In analyzing the study background information, it is worth identifying the main goal on which the research is based on. Therefore, the use of stem cell transplant in myelin repair is something being considered a therapeutic goal in chronic demyelinating diseases such as leukodystrophies. In this article, it very clear that the relatively slow human oligodendrocyte differentiation rate limits the therapeutic repair of myelin disorders ( Abiraman et. al , 2015) . Also, the study postulates that the process of myelination and demyelination in interspecies differentiation is known. With this knowledge, the molecular pathways are some of the elements known to regulate the differentiation of human oligodendrocyte progenitor cells. Therefore, it is very evident that the classification of these cells will provide a channel through which human transplanted cells can myelinate axons in a hypomyelinated rodent model.
However, the researchers are seeking to know whether the techniques used in augmenting rodent repair can be applicable in human patients. Based on the advanced knowledge in species variations in myelination mechanisms, the researchers are seeking to experiment this concept using a rodent model. Furthermore, the study is also seeking to identify some of the appropriate pharmacological resources that can be used to catalyze human oligodendrocyte progenitor cell differentiation. These assertions are anchored on the main question whether muscarinic adjunction therapy can be used to promote oligodendrocyte repair of a human.
Delegate your assignment to our experts and they will do the rest.
In this article, it has been hypothesized that the O4 antigens can be used for the isolation of human oligodendroglia populations. This hypothesis was based on the fact that O4 antigen can be used effectively with CD14a and it can also identify human oligodendroglia stages ( Abiraman et. al , 2015) . Because O4 antigen is capable of identifying an immature oligodendrocyte population in the trimester, it is worth using it in this hypothesis. The main motive behind the use of O4 antigen was to integrate it with CD14a to classify the three glial lineage stages of a human. Furthermore, it was also hypothesized that the various stages of human glial cells differentiation could well be represented by some subfraction of CD14a. This concept is based on the fact that O4 antigens are widely used in rodent models to identify oligodendrocytes. Based on this relationship, the researchers hypothesized that the same relationship could be used with human tissues.
The process of testing these hypotheses was thorough since it involved the assembly of numerous materials for the study. A fetal brain sample was assembled from some of the patients who had consented to have such materials used for scientific purposes. The brain tissue was immersed in cold paraformaldehyde, washed then stored in 4 degrees Celsius before processing ( Abiraman et. al , 2015) . The colocalization of the cells was then evaluated by having the CD14a, and O4 stained nucleus counted. With this data, the various proportional positive cells among the O4 and CD14a cells were determined. This process was necessary since it provided a means through which glial populations could be counted as per the hypothesized design.
The colocalization process was then followed by FACS and magnetic cell sorting. This process involved staining the regained cells with CD14a conjugate antibody, then sorting the dead cells from the doublets using gate control techniques. After that, the sorted cells were placed in 24-well plates for fixation. The main motive behind this process was to use the FACS to identify the glial stages of a human lineage. The genome microarray was then used to profile and compare them with one another. This process was achieved by having the EZNA Total Kit isolate the RNA. However, before, getting the RNA analyzed, it was prudent to amplify it based on the manufacturer instructions. Thereafter, the amplified product was then analyzed and the complete array of data recorded.
Also, the RT-PCR quantitative analysis was carried out through the reverse transcriptase process. In this technique, the IDT and Invitrogen were used to provide means through which human-specific primers could be obtained for the experiment. Thereafter, the fetal neuron culture was initiated. This process was archived by having the cells cultured in neurobasal liquid to enhance the formation of aggregates. The resulting products were then seeded for seven days to limit the expansion of both glia and progenitor structures. The cultured and seeded cells were then treated before being counted to assess the differentiation mechanism. The main idea in this process was to identify and validate the functionality of muscarinic receptors in hOPC. This process was then followed by having the products treated by solifenacin in vivo. However, it is important to note that solifenacin was used objectively to identify the impact of adjunct therapy on the myelination acceleration. Furthermore, animals treated with solifenacin did not show any influence of the drug on growth rate ( Abiraman et. al , 2015) .
The study also involved carrying out a western blotting process which involved homogenizing the forebrain tissues in a lysis buffer. The main motive behind this step was to determine the protein concentration levels. Finally, the consecutive transplantation of the products using the shiverer mice was conducted based on the stipulated protocols. In this study, it is very evident that the researchers only used fetal brain sample tissues that were within 17 to 22 weeks of gestation ( Abiraman et. al , 2015) . On the other hand, the recipient animals were subjected to either a solifenacin or saline treatment. Before surgery, the CD14a products were cultured for about one week and respective cells prepared for injection in 48 hours. Anesthesia was used before injecting the pups with the respective cells. However, this process was done carefully by having the injection target the corpus callosum. The remaining cells were carefully stored to identify the exact time oligodendrocyte differentiation is initiated.
Regarding the vivo analysis, a section of the mouse forehead was cut and a brain tissue sampled for evaluation. With this tissue, the rat anti-MBP was used to determine the myelination that had taken place. It also provided a way through which, oligodendrocyte differentiation was assessed. The resulting auditory brainstem response was evaluated and viable results recorded. This process was carried to improve on the viability of the cell-based therapy. Even though the hypotheses testing procedure was long, it provided positive insights into the debatable topic.
Initially, the researchers had hypothesized that subfraction of CD14a could be used to represent some glial stages of a human. The researchers also had hypothesized that O4 antigen of a human is restricted in the postmitotic cells. The results of the study indicated that both O4 and CD14a were abundant ( Abiraman et. al , 2015) . More so the O4 negative and CD14a positive cells were the most dominant among samples with 18-22 weeks of gestation. Also, the results corroborated that the different cell proportions varied with age advancement of the specimen.
Therefore, the attained results were within the expectations of the research question of whether adjunction therapy can be used to elevate the oligodendrocyte repair of a human. Based on the obtained results, this study provides insights into the identification of a new drug that will facilitate mitigation of myelin-associated illnesses. For instance, the FACS used in this process provide a way through which pharmacological targets can be obtained. Also, the comparison of both mouse and human data sheet differentiation with the obtained gene-expression analysis data helps to identify some of the CD14a and O4 positive selected receptors ( Imamura et. al , 2015) . With this information, the researchers can be in a position to expressively rate the oligodendrocyte differentiation in a human without difficulties.
Furthermore, the researchers were able to identify the oligodendrocyte development stages using human dissociates. This concept provides answers to the hypothesized relation of O4 representation of human glial stages. Also, based on the FACS array analysis, the study postulated that there are some receptor elements whose expression is first confined within oligodendrocyte stages before advancing to the postmitotic platforms ( Abiraman et. al , 2015) . This investigation is very important since it provides untold reasons to the practical stalling of oligodendrocyte differentiation in humans. Initially, myelin lesion was considered a place where the stalling of oligodendrocyte differentiation was initiated. Therefore, the results obtained in this research provided a scientific solution to this problem. Also, based on the generated hypothesis, this result provided insights into the explanation of human oligodendrocyte stages and their association to the respective CD14a and O4 antigens of humans. Also based on the gleaned information on acetylcholine candidate, it was evident that the product can be used to initiate myelination in human and mouse glia. However, this process is archived through muscarinic signaling which acts to represent the acetylcholine candidate. With this knowledge, the study corroborated that the candidate can be used to enhance the viability of human transplantation. Based on this contributions, the researchers were able to investigate means through which pharmacological modification can be used to influence myelination and OPC differentiation in human transplants. These assertions help to confirm the hypothesized assertion. Also, the pharmacological modification techniques gleaned in this study help to provide solutions on whether adjunct therapy can be used to accelerate human oligodendrocyte repair. In conclusion, the results obtained in study supported the hypothesis.
References
Abiraman, K., Pol, S. U., O'Bara, M. A., Chen, G. D., Khaku, Z. M., Wang, J., ... & Salvi, R. J. (2015). Anti-muscarinic adjunct therapy accelerates functional human oligodendrocyte repair. Journal of Neuroscience , 35 (8), 3676-3688.
Imamura, O., Arai, M., Dateki, M., Ogata, T., Uchida, R., Tomoda, H., & Takishima, K. (2015). Nicotinic acetylcholine receptors mediate donepezil ‐ induced oligodendrocyte differentiation. Journal of neurochemistry , 135 (6), 1086-1098.
