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This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. 
 

Targeting the Epigenetic Factors of 

Neuroinflammation 

Anusha Patil 

Written under the direction of Dr. David P. Crockett 

 

Abstract 

 

Currently, there are no effective treatments for traumatic brain injury (TBI). This is because the 

mechanisms behind post-injury neuroinflammation are not well understood. This project 

studies a novel signaling pathway responsible for the activation of microglia post-TBI. Its goal 

is to identify epigenetic factors of neuroinflammation that may be targeted with future 

therapies. 

 

We are examining the possibility that class IIa Histone Deacetylases (HDACs), particularly 

HDAC7, are responsible for initiating the inflammatory response after TBI. In addition, we plan 

to explore what its upstream regulation factors may be. One possible upstream regulation 

factor explored is regulating Kinase 2, also known as Par1b (Par1b/MARK2), since prior 

research indicates that a deficiency in Par1b/MARK2 increased the inflammatory response of 

microglia in a mouse model of TBI. In our experiments, we examined brains from sham mice 

(i.e., without head injury) and mice subjected to closed head injury (CHI). Our experiments 

made use of wild-type mice and mice deficient in Par1b/MARK2. Qualitative analyses were 

conducted using fluorescent microscopy imaging of immunohistochemistry. Using cell-specific 

markers of inflammation, we found an increase in astrocytic marker GFAP (glial fibrillary acidic 

protein) and microglial protein IBA1 (ionized calcium binding protein) expression in the cortex 

of the mice after CHI. These increases were dramatic ipsilaterally (same side) to the injury, but 

only moderate contralaterally (opposite side). In the control brains (sham operates), little to no 

increase in these markers were detected. 

 

In our experiments, we observed increased expression of HDAC7 in post-TBI microglia as well 

as in reactive GFAP-expressing astrocytes. Others have observed that Par1b/MARK2 may 

negatively regulate HDAC7 activity and there is evidence that HDAC7 is an inhibitor of anti-

inflammatory genes.    

 

 

 

 

 

https://creativecommons.org/licenses/by-nc-sa/4.0/
https://creativecommons.org/licenses/by-nc-sa/4.0/


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Introduction 

 

Traumatic brain injury (TBI) has been dubbed the “silent epidemic.” It is estimated that 69 

million people worldwide suffer from death and disability caused by TBI, which is more than any 

other traumatic insult (Dewan et al 2019). In the U.S. alone, approximately 1.7 million people 

sustain a TBI each year, which leads to at least 52,000 deaths annually. The financial burden on 

society in both direct and indirect costs adds up to an astounding $60 billion, not to mention 

the cost of the individual’s life or independence. Unfortunately, there is currently no effective 

treatment for TBI. One major reason for this is the body’s inflammatory response that follows 

damage to the central nervous system (e.g., Loane and Byrnes 2010; Faden and Loane, 2015). 

Neuroinflammation is a double-edged sword because it has both beneficial and detrimental 

effects post-TBI, and general immunosuppression has not proven helpful (Russo and McGavern, 

2016; Loane and Byrnes 2010). Neuroinflammation has also been implicated in many 

neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease (Faden and Loane, 

2015). Fully understanding the mechanisms behind the body’s neuroinflammatory response will 

not only allow for the development of targeted treatments for TBI, but also will likely allow for 

better treatments for those with neurodegenerative diseases (Ransohoff, 2016) and other 

diseases that may have a neuroinflammatory component such as diabetes (Wang et al 2014).  

 

The epigenetic mechanisms that control gene expression of neuroinflammation have been 

studied, specifically the mechanism of histone deacetylases (HDACs). HDACs are a class of 

enzymes that modify chromatin conformation by removing acetyl groups from the histones of 

chromatin. This condenses chromatin, making it more difficult for the DNA to be accessed by 

transcriptional machinery and therefore repressing transcription (Allfrey et al 1964). In recent 

years, there has been a focus on HDACs as a therapeutic target to treat a variety of diseases 

including cancer, cardiovascular disease, stroke, injury and diabetes (Yoon and Eom, 2016; 

Wang et al, 2014; Mihylova et al, 2011; Baltan et al, 2011). Of the HDAC enzymes, class IIa 

HDACs receive particular attention given that they have higher expression in the brain and are 

involved in neuronal activity, degeneration, and apoptosis. Class IIa HDACs have been 

implicated as important factors in regulating the immune response (Altman and Kong, 2011; 

Poralla et al, 2015; Shakespear et al, 2013; Wang et al, 2014; Gupta et al, 2016). For example, 

HDAC5 and HDAC7 have been detected in macrophages, immortalized microglia known as BV2 

cells, and microglia (Gupta et al 2016; Shakespear et al 2013; Crockett et al 2017). HDAC7 

serves as a negative regulator of the anti-inflammatory gene, Nur77 (Dequiedt et al 2003; 

Hanna et al 2011; Liu et al 2017), and there is preliminary evidence of increased expression of 

HDAC7 in microglia following TBI (Crockett et al 2017).  

 

It has been hypothesized that class IIa HDACs may play an important role in initiating the 

neuroinflammatory response. Because of this, it is important to determine possible upstream 

factors that regulate class IIa HDAC function. Following this, it is important to examine the 

possibility that polarity proteins Par1 (partitioning defective 1), also known as MARK 



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(microtubule affinity regulating kinase), act as functional regulators of HDAC IIa. This is because 

MARK2/Par-1b has been observed to play a vital role in neuronal migration and axon 

formation. It was previously observed that animals deficient in Par-1b/MARK2 seemed to be 

“primed” for an inflammatory response; Par-1b/MARK2 deficient mice that had sustained TBI 

displayed a heightened inflammatory response (DiBona et al 2019). It was therefore proposed 

that one of MARK2’s downstream functions is to regulate class IIa HDACs within microglia and 

their response to injury. By determining whether such a signaling pathway exists within 

microglia, it is possible that this pathway could be therapeutically targeted in the future with 

the hope of regulating the runaway inflammatory response associated with brain injury.  

 

Materials and Methods 

 

To ensure the rigor and reproducibility of the studies, the following measures were taken. First, 

data collection and analyses were completely blinded to the experimenter. Second, rigorous 

control experiments were performed to ensure the validity of the conclusions—all antibodies 

were positive and negative control tested with immunohistochemistry.  

 

Our TBI studies were conducted using a controlled cortical impact (CCI) injury model in mice 

over the course of several years (e.g., DiBona et al 2019). Sham mice (those who just had a 

craniotomy performed without significant injury) as well as CCI-injured mice consisting of both 

wild-type (WT) and Par1b/MARK2 knockout (KO) (−/−) mice (B6.129X1-Mark2tm1Hpw/J; stock 

009365) were used. Knockout mice (without Par1b/MARK2) were used to demonstrate an in 

vivo model of changes in morphology and density of astrocytes and microglia. All animal 

procedures were approved by the Rutgers-RWJMS Institutional Animal Care and Use Committee 

prior to the start of experimentation. All animals had free access to food and water and were 

housed in a clean, temperature-regulated facility that maintained a strict 12-hour light/dark 

cycle. Animals were anesthetized prior to being sacrificed by cardiac perfusion. Once the mice 

were phosphate-buffered saline (PBS) cleared and fixed with paraformaldehyde (PFA), the brains 

were extracted and submerged in PFA for 1–2 hours post-fixing. Each brain was washed with 

PBS and transferred through sucrose gradients (10%, 20%, and 30%) for one day each and stored 

in a 4 °C refrigerator.  

 

Brains from these mice were mounted with O.C.T. Compound embedding medium, and 60-μm 

sections were obtained with a frozen sliding microtome. Brains were pierced on the right side 

(injury was made on the left) to serve as a landmark and maintain orientation throughout the 

later steps of the process. Sections were stored in cryoprotectant solutions (25% Glycerol, 25% 

Ethylene Glycol, 30% Sucrose in 0.1 M PBS) until immunohistochemistry was performed. For 

immunohistochemistry, two intact hippocampal sections per animal were taken. Cells were 

stained with DAPI, GFAP, and IBA1. DAPI is a stain for gross cell morphology and is used to label 

nuclei. GFAP is a stain used to determine astrocyte activation, as astrocytes are specialized cells 



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that function to support the central nervous system (CNS). IBA1 is a stain used to determine 

microglia activation, as microglia are specialized macrophages of the CNS.  

 

For the CCI-TBI experiment, tissue was washed 3 times for 5 minutes each with 1× PBS to 

remove cryoprotectant residue. Following thorough washing, tissue was permeabilized in 

Blocking Solution (2 mL Donkey Serum, 50 mL PBS-T) for 30 minutes at room temperature. 

After blocking Solution was removed from each tissue, the unrinsed sham-operated control and 

CCI-TBI tissues were then incubated with 0.200 mL of donkey-anti-mouse Fab fragment IgG 

(Jackson ImmunoResearch, West Grove, PA, 1:60 dilution) for at least 1 hour at room 

temperature. This solution was removed from each tissue, and primary antibodies were then 

applied at the desired dilution to incubate overnight at room temperature. The following day, 

tissue was washed again using the process above, secondary antibody at desired dilution was 

applied, and the tissues were incubated at room temperature for at least 2 hours. The tissue 

was then washed and mounted on slides, before being sealed in with DPX. To minimize bias in 

imaging acquisition and quantification, all slides were blinded prior to imaging, and unblinding 

occurred after image quantification.  

 

Results 

 

These figures are fluorescent microscope images taken after immunohistochemistry was 

performed on brain sections (containing the hippocampus) taken from different mice. Figure 1 

acts as a summary of findings in the ipsilesional cortex taken from brain of a mouse with closed 

head injury (CHI) performed at 21 days and euthanization performed at 28 days. In Figure 1A, 

the DAPI stain demonstrates that there are no obvious changes to the structure of the cortex or 

hippocampus other than the injury site, which negates the possibility of increased expression 

being due to structural changes. However, in Figure 1 B and C, the results qualitatively indicate 

an increase in expression of GFAP (Figure 1 B) and IBA1 (Figure 1 C). This is made more evident 

when looking at Figure 2, which serves as a negative control and shows the baseline levels of 

expression for both GFAP and IBA1 in CHI092319-1, a MARK2 wild-type sham mouse (only 

craniotomy) operated on at 20 days and euthanized at 28 days.  

 



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Figure 3 shows the differences in expression levels on each side of the cortex: the same side as 

the injury (ipsilesional) and the opposite side of the injury (contralesional). There is increased 

expression of GFAP in both the ipsilesional (Figure 1 B) and contralesional (Figure 1 C) sides of 

the cortex; however, the ipsilesional side has a much greater increase in expression in GFAP, 

especially in the area immediately surrounding the injury site. There is also increased 

expression of IBA1 in both the ipsilesional (Figure 1 E) and contralesional (Figure 1 F) sides of 

the cortex; however, the ipsilesional side has a much greater increase in expression in IBA1, 

especially in the area immediately surrounding the injury site.  



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Discussion 

 

Purpose 

 

The purpose of this project was to determine the role that class IIa HDACs may play in initiating 

the inflammatory response after TBI. Now that it is understood that class IIa HDACs, particularly 

HDAC7, do in fact play a role in initiation of the inflammatory response after TBI, upstream 

factors can be explored to unearth the molecular mechanism behind the subsequent 

neuroinflammation after TBI. Gaining knowledge of the pathways involved will facilitate 

attempts to discover therapy or even cures for TBI and spinal cord injury (SCI). 

 

Though it took several tries to obtain intact sections and perform operational IHC staining, the 

experiment was successful since the high expression of GFAP and IBA1 supported the 

hypothesis that HDAC plays a role in mediating neuroinflammation. This is evident through the 

IHC staining, which involved mixing HDAC7 (a class IIa HDAC) with GFAP and IBA1 secondary 

antibodies. When comparing injury in Figure 1 vs sham in Figure 2 results, there is a higher 

expression of GFAP and IBA1 in the closed head injury mouse compared to the baseline levels in 

the sham operation mouse. The results also suggest something that had no clearly defined 

expectations, which was a global increase in expression of GFAP and IBA1. This is evident in 

Figure 3, when comparing the contralesional and ipsilesional sides of the cortex. Not only was 

there increased expression of GFAP and IBA1 in the same side of the cortex as the injury, but 

also on the opposite side of the brain (albeit in lower concentration). This indicates that the 

inflammation doesn’t just take place at the injury site, but spreads throughout the rest of the 

brain at the molecular level. The reason this increased staining is important is because of the 

roles these cells play in the CNS. If there is disease or injury (such as SCI or TBI) of the CNS, one 

would expect an activation in astrocytes (given their role of support) and microglia (given their 

role of maintaining CNS health by phagocytizing infection and damaged neurons). 

 



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Future directions 

 

The next stage of this experiment is to compare the expression level differences between the 

contralesional and ipsilesional sides of the cortex in a MARK2-deficient mouse with CHI, as well 

as expression levels in a CHI MARK2-deficient mouse vs sham MARK2-deficient mouse.  

When these molecular results are obtained and compared to the results from these wildtype 

(WT) mice, it can be determined whether Par1b/MARK2 is a possible upstream factor for class 

IIa HDACs.  

 

After this is completed, the next steps will be to test possible therapies from both behavioral 

and molecular perspectives. The potential therapies for TBI that will be focused on are the 

diabetic drug Metformin and class IIa HDAC inhibitors. The diabetic drug Metformin has been 



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identified as a potential candidate because of its anti-oxidative, anti-inflammatory, and anti-

ischemic properties, which may lend itself to neuroprotective effects. There is evidence that 

Metformin can improve the cognitive outcome of mice following TBI (DiBona et al, under 

review). These studies suggested that Metformin’s pleiotropic effects (Bridgeman et al, 2018) 

may affect the neuroinflammatory response through stimulation of Par1b. Even a 50% decrease 

in Par1b/Mark2 can lead to an apparent increase in neuroinflammation (DiBona et al 2019). 

Metformin treatment has shown positive effects in several animal studies dealing with spinal 

cord injury and brain trauma (Wang et al 2016; Zhang et a 2016; Tao et al 2018; DiBona et al 

2019 under review) and in human clinical trials (Taheri et al 2019). In addition, Metformin has 

been shown to influence HDAC7 functioning (Mihaylova et al 2011; Bridgeman et al; 2018). 

Metformin has been shown to alter microglial functioning in an animal model of stroke (Ji et al, 

2018). Although the mechanism by which Metformin creates positive effects in injury models is 

not fully understood, it has been hypothesized that Metformin dampens the inflammatory 

response by decreasing the availability of nuclear HDAC7 by enhancing Par1b/MARK2 activity in 

microglia. 

 

Possible flaws 

 

One of the potential problems with this protocol is the sham operation animal as a negative 

control. According to our experimental setup, the sham mouse receives a craniotomy in place 

of a closed head injury. Though the craniotomy did function as a negative control in this 

experiment, we cannot be sure that a craniotomy procedure does not cause unique trauma 

different from that of CHI and accompanying unique neuroinflammation. As a result, this may 

not be the best representation of baseline expression levels of GFAP and IBA1. To prevent such 

confounding data disrupting the results of other experiments, a future experiment could be 

done comparing an animal only under anesthesia with the conventional negative control of 

sham operation. This experiment would demonstrate whether the sham is an accurate 

representation of baseline expression levels without conferring distinct damage and, therefore, 

altered expression levels.  

 

Acknowledgements 

 

Special thanks to Dr. David Crockett for serving as a wonderful mentor and providing many 

great learning opportunities. In addition, many thanks to the Aresty Reseach Center for funding 

this research project and to Robert Wood Johnson Medical School for providing resources. This 

paper was a progress report submitted to The Department of Cell Biology and Neuroscience 

Rutgers University. 

 

 

 

 



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