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The potential translation of mesenchymal stem cell (MSC) therapy into a multimodal protocol for traumatic brain injury requires evaluation of viability and cytokine production in a hyperosmolar environment. Optimization of MSC therapy requires delivery to the target area without significant loss of cellular function or viability. No model evaluating the potential efficacy of MSC therapy at varying osmolarities currently exists.
Rat MSCs were characterized with flow cytometric immunophenotyping. MSCs (passage 3) were placed in culture with multipotent adult progenitor cell media at varying osmolarities (250, 270, 290, 310, 330, 350 and 370 mOsm) potentially found with hypertonic saline infusion. After culture for 24 h, cellular viability was measured using flow cytometry (n = 6). Next, brain tissue supernatant was harvested from both normal rat brains and injured brains 6 h after cortical injury. Subsequently, MSCs were placed in culture with multipotent adult progenitor cell media ± 20% normal brain or injured brain supernatant (at the aforementioned osmolarities) and allowed to remain in culture for 24 h (n = 11). At this point, media supernatant cytokine levels were measured using a multiplex cytokine assay system.
MSCs showed no clinically significant difference in viability at 24 h. MSCs cultured with 20% injured brain supernatant showed an decrease in proinflammatory cytokine production (IL-1α and IL-1β) with increasing osmolarity. No difference in anti-inflammatory cytokine production (IL-4 and IL-10) was observed.
Progenitor cell therapy for traumatic brain injury may require survival and activity in a hyperosmolar environment. Culture of MSCs in such conditions shows no clinically significant effect on cell viability. In addition, MSC efficacy could potentially be enhanced via a decrease in proinflammatory cytokine production. Overall, a multimodal traumatic brain injury treatment protocol based upon MSC infusion and hypertonic saline therapy would not negatively affect progenitor cell efficacy and could be considered for multicenter clinical trials.
In the USA, 1.5 million patients suffer a traumatic brain injury (TBI) each year, resulting in 50,000 deaths with an additional 230,000 patients requiring hospitalization . The overall prevalence of TBI is estimated to be 6.5 million people . Of those patients affected, up to 48% are impaired by physical, cognitive and psychosocial deficits . Beginning aggressive rehabilitation early in the patient’s hospital course has been shown to lead to improvement in functional status [4,5]; however, neurons show little ability to repair and no treatment modality is currently available to reverse acute brain injury. A large body of work has failed to show significant efficacy from single-agent pharmacologic neuroprotective therapies. Therefore, the NIH recently convened a meeting to discuss the complex pathophysiology of neuronal injury and the failure of all trials based on current monotherapies (controlled hypothermia, hyperosmotic infusion) to date. Recommendations included the development of in vitro models to research multimodality treatments that could target several mechanisms of TBI’s complex pathophysiology [6,101]. Two therapeutic modalities currently under investigation for the treatment of TBI are the infusion of hypertonic saline (HTS) and progenitor cell therapeutics.
Preliminary research has shown that HTS infusion after TBI could offer potential neuroprotection. One possible pathway could be via a decrease in intracranial pressure (ICP). Both preclinical research using a dog model  and prospective randomized trials  have shown decreased ICP after HTS infusion without significant effect on cerebral blood flow (CBF). Additional investigation completed by Khanna et al. demonstrated that HTS infusion in a pediatric population was associated with improved CBF in accordance with a decrease in ICP . HTS resuscitation also increases mean arterial pressure, thereby attenuating a significant increase in poor neurological outcomes seen with hypotension within 6 h of TBI . Furthermore, Vialet et al. have shown HTS infusion to be more effective than mannitol as a hyperosmolar agent after TBI . Despite promising initial results, the effect of HTS infusion upon ICP remains controversial as alternate prospective trials have failed to show improvement in ICP control when compared with crystalloid (lactated ringer’s) infusion [12,13]. HTS infusion decreased the number of complications, the number of required interventions and length of intensive care unit stay after TBI in a pediatric population ; however, additional studies completed with an adult population failed to show a favorable effect on the required interventions . Despite the promising results noted in the acute and subacute setting, some trials have failed to show sustained improved neurological outcomes 6 months after TBI [6,15]. As a result of the controversial results derived from the initial preclinical and clinical trials, the Joint Section on Neurotrauma and Critical Care did not make any recommendations for the use HTS infusion for TBI ; however, the potential neuroprotection observed specifically in the pediatric population requires additional investigation.
A large amount of research has been completed to investigate the potential role of progenitor (stem) cell therapeutics for the treatment of TBI. While initial preclinical research has shown a potential benefit from progenitor cell therapeutics [17–20], the precise mechanism remains intensely controversial. Furthermore, the secondary injury observed with TBI is associated with an induction of the systemic inflammatory response. Analysis of rat brain supernatant from a TBI model has shown significant increase in the proinflammatory cytokines IL-1α, IL-1β, IL-6 and TNF-α in both the direct injury and penumbral areas . In addition, previous preclinical work has shown progenitor cells to migrate towards the site of injury and potentially modulate cytokine production. Co-culture of mesenchymal stem cells (MSCs) with purified immune cells has shown a decrease in proinflammatory cytokine production (TNF-α and IFN-γ) with an increase in production of the anti-inflammatory cytokines IL-4 and IL-10 . Therefore, MSCs could offer neuroprotection via modulation of locoregional proinflammatory cytokine production in accordance with the production of anti-inflammatory cytokines.
Previous research has shown HTS infusion and progenitor cell therapeutics to be ideal potential candidates for multimodal TBI treatment regimens. The potential translation of MSC therapy into such a multimodal protocol requires evaluation of cell viability and cytokine production in a hyperosmolar environment. Previous research has shown osmotic stress to induce apoptotic cell death in multiple cell lines including mononuclear cells , cardiomyocytes  and fibroblasts . Furthermore, hyperosmotic stress has been shown to increase proinflammatory cytokine production from human peripheral blood mononuclear cells . No model evaluating the potential efficacy of MSC therapy at varying osmolarities currently exists. We designed a series of in vitro experiments to investigate MSC viability and cytokine production while cultured at the osmolarities observed with HTS infusion for the treatment of TBI.
Mesenchymal stem cells (passage 3) were placed in culture with multipotent adult progenitor cell (MAPC) media with the varying osmolarities (250, 270, 290, 310, 330, 350 and 370 mOsm) potentially found with HTS infusion. After 24 h in culture, cellular viability was measured using flow cytometry (n = 6 samples per media osmolarity). Next, brain tissue supernatant was harvested from both normal rat brains and injured brains 6 h after cortical injury. Subsequently, MSCs were placed in culture with MAPC media ± 20% normal brain or injured brain supernatant (at the aforementioned osmolarities) and allowed to remain in culture for 24 h (n = 11 samples per media osmolarity). At this point, media supernatant cytokine levels were measured using a Bio-Plex cytokine assay system (Bio-Rad Laboratories, Hercules, CA, USA).
All protocols involving the use of animals were in compliance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Texas Institutional Animal Care and Use Committee (protocol HSC-AWC-07–055).
Unless otherwise indicated, all values are represented as mean ± standard error of the mean. Values were compared using analysis of variance (ANOVA) with a post-hoc Dunnett analysis using an osmolarity of 290 as the control. A p-value of ≤ 0.05 was used to denote statistical significance.
Mesenchymal stem cells were isolated from the bone marrow of Sprague-Dawley rats and expanded in MAPC media as previously described . Flow cytometric immunophenotyping was used to ensure that the MSCs were CD11b−, CD45−, CD29+, CD49e+, CD73+, CD90+, CD105+ and Stro-1+. Passage 3 cells were used for all experiments.
The osmolarity of MAPC media was measured using the 5500 vapor pressure osmometer (Wescor Inc., Logan, UT, USA). Adjustments in media osmolarity were completed by addition of sterile distilled water or sodium chloride.
Mesenchymal stem cells were removed from the culture plates and incubated with propridium iodide for 10 min. Using flow cytometry, the fraction of dead cells was calculated.
A controlled cortical impact (CCI) device (eCCI Model 6.3; VCU, Richmond, VA, USA) was used to administer a unilateral brain injury as described previously . Male Sprague Dawley rats weighing 225–250 g were anesthetized with 4% isoflurane and a 1:1 mixture of N2O/O2 and the head was mounted in a stereotactic frame. With the head held in a horizontal plane, a mid-line incision and subsequent 7- to 8-mm craniectomy was performed on the right cranial vault. The center of the craniectomy was placed at the midpoint between bregma and lambda, 3 mm lateral to the midline and overlying the tempo-parietal cortex. Animals received a single impact of 3.1-mm depth of deformation with an impact velocity of 5.8 m/s and a dwell time of 150 ms (moderate-to-severe injury) at an angle of 10° from the vertical plane using a 6-mm diameter impactor tip, making the impact orthogonal to the surface of the cortex. The impact was delivered onto the parietal association cortex. Sham injuries were performed by anesthetizing the animals, making the midline incision, and separating the skin, connective tissue and aponeurosis from the cranium. The incision was then closed. The body temperature was maintained at 37°C by the use of a heating pad. Previously obtained serial arterial PaO2 and PaCO2 measurements have shown that animals do not become hypoxic or hypercarbic during this procedure .
Rats were sacrificed 6 h after CCI or sham injury. Their brains were extracted and four regions, relative to the injury, were isolated: the site of direct injury, penumbral region, ipsilateral frontal region and contralateral region, as we have previously described . The sections were weighed to ensure each section was 120 mg, gently minced with a pellet pestle, diluted in 1 ml (low-glucose Dulbecco’s modified Eagle medium with 10% fetal bovine serum; Gibco, Carlsbad, CA, USA), vortexed for 30 s and centrifuged for 6 min at 1000 g. The supernatant, containing intracerebral, interstitial fluid, was collected . Of note, only the supernatant harvested from the direct injury region was used for stimulation of MSC cultures.
Cytokines were detected in the MSC media supernatant using the Bio-Plex cytokine assay system (Bio-Rad Laboratories, Hercules, CA, USA). Concentrations of IL-1α, IL-1β, IL-4, IL-6, IL-10 and TNF-α were simultaneously evaluated using a commercially available multiplex bead-based immunoassay (Rat 9-Plex; Bio-Rad Laboratories). The assay was performed per the manufacturer’s instructions and the details have been previously published by our group and others [29,30]. High standard curves (low RP1 target value) for each soluble cytokine were used, ranging from 2 to 32,000 pg/ml. A minimum of 100 beads per cytokine region were evaluated and recorded. Values with a coefficient of variation beyond 10% were not included in the final data analysis. All samples were run in duplicate.
Mesenchymal stem cells placed in culture with MAPC media at the various potential osmolarities observed with HTS therapy (250–370 mOsm) showed a narrow range of viability (91.4–93.9%). Statistical analysis using an osmolarity of 290 to represent a physiologic control showed difference for osmolarities of 250, 330, 350 and 370. Figure 1 outlines MSC viability at the varying osmolarities.
Table 1 shows the proinflammatory cytokine production (IL-1α, IL-1β, IL-6 and TNF-α) measured in the media (with and without 20% normal/injured brain supernatants) of MSC cultures after 24 h. Table 1 shows a significant (p = 0.004) decrease in IL-1α production at an osmolarity of 370 for MSCs cultured with injured brain supernatant (compared with the control value of 290). In addition, for MSCs cultured with injured brain supernatant there was a decrease in IL-1β production at serum osmolarities of 270 and 370 (p = 0.02) (Table 1). Further analysis failed to show a difference in the production of TNF-α or IL-6 when cultured in injured brain supernatant. In addition, no difference was observed for MSCs cultured in media alone or with 20% normal brain supernatant.
Table 2 shows anti-inflammatory cytokine production (IL-4 and IL-10) measured in the media (with and without 20% normal/injured brain supernatants) of MSC cultures after 24 h. No difference was observed for IL-4 or IL-10 at any osmolarity.
Multimodal protocols for the treatment of TBI could require the intravenous infusion of MSCs with HTS therapy. Optimal cell efficacy requires adequate viability and function at elevated serum osmolarities. Our data show that MSC viability (91.4–93.9%) is largely unaffected by the range of serum osmolarities (250–370 mOsm) that could be observed in a traumatically injured patient. While a significant increase in MSC viability is observed with increasing osmolarity, difference in viability fails to reach clinical significance.
The potential neuroprotection observed with HTS therapy after TBI could be derived from a decrease in proinflammatory cytokine production. Recent investigation has shown an increase in plasma matrix metalloproteinases (MMPs) as well as IL-6 after TBI in both animal models and injured patients [31,32]. MMPs are believed to contribute to further ischemic brain injury via breakdown of the blood–brain barrier. While initial work has shown controlled hypothermia to decrease the levels of MMP after TBI [32,33], little investigation into the effect of HTS has been completed. However, HTS therapy has been shown to mediate hepatic MMP production in a pancreatitis model and could offer neuroprotection via a similar pathway after TBI .
The observed benefit from MSC therapy for TBI could be explained by modulation of the locoregional or systemic inflammatory milieu . While still controversial, the implanted MSCs could be responsible for the production in anti-inflammatory cytokines that lead to enhanced neuroprotection. Our data show a decrease in production of the proinflammatory cytokines IL-1α and IL-1β with increasing osmolarity. In addition, the production of the anti-inflammatory cytokines IL-4 and IL-10 remains unaffected with an increase in osmolarity. These data show that increasing osmolarity does not affect MSC function in the form of anti-inflammatory cytokine production. In addition, increasing osmolarity may be of some benefit as evidenced by a decrease in the pro-inflammatory cytokine concentration, which could lead to enhanced neuroprotection.
Traumatic brain injury is a major burden on the healthcare system worldwide. Current therapy in the acute setting is supportive with no pharmacologic treatment to attenuate neuronal cell death available today. The development of multimodal treatment protocols to attack TBI’s complex pathophysiology at multiple points could offer the most effective form of neuroprotection. The combination of HTS infusion and progenitor cell therapies could lead to improved CBF as well as modulation of the inflammatory response leading to reduced neuronal death. Our data have shown that the intravenous infusion of MSCs into the hyperosmolar environment seen with HTS therapy has no clinically significant effect on cell viability. In addition, MSC efficacy could potentially be enhanced via a decrease in pro-inflammatory cytokine production. Overall, a multimodal TBI treatment protocol based upon MSC infusion and HTS therapy would not negatively affect progenitor cell efficacy and could be considered for multicenter clinical trials.
Traumatic brain injury
Hypertonic saline therapy
Progenitor cell therapy
Inflammatory cytokine production
Financial & competing interests disclosure
The authors are supported by grants NIH T32 GM 08 79201, M01 RR 02558 R21, Texas Higher Education Coordinating Board, Children’s Memorial Hermann Hospital Foundation and The Brown Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
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