In Vitro Neural Injury Model for Optimization of Tissue-Engineered Constructs

Cullen, D.K., Stabenfeldt, S.E., Simon, C.M, and C. Tate. 2007. In Vitro Neural Injury Model for Optimization of Tissue-Engineered Constructs. Journal of Neuroscience Research 85: 3642-3651.

Summary:
Traumatic brain injury (TBI) is a common form of neurological damage caused by physical deformation of the brain. This particular type of injury can cause permanent damage to an individual’s cognitive, motor, or sensory functions. Current attempts to restore brain function via therapeutic means have shown only limited success, particularly because of our limited understanding of the intricate pathological mechanisms initiated by the body in response to TBI. It is known that a hostile environment is created as a result of trauma-related destruction of neural cells. One aspect of the body’s response can be reactive astrogliosis in which a glial scar is formed to effectively cut off injured tissue from the rest of the brain. This glial scar is composed of hypertrophic astrocytes that are characterized by an abundance of chondroitin-sulfate proteoglycans (CSPGs) and increased expression of intermediate filaments like glial fibrillary acidic protein (GFAP). Another main feature of post-injury environment is that cell death may be delayed for months after injury onset. Tissue engineering provides one potential approach for addressing such issues, as it could enable researchers to optimize cells to be transplanted into such a hostile environment as well as allow for prolonged treatment of this injury. As of now, however, the biggest obstacles to implementing effective methods in vivo have been the abundance of donor cell death and lack of integration of these cells with host brain tissue following transplantation.



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To confront the complications associated with in vivo cell transplantation in treating TBI, the authors of this paper developed a 3D in vitro model to mimic specific aspects of the hostile post-injury environment consistent with TBI. Cell cocultures were generated using neurons from embryonic day 17-18 rat fetuses and astrocytes from postnatal day 0-1 rat pups. These neuronal-astrocytic cocultures were initially plated at a neuron:astrocyte ratio of 1:1 and allowed to culture in vitro for 21 days in order to allow for maturation of the cells and formation of neural networks (Figure 1). The cocultures were then separated into three groups and either treated with transforming growth factor-β1 (TGF-β1, a cytokine shown previously to induce astrogliosis), subjected to mechanical deformation, or left untreated (control conditions). Mechanical deformation of cocultures was performed using a 3D cell-shearing device. The resulting heterogenous distribution of strains on cells was characteristic of the sort of mechanical injury that occurs in vivo via TBI. The cocultures that received TGF-β1 served as experimental controls by mimicking TBI-induced astrogliosis but without the associated precursor of cell death. This set-up allowed researchers to isolate how astrogliosis alone affects transplanted neural stem cells (NSCs). 48 hours post-insult (i.e. after treatment with TGF-β1 or mechanical loading), NSCs were delivered in medium to each of the cocultures.

First, the viability of astrocytes and neurons in the cocultures was assessed using a live-dead assay (Figure 2). A significant (i.e. P<0.05) decrease in the percentage of viable cells accompanied by increase in spatial density of dead cells following mechanical loading was found (as revealed by fluorescent confocal microscopy), but no significant change in cell viability amongst cocultures treated with TGF-β1 was observed. Immunochemistry was also employed to detect reactive astrogliosis by tracking markers for GFAP and CSPG, two substances known to be in abundance when astrocytes are undergoing this process of growth due to local neuron death. In fact, a significant increase in CSPG expression in the matrix was found post-insult only in TGF-β1-treated cultures (relative to controls). GFAP expression was used to examine both astrocytic reactivity and hypertrophy. In both the TGF-β1-treated cocultures and mechanically-deformed cocultures, the authors found a significant increase in GFAP immunoreactivity accompanied by an increase in astrocyte process density compared to cell in the control cocultures. Additionally, the viability of donor cells post-transplantation was examined using TUNEL staining, an assay that interacts with fragmented DNA of dying cells. A significant increase in the proportion of TUNEL+ NSCs was observed upon transplantation into the mechanically injured cocultures compared the other two coculture types (Figure 3). Thus factors specific to the mechanically-damaged environment but not the strictly astrogliotic environment were found to negatively impact NSC survival; but this same environment yielded no abnormal NSC differentiation patterns relative to the other two types of cocultures (Figure 4).

Based upon these findings, the researchers decided to further explore NSC viability upon transplantation into a mechanically-damaged environment. Specifically, they hypothesized that the method with which cells are delivered to the injury site could affect post-injury survival of donor cells. Furthermore, they suspected that tissue-engineered bioactive scaffolds could improve donor cell survival by adding structural and adhesive support for the cells. NSCs were delivered to both mechanically-damaged and control 3D cocultures via three different delivery methods (a media vehicle, a methylcellulose (MC) scaffold, or a methylcellulose-laminin (MC-LN) scaffold). Frequency of caspase activation was used to gauge the efficacy of delivery, since caspase activation signals the initiation of caspase-related apoptotic pathways. When NSCs were delivered in either the MC or MC-LN scaffolds to the mechanically-injured cocultures, a significant reduction in caspase activation was observed. The authors also simultaneously examined growth of donor cell processes and found that growth was significantly improved by the use of an MC-LN scaffold relative to use of a media vehicle. These results suggest that delivery of NSCs to mechanically-injured cocultures using MC-LN scaffolds decreases donor cell death attributable to caspase-apoptotic mechanisms and also promotes integration of these cells with the host environment.

Overall, the results of this paper showed that according to 3D in vitro models of brain tissue post-TBI, other factors affecting mechanical injury (beyond just astrogliosis) are responsible for donor cell death following delivery to injured brain tissue. Additionally, the use of bioactive scaffolds for donor cell delivery provide a promising option for improving donor cell survival and success post-transplantation.

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Significance:
According to current figures published by the U.S. Department of Defense, 10 to 20 percent of returning soldiers present symptoms characteristic of mild TBI, so one can see that this is a pressing medical issue that demands the attention of today's scientific and medical communities. This paper demonstrates the promising development of a 3D in vitro model to serve as a surrogate environment that mimics the biological response of brain tissue damaged via TBI and its possible contribution to engineering more optimal conditions for improved NSC survival upon transplantation. Typically, finding optimal donor cell conditions prior to therapeutic attempts has required expensive and time-consuming in vivo studies. The model developed by these authors, however, could possibly serve as a better alternative in the early stages of testing. Additionally, this paper provides the framework for the development of a model that is flexible enough to allow for trialwise elucidation of other factors hypothesized to affect NSC survival in vivo. Another aspect of this research is that it reinforced the possible contribution of optimized tissue-engineered scaffolds for the transplantation of donor cells to areas affected by mechanical injury. Overall, this paper provides a more feasible method for further improvement of transplantation strategies, strategies which might someday results in proven methodology for the treatment of TBI and other neurological deficits.

I chose this paper, because it suits my interest in traumatic brain injury research while also expanding upon many of the cell culture techniques/concepts we’ve learned in this class. As an undergraduate researcher, I conduct brain imaging and rehabilitation research and work primarily with individuals who have TBIs. But prior to discovering this article, I was virtually unaware of efforts being made in the tissue engineering field to regenerate damaged tissue crucial to our most important neural networks. So I found this paper very stimulating and promising!