disorders. In retinal ischemia, we show early, transient upregulation of TNF, TNF receptor 1 (TNF-R1) and TNF-R2 6 hours after reperfusion preceding neuronal cell loss." (Fontaine, 2002) The specific role of TNF and its receptors were assessed through comparison of "ischemia-reprefusion-induced retinal damage in mice deficient for TNF-R1, TNF-R2or TNF by quantifying neuronal cell loss 8 days after the insult." (Fontaine, 2002) It is stated to be surprising that TNF deficiency did not affect overall cell loss, yet absence of TNF-R1 led to a strong reduction of neurodegeneration and lack of TNF-R2 led to an enhancement of neurodegeneration, indicative of TNF-independent and TNF-dependent processes in the retina, with TNF-R1 _/_ animals correlated with the presence of activated Akt/protein kinase B (PKB)." (Fontaine, 2002)

III. IMMUNOHISTOCHEMISTRY

Immunohistochemistry is stated to be "the localization of antigens in tissue secretions by the use of labeled antibody as specific reagents through antigen-antibody interactions that are visualized by a market such as fluorescent dye, enzyme, radioactive element or colloidal gold." (IHC World, 2003) The first to label antibodies with a fluorescent dye was Albert H. Coons and colleagues and it was used for identification of antigens in tissue sections. As the technique of immunohistochemistry developed and expanded "enzyme labels have been introduced such as peroxidase (Nakane and Pierce 1966; Avrameas and Uriel 1966) and alkaline phosphatase (Mason and Sammons 1978). Colloidal gold (Faulk and Taylor 1971) label has also been discovered and used to identify immunohistochemical reactions at both light and electron microscopy level. Other labels include radioactive elements, and the immunoreaction can be visualized by autoradiography." (IHC World, 2003)

Immunohistochemistry has advantages over the traditionally techniques for staining enzymes because it "involves antigen-antibody reactions as the traditional techniques "identify only a limited number of proteins, enzymes and tissue structures."(IHC World, 2003) For this reason immunohistochemistry has become a technique that is critical and used widely in medical research and clinical diagnostics." (IHC World, 2003)

The work of Laura A. Volpicelli-Daley and Allan Levey (2003) published in the Journal of Current Protocols in Neuroscience and entitled: "Immunohistochemical Localization of Proteins in the Nervous System" relates that immunohistological method can be used in the visualization of "nervous system proteins, receptors and neurochemicals." Immunoperoxidase reactions involving a benzidine derivative and light microscopy are the methods generally used in visualizing the "distribution of a single primary antibody directed to an antigen of interest." (Volpicelli-Daley and Levey, 2003)

Additionally it is stated that double-labeling immunifluorescence and confocal microscopy techniques detect the localization of a protein relative to another protein and allow analysis of colocalization at a cellular and subcellular level." (Volpicelli-Daley and Levey, 2003) If precise localization is… READ MORE

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