The Effect of Chronic CRH Treatment on Astrocytes and Brain Tumors

This report presents a study investigating the impact of chronic corticotropin-releasing hormone (CRH) treatment on astrocytes and its implications for brain tumor development. The research examines the hypothesis that in-vitro treatment of astrocytes with CRH can promote brain tumor growth. The study utilizes cultured cells from mice, comparing CRH-treated astrocytes with a control group. The methodology includes cell culture, treatment, and various assays to assess cell proliferation and viability. The report explores the role of astrocytes in brain tumor progression and the influence of CRH on astrocytic calcium response, potentially affecting cancer cell invasion. The study aims to answer research questions regarding the effects of CRH on astrocytes and its subsequent impact on the incidence and progression of brain tumors. The findings are expected to support the hypothesis, highlighting the significance of CRH and astrocytes in the development of brain tumors. The report concludes by emphasizing the need for further research in this area, particularly regarding the complex relationship between astrocytes and brain tumors, and the potential therapeutic targets related to the study's findings.

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The Effect of In-Vitro Chronic Corticotropin-Releasing Hormone Treatment on Astrocytes;
Implications for Brain Tumor
Abstract
Corticotropin-releasing hormone (CRH) is mainly found in the brain and the spinal cord. Other
areas where this hormone can be distributed include the endometrium, the myometrium, and
several inflammatory sites. It has been established that CRH exerts an indirect inflammatory
effect on the immune cells. Additionally, CRH acts directly on a proinflammatory fashion to
affect immune cells. CRH is an important neuro-regulator in the brain. This is because of the
many influential effects that CRH exerts in the brain. It is also worth noting that the neuron
activation of CRH can interfere with the response of calcium on astrocytes, thus playing a vital
role in the development of brain tumors. This paper examines the hypothesis that the treatment of
astrocytes with chronic CRH can lead to brain tumors. A study will be performed on cultured
cells from 6-week old mice. The results are expected to agree with the hypothesis of the research.
Background
Brain tumors are serious conditions that have massively affected people in recent years. A lot of
people suffer from malignant brain tumors. The most common malignant brain tumors are
malignant gliomas that account for approximately 80% of the total number of malignant brain
tumors (Placone et l., 2016, p. 62). Averagely, the time required for survival in cases of gliomas
is approximately 15 months. The treatment of gliomas normally involves surgery to the brain.
This resection surgery is then followed by radiotherapy or chemotherapy (Hervey-Jumper and
Berger, 2014, p. 248). Studies have revealed that only less than 10% of patients stand a chance of

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survival five years after diagnosis (Chen et al., 2012, p. 38). These low survival rates indicate
that brain tumors are very serious conditions and are mostly terminal.
The tumors in the brain are difficult to treat and eliminate because of the cells that surround the
brain. These cells are known to facilitate the invasion and proliferation of cancer. Some types of
cells that promote cancer proliferation are microglia cells (Placone et al., 2015, p. 64). They
constitute approximately 15% of all the cells that support the brain. These cells are believed to
enhance the invasion and growth of malignant glioma. Another set of cells that support the brain
are the astrocytes. They constitute approximately 50% of cells in the brain. The significance of
microglia in the progression of brain tumors has been extensively researched and reported.
Astrocytes, on the other hand, have not been studied extensively even though most inflammatory
cytokines and growth factors that are associated with microglia are also associated with
astrocytes.
These inflammatory cytokines that are expressed by the two types of cells expressed above are
affected by a hormone known as corticotropin-releasing hormone (CRH). The inflammatory
cytokines are primarily produced by macrophages. This hormone increases the production of
proinflammatory cytokines. Studies have shown that CRH indirectly and directly exerts an anti-
inflammatory and proinflammatory influence respectively on immune cells (Hu et al., 2016, p.
543). It is, however, important to note that CRH plays a lot of other very vital functions in the
brain. Studies indicate that CRH helps in the regulation of several physiological responses to
various stressors in the body, including a stroke. For example, after ischemia, inflammation and
excitotoxic damage lead to the development of a stroke. The role played by CRH in this
development cannot be understated. CRH increases the excitotoxic damage that occurs in the
brain. Additionally, it modulates peripheral inflammatory responses (Karve et al., 2016, p. 694).

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As a result, the pathology of stroke is massively influenced. The action of CRH has been found
to influence the operation of astrocytes, especially in the event of a brain tumor.
Astrocytes are the most predominant glial cells that are found in the brain. For a very long time,
they have been considered to only support neural functions in the body. Research has, however,
revealed that astrocytes play other vital functions that include the maintenance of the blood-brain
barrier and the regulation of the microcirculation in the brain (Cabezas et al., 2014, p. 211). It is
also worth noting that astrocytes play another crucial role, which involves neurogenesis. In the
presence of tumors, the astrocytes undergo reaction gliosis. This is the process in which the glial
fibrillary acidic protein and inflammatory cytokines are upregulated. Reaction gliosis is a process
that aims at repairing the damaged brain cells. However, this may not be the case in the presence
of cancer or malignant tumors. This is because in tumors, astrocytes, especially those that
surround the brain become reactive. As a result, some growth factors are secreted, thus leading to
the proliferation of cancer can occur (Wang et al., 2013).
In vitro chronic corticotropin-releasing hormone treatment, the presence of astrocytes contributes
to an increase in the proliferation of cancer and tumors. In a study done by Placone et al. (2016),
the metabolic activity of astrocytes in rats was stimulated and then intubated alongside breast-
metastatic brain cancer. 18 hours after the intubation, it was noticed that the proliferation of
tumor cells had increased by approximately 400% (p. 66). This massive increase just shows the
effect of astrocytes on brain tumors. Additionally, it has been determined that astrocytes play a
major role in the initial invasion of cancer cells. According to Chen et al. (2018), CRH affects
the stimulation of astrocytes by affecting the astrocytic calcium response thus influencing the
reaction of astrocytes around the brain. As a result, the brain becomes more vulnerable to the
invasion and proliferation of cancer and tumor cells. This shows that CRH treatment on

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astrocytes can bear serious implications for brain tumors. This is because high levels of CRH
around the brain tissues can lead to serious anti-inflammatory actions.
Aims and Objectives
This research aims to determine the effect of chronic CRH treatment on astrocytes and the
implications of this treatment on brain tumors. We will compare and evaluate findings from
different studies to help in justifying our hypothesis. This research also aims to understand the
function of astrocytes in the brain and how they contribute to the invasion and proliferation of
brain cancer and brain tumor cells. To determine the effect of CRH on astrocytes, this study will
answer two important questions.
Research Question 1: What is the effect of in vitro chronic corticotropin-releasing hormone
treatment on astrocyte cell culture?
Research question 2: How will this effect affect the incidence and progression of brain tumors?
Methodology
Our hypothesis that we tend to prove is whether the treatment of astrocytes with chronic CRH
leads to the proliferation of cancer cells in brain tumors. The independent variable in the study is
the corticotropin-releasing hormone, which is widely known to help in releasing stress. The
dependent variable in the study is the invasion and proliferation of cancer cells, thus leading to
the formation or progression of brain tumors. Another dependent variable is tumor suppressor
genes. Our control group is astrocytes with no treatment with a chronic corticotropin-releasing
hormone. We will, therefore, compare astrocytes that have been treated with CRH and those that
have not been treated. The experimental group will be astrocytes with treatment with a chronic
corticotropin-releasing hormone.

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Inclusion and Exclusion Criteria
Several necessary data was collected from various research journals to help in coming up with
this research proposal. The inclusion criterion for necessary articles involved using key terms
such as corticotropin-releasing hormone, astrocytes, and brain tumors, the effect of corticotropin-
releasing hormone on astrocytes, and corticotropin-releasing hormone in the brain. This search
method brought very many journals that again had to be filtered. Another inclusion criterion
involved including only articles published in the last 10 years. We then narrowed down our
search to include only articles that had been published in English. We had to narrow down the
search even further by including only peer-reviewed journals. Out of the results that were
returned, we picked only 8 research articles that we felt, satisfied our research objectives. These
are the papers that we used to inform the discussion in this research proposal. All articles that did
not meet the above criteria were excluded from the research.
Mice
Necessary ethical approvals will be sought from the Ethical Committee of Experimental animals
before any mice can be used for this study. This committee will place protocols in place to
reduce the number of mice used in this experiment and to minimize their suffering. Mice will
then be randomly acquired from Mikaelson Laboratories to aid in the study. The handling of
these mice will be done according to all the regulations and procedures outlined by an approved
ethical committee. The mice will be kept at the institution’s animal facility for 1 week before the
study. This will help them in adjusting to the new environment. Additionally, this 1 week period
will be used to confirm the health status of the mice. Between 8 and 10 randomly picked rats will

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be used in each culture. Some rats will be injected with the chronic corticotropin-releasing
hormone, while others will be used as originally obtained from Mikaelson Laboratories.
Cell Culture
Astrocyte cells will be obtained from 8-week old mice and treated with the appropriate reagents
for this study. The cells will be cultured in Dulbecco’s Modified Eagle Medium. The metabolic
activities of these astrocyte cells will then be stimulated using phytohemagglutinin. Cells from
one set of cultures will then be treated with the chronic corticotropin-releasing hormone, while
cells from another culture were not treated. The treated stimulated cells will then be placed in a
conditioned environment and incubated alongside brain-metastatic breast cancer cells. This will
be done to investigate the proliferation of cancer cells. It is expected that the proliferation will
increase massively in the set of astrocyte cells with treatment with chronic CRH after
approximately 18 hours. The proliferation of the untreated cells will also increase due to reactive
gliosis of the astrocyte cells. However, the proliferation will not be as high as the one in cells
treated with chronic CRH.
Measurement of Results
The astrocytes cells will be examined after 18 hours of intubation to determine any notable
changes. An MTT assay will be used to identify and isolate the cells that will be considered
viable. DAPI fluorescence staining solution will then be used as a staining agent to stain these
viable cells. We will then fix the stained cells and observe them under a digital microscope to see
the finer details of the cultured cells. We will then perform a fluorescent activated cell sorting to
understand the orderly programmed cell death popularly known as apoptosis. We will conclude

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by performing a cell fusion assay and a reverse transcription-polymerase chain reaction (RT-
PCR). The obtained results will then be analyzed statistically to determine whether our
hypothesis has been confirmed.
Conclusion
In this research proposal, we have tried to investigate the effect of chronic CRH treatment on
astrocyte cells and the impact of this treatment on brain tumors. From this paper, it is noticeable
that astrocyte cells play a very vital role in the progression of cancer, especially brain cancer and
brain tumor. This paper has also attempted to explain the relationship between CRH and
astrocytes that causes cancer cell proliferation. The study findings from this proposal should be
able to demonstrate how CRH stimulates cytokines inflammatory and reaction gliosis of the
astrocytes in the progression of brain tumors. It is also worth noting that this paper pointed out
other important roles of CRH in the body. More research should, however, be done regarding the
relationship between astrocytes and brain tumors because it is an area that causes a lot of
confusion. One instance of this confusion is stated in this research proposal; the astrocyte cells
help in the regeneration of cells in traumatic head injuries but cannot do the same in cases of
cancer.

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References
Cabezas, R., Ávila, M., Gonzalez, J., El-Bachá, R.S., Báez, E., García-Segura, L.M., Jurado
Coronel, J.C., Capani, F., Cardona-Gomez, G.P. and Barreto, G.E., 2014. Astrocytic modulation
of blood brain barrier: perspectives on Parkinson’s disease. Frontiers in cellular neuroscience, 8,
p.211.
Chen, C., Jiang, Z., Fu, X. and Tasker, J.G., 2018. Astrocytes amplify neuronal dendritic volume
transmission. bioRxiv, p.361014.
Chen, J., McKay, R.M. and Parada, L.F., 2012. Malignant glioma: lessons from genomics,
mouse models, and stem cells. Cell, 149(1), pp.36-47.
Hervey-Jumper, S.L. and Berger, M.S., 2014. Role of surgical resection in low-and high-grade
gliomas. Current treatment options in neurology, 16(4), p.284.
Hu, Y., Li, M., Lu, B., Wang, X., Chen, C. and Zhang, M., 2016. Corticotropin-releasing factor
augments LPS-induced immune/inflammatory responses in JAWSII cells. Immunologic
research, 64(2), pp.540-547.
Karve, I.P., Taylor, J.M. and Crack, P.J., 2016. The contribution of astrocytes and microglia to
traumatic brain injury. British journal of pharmacology, 173(4), pp.692-702.
Placone, A.L., Quiñones-Hinojosa, A. and Searson, P.C., 2016. The role of astrocytes in the
progression of brain cancer: complicating the picture of the tumor microenvironment. Tumor
Biology, 37(1), pp.61-69.

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Wang, L., Cossette, S.M., Rarick, K.R., Gershan, J., Dwinell, M.B., Harder, D.R. and
Ramchandran, R., 2013. Astrocytes directly influence tumor cell invasion and metastasis in
vivo. PLoS One, 8(12).