Glioma

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 4  |  Issue : 3  |  Page : 42--48

Establishment and evaluation of a Sprague-Dawley rat model of intramedullary spinal cord glioma


Dongkang Liu, Guo Yi, James Jin Wang, Guihuai Wang 
 Department of Neurosurgery, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing, China

Correspondence Address:
Dr. Guihuai Wang
Department of Neurosurgery, Beijing Tsinghua Changgung Hospital, No. 168 Litang Road, Dongxiaokou Town, Changping District, Beijing 102218
China

Abstract

Background and Aim: Intramedullary spinal cord glioma has no evident boundary with normal spinal cord tissue. The rate of successful surgical resection of intramedullary spinal cord glioma is low. Well-established animal models for intramedullary spinal cord glioma can help promote translation from related basic therapy research to clinical applications. In this study, we established a rat model of intramedullary spinal cord glioma. Materials and Methods: A total of 23 male Sprague-Dawley (SD) rats were randomized into blank control (n = 3) and experimental (n = 20) groups. The blank control group received intramedullary injection of Dulbecco's modified Eagle medium (DMEM) and the experimental group was injected with DMEM containing C6 glioma cells. The neurological states of these rats were evaluated using the Basso, Beattie, and Bresnahan scale (BBB). Tumor sizes were measured by magnetic resonance imaging. The histopathological analysis was performed to observe the growth of infiltrating tumors. All procedures involving animals were approved by the Ethics Committee of the Laboratory Animal Facility Biomedical Analysis Center, Tsinghua University (Beijing, China; approval No. 17-WGH1). Results: On postoperative 7 days, the experimental group presented with a significant progressive decrease in motor function (mean BBB score 15.00 ± 1.20) compared with the blank control group (20.67 ± 0.47, P < 0.01). All rats in the experimental group showed exponential tumor growth and had an average survival of up to 5 weeks after tumor cell implantation. The tumor sizes were 3.18 ± 0.21 mm3, 68.55 ± 3.38 mm3, and 345.28 ± 22.57 mm3 on postoperative 7, 14, and 28 days. The histopathological analysis illustrated that the growth of infiltrating tumors followed the longitudinal axis of the spinal cord. Conclusions: Thus, we have established a SD rat model of intramedullary spinal cord glioma, and we found that our findings are reproducible and homogeneous. These positive results provide solid bases for further studies of intramedullary spinal cord glioma.



How to cite this article:
Liu D, Yi G, Wang JJ, Wang G. Establishment and evaluation of a Sprague-Dawley rat model of intramedullary spinal cord glioma.Glioma 2021;4:42-48


How to cite this URL:
Liu D, Yi G, Wang JJ, Wang G. Establishment and evaluation of a Sprague-Dawley rat model of intramedullary spinal cord glioma. Glioma [serial online] 2021 [cited 2023 Feb 3 ];4:42-48
Available from: http://www.jglioma.com/text.asp?2021/4/3/42/330197


Full Text



 Introduction



Intramedullary spinal cord glioma, which accounts for approximately 30% of all primitive intramedullary tumors, has no evident boundary with normal spinal cord tissue.[1],[2],[3] Due to its infiltrative growth behavior, the rate of successful surgical resection of intramedullary spinal cord glioma is low, and the recurrence rate is high, whereas the rate of long-term survival is low.[1],[4],[5] Its current clinical treatment is mainly surgery supplemented by postoperative radiation and chemotherapy.[6],[7] However, the efficacy of adjuvant radiotherapy and chemotherapy for intramedullary spinal cord glioma remains controversial and has questionable survival benefits, and a definite standard chemotherapy regimen has not been established.[4],[8],[9],[10]

With the progress in molecular oncology, immunotherapy and targeted therapy are opening new possibilities for treating intramedullary glioma.[5],[6],[11],[12],[13] Therefore, well-established animal models for intramedullary spinal cord glioma can help promote translation from related basic therapy research to clinical applications.

In this study, we selected Sprague-Dawley (SD) rats and the C6 glioma cell line to establish a rat model of intramedullary spinal cord glioma, and we hope these studies can promising for future research of intramedullary spinal cord glioma.

 Materials and Methods



Experimental design

A total of 23 male SD rats were randomized into a blank group (n = 3) and an experimental group (n = 20) by using the method of random number table. The blank control group received intramedullary injection of complete Dulbecco's modified Eagle medium (DMEM), and the experimental group received intramedullary injection of DMEM containing C6 glioma cells. The neurological state of each rat was evaluated daily using the Basso, Beattie, and Bresnahan scale (BBB).[14] Magnetic resonance imaging (MRI) was performed to assess tumor sizes and, dynamic changes in tumor growth on postoperative 7, 14, 28, and 35 days. The maximal length, width, and height of the tumor were measured and the tumor volume was calculated as follows: Volume = (length × width × height)/2. The maximal length of tumor was <6 vertebral segments permitted by the ethics committee, and the maximal tumor length in our rat models was not exceeded.

Tumor line

The C6 glioma cell line (RRID: CVCL_0194) was gifted by the Central Laboratory of Beijing Tsinghua Changgung Hospital, China. The cells were cultured in DMEM (Hyclone Corporation, Shanghai, China) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Beijing Solarbio Science and Technology Co., Beijing, China) in an incubator at 37°C with 5% CO2 and saturated humidity.[15] On the day of operation, C6 glioma cells were harvested and diluted to a concentration of 1 × 108 cells/mL.

Animals

Twenty male SD rats (age 6 weeks, weight 220 ± 10 g) were purchased from Beijing Huafu Experimental Animals Inc. (China). The SD rats were bred and maintained under defined conditions at the Animal Experiment Center of the College of Medicine (specific pathogen-free grade) of Tsinghua University, China.

All procedures involving animals were approved by the Ethics Committee of the Laboratory Animal Facility Biomedical Analysis Center, Tsinghua University (Beijing, China; approval No. 17-WGH1) and reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments).

Surgical technique

The SD rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg, Huayehuanyu, Beijing, China) and placed in the prone position in a sterile area. Their backs were cleaned, shaved, and sterilized with betadine solution. The T9 spinous process was distinguished, and a 20 mm midline skin incision was made above the spinal process [Figure 1]. The T8–T10 spinous process was used as the reference point for the vertical line, and a micro-syringe needle (Hamilton Company, Reno, NV, USA) assisted by a stereotactic guidance system (Beijing Jiandeer Science and Technology Co., Beijing, China) was advanced approximately 1.5 mm through the dura. Then, a 5 μL cell suspension of DMEM containing C6 glioma cells (approximately 5 × 105 cells/mL) was slowly injected into the spinal cord over 3 min in the experimental group. The blank control group received a 5 μL intramedullary injection of complete DMEM. Finally, the muscles and skin were closed layer by layer.{Figure 1}

Functional testing

Functional testing of the hind limb motor function in SD rats was assessed using the BBB,[14] which was used to assess motor function recovery, ranging from 0 to 21. Higher scores represent better neurological function. Once the SD rats walked continuously in an open field, the examiner conducted a 4 min testing session using the BBB. To ensure that all SD rats had an initial score of 21, all were tested before surgery. Postoperatively, the rats were tested once daily for consecutive 35 days.

Radiographical assessment

The SD rats underwent MRI on postoperative 7, 14, 28, and 35 days. All images were collected with a Philips Achieva 3.0 TX (Royal Philips Electronics, Eindhoven, The Netherlands) using 16-channel flex coils and including axial and sagittal T1- and T2-weighted images (T1WI and T2WI, respectively), and gadolinium-diethylene triamine pentaacetic acid was used to enhance T1WI. The acquisition parameters were as follows: Repetition time, 3 s; echo time, 20 ms; section thickness, 3 mm.

Histopathological analysis

After MRI scanning on postoperative 35 days, the rats were sacrificed under anesthesia with an intraperitoneal injection of sodium pentobarbital, and the entire spinal cord was removed from the spinal canal. Tumors located by the position of intramedullary injection were taken and fixed in 4% paraformaldehyde for 24 h. Tissues samples were embedded in optical coherence tomography compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and stored at – 80°C. Serial 6 μm-thick frozen sections were prepared for histological analysis. Images were captured and analyzed using a digital slide scanner (AperioTechnologies, Vista, CA, USA).

Statistical analysis

Biochemical and histological analyses were conducted blinded to treatment, whereas microscopy was not performed blinded to the conditions of the experiments. No animals or data points were excluded from the analysis. No statistical methods were used to predetermine sample sizes; however, our sample sizes were similar to those reported in previous publications.[16],[17],[18] All data were statistically analyzed with SPSS 24.0 (IBM, Armonk, NY, USA). One-way analysis of variance, followed by the least significant difference test, was used for group comparisons. Survival analysis was performed using the Kaplan–Meier survival analysis, and a P < 0.01 was considered to indicate statistical significance.

 Results



Motor function and survival of rats

The experimental group showed a progressive decrease in motor function (BBB score 15 ± 1.2), compared with the blank group (20.67 ± 0.47) since postoperative day 7 [Figure 2]. The difference in BBB score between the experimental and blank groups was significant (P < 0.01). The SD rats in the experimental group started to die on postoperative day 14, and all died by postoperative day 35. However, all rats in the blank group survived at these time points. Thus, there was a significant difference between the groups in survival time (P < 0.01) [Figure 3].{Figure 2}{Figure 3}

Tumor sizes assessed by radiography

Efficacy evaluation was performed by MRI (Royal Philips Electronics, Eindhoven, The Netherlands), tumor volume was calculated using the three-dimensional imaging software. No tumor was seen in any of the MR images of the rats in the blank group. On postoperative day 7, the experiential group presented with mildly hypointense signals in the spinal cord on T1WI as well as hyperintense signals on T2WI with normal subarachnoid spaces. The contrast enhancement of T1WI showed local enhancement of an intramedullary lesion (3.18 ± 0.21 mm3) [Figure 4] and [Table 1]. On postoperative day 14, spinal cord transverse myelitis was seen in the experimental group with the disappearance of subarachnoid spaces. The contrast enhancement of T1WI showed obvious intramedullary lesions, and the mean tumor size was 68.55 ± 3.38 mm3 [Figure 5] and [Table 1]. On postoperative day 28, MRI revealed central canal dilatation, and diffuse infiltration of tumor cells followed the longitudinal axis of the spinal cord (345.28 ± 22.57 mm3) [Figure 6] and [Table 1]. On postoperative day 35, MRI revealed intraspinal metastasis in two rats in the experimental group [Figure 7].{Figure 4}{Figure 5}{Figure 6}{Figure 7}{Table 1}

Tumors assessed by histopathology

Histopathological examination of the blank group showed no evidence of tumors, and the rats in the experimental group progressively developed intramedullary glioma [Table 1]. The histopathological analysis illustrated that the growth of infiltrating tumors followed the longitudinal axis of the spinal cord [Figure 8]. Within the tumors, the cells were pleomorphic with enlarged nuclei and prominent nucleoli, and mitotic cells were also observed. In addition, extensive areas of the tumor had undergone necrosis at later time points.{Figure 8}

 Discussion



The few existing studies about animal models of intramedullary spinal cord tumors in the literature were established from different tumor cell lines. The first animal model of the intramedullary tumor was reported by Salcman et al. in 1984,[16] which was a canine model that involved intramedullary injection of brain tumor cells that were generated using the respiratory syncytial virus. Rabbit models with the intramedullary injection of VX2 squamous cell carcinoma cells have also been reported.[17] In 2006, Caplan et al.[18] established a Fischer rat model of spinal cord tumors by injection of 9 L gliosarcoma and F98 glioma cells. In 2012, Hsu et al.[19] established a novel rat model of spinal cord glioma using a cell line derived from human glioblastoma. Subsequently, the Fischer rat model of spinal cord glioma was further optimized by Zhuang et al.[20] Studies of these models demonstrate the feasibility of direct injections into the spinal cord. However, the lack of uniform size criteria for implantation sites and the number of different cell lines used has resulted in poor stability and reproducibility of models of intramedullary spinal cord glioma and has seriously limited the experimental value of these animal models. Establishing homogeneous tumor models is for basic research of intramedullary glioma.

In this study, we used the rat C6 glioma cell line for implantation in SD rat models to establish a new model of intramedullary spinal cord glioma, and we assessed tumors by histopathological examination and MRI. The procedures for establishing and evaluating this disease model have been optimized.

When constructing our animal models, a limited laminectomy was performed at the T9 level to expose the dura mater under an operating microscope to avoid spinal cord injury. The micro-syringe needle fixed on a brain stereo positioning instrument was advanced approximately 1.5 mm through the dura. Suspensions were slowly injected into the spinal cord over a 3 min period. Neither severe nor minor postoperative complications occurred in any SD rat. All rats in the experiential group had confirmed intramedullary glioma as illustrated by pathological analysis and MRI, without any signs of extramedullary lesions in the early stage, and all experiential rats showed exponential tumor growth and had an average survival of up to 5 weeks after tumor cell implantation.

Limitations

This study also has potential limitations. First, we selected only one glioma cell line and only SD rat to establish the rat model of intramedullary spinal cord glioma, the absence of more control groups; the lack of the small sample size may be considered limitations. Second, we did not verify the potential mechanism of tumorigenesis and glioma development in the SD rat model and need more in-depth studies.

 Conclusions



In summary, our study showed highly reproducible results for tumor development in all SD rats injected with C6 glioma cells. Furthermore, the highly synchronous and homogeneous results of our SD rat model could help guide future research of intramedullary spinal cord glioma.

Financial support and sponsorship

This project was supported by Beijing Municipal Science and Technology Commission (No. Z171100001017199) and the Chinese Society of Neuro-Oncology (No. CSNO-2016-MSD05). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional review board statement

All procedures involving animals were approved by the Ethics Committee of the Laboratory Animal Facility Biomedical Analysis Center, Tsinghua University (Beijing, China; approval No. 17-WGH1).

Conflicts of interest

There are no conflicts of interest.

Editor note: GW is an Editorial Board member of Glioma. He was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer review handled independently of this Editorial Board member and their research groups.

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