Expression of Gap Junctions on Retinal and Its Effect on Vision

Expression of Gap Junctions on Retinal and Its Effect on Vision
Name:

Abstract
The Retinitis pigmentosa (RP) refers to a myriad of retinal neurodegenerative diseases that come as a result of heredity. As a matter of fact, the agglomeration of diseases is apposite to the formation of rod-specific mutations that result in the death of both horizontal and bipolar rods (Paschon, Higa, Resende, Britto & Kihara, 2012). There are varied explanations to the phenomenon behind the spread of the mutation from rods to cones and subsequent death of the latter. This research proposal discusses the role of the bystander effect in the degeneration of vision in mice. According to Abd-El-Barr et al. (2009), the effect is as a result of toxic molecules that result in the death of rods. Afterward, it spreads through gap junctions and affects other healthy cone photoreceptors. The research proposal employs immunohistochemistry to make a progressive comparison of the degeneration of cones between connexin36-expressing mice and connexin36-deficient mutants at various stages in their life cycle (Heikkinen, Vinberg, Nymark, Koskelainen, 2011). An assessment of the changes in morphology by the use of the rhododendron knockout on the onset and rd1 mutant models at later stages of the secondary death of cones is indispensable (Abd-El-Barr et al., 2009). The results indicate that the disruption of the connexin36-originated coupling of rods and cones due to mutagens neither delays nor restricts the degeneration of cones that results in retinitis pigmentosa.
Hypothesis: cell death signals from both horizontal and bipolar rods do not spread to the extracellular space in the retina but propagate to healthy cones through the gap junction.

Expression of Gap Junctions on Retinal and Its Effect on Vision
Retinitis pigmentosa is an amalgam of diseases that results from mutations in over forty different genes. The principal cells that experience the effect are rods. RP causes the death of rods and leads to both tunnel vision and night blindness. The loss of central vision and total blindness can result due to the subsequent death of cones . Although it is unclear how the propagation of death from rods to cones occurs, the loss of rods leads to the disruption of the physical interaction between them and cones . For this reason, cones lack a direct link to nutrients and die . However, the gap-junction bystander effect offers the best explanation for the phenomenon. As mentioned, the death of rods propagates to healthy cones through gap junctions. For instance, retinal development is apposite to dying neuroblasts. These cells generate apoptotic signals that permeate the gap junction and result in bystander killing of healthy cones. The gap-junction bystander effect fails to explain how connexin36-expressing mutants differ from connexin36-deficient counterparts.
Problem Statement
• To investigate the effect of rod-cone photoreceptor coupling on different stages of cone degeneration by the use of Rhodopsin knockout and rd1 mice.
Literature Review
Gap junctions are apposite to the propagation of retinal neurons and cell lines due to the introduction of cell death-inducing signals or mutagens. According to Cusato et al. (2003), carbenoxolone is a gap junction inhibitor that reduces the frequency of dying photoreceptor cells in the growing retina. It is obligatory to note that these cells die as the retina develops (Abd-El-Barr, Pennesi, Saszik, Barrow & Lem, 2009). Their hypothesis is conclusive of the fact that these cells communicate via gap junctions (Cusato et al., 2003). Such communication includes the apoptotic death of rod cells (Cusato et al., 2003). According to Cusato et al. (2003), there is passage of a cytotoxic, or cellular toxic byproduct of metabolism, from the affected rods to the healthy cones.
Similarly, the apoptotic-oriented acute trauma retinal model is the work of Paschon et al. (2012). Apparently, they tried to use the model to indicate a reduction in spread of apoptosis from rods to cones (Paschon et al., 2012). The researchers tested for the effects of Cx36-specific junction blockers to the propagation of retinitis pigmentosa (Paschon et al., 2012). Accordingly, the model is applicable to cells that have a neuronal injury (Paschon et al., 2012). However, it is very unlikely that the death of cones is as a result of an apoptotic origin (Abd-El-Barr et al., 2009). These models are inadequate in their explanation of how the death of cones photoreceptors in mice that have retinitis pigmentosa occurs (Abd-El-Barr et al., 2009). Therefore, the belief in a classical apoptotic approach to gap junction mediated retinitis pigmentosa undermines scientific research (Abd-El-Barr et al., 2009). For this reason, an alternative non-apoptotic approach is imperative to the study of RP mice and the effect of Cx36 mutagens and the associated mutagenesis on the degeneration of cones (Abd-El-Barr et al., 2009). Similarly, it is indispensable to consider the excessive activation of proteases that depend on calcium, deregulation of the metabolism of cGMP metabolism, and the activity that pertains to certain histone deacetylases of the retina.
The research does not postulate the fact that the metabolism inherent in retinitis pigmentosa produces metabolites that are cytotoxic and permeate the gap junction between rods and cones (Abd-El-Barr et al., 2009). Therefore, it might prove impossible to assert that there are effects of an apoptotic origin that result in the degeneration of the neurons and retinitis pigmentosa. It is especially true for the gap junction that has Cx36 proteins in the cones (Abd-El-Barr et al., 2009). In addition, a non-apoptotic explanation cannot assert with certainty that Cx36 gap junctions contain rods that produce these cytotoxic metabolites (Abd-El-Bar et al., 2009). It is also paramount to note that the gap junction might close as soon as the rods begin to die.
Methodology
The basis of the bystander hypothesis is the electrical coupling between cone and rod photoreceptor cells (Abd-El-Barr et al., 2009). The demonstration of the thesis involves the use of the mouse (Abd-El-Barr et al., 2009). However, it is notable that scientists are cognizant of only the Cx36 that occurs on the cone surface (Abd-El-Barr et al., 2009). As mentioned earlier, research indicates that the disruption of the rod-cone coupling, which results in the secondary death of cones, may result from the disruption of Cx36 mediated coupling (Abd-El-Barr et al., 2009). If this is the case, mouse models with retinitis pigmentosa should express the phenomenon (Abd-El-Barr et al., 2009). Therefore, it is mandatory to breed Rho, that is Cx36, and rd1 mice to avoid the unnecessary predominance of the connexin36 trait (Abd-El-Barr et al., 2009). The breeding will aid in the complete testing of the bystander hypothesis.
One will carry out the experiment that pertains to this study appropriate to all the institutional laws, statutes, regulations, and guidelines for animal welfare. Similarly, it must follow all the standards that the federal law for animal protection describes (Abd-El-Barr et al., 2009). It is imperative to note that the killing of mice is not against the law as long as it is on the basis of research. In addition, the local and state authorities approve of the act of harvesting tissues of mice for scientific analysis. What is more, the killing does not become illegal unless the researcher applies some additives and needs no further clarification beyond this point (Abd-El-Barr et al., 2009). The research involves the studying of conical degeneration in rd1 mice and Rho mice and rd1 mice (Trümpler, Dedek, Schubert, de Sevilla Müller & Seeliger, 2008). As mentioned, these mice suffer from retinitis pigmentosa. Afterward, there needs to be a crossbreeding process of both retinitis pigmentosa mice with Connexin36 mice.
It is imperative to note that both models are homozygous in their traits. Therefore, the resultant rd1 and Rho mice possess a heterozygous connexin36 deletion (Abd-El-Barr et al., 2009). Afterward, the research mandates the intercrossing of the progeny of the connexin36 rd1 mice with the connexin36 Rho mice. Therefore, the resultant of the retinitis pigmentosa models is Connexin36-deficient mice and connexin36-expressing mice (Abd-El-Barr et al., 2009). Apparently, these mice are homozygous in their expression of their traits. The next step involves the identification of the genotype of the two mice, that is, the connexin36-expressing homozygous and connexin36-deficient homozygous models. It is important to note that homozygous traits reduce the errors inherent in the retinitis pigmentosa research (Abd-El-Barr et al., 2009). It involves the use of primers to test for gene alterations in the genes that code for three proteins. These include connexin36, Pde6β or rod cGMP phosphodiesterase beta-subunit, and Rhodopsin. The study for the genetic code alterations involves the analysis of a polymerase chain reaction of the deoxyribonucleic acid tails from the tissues of both the connexin36-expressing and connexin36-deficient mice.
Primers used for mouse genotyping

Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584123/table/pone-0057163-t001 /

There will be a comparative study of the background of the genetic makeup of connexin36-deficient mice to help minimize the disparities in the samples (Abd-El-Barr et al., 2009). The researcher will perform a weeks’ analysis of the Rho-deficient connexin36-expressing mice and the Rho-deficient connexin36-deficient mice (Abd-El-Barr et al., 2009). These weeks will vary from 5 to 17 with weeks 9 and 12 in between. Similarly, the researcher will consider weeks fifteen, twenty-one, and thirty for mice with rd1 connexin36-deficient mice and their rd1 connexin36-expressing counterparts . Additionally, a control experiment is indispensable, in this case. The researcher will need pure strains that manifest the same genotypic background as the rodents in the experiment.
The preparation of immunohistochemistry and the retinal Tissue involves a series of steps. The first step requires anaesthesia using carbon (II) oxide and killing through a dislocation of the cervix . The next step involves the enucleation of eyes and preparation in Ringers solution. Ringeŕs solution contains sodium chloride, potassium chloride, calcium chloride, magnesium chloride, pH 7.4, 5 HEPES, and 10 D-glucose.
The next step involves the removal of the vitreous body, lens, and cornea from the eyecup. Afterward, the researcher fixes the vertical retina sections from the eyecup with one of the two solutions. These are either four percent paraformaldehyde in pH 7.4 buffered saline phosphates for an hour or twenty minutes in two percent paraformaldehyde (Abd-El-Barr et al., 2009). The next step requires thorough washing in PBS (Abd-El-Barr et al., 2009). Cryoprotection of the eyecups follows with the application of a PBS solution containing sucrose, which is ten percent, twenty percent, and thirty percent sucrose solution (Abd-El-Barr et al., 2009). The resultant solution needs a subjection to negative twenty degrees Celsius that is apposite to its embedding in cryoblock (Haverkamp, Specht, Majumdar, Zaidi & Brandstätter, 2008). The vertical sections of the retina are subject to both qualitative and morphological analysis (Abd-El-Barr et al., 2009). The latter mandates between eighteen and twenty nanometer sections, while the former requires twelve nanometer sections (Pang, Gao, Lem, Bramblett & Paul, 2010). Therefore, the researcher will use a cryostat to cut the sections.
The researcher must ensure that the rodents have the same age and are from the same background (Sonntag, Dedek, Dorgau, Schultz & Schmidt, 2012). Similarly, the sections must be of the same age for each category, and the researcher should subject them to the same treatment. The next step involves the rinsing of the cryosections in TBS. TBS is Tris Buffered Saline (Mataruga, Kremmer, Müller, 2007). It contains 0.3 percent Triton X-100 (Cangiano, Asteriti, Cervetto & Gargini, 2012). The researcher can also use 0.1M phosphate buffer (Abd-El-Barr et al., 2009). The next step involves blocking in ten percent normal serum from a donkey or ten percent of normal serum from a goat (Abd-El-Barr et al., 2009). The researcher should apply primary antibodies at four degrees Celsius (Abd-El-Barr et al., 2009). The researcher should wash the site and incubate for about three hours in secondary antibody solution. Consequently, the researcher undergoes rinsing and mounting in Vectashield (Cangiano et al., 2012). A blocking solution aids in the dilution of secondary antibodies. The next step involves the application of a TUNEL assay on vertical cryostat sections; this helps establish the dying cells’ number (Pennesi, Michaels, Magee, Maricle, Davin, 2012).
Results
The researcher must possess an in situ cell detection kit for dead tissues. (Abd-El-Barr et al., 2009) Next, the researcher performs a morphological analysis of the tissues by use of a microscope, preferably a confocal. The researcher should analyze the confocal images in their full representation (Abd-El-Barr et al., 2009). One of the most typical reasons is the effect of dying rods that propagate through gap junctions to result in a spectator agent. The agent causes the death of healthy photoreceptor cones. This document will investigate the effect of the disruption of the red-crested coupling, prevent the secondary death of cones, and minimize the degenerative effect. The testing of the hypothesis involves the use of two mice (rhodopsin knockout, or Rho, and rd1) (Sonntag et al., 2012). The research involves making a crossbreed between the mice with connexin36-deficient mice. It is imperative to note that connexin36 is a representation of the proteins on the cones (Abd-El-Barr et al., 2009). Therefore, lack of it equates to a disruption in rod-cone coupling.
Quantification and Statistical Analysis
Previous studies involved the physiological and structural analysis of retinas. However, such analysis does not bring a distinction between rd1 and rd1 Cx36-deficient mice. Therefore, quantitative and statistical analysis is imperative. The retinas of each experimental group will undergo processing that entails incubation, tissue dissection, and microscopic evaluation. Afterwards, the quantification of TUNEL-positive cells and cone photoreceptors is imperative. The quantitative analysis involves the adjusting and assembling of confocal maximum projections of the outer segment region, from the optic nerve up to the dorsal-ventral end of the retina in Photoshop CS4. The researcher will also carry out TUNEL quantification and cone photoreceptor was in the central retina. The area ranges from the optic nerve towards the periphery of the retina. A summary of the results displays the number of TUNEL-positive cells and cones per 100 µm retina length. Statistical analysis involves intraspecific and interspecific genotypic by us of two-tailed Students t-test. The analysis of these results will aid in the determination of whether cell death signals from both horizontal and bipolar rods spread to the extracellular space in the retina or propagate to healthy cones through the gap junction. It is the culmination of the bystander effect that will investigate the effect of rod-cone photoreceptor coupling on different stages of cone degeneration. It will answer the long-standing argument about the role of gap junctions in the propagation of apoptotic cell-death signals from rods to cones.
Figure legend

The reorganization of the horizontal rods does not occur as a result of the connexin36 deficiency or mutation.
Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584123/figure/pone-0057163-g003/

Figure legend

The organization of the bipolar rods does not change due to the deletion of connexin36.
From the diagram, there was counterstaining of the nuclei of bipolar rods with blue acid stain and the green coloring indicates antibodies. There was no difference between connexin36-expressing and connexin36-deficient mice in both cases, that is, bipolar and horizontal cells.
Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584123/figure/pone-0057163-g003/

There is a lack of a wrap up paragraph that should indicate conclusions and future directions is needed…..

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