The findings presented here address a significant gap in the microglia literature in the context of TBI, specifically the consequences of the primary-injury on microglia viability, morphology, and function. We demonstrate that rat primary microglia remain viable while displaying significant functional and morphological perturbations in response to a stretch-injury with a 20% strain magnitude.
The longitudinal assessment of media LDH from stretch-injured cells showed no significant changes at any post-injury time point examined compared to controls, demonstrating that stretch-injury does not result in altered membrane integrity or necrosis. Media levels of the intracellular enzyme LDH are an indicator of compromised membrane integrity and necrosis (26, 27). For example, stretch-injured rat cortical astrocytes exposed to injury parameters similar to those used in this study exhibit increased media LDH up to twenty-four hours after injury, suggestive of injury-induced membrane permeability (27). Additionally, stretch-injury does not induce apoptosis, as indicated by no changes in cleaved caspase-3 expression between injured and control cells. These findings are counter to what is found in stretch-injured astrocytes (12) and neurons (28). They can be attributed to differences in cellular size, morphology, and functional roles in the brain. Astrocytes are significantly larger than microglia and, as a consequence, have more surface area exposed to the strain forces, leading to increased membrane alterations (29). Similar to astrocytes, neurons vary significantly in cell diameter and morphology compared to microglia, with a larger cell body and long, delicate axons that are susceptible to damaging strain forces (10). Furthermore, microglia may be less sensitive to injurious strain forces than astrocytes and neurons because they are constantly surveying the brain through migratory and motile behavior. In contrast, astrocytes and neurons are adherents, stationary and non-migratory in non-pathological conditions, rendering them more susceptible to mechanical deformations. Taken together, these results suggest that stretch-injury does not alter viability or membrane permeability.
Stretch-injury significantly downregulated iNOS expression and decreased media levels of NO. These findings contrast to what is observed in patients and experimental models of TBI. They can be attributed to fundamental modeling differences and may indicate an innate response of microglia to the injury itself. To assess the effects of primary injurious forces on microglia, mono cultured are exposed to damage in a sterile environment, free from other damaged cells and damage-associated molecular patterns (DAMPS). Because of this unique environment, microglia activation and subsequent up-regulation of iNOS and NO production are not observed. Microglia upregulate the immune-response enzyme iNOS increasing the production of NO, an inflammatory mediator. Multiple experimental models of TBI (30–33) show a significant upregulation of iNOS following injury. Continuous production of NO, through increased iNOS expression and activity, can lead to cytotoxic oxidants, such as peroxynitrite, that contribute to oxidative stress (34–36). Multiple aspects of the process involved in NO production were evaluated in the current study. Media NO levels were significantly down-regulated at six, twelve, eighteen, and forty-eight hours after injury compared to uninjured cells.
Consistent with the decreased NO in the media, the expression of iNOS protein was significantly down-regulated twelve hours following injury. However, mRNA transcripts were unaffected by injury at the same time points compared to non-stretched cells. Interestingly, a rat model using a controlled cortical impact (CCI) showed that tissue NO is significantly decreased at two, six, and twelve hours following injury near the injury site. However, opposite to our iNOS findings, there was a significant increase in NADPH-diaphorase-positive cells, indicative of NOS activity, in the same regions where the NO decrease was detected. The authors attributed the reduction in NO to it being converted into perioxynitrite {Ziaja, 2007 #76}. Additionally, consistent with the findings of the in vivo study, we observed a significant decrease in media NO levels following injury; however, in contrast to the animal model, we found that the protein expression of iNOS is significantly decreased. This can be explained by the context of the in vitro environment, which lacks damaged associated factors that lead to microglial activation and increase iNOS expression. Future studies will evaluate the mechanism underlying the marked reduction in iNOS expression and determine if stretch-injury alters NO production in activated microglia. Our findings show that a stretch-injury suppresses media NO levels and down-regulates iNOS protein expression, suggesting that basal level NO production is affected by stretch-injury
The finding that stretch alters media NO and iNOS expression, but not iNOS mRNA transcription, is puzzling. A possible explanation could be that stretch-injury disrupts protein synthesis by inducing endoplasmic reticulum stress, which has been observed in blast (37) and fluid percussion injury (38) animal models of TBI. Additionally, rats exposed to a CCI model of TBI showed differentially methylated mRNA transcripts (39), a prominent post-transcriptional modifier of RNA, and has been shown to inhibit translation (40). Collectively, the data indicate that stretch-injury significantly down-regulated NO media production at multiple time points, and that this is mediated by a significant decrease in iNOS expression, which demonstrates that stretch-injury produces functional deficits in immune function.
Experimental models of TBI show that chronically activated microglia contribute to neuronal damage and dysfunction by producing free radical oxygen species that lead to cytotoxic oxidative stress (36, 41, 42). Following activation, microglia upregulate NOX2 and increase the production of ROS, that when combined with RNS, can form cytotoxic factors, such as peroxynitrite, that lead to protein nitrosylation resulting in cellular dysfunction or death. To evaluate the effect of stretch-injury on microglial ROS generation and oxidative stress, assessments of ROS, oxidative stress and the NOX2 enzyme were performed. Production of ROS was conducted by evaluating media and cellular levels twelve hours after injury. Interestingly, there was no effect of stretch-injury on ROS production compared to control cells. Next, western blot analysis showed that the expression of gp91, the membrane-bound component of the NOX2 complex, was unaffected by stretch-injury. Lastly, oxidative stress was evaluated by western blot for 3-NT, a marker of protein nitrosylation, specifically tyrosine residues. Consistent with the other ROS-related outcome measures, stretch-injury did not affect the oxidative stress response; specifically, protein nitrosylation, indicated by no changes between control and injured cells using western blot analysis of 3-NT. These results are surprising, given that multiple lines of research show both NO and ROS production are increased following microglial activation (43–45). Contrary to the RNS data, ROS does not appear to be affected by stretch-injury.
Following a TBI, activated microglia release both pro and anti-inflammatory associated cytokines (46). Multiple converging lines of research show that the post-insult inflammatory response in the brain presents with a pro and anti-inflammatory phenotype, and this duality varies temporally. For example, a rodent model of diffuse brain injury, where strain-induced injuries are prevalent, showed an increase in both pro (IL-1β, CD14 and iNOS) and anti (arginase-1) inflammatory mRNA transcripts at twenty-four hours post-injury (47). The effect(s) of the primary forces on the expression of microglial inflammatory-related genes is undefined. To address this gap in the literature, mRNA transcripts of inflammatory-associated genes were evaluated using RT-PCR at twelve hours post-stretch-injury. Interestingly, we found that both pro-inflammatory (IL-6) and anti-inflammatory (IL-10 and arginase1) associated genes were down-regulated following injury. This is counter to what is observed in experimental models and patients with TBI, where these genes are commonly upregulated. For example, plasma levels of IL-6 are significantly elevated acutely in patients who sustain a mTBI (48). In a rat model of experimental TBI, there was an immediate increase in brain IL-10 mRNA transcripts, peaking at four hours post-injury and continuing to rise until 20 hours post injury (49). In a mouse model of TBI, arginase-1 mRNA transcripts were significantly upregulated twenty-four hours following injury in both young (three- month old) and old (24-months old) animals (50). However, an important distinction between our findings and these in vivo observations is that, in our study, 1) only microglia are exposed to the stretch-injury, and 2) they do not interact with DAMPs or damaged cells and do not appear to be activated, either morphologically or functionally. Therefore, the upregulation of inflammatory genes would not be expected. Stretch-injury suppresses multiple genes associated with the inflammatory response following injury; the consequence and long-term ramification of this effect on microglial inflammatory response is an important question since dysfunctional inflammatory responses are observed in the chronically injured brain.
Stretch-injury decreases basal level microglial migration. Following the initial injury, microglia sense factors from the trauma and migrate to the injury site to remove debris and damaged cells (51). To date, the effect of stretch-injury on migration in the biomechanically injured brain is unexplored in the literature. To address this gap in the literature, we assessed migration following stretch-injury by using a cell exclusion assay and found that stretch-injury significantly reduced the number of cells that migrated into the exclusion zone at forty-eight and seventy-two hours post-injury. These findings show that a mechanical stimulus can reduce an essential functional behavior integral to the response to injury and necessary for homeostatic function. Future studies will elucidate the extent these migratory deficits play in a physiologically relevant setting.
Microglial morphology is significantly altered following exposure to stretch-injury. Following the primary injury, microglia undergo a morphological transformation characterized by a significant increase in soma diameter and reduction of processes to take on an amoeboid phenotype (52). Phase-contrast imaging showed that stretch-injury significantly reduced process length twelve hours after injury, demonstrating that a deformation event alone can rearrange the cytoskeleton. This is consistent with morphological alteration in neurons (53, 54), astrocytes (55), and endothelial (56) cells exposed to stretch-injury. The stretch-induced reduction in process length may contribute to the decreased number of cells migrated into the exclusion zone. Since migration involves remodeling of the cytoskeleton, one explanation could be that stretch-injury alters the cytoskeleton and impairs its remodeling during migration.
The microglial inflammatory response is not altered following treatment with LPS post-stretch-injury. To extend our model into a more physiologically relevant setting, where to mechanical forces are followed by exposure to inflammatory challenges, stretch-injured microglia were treated with LPS, a potent microglial activator, fifteen minutes following injury. Then media levels of NO and LDH were assessed at twenty-four, forty-eight, and seventy-two hours post-treatment to determine viability and activation. Surprisingly, the suppressive effect of stretch-injury on NO media levels was not observed in the post-stretch, LPS treated cells. Consistent with the LDH results from the time course assessment, LDH media levels were unchanged between any groups. The unexpected NO media result could be attributable to multiple issues. The first could be the potency of LPS as an inflammatory challenge.
Given that it is an endotoxin found on the outer membrane of gram-negative bacteria, it is expected that elicit a more robust inflammatory response than factors that are released during a TBI. For example, primary rat microglia treated with interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα), factors associated with brain trauma, display a varied inflammatory response compared to LPS. Compared to the IFNγ and TNFα treatment, LPS treated cells expressed significantly more NOS and arginase-1 mRNA transcripts and protein expression. Additionally, media NO levels were also considerably higher. The second issue could be the timing of the challenge post-injury. The effects of stretch-injury may not have enough time to alter the response to LPS. Future studies will challenge stretch-injured cells at immediate and delayed time points. These results suggest that stretch-injury does not change microglial inflammatory response when subsequently challenged; however, as noted above, multiple factors could account for these findings.
Phagocytosis is unaffected following stretch-injury. Multiple studies show that activated microglia significantly increase their phagocytic function compared to their resting cells {Fan, 2018 #78}{Hornik, 2016 #80}{Yi, 2020 #81}. A critical role of activated microglia in the post-injured brain is phagocytosing debris and dead and damaged cells {Loane, 2016 #5}. The assessment of phagocytosis on stretch-injured cells showed no difference in fluorescently label conjugated beads uptake between the control and stretch-injured group, twelve hours post-injury. Given that basal level phagocytosis in resting microglia is already low, it was not expected to see an effect of a stretch injury. An interesting observation was the relatively low number of phagocytosed beads in the non-activated cells cultured on Pronectin. This finding is consistent with a study showing that when primary rat microglia are incubated with fibronectin, there is a reduction in phagocytosis, as measure by the engulfment of zymogen particles. Furthermore, the same study showed that morphological phenotypes were consistent with our control cells, with cells displaying a reduced cell body diameter and an increased number of processes {Chamak, 1991 #79}.