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Tissue Engineering

Tissue Engineering - Poster Session C

Poster L17 - Investigating Effects of Fibrous Environment on Metabolic Activity of Reactive Astrocytes Under Tissue-Engineered Model of Traumatic Spinal Cord Injury

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Traumatic spinal cord injury (tSCI) is a life-altering condition resulting from brunt force to the vertebrae, often leading to severe impairment and disability. In the United States alone, there are approximately 18,000 new cases of tSCI every year with first year expenses of treatment costing from $429,348 to $1,315,554 (1,2). The chronic effects do not arise from the initial, acute injury, but from the cascade of reactions which ultimately forms a fibrotic core surrounded by reactive astrocytes. This shift in glial behavior is necessary for the control of site inflammation and facilitating wound healing, however, the glial scar’s physical presence disrupts adjacent neural activity. The glial scar is mainly composed from increased deposition of collagen I into the extracellular matrix (ECM). The integrin-β1 (ITGβ1) subunit in astrocytes then interacts with the increased density of collagen I fiber and initiates astrogliosis, which converts the non-reactive astrocytes into reactive (3). ITGβ1 is involved in the FAK/PI3K/AKT pathway which upregulates glucose metabolic activity, however, the specific metabolic pathways that they affect are not well understood (4). For better understanding of the specific changes to glucose metabolism in reactive astrocytes, a biomimetic model is constructed to simulate the high-density fibrotic environment of the glial scarring.

Materials and

Methods:  Collagen I isolation and spinal cord decellularization were both prepared following previously established methods (5,6).
The tSCI model was prepared by mixing collagen I and decellularized spinal cord pregel at a 1:1 ratio. This pregel was neutralized to 7.4 pH and embedded with normal human astrocytes (NHA). These pre-gel solutions were subject to cold- and warm-casting mimicking fibrous and healthy ECM microenvironment (7). Warm-cast gels were solidified at 37oC, and cold-cast gels were solidified at a gradual temperature ascent from 4oC, 22o, then 37oC. Embedded NHA in collagen I-spinal cord gel was cultured for 7 days in astrocyte growth media which was changed every 2 days.
Collagen I fiber thickness and markers of reactive astrocytes N-cadherin and alpha smooth muscle actin (α-SMA) were analyzed via immunofluorescence and confocal reflectance microscopy imaging. Average fiber thickness was measured by utilizing ImageJ, and the activity of the markers was measured from immunofluorescence via RGB analysis using custom MATLAB codes.
For observation of aerobic glucose metabolic activity, function blocking ITGβ1 antibody was added to half of the cold- and warm-cast samples. Optical redox imaging of astrocytes was performed to capture autofluorescence signals of metabolic cofactors NADH and FAD (8). The fluorescence intensity was then quantified and normalized to cell area using MATLAB. Optical redox ratio is calculated by the following formula: FAD/(FAD+NADH). A higher ratio causes cells to rely more on oxidative-phosphorylation while a lower ratio causes cells to rely more on glycolysis.

Results, Conclusions, and Discussions: Thickness of the collagen I fibers in the gel were measured from a reflectance image under the confocal microscope. Figure 1 presents that fiber diameter was significantly higher in the cold-cast sample compared to the warm-cast sample. The greater diameter is due to the gradual temperature ascent of the cold-cast samples which allow collagen I fibers to assemble to a greater thickness and density compared to the warm-cast samples. Therefore, compared to the warm-cast gel, the cold-cast gel better mimics tSCI lesions, which have high-density fibrotic environments due to glial deposition.
To assess the reactivity of the astrocytes in the cold- and warm-cast gels, markers α-SMA and N-cadherin were quantified under immunofluorescence and confocal microscopy. Figure 2 shows that the cold-cast, injury-mimicking sample had significantly higher fluorescence values for both α-SMA and N-cadherin. With a higher fluorescence of reactive astrocyte markers, the results suggest that the cold-cast gel has an increased presence of reactive astrocytes when compared to the warm-cast controls. The results overall suggest that when compared to the warm-cast sample, the cold-cast sample can mimic the denser fibrotic environment and astrocyte reactivity of a tSCI lesion.
For the quantification of NADH/FAD, ITGβ1 function-blocking antibody was introduced to inhibit ITGβ1 activity. The inhibition of ITGβ1 allows for the observation of ITGβ1’s involvement in the aerobic glucose metabolic activity through the FAK/PI3K/AKT pathway. Figure 3 shows that there is no significant change between the four conditions which suggests that both reactivity and ITGβ1 does not affect the aerobic glucose metabolic activity of astrocytes.
With the presence of reactive astrocytes in the cold-cast gel, further studies can be conducted to continue investigation into reactive astrocyte’s glucose metabolic activity. The phosphorylation of AKT in the ITGβ1/FAK/PI3K/AKT pathway should be compared with reactive and non-reactive astrocytes to observe whether reactivity affects the pathway. Additionally, more specific glucose metabolic pathways such as glycolysis, HMP shunt, and glycogenesis should be explored while testing with function-blocking ITGβ1 antibody. Investigation of these pathways under the tSCI biomimetic model will contribute to a better understanding of the specific effects of ITGβ1 on the glucose metabolic behavior of astrocytes.

Acknowledgements (Optional):  This work was supported by the National Institute of Health (R15NS121884, R37CA279722, P20GM139768), Arkansas Biosciences Institute, and the University of Arkansas Honors College Research Grant.

(1) Jain, N. B. et al. Traumatic Spinal Cord Injury in the United States, 1993-2012. JAMA 313, 2236 (2015).
(2) Traumatic Spinal Cord Injury Facts and Figures. National Spinal Cord Injury Statistical Center (2022).
(3) Hara, M. et al. Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury. Nat Med 23, 818–828 (2017).
(4) Ge, H., Tian, M., Pei, Q., Tan, F. & Pei, H. Extracellular Matrix Stiffness: New Areas Affecting Cell Metabolism. Front Oncol 11, (2021).
(5) Rajan, N., Habermehl, J., Coté, M.-F., Doillon, C. J. & Mantovani, D. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nature News (2007).
(6) Medberry, C. J. et al. Hydrogels derived from central nervous system extracellular matrix. Biomaterials 34, 1033–1040 (2013).
(7) Seo, B. R. et al. Collagen microarchitecture mechanically controls myofibroblast differentiation. Proceedings of the National Academy of Sciences 117, 11387–11398 (2020).
(8) Alhallak, K. et al. Optical imaging of radiation-induced metabolic changes in radiation-sensitive and resistant cancer cells. Journal of Biomedical Optics 22, 060502 (2017).