CONNEXIN43 Expression Following Retinal Ischemia

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 required immunogold and electron microscopy techniques also may be used." (Volpicelli-Daley and Levey, 2003)

Prior to visualization of the molecule of interest it is necessary to "fix and section the brain tissue. Double-labeling immunofluorescence is stated to detect "localization of a protein of interest as well as the distribution of the protein relative to another marker such as a neurochemical or organelle marker." (Volpicelli-Daley and Levey, 2003 Fluorescence imaging labeled tissue through use of confocal makes provision of "high-resolution analysis of the extent of colocalization, with a theoretical limit of resolution of 0.1 to 0.2 um." (Volpicelli-Daley and Levey, 2003) Immunofluorescence techniques are stated to "in general...utilize secondary antibodies conjugated to a flurosphore." (Volpicelli-Daley and Levey, 2003) It is important according to Volpicelli-Daley and Levey to choose flurosphores with "minimal background staining and a minimum overlap of excitation/emission spectra...when performing double labeling experiments." (2003)

IV. FLUORESCENCE MICROSCOPY

The work of Coling and Kachar (1997) entitled: "Theory and Application of Fluorescence Microscopy" states that fluorescence is the luminescent emission that results form absorption of photons. Fluorescence is distinguished form its counterpart, a longer-lasting afterglow call phosphorescence, by the magnitude of the decay time." Coling and Kachar report that there is an abrupt ceasing of fluorescent emission at the time the "exciting energy is shut off." (Coling and Kachar, 1997)

Fluorescent imaging is used in various spectroscopy techniques and is stated to have particular usefulness in fluorescence microscopy." (Coling and Kachar, 1997) The primary use of fluorescent microscopy is the examination of specimens that have been treated with special fluorescent reagents which have the ability to absorb a certain wavelength of light and emit light "...of a certain wavelength slightly shifted toward the red end of the spectrum from the absorbed light." (Coling and Kachar, 1997) Selective examination of a specific component of a complex bimolecular assembly is enabled by fluorescence microscopy." (Coling and Kachar, 1997)

Coling and Kachar report that the importance in biology and in neurobiology of florescence microscopy is because of:

(1) the extraordinary development of new fluorescent molecular probes; and (2) the development of improved low light level imaging systems and confocal microscopy techniques." (1997)

V. CONFOCAL MICROSCOPY

Confocal microscopy is reported to produce "sharp images of structures within relatively thick specimens" or those up to several hundred microns. (Paddock, Fellers, and Davidson, 2009) Confocal microscopy is stated to be especially useful in the examination of specimens that are fluorescent. When viewing thick fluorescent specimens from a conventional widefield fluorescent microscope they appear fuzzy and lacking in contrast since fluorosphores within the specimens' entire depth are "illuminated and fluorescence signals are collected not only from the plane of focus but also from areas above and below." (Paddock, Fellers, and Davidson, 2009)

Advantages of confocal microscopy overconventional optical microscopy include those of:

(1) shallow depth of field;

(2) elimination of out-of-focus glare; and (3) the ability to collect serial optical sections from thick specimens. (Paddock,

Fellers, and Davidson, 2009)

In the biomedical sciences a major application of confocal microscopy is stated to involve "imaging either fixed or living cells and tissues that have usually been labeled with one of more fluorescent probes." (Paddock, Fellers, and Davidson, 2009) The following illustration shows the principal light pathways in confocal microscopy.

Figure 1

Source: Paddock, Fellers, and Davidson (2009)

The majority of confocal microscopes are reported to be "...relatively easy to operate" and it is stated that these have "...become part of the basic instrumentation of many multi-user imaging facilities." (Paddock, Fellers, and Davidson, 2009)

The laser scanning confocal microscope (LSCM) is stated to be superior to that in the conventional widefield optical microscope however, it is still "...considerably less than that of the transmission electron microscope, it has in some ways bridged the gap between the two more commonly used techniques." (Paddock, Fellers, and Davidson, 2009)

While in a conventional widefield microscope "...the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected directly onto an image capture device or photographic film. In contrast, the method of image formation in a confocal microscope is fundamentally different. The illumination is achieved by scanning one or more focused beams of light, usually from a laser, across the specimen. The images produced by scanning the specimen in this way are called optical sections. This terminology refers to the noninvasive method by which the instrument collects images, using focused light rather than physical means to section the specimen." (Paddock, Fellers, and Davidson, 2009) This is shown in the following illustration labeled Figure 2.

Figure 2

Source: Paddock, Fellers, and Davidson (2009)

More useful imaging of living specimens has been facilitated by the confocal imaging approach and also enabled has been the automated collection of three-dimensional data. Confocal imaging has further improved the images obtained of specimens using multiple labeling." (Paddock, Fellers, and Davidson, 2009) The following illustration labeled Figure 3 shows a comparison of a conventional epifluorescence image with a confocal image of similar regions of a whole mount of a butterfly pupal wing epithelium stained with propidium iodide." (Paddock, Fellers, and Davidson, 2009)

Figure 3

Source: Paddock, Fellers, and Davidson (2009)

The most widely used confocal variation in biomedical research is the laser scanning confocal microscope. The confocal microscope was invented by Marvin Minsky in 1955 with the development of this approach stated to be driven by "the desire to image biological events as they occur in living tissue (in vivo)..." (Paddock, Fellers, and Davidson, 2009) The original configuration utilized by Minsky was one in which a "...pinhole was placed in front of a zirconium arc source as the point source of light. The point of light was focused by an objective lens at the desired focal plane in the specimen, and light that passed through it was focused by a second objective lens at a second pinhole having the same focus as the first pinhole (the two were confocal). Any light that passed the second pinhole struck a low-noise photomultiplier, which generated a signal that was related to the brightness of the light from the specimen. The second pinhole prevented light originating from above or below the plane of focus in the specimen from reaching the photomultiplier. The use of spatial filtering to eliminate out-of-focus light or flare, in specimens that are thicker than the plane of focus, is the key to the confocal approach." (Paddock, Fellers, and Davidson, 2009)

Paddock, Fellers, and Davidson (2009) report that the focused spot of light "must be scanned across the specimen" if one is to build an image through use of the confocal principle. Paddock, Fellers, and Davidson (2009) state that in the original instrument that Minsky built:

"...the beam was kept stationary and the specimen itself was moved on a vibrating stage. This arrangement has the advantage that the scanning beam is held stationary on the optical axis of the microscope, which can eliminate most lens defects that would affect the image. For biological specimens, however, movement of the specimen can cause wobble and distortion, resulting in a loss of resolution in the image. Furthermore, it is impossible to perform various manipulations on the specimen such as microinjection of fluorescently labeled probes when the stage and specimen are moving." (Paddock, Fellers, and Davidson, 2009)

In actuality, there was not the necessary technology available in 1955 for Minsky to fully develop and demonstrate "the potential of the confocal approach, especially for imaging biological structures." (Paddock, Fellers, and Davidson, 2009) Paddock, Fellers, and Davidson report that the information flow in the modern laser scanning confocal microscope is shown in the following figure which has been adapted from their work.

Figure 4

The information flow in a modern laser scanning confocal microscope

Source: Paddock, Fellers, and Davidson (2009)

The basic optics of the optical microscope are reported in the work of Paddock, Fellers, and Davidson to have "remained fundamentally unchanged for decades because the final resolution achieved by the instrument is governed by the wavelength of light, the objective lens, and the properties of the specimen itself. The dyes used to add contrast to specimens, and other technology associated with the methods of optical microscopy, have improved significantly over the past 20 years." (2009)

Modern technology has served to bring about both "growth and refinement" in the confocal approach largely due to optical microscopy renewal driven by modern technology advances. Confocal microscope classification of designs is one the "basis of the method by which the specimens are scanned." (Paddock, Fellers, and Davidson, 2009) Two basic means of scanning include:

(1) to scan the stage beam; or (2) to scan the illumination beam. (Paddock, Fellers, and Davidson, 2009)

Stated to be an alternative method for imaging biological systems and one that is more practical for use is "to scan the illumination beam across a stationary specimen. This approach is the basis of many of the systems that have evolved into the research microscopes that are in vogue today." (Paddock, Fellers, and Davidson, 2009)

The most popularly used method is single-beam scanning in which the scanning of the beam is generally accomplished through use of mirrors that are computer-controlled and "drive by galvanometers at a rate of one frame per second. To achieve faster scanning, at near video frame rates, some systems use an acousto-optical device or oscillating mirrors." (Paddock, Fellers, and Davidson, 2009)

There are stated to be only two alternative methods to confocal microscopy that are in use for producing optical sections and these are stated to be those of:

(1) deconvolution; and (2) multiphoton imaging. (Paddock, Fellers, and Davidson. 2009)

Deconvolution uses computer-based algorithms for the purpose of calculation and removal of information that is out-of-focus form florescence images. The more efficient algorithms and faster mini computers have rendered this technique a more practical choice for imaging. (paraphrased) It is reported that deconvolution utilizes computer-based algorithms for calculation and removal of out-of-focus information from fluorescence images.

This technique has "due to more efficient algorithms and much faster mini computers" become an option for imaging that is flexible in nature. It is additionally reported that multiphoton microscopy uses the same scanning system that the LSCM uses however it does not require a "pinhole aperture at the detector...because the laser excites the fluorochrome label only at the point of focus, eliminating the out-of-focus emission." (Paddock, Fellers, and Davidson. 2009)

Stated as another benefit of imaging living tissues is the advantage provided in the reduction of the specimen when imaging living tissues by photobleaching because of the reduced energy absorption from the laser beam. An additional benefit in the imaging of living tissues is that photobleaching is reduced in the specimen due to the reduced energy absorbed from the laser beam.

VI. WESTERN BLOTTING

Immunoblotting is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Protein samples are solubilized and generally through use of dosium dodecyl fulfate (SDS) and reduction agents including "dithiothreitol (DTTA) or 2-mercaptoethanol (2-ME)." (Gallagher, Winston, Fuller, Hurrell, 2004) After solubilization DSD-PAGE separates the material and the antigens are transferred "electorphoretically in a tank or semidry transfer apparatus to a nitrocellulose, VDF or nylon membrane, a process that can be monitored by reversible staining or by Poncdeau S. staining. Previously stained gels may also be blotted." (Gallagher, Winston, Fuller, Hurrell, 2004)

The proteins that have been transferred are then bound to the membrane's surfaces, "providing access for reaction with immunodetection reagents." (Gallagher, Winston, Fuller, Hurrell, 2004) It is stated that the binding sites that are remaining are then block through immersal of the membrane is a solution that contains a detergent blocking agent or a protein. Following a probe with the main antibody, the membrane is then washed and identification made of the antibody-antigen complexes with horseradish peroxidase (HRP) or alkaline phosphate enzymes coupled to the secondary anti-IgG antibody or via an avidin-biotin bridge to the secondary antibody." (Gallagher, Winston, Fuller, Hurrell, 2004) The activity is then visualized through use of chromogenic or luminescent substrates.

Protein blotting with tank transfer systems is stated to be a method of blotting that is conducted "in a tank of buffer with the gel in a vertical orientation. This is completely submerged located in the midst of two large electrode panels. Some systems allow a total of four gels to be transferred simultaneously and tank blotting is the preferred method as compared to semidry systems in transferring proteins that are difficult for transfer since "transfers are possible without buffer depletion." (Gallagher, Winston, Fuller, Hurrell, 2004)

VII. REAL-TIME QUANTITATIVE PCR

It is related in the work of 'invitrogen' that the polymerase chain reaction (PCR) is "one of the most powerful technologies in molecular biology. Using PCR specific sequences within a DNA or cDNA template can be copied or 'amplified' many thousand-to a million-fold in traditional [endpoint] PCR detection and quantitation of the amplified sequences are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophonesis and image analysis." (Invitrogen, nd)

Measurement of the amount of PCR product is taken each cycle and it is this ability to closely monitor the reaction during its "exponential phase" that serves to assist users in determining the beginning target with greater accuracy." (Invitrogen, nd) Theoretically DNA is exponentially amplified by PCR resulting in the number of molecules present being doubled with each cycle of amplification. Determining the number of cycles and PCR end-product amount can be used in theory for calculation f the quantity of genetic material however, this calculation is impacted by several factors.