Investigating the Mechanisms of Microglia/Macrophage Activation in Mediating Inflammatory Responses following Distraction Spinal Cord Injury

Table: MED3
Experimentation location: Reseach Institution, School
Regulated Research (Form 1c): Yes
Project continuation (Form 7): No

Abstract:

Background and Purpose: Scoliosis is a disease in which the 3D sequence of the spine is abnormal. Distraction spinal cord injuries (DSCIs) are a common complication during severe scoliosis correction surgery, and severe cases may appear as paralysis. Inflammation is an important mechanism for the aggravation of DSCI, but the inflammatory pathway and microglial/macrophage activation mechanisms of DSCI are still unclear. Therefore, this study aimed to investigate the role of microglia/macrophages, along with TLR4-related inflammatory pathways, in DSCIs of pig animal models.
Methods: All experimental Bama pigs were randomized into the sham, incomplete, and complete distraction spinal cord injury (CDSCI and IDSCI) groups. Behavioral changes were assessed using the Tarlov scoring at the post-operation 1, 3, and 7 days, as well as the individual limb motor scale (ILMS). Histopathological examinations were conducted after seven days. Immunofluorescence was used to assess the expression of M1/M2 microglia/macrophage-related makers and inflammatory proteins. Western blotting was used to detect the expression of the TLR4 pathway.
Results: It was demonstrated significant decreases in Tarlov and ILMS scores in the pigs of both injury groups versus the sham group. H&E and Nissl staining showed substantial disruption in the white and gray matter structures, blooding, and neurons with atrophy and reduced number of Nissl bodies within the injured tissues of the two DSCI. Immunofluorescence staining showed enhanced expression of CD16 and CD206 in tissues of both injury groups. Furthermore, the CDSCI group exhibited higher CD16 and lower CD206 expression compared to the IDSCI group. Additionally, the intensity of NF-κB P65 fluorescence was significantly increased in the tissues of the two injured groups. Western blotting results showed increased total and phosphorylated protein expression of TLR4/NF-κB/MAPK pathway in injured spinal cord tissues.
Conclusions: The results showed that continuous mechanical distraction stress could lead to nerve function decline, Inflammatory infiltration, and neuron apoptosis, which increased as the distracted degree of the DSCI increased. The injury mechanism results revealed that the inflammatory TLR4/MAPK/NF-κB pathway and microglia/macrophage activation play a crucial role in inflammatory tissue injury development after DSCI. This study successfully established a large-animal model to simulate clinical DSCI, and the research results provide experimental evidence for further investigating the DSCI mechanisms and potential anti-inflammatory targets.

Bibliography/Citations:

1.    Xie J, Wang Y, Zhao Z, et al. Posterior vertebral column resection for correction of rigid spinal deformity curves greater than 100°. 2012;17(6):540-51. doi:10.3171/2012.9.Spine111026

2.    Schwartz DM, Auerbach JD, Dormans JP, et al. Neurophysiological detection of impending spinal cord injury during scoliosis surgery. 2007;89(11):2440-9. doi:10.2106/jbjs.F.01476

3.    Ju G, Wang J, Wang Y, research XZJNr. Spinal cord contusion. 2014;9(8):789-94. doi:10.4103/1673-5374.131591

4.    Alizadeh A, Dyck SM, neurology SK-AJFi. Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. 2019;10:282. doi:10.3389/fneur.2019.00282

5.    Xu L, Wang J, Ding Y, Wang L, neurology Y-JZJFi. Current Knowledge of Microglia in Traumatic Spinal Cord Injury. 2021;12:796704. doi:10.3389/fneur.2021.796704

6.    Impellizzeri D, Ahmad A, Paola RD, et al. Role of Toll like receptor 4 signaling pathway in the secondary damage induced by experimental spinal cord injury. 2015;220(9):1039-49. doi:10.1016/j.imbio.2015.05.013

7.    Rahimifard M, Maqbool F, Moeini-Nodeh S, et al. Targeting the TLR4 signaling pathway by polyphenols: A novel therapeutic strategy for neuroinflammation. 2017;36:11-9. doi:10.1016/j.arr.2017.02.004

8.    Kasuya Y, Umezawa H, sciences MHJIjom. Stress-Activated Protein Kinases in Spinal Cord Injury: Focus on Roles of p38. 2018;19(3). doi:10.3390/ijms19030867

 


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Research Plan:

1. Purpose and hypotheses

Distraction spinal cord injuries (DSCIs) frequently manifest as neurological complications during scoliosis correction surgery, which produces a hefty socioeconomic load. (1, 2) The mature animal model types of SCIs being studied include spinal cord dislocation, contusion, and cutting, while there are few studies on DSCIs.(3) The injury mechanism varies for different types of SCIs; therefore, it is necessary to conduct in-depth research on the injury itself, as well as potential treatment mechanisms for DSCIs.

The pathophysiological process behind SCIs consists of a primary injury caused by mechanical factors (distraction, trauma, etc.), along with a secondary injury. Secondary SCIs are series of pathological reactions which occur after the initial SCI, further increasing the degree and scope of the injury. (4) Microglial activation and the release of proinflammatory cytokines are the primary manifestations of the inflammatory response during the secondary SCI period, such as tumor necrosis factor-α (TNF-α), interleukin- 1 beta (IL-1β), and interleukin-6 (IL-6), which can directly promote neuronal death. (5)

The Toll-like receptor 4 (TLR4) pathway is crucial in activated microglia-induced neuroinflammatory responses, and TLR4 is expressed with increased expression levels on the microglial membrane after an SCI, which further activate nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs). (6) The MAPK pathway are phosphorylated during the inflammatory response to promote the production of inflammatory mediators.(7) Many studies have shown that the TLR4-mediated activation of NF-κB and MAPK in microglia after an SCI is an important mechanism of inflammatory injury in spinal cord tissue; however, its role in DSCIs remains unclear. (8)

This study aims to explore the activation of microglia and macrophages, along with alterations in TLR4-mediated NF-κB and MAPK pathway activity following DSCIs. We hypothesize that the activation of the TLR4/NF-κB/MAPK pathway prompts the polarization of microglia into the M1 subtype, thereby inducing neuroinflammation in spinal regions.

This study intended to clarify DSCI-associated neuroinflammation mechanisms, in turn providing evidence for identifying potential anti-inflammatory targets.

2. Subjects

In this study, Bama miniature pigs were utilized as the experimental subjects. These pigs are considered the most widely accepted large vertebrate animal model, serving as an alternative to human subjects in research settings.

3. Methods

3.1 Animal caring

We procured SPF Bama miniature pigs aged 3 months (n = 9, weighing 11.40 ± 1.68 kg, sourced from China) to establish DSCI animal models, and they were acclimatized through adaptive feeding for 1 week prior to experimentation. Throughout the study, all animals were housed in environmentally controlled rooms with a 12:12-hour light-dark cycle within isolation units.

During the surgical procedures, the pigs were anesthetized to minimize discomfort. Initially, each pig received an intramuscular injection of 3% pentobarbital sodium solution, followed by maintenance under continuous inhalation anesthesia with isoflurane. Endotracheal intubation was performed for each pig, and the depth of anesthesia was closely monitored using pain and corneal reflex tests. Simultaneously, the vital signs of the pigs were continuously monitored using a multifunctional bedside physiological monitor (Nihon Kohden, Japan).

All animal-related experimental protocols adhered to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 8523, revised 2011, United States).

3.2 Establishment of Porcine DSCI Model

The steps involved in DSCI surgery were as follows: Following the administration of anesthesia to the animals, a midline longitudinal incision was made, spanning from thoracic vertebra 13 (T13) to lumbar vertebra 2. The laminae from T14 to L1 were exposed, and temporary fixation was achieved by inserting short caudal pedicle screws (4.0 mm × 25 mm, Weigao, Shandong, China) and SINO rods into T14 and L1. Subsequently, a global column osteotomy was conducted at T15, accompanied by the removal of adjacent intervertebral discs. Finally, a spreader was utilized to gradually widen the space between T14 and L1 by 1 mm. Throughout the procedure, an electrophysiological monitor (Cadwell, United States) was employed to track changes in MEPs, enabling the detection of DSCI occurrence. 

The pigs were randomly divided into three groups (n = 3 per group) as follows: (1) Sham group: pigs underwent T15 osteotomy and pedicle screw fixation, exhibiting normal motor evoked potential (MEP) responses; (2) Complete Distraction Spinal Cord Injury (CDSCI) group: pigs underwent T15 osteotomy along with DSCI surgery, resulting in a complete loss of MEP amplitude; (3) Incomplete Distraction Spinal Cord Injury (IDSCI) group: pigs underwent T15 osteotomy combined with DSCI surgery, experiencing a decrease in MEP amplitude of approximately 75%.

3.3. Behavior assessment

To evaluate the effectiveness of DSCI modeling and its impact on the nerve function of the pigs after surgery, we conducted evaluations of the motor function in their bilateral limbs. These assessments encompassed the traditional Tarlov scale, which measures movement, weight-bearing capacity, and walking abilities of the hind limbs, along with the individual limb motor scale, which evaluates muscle strength, limb positioning, joint angles, weight support, and stepping while walking for each hind limb. Throughout the evaluation period, spanning post-surgery days 1, 3, and 7, all animals underwent assessment using both scales.

3.4. Histology evaluation

The spinal cord samples in the central DSCI lesions were acquired after sacrifice and stained with hematoxylin and eosin (HE) Staining Kit (Solarbio, Beijing, China) and Nissl Staining Kit (Solarbio)according to the manufacturer’s instructions. The histological features of DSCI regions were further evaluated using a NanoZoomer S60 digital slide scanner (Hamamatsu, Japan) and NDP. view2 viewing software. (Hamamatsu, Japan) 

3.5. Immunofluorescence Staining

To further explore the polarization of microglia in spinal cord injury lesions, we detected the expression level of CD16, CD206, Iba-1, and NF-κB P65 in spinal cord paraffin sections. Sections (4 µm thick) from each specimen were dewaxed, hydrated, boiled to repair the antigen, and blocked with 0.1% Triton X-100 and 10% normal goat serum at room temperature for 2 h. Sections were incubated overnight at 4◦C with the primary antibodies.The sections were washed 3 times using PBS, 5 min each time, after which the sections were incubated with secondary

antibodies for 1 h at room temperature. Following the PBS rinsing, the nuclei were stained blue with 40 ,6-diamidino-2- phenylindole (DAPI) (Solarbio). The sections were sealed with an anti-fluorescence quenching agent after processing, and immunofluorescence imaging was performed using a digital slide scanner. ImageJ software (National Institutes of Health, United States) was utilized to measure the mean fluorescence

intensity of specific proteins.

3.6. Western blotting

The spinal cord samples were stored at −80°C until manually homogenized and combined with RIPA lysis and extraction buffer. The protein concentration was determined using the BCA method. Subsequently, proteins were separated via SDS-PAGE and electrophoretically transferred onto PVDF membranes. These membranes were then blocked with 5% BSA Blocking Buffer for 1 hour, followed by overnight incubation with primary antibodies at 4°C. The primary antibodies utilized were: anti-TLR4, anti-p-IκBα, anti-NF-κB P65, anti-p-JNK, anti-p-ERK, anti-p-P38, and anti-β-actin. Following primary antibody incubation, the membranes were further incubated for 1 hour with either goat anti-rabbit or goat anti-mouse IgG HRP-conjugated secondary antibodies (dilution 1:5000). Subsequently, PVDF membranes were visualized using a Tanon 5200 chemiluminescence image analysis system. After exposure, ImageJ software was utilized to analyze the gray values of the bands.

3.7. Statistical Analysis

All data are expressed as mean ± standard deviation (SD). Statistical comparisons were conducted using either Student’s t-test or one-way analysis of variance (ANOVA) with a Bonferroni adjustment for multiple comparisons among the three groups. GraphPad Prism software (United States) or SPSS 22.0 (United States) was utilized for statistical analyses. Results were deemed statistically significant at P < 0.05.

 

Questions and Answers

1. What was the major objective of your project and what was your plan to achieve it? 

       a. Was that goal the result of any specific situation, experience, or problem you encountered?  

This project aimed to explore the injury mechanisms and potential treatments for distraction spinal cord injury (DSCI). In order to solve this problem, I plan first to conduct a detailed literature review to understand the common characteristics, occurrence mechanisms, research hot spots, and other essential knowledge of spinal cord injury. At the same time, I organized research methods related to spinal cord injury based on the literature and initially formulated my research plan based on previous research. Ultimately, I plan to focus on the impact of microglial polarization on DSCI, a research direction that has been extensively studied and discussed in recent years. After initially formulating the plan, I tried to contact Professor Xianjun Qu from the Department of Pharmacology at Capital Medical University. He provided generous guidance and professional modifications for this project, fully guaranteeing the professionalism of this research. With the help of Professor Qu, I plan to conduct relevant experiments with the graduate students in Professor Qu’s research group in my spare time and solve problems encountered in the research with regular discussions.       

I have had the idea of further exploring the mechanisms of DSCI for many years. Since junior high school, I have followed the Orthopedic Spine Team of Beijing Chaoyang Hospital to Qinghai Province, China, during summer vacations to approach Tibetan children with scoliosis as a volunteer service for five consecutive years. With the help of the charity foundation, these children finally got the opportunity to get out of the plateau and come to Beijing, the capital of China, to receive advanced scoliosis correction surgery. DSCI is the most severe complication in spinal deformity correction surgery. Once it occurs, it may cause paralysis or even death. It is like a dark cloud shrouding every Tibetan child who comes for surgery. Since the specific pathogenesis is not clear, once it occurs during surgery, doctors can only use two methods: decompression as soon as possible and high-dose hormone administration. The clinical effect is far from satisfying. My testimony over the past few years has given me a deep understanding of the helplessness this complication brings to surgeons and the irreparable harm it causes to patients. Although my professional knowledge in related fields needs to be improved, I have been learning step by step and hope that one day I can join the research on DSCI. This is also the fundamental reason I established this project's theme.

       b. Were you trying to solve a problem, answer a question, or test a hypothesis?

With the help of molecular biology, my project attempts to test the hypothesis that the activation of the TLR4/NF-κB/MAPK pathway prompts the polarization of microglia into the M1 subtype, thereby inducing neuroinflammation in spinal regions.

2. What were the major tasks you had to perform in order to complete your project?

       a. For teams, describe what each member worked on.

          The research plan to complete this project is divided into three parts: research design, experimental operation, and result analysis. After an in-depth search of relevant literature, I chose the Bama pig, a vertebrate animal closer to humans, as the research object. With the guidance of Prof. Qu, I designed a research plan consisting of three parts: large animal modeling, behavioral testing, and histopathological analysis. I also entered the team of Professor Qu Xianjun from the Department of Pharmacology of the Medical University implemented this project while learning relevant experimental techniques. Through six months of study, I have mastered basic biological experimental techniques such as animal behavior evaluation, western blotting, immunofluorescence staining, and the relevant principles of statistical analysis. These experimental methods will help us complete this part of the study in our spare time.

3. What is new or novel about your project?

      The main innovation of this study is the establishment of a large animal Bama pig DSCI model and the potential exploration of the injury mechanism. Through a large amount of literature reading, I found that due to different trauma mechanisms, the pathological classification of spinal cord injury is mainly divided into dislocation, contusion, cutting, and distraction. Among them, the research on dislocation, contusion, cutting, and other models is relatively mature, but only some researchers have paid attention to DSCI. In scoliosis correction surgery, the most common mechanism of spinal cord injury is the stretch caused during the correction. Therefore, focusing on the type of spinal cord injury caused by the stretch mechanism during surgery is meaningful. Compared with mice, the most commonly used animal model for spinal cord research, using Bama pigs for spinal cord injury modeling is also an innovation of this study because the large animal model allows us to use the screw-rod system to restore the actual surgical model. Compared with mouse and rat models, the Bama pig model is more convincing to restore intraoperative DSCI.

4. What was the most challenging part of completing your project?

       Establishing the Bama pig DSCI model and the experiment design was the most challenging part of this study. The advantage and difficulty of large vertebrate animal modeling is that it can highly simulate the human surgical process, which requires rich surgical experience, animal experiment qualifications, relevant animal experiment ethical review, and financial support. As a high school student, I need more technology and experience. The only way to advance the experiment is to find a suitable team for related cooperation. Through email contact, Professor Qu Xianjun from the Department of Pharmacology at Capital Medical University was interested in my experimental plan, and we conducted many online and offline communications. Fortunately, Professor Qu's team has relevant experience modeling large vertebrate animals and is conducting research on spinal cord injury. After repeated research and discussions, I was fortunate to receive technical and financial support from Professor Qu’s team. After weeks of communication, we determined the modeling protocol for the Bama pig model. I was very fortunate to observe the practical operations and take part in the postoperative care of animals. Finally, after sacrificing all the animals, we got the spinal cord samples that needed further evaluation.

This experience taught me I should keep going even when encountering difficulties. You need to analyze and solve problems calmly through persistent efforts. It would help if you firmly believed that dogged persistence would bring more luck. In addition, professional matters require the most professional team to cooperate. As a project leader, I cannot blindly solve everything by myself. The wisest choice is to learn and improve oneself continuously during the cooperation process.

5. If you were going to do this project again, are there any things you would you do differently the next time?

If I do this project again, I may communicate with the cooperation team earlier to improve the project's efficiency. I initially conceived the idea of exploring the process of tension being transmitted into spinal cord tissue to cause DSCI injury. However, since there is still a lack of relevant mechanisms in the DSCI research field, Professor Qu suggested that I start with neuroinflammation to describe the phenomenon of secondary injury in DSCI. The research plan became relatively more feasible after revision under his guidance. Due to my lack of understanding of the mechanisms related to spinal cord injury, I initially wasted much time exploring what I could do independently without profoundly considering how to start a new research project. The advice of the professional team is key to ensuring the project’s feasibility. In future project planning, I should listen more to other professional's opinions and work in combination with my abilities and expertise.

6. Did working on this project give you any ideas for other projects? 

Through preliminary analysis of the results of this study, the research hypothesis, that is, the activation of the TLR4/NF-κB/MAPK pathway prompts the polarization of microglia into the M1 subtype, thereby inducing neuroinflammation in spinal regions, has been initially verified. The result brings us the following tips:

1. The research mechanism on DSCI can be further discussed. Since this is the first time I have come in contact with biology-related experiments, the rigor of this study's derivation of signaling pathways within tissues still needs further improvement. In vivo animal experiments can be optimized by introducing specific inhibitors and other methods to clarify the upstream and downstream relationships of the signaling pathways explored this time, or cell experiments can be introduced for in vitro verification work. Further in-depth exploration of the injury mechanism of DSCI can become a new research project.

        2. Current drug treatment for spinal cord injury still relies on high-dose hormone administration, whose effectiveness is uncertain and prone to many complications. The current research hotspot is to explore a targeted therapy drug. In this study, the microglial polarization process exacerbated the occurrence of neuroinflammation, and finding targeted drugs that inhibit the polarization of microglia toward the M1 type may be one of the keys to inhibiting secondary injury. Since I got a good place in the chemistry contest, I have a particular ability to participate in synthesizing and modifying targeted compounds. It will be a meaningful research project if I can screen and verify the efficacy of improved targeted drugs in existing models.

7. How did COVID-19 affect the completion of your project?

My main experimental project was carried out in the SPF animal center. All relevant experimental personnel and animals passed the COVID-19 test, and all experiments were completed on time.