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Figure 3.1: Protein standard curve. This figure shows the graph generated from the concentration and absorbance readings of bovine serum albumin taken at 570 nm which was used as a known standard used in determining the protein concentration of the rat brain tissue
Concentration of Unknown samples
Average absorbance reading of pellet
Average absorbance reading of supernatant
At 1: 100
Protein in pellet = 0.491 - 01565 = 340.67 Âµg/ml
Protein supernatant = 0.457 - 0.3254 = 130.67Âµg/ml
At 1: 10
Protein in pellet = 0.587 - 0.3254 = 624 Âµg/ml
Protein supernatant = 0.652 - 0.3254 = 410.67 Âµg/ml
Protein in pellet = 1.051 - 0.3254 = 3030.67 Âµg/ml
Protein in supernatant = 1.057 - 0.3254 = 1787.33 Âµg/ml
3.1.1 BCA QUANTIFICATION ASSAY FOR THE HUMAN EPILEPTIC AND CONTROL TISSUE SAMPLES.
TABLE 1: The table above shows the average mean absorbance reading of bovine serum albumin at varying concentrations, this served as the known standard to which the unknown protein in the Human hippocampal samples are measured against.
FIGURE 3.1.1: Shows the graph generated from the concentration and absorbance readings of bovine serum albumin which served as the known standards taken at 570 nm. The formula derived from the graph after using the microsoft excel software was used to determine the amount of protein in the unknown sample.
The tables above shows the average absorbance readings for the human control and epileptic tissues for both pellet and supernatant samples at a concentration of 1 in 100, 1 in 10 and 1 in 2.
The estimated weight of the protein samples at varying concentrations was calculated using the method previously described in the BCA assay for the rat tissue to calculate that of human, the estimated weight derived from the 1:2 absorbance values was subsequently used to determine the amount of protein that was loaded in the wells.
3.2 The Expression of GABA B Receptor 2 and Î²-Actin in Rat and Human Brain Tissues.
Figure 3.2.1 : The image above shows the licor image scanner detection of the expression of GABAB2 receptor in a rat brain tissue after its localisation by GABAB2 anti-rabbit primary antibody (1:1000) Abcam 5228 and its amplification by Secondary antibody goat anti rabbit IR dye (Odyssey, 1:2000) on a nitrocellulose membrane.
Protocol: 15Âµg of protein for both pellet and supernatant were loaded into separate wells and they were separated with 9% SDS polyacrylamide gel followed by blotting onto a nitrocellulose membrane for 1 hour. The membrane was blocked in blocking solution (5% non-fat dried milk in TBS) for 1 hour followed by overnight incubation with the GABAB2 anti-rabbit primary antibody for receptor detection, it was amplified with IR Dye 800 goat anti-rabbit secondary antibody. The image was captured at the 700 and 800 channels at an intensity of 5. A faint band was detected at around the 100 kDa marker.
Figure 3.2.2 : The image above shows the licor detection of Î²-actin receptor (A house keeping receptor) in the rat brain tissue following its localisation by the primary licor anti-rabbit IR dye (1:200, Odyssey) and its amplification by a goat anti-rabbit IR dye (Odyssey, 1:1000) on a nitrocellulose membrane. The localisation of the Î²-actin was to serve as a control that will check if the rat brain tissue was viable for experimental analysis.
15Âµg of pellet and 20Âµg of supernatant protein samples from rat brain tissue was separated using the previously described protocol, the difference been the change in the blotting time from 1 hour to 2 hours and the increased concentration of primary antibody used.
Figure 3.2.3: The image shows the licor scanner detection of the GABAB2 receptor in a rat brain tissue after its localisation by GABAB2 anti-rabbit primary antibody ((1:1000), Abcam 52248) and a secondary goat anti rabbit IR antibody (1:1000, Odyssey).
12Âµg of pellet and 15Âµg of pellet and supernatant protein samples were separated and transferred by western blotting unto a nitrocellulose membrane,. Faint bands can be seen at around the 100 kDa of the pellets of the brain sample while clear bands can be seen in the supernatants.
Figure 3.2.4: The figure showing the image of human Î²-actin receptor detected in normal human hippocampal tissue by the licor scanner following its localisation by a primary anti Î²-actin IR dye antibody (1:200) after western blotting and its amplification with Odyssey goat anti-rabbit secondary antibody (1:1000). The Î²-actin is detected around the 50 kDa band because its estimated molecular weight is 47 kDa, it was detected on 800 band at an intensity of 5.0.
The Î²-actin protein serves as a house keeping protein, 15Âµg the protein samples for both pellet and supernatant was transferred unto the nitrocellulose membrane and detected by the anti actin IR dye from licor. A clear expression of the protein bands are clearly seen.
Figure 3.2.5: The image showing the licor detection of the expression of GABAB2 receptor and Î²-actin protein derived from the human epileptic tissue sample and localised on the same membrane, they are clearly seen around the 100 kDa and 50 kDa marker respectively following their antibody localisation with (1:1000) GABAB2 anti rabbit primary antibody Abcam 52248 and Î²-actin primary (Odyssey) IR antibody (1:200) as well as the amplication of the primary antibody detection by goat-anti rabbit secondary antibody.
15Âµg of protein for both pellet and supernatant were seperated by electrophoresis in 9% SDS polyacrylamide gel for 1 hour 30 minutes at 130 volts after which they were transferred onto a nitrocellulose membrane at 80 volts for two hours. They were blocked in blocking solution for 1 hour followed by overnight incubation with primary antibody in a cold room, 10 minutes triplicate washing with TBST and one incubation with secondary antibody at room temperature followed by their detection on the Odyssey licor scanner.
Figure 3.2.6 : The image showing the licor scanner detection of GABAB2 receptor and Î²-Actin house keeping protein from human control hippocampal brain tissue sample after its localisation by Abcam 52248 anti rabbit GABAB2 (1:1000) and anti-actin(1:200) primary antibodies and their subsequent amplification by the goat anti rabbit IR secondary antibody on a nitrocellulose membrane.
Figure 3.2.7: The expression of GABAB2 receptor in human epileptic hippocampal tissue and in normal human hippocampal tissue sample detected by licor image scanner after their analysis and transfer by western blotting analysis and their localisation by the Abcam 5228 GABAB2 anti-rabbit primary antibody (1;1000) followed by their amplification by goat anti rabbit IR dye secondary antibody (1:1000).
20Âµg of protein for both pellet and supernatant human epileptic and control protein sample were loaded into 9% SDS gels and separated by electrophoresis at 130 volts for two hours followed by electro transfer unto a nitrocellulose membrane for two hours at 80 volts by western blotting.The bands showing the expression of the GABAB2 receptors can be clearly seen at around the 100 kDa molecular weight marker. The expression of GABAB2 receptors is seen to be slightly higher in the epileptic hippocampal brain sample than the normal hippocampal brain sample but this cannot be authenticated just by normal eye viewing therefore densitometry was required to be able to quantify the abundance of the receptor in each hippocampal group.
Figure 3.2.8: Another image showing the expression of GABAB2 receptor detected on a nitrocellulose membrane after a two hour western blotting transfer by a licor image scanner after its localisation and detection by the use of (Abcam 5280) GABAB2 anti rabbit primary antibody (1:1000) and an (Odyssey) IR dye goat anti-rabbit secondary antibodies respectively.
As seen from the image, the protein bands of the GABAB2 receptors are clearly visible in both epileptic and control human tissue samples at both pellet and supernatant samples. However, another set of bands are clearly seen above the 50 kDa molecular weight marker.
Densitometry analysis of GABA receptors in epileptic and normal tissues to quantify the amount of neurons in each sample.
Due to the fact that the similarities and differences between GABAB expression levels seen in the images for both the control and epileptic tissue samples cannot be easily quantified with the naked eye, densitometry analysis for both epileptic and control samples was performed on some selected images to quantify the amount of GABAB receptors present in them in order to check if there was significant differences in the expression levels of GABAB2 receptors in epileptic and control sample tissues.
Densitometry values in Figure 3.7
Pellet : 13.89
Supernatant : 17.36
Pellet : 8.74
Densitometry Values for Figure 3.8
Pellet : 5.40
Supernatant : 5.58
Pellet : 4.19
Supernatant : 5.12
The result of the densitometry analysis showed that there were more GABAB2 receptor expression in the supernatant samples than in the pellet samples for both epileptic and control hippocampal tissues. The expression of GABAB2 receptors was clearly seen to be higher in the epileptic hippocampal tissue samples than in the control hippocampal samples in all the tissue samples examined.
This research work happens to be a novel one because there has never been a published scientific literature on the use of western blotting analysis to investigate the expression of GABAB2 in human hippocampal temporal lobe epileptic tissue samples.
As observed from the BCA protein assay for the human hippocampal tissues, the expression of protein in the human control hippocampal tissue was lower than that of the human hippocampal epileptic tissue, this is due to the fact that the tissues were derived from different human beings and therefore the size of the temporal lobe in the epileptic tissue may be higher than that of the control tissue.
The high level of GABAB expression following western blotting analysis further gives credence to previous studies by (Princivalle et al., 2003b) and (Furtinger et al., 2003b) that have shown a high presence of GABAB2 in the hippocampal human tissues, these authors also showed that the increased expression was highest at the subiculm, dentate gyrus and in some parts of the Amon's horn.
Furtinger et al., (2003b) and Ben-Ari., in 2006 highlighted several factors that have been implicated in pathology of epilepsy and they include: change in the amount of GABA-ergic neurons, change in the expression of GABA-transporters and glutamate carboxylase, altered subunits of GABA receptors as well as higher levels of the glutamatergic production leading to increased functional mechanisms that reduces or overcome the threshold of inhibitory mechanism produced.
As seen in most of the images, there was expression of the GABAB2 receptor both in the supernatant and pellet rat and human tissues and it was higher in the supernatant than in the pellet, in some instances a lower amount of pellets in comparison to supernatants were separated and transferred as shown by figure 3.4 and figure 3.5 these alterations in the levels of pellet and supernatant loaded is part of the reasons why there was a lower expression of pellets in comparison with supernatants but in making a general conclusion, the level of GABAB2 in supernatant samples was still generally higher in the supernatant samples as seen in the instances in which the same amount of supernatant and pellet were loaded and transferred.
Figure 3.2.1 showed a westen blot image with blurred and faint band at the pellet end with nothing to show for the supernatant, this was perharps due to a low blot transfer time as subsequent images from other blots in which there was a longer blotting time had nicer images.
As seen from figures 3.2.7 and 3.2.8, expression of the receptor in the epileptic tissue sample was higher than that of the control sample although this was difficult to substantiate, this lead to the idea of quantifying the amount of GABAB2 receptors expressed in each sample by way of densitometry which confirmed the previous assertion that the levels of GABAB2 was indeed increased in the epileptic tissue in comparison with the control, this result correlated with previous experimental work done by (Furtinger et al., 2003b); (Princivalle et al., 2002); (Princivalle et al., 2003b) and Straessle et al., (2003)
According to Furtinger et al., (2003), the increase in the expression levels of GABAB2 receptors can be attributed to the creation of a protective mechanism by the gabaergic system in temporal lobe epilepsy by way of presynaptic inhibition so as to prevent glutamate release.
(Princivalle et al., 2002) hypothsized that the increase in GABAB2 receptor expression in temporal lobe epileptic tissues might be due to the creation of a system that helps limit the level of the inhibitory mechanisms of the receptor in patients affected with TLE while at the same time aiding the pathogenesis of the disease. Lanerolle and Lee., in 2005., also give credence to the compensatory mechanism hypothesis using the hyper excitability of granule cells seen in the hippocampus as a reference and they did suggest that the excitability neurones is not due to an absence of inhibition but rather, it is due to an upregulation in excitatory mechanisms which the inhibitory mechanisms cannot nullify.
Depending on whether GABAB receptors are situated in glutamatergic or gabaergic neurones, they are known to exhibit pro-convulsant or anti-convulsant activities. (Sharma et al., 2007)
The increase corroborate a previous study by (Princivalle et al., 2003) which showed an increase in GABAB2 expression, where they showed that the increase is pronounced in the hilus and dentate gyrus as well as in the Amon's horn of temporal lobe epileptic tissues. These increases observed can be associated with reduced inhibition seen in pathological progression of TLE in patients. Also (Esclapez and Houser., 1999) showed an upregulation in the level of GAD 65 and GAD 67 gene and protein products where seen in an amino acid transporter known as EEAT3 especially in the granular and pyramidal hippocampal layers.
According to Princivalle et al., (2003) although there was an upregulation in the level of enzymes producing GABA, this shows a direct correlation with the increased levels of GABAB receptors expression, but a reduction in the levels of GABA transporters was also noted which makes it logical to propose that even with the upregulationamount of GABA inadequate. This can be explained by two ways, it can either be said that the GABA receptors upregulation can be due to a method by which the reduction in GABAergic mechanisms is compensated for while the second reason can be hypothesized to be the fact that glutermegic excitatory mechanisms easily overide inhibitory mechanisms produced by the receptors due to the loss of GABA transporters.
The theory that the plasticity of GABAB2 receptor function can result in increased excitation of the hippocampal neurones in temporal lobe epilepsy can also be explained by the findings by Palma et al., in 2006 that discovered that there was an increase in chlorine ion transporter NKCC1 and decrease KCC2 levels that enabled a higher influx of chlorine into the subiculum of patients with TLE resulting in a change in the functions of GABAergic mechanisms from an inhibitory one to an excitatory one, this change was said to be highly influenced by the increased NKCC1 especially in the subiculum. Although the study was mainly carried out on GABAA, the fact that GABAB2 also prevent the influx of chloride ions into inwardly rectifying channels suggests that these findings can also be true for GABA B receptor 2.
There is increasing body of evidences that indicates that GABAergic mechanims associated with GABA receptors in general and GABAB receptor 2 in particular is not only involved in the inhibition of neuronal excitability but also in neuronal excitation, these emerging heterogeneous roles for GABAB2 receptors indicates that the " Loss of GABAergic activities leading to pathogenesis and pathological progression of epilepsy" concept needs to be reviewed.
Also more clinical and laboratory investigations needs to be performed to fully ascertain whether the over expression of GABAB2 in temporal lobe epilepsy is due to the development of a protective mechanisms for the surviving neurones or if it is part of the pro-excitatory mechanisms that contributes to the pathological progression and therapeutic resistance associated with temporal lobe epilepsy.
This will help to develop novel therapies or modify existing ones in order to come up with theraupetic substances that this form of epilepsy cannot resist.
This study is probably the first to detect multiple bands of proteins at different molecular weights after the antibody mediated localisation of GABAB2 receptor on a membrane, further research can be carried out on this unique protein that should involve its separation and analysis using Matrix Assisted Laser Desorption Ionisation (MALDI) to check if the protein is an isoform of GABAB2 or if its presence was due to degradation of the protein.