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Basic Science Aspects

Remote Ischemic Conditioning Reduced Acute Lung Injury After Traumatic Brain Injury in the Mouse

Saber, Maha∗,†; Rice, Amanda D.; Christie, Immaculate∗,†; Roberts, Rebecca G.; Knox, Kenneth S.; Nakaji, Peter§; Rowe, Rachel K.∗,†,||; Wang, Ting; Lifshitz, Jonathan∗,†,||

Author Information
doi: 10.1097/SHK.0000000000001618

Abstract

INTRODUCTION

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide (1). It is a non-discriminatory event that can occur to an individual of any age, sex/gender, or socioeconomic background. Those at highest risk of TBIs are young males (compared with young females), soldiers, children between 0 and 4, and aged populations (>65) (1–3). Though research has focused largely on the effects of a TBI on the brain, an isolated TBI can also present with peripheral pathology and inflammation; TBI-induced pathophysiology can include increases in peripheral inflammation and damage to peripheral organs (4–7). Specifically, TBI can increase neutrophils and monocytes in peripheral organs and in the blood stream within hours and can last up to days or months post-TBI (4, 7, 8). Approximately 20% to 25% of severe TBI patients develop acute lung injury (ALI) within the first 24 h after injury in clinical settings, where the lungs are particularly susceptible to injury and inflammation (9, 10). With a ∼30% mortality, ALI worsens overall clinical outcome and decreases quality of life in TBI patients (11, 12). However, the mechanism of TBI-induced ALI remains poorly understood, which narrows treatment options (9).

ALI alone affects 200,000 patients each year in the United States, with a mortality rate of 29% to 42% and can manifest as acute respiratory distress syndrome (ARDS) in severe cases, though with the recent outbreak of coronavirus 2019 this rate may have increased (11). Variability in the mortality rate is due to the geographical variation, inadequate documentation, and under recognition of ALI (11). ALI is an inflammatory condition that disrupts the lung endothelial and epithelial integrity. It is characterized by increased permeability of the alveolar–capillary barrier, an influx of neutrophils, and increased lung edema (13). Though ALI can be caused by direct mechanical stress, such as aspiration, ventilation, or trauma, the underlying mechanism of ALI is increased inflammation in the lung (11). In ALI, neutrophils are the first immune cells to be recruited to the lung and play a key role in disease progression (13). Patients who survive ALI have a significant increase in long-term psychological and respiratory symptoms and reduced quality of life (12). Therefore, pulmonary edema and inflammation have been principal targets for treatment and improved outcomes of ALI.

Among the circulating active mediators of ALI is sphingosine-1-phosphate (S1P), a bioactive metabolite that regulates lymphocyte trafficking and endothelial barrier function (14). Activation of the predominant S1P receptor 1 (S1PR1) enhances endothelial integrity and reduces acute lung injury-associated edema (15, 16). As such, S1PR1 agonists reduced pulmonary edema during lung transplantation in patients (15). S1PR3, a less abundant S1P receptor, reduces endothelial integrity and promotes pulmonary edema (14). Though S1PR1 activation is associated with improved outcomes in ALI, a ratio of S1PR1/S1PR3 receptor activation may be necessary for the protective effects of S1P (17). Further, S1P and S1PR1 agonists can reduce ALI by increasing skeletal muscle released cytokines, known as myokines (18). Exogenous S1P increased myokine production and decreased hypoxia-induced metabolic dysfunction in a mouse model of lung injury (19). In contrast, antagonism of S1P-dependent pathways reversed the protective effects of myokines in a mouse model of myocardial ischemic damage (18). Myokines are released during exercise and have been used to prevent ischemia/reperfusion damage to the heart and lungs, increase brain function and neuroplasticity, and decrease inflammation (18–21). The myokine irisin, in particular, reduced lung damage in a mouse model of ALI (20). Irisin is cleaved from fibronectin type III domain-containing protein 5 (FNDC5) and can form heterodimers varying in molecular weight (22). Neonatal patients with ARDS have decreased irisin in serum compared with healthy controls, but increased levels of irisin in broncho-alveolar lavage fluid, liquid collected from the lungs for diagnosis of lung injury (20). These data suggest irisin, compared with other myokines, may be specifically involved with ALI recovery. Therefore, the induction of irisin in association with S1P/S1PR pathway activation holds potential to mitigate detrimental outcomes associated with TBI-induced ALI.

Remote ischemic conditioning (RIC) has been used as an intervention for lung injury by mimicking exercise-induced increases in irisin (20). RIC is the repeated cessation and reperfusion of blood flow to a distal limb that has been used to reduce ischemic damage from stroke, myocardial infarctions, organ transplant, and other inflammatory conditions (23–26). RIC is most commonly administered in three or four episodes of 5 or 10-min ischemia and reperfusion (23). In clinical settings, the use of RIC on patients has been associated with a blunted increase in TBI-induced biomarkers 24 h after treatment (27). Furthermore, RIC was effective in increasing survival rates and improved lung function in a mouse model of lung injury (20). With efficacy in both TBI and ALI, determining the therapeutic efficacy of RIC on TBI-induced ALI would be a natural extension of the field.

In this study, 6 to 8-month-old male mice were used to investigate ALI induced by diffuse TBI and whether RIC could reduce postinjury histopathology and inflammation in the lung. We hypothesized that TBI would increase neutrophil infiltration and edema in the lungs within 1-h postinjury and that RIC would reduce TBI-associated damage and inflammation by regulating S1P signaling and irisin expression in lung and plasma. We further hypothesized that evidence of lung injury would be evident in TBI mice up to 7 days postinjury (DPI), but not in uninjured sham or RIC treated mice.

MATERIALS AND METHODS

Rigor

Animal studies were conducted in accordance with the guidelines established by the internal Institutional Animal Care and Use Committee and the NIH guidelines for the care and use of laboratory animals. The Animal Research: Reporting In Vivo Experiments guidelines were followed while conducting and reporting this study (28). Surgeries were performed on a total of 72 animals. A total of 10 animals died from the injury. Furthermore, 11 animals were excluded from this study, where five animals were euthanized for technical failures (e.g., dura damage during surgery, loose hubs, etc.) and six were euthanized for falling outside of the predetermined inclusion criteria for righting reflex times (4–9 min). A total of 51 mice were used for data analysis. One cohort of 25 mice was euthanized at 1-h postinjury and the other cohort of 26 mice was euthanized at 7 DPI. One TBI RIC mouse was excluded from broncho-alveolar lavage (BAL) analysis and one TBI mouse was excluded from lung histology analysis at 7 DPI due to technical failure.

Animals

Male 6 to 8-month-old Hsd:ICR(CD-1) mice (Envigo Laboratories, Indianapolis, Ind) were used in all the experiments. Animals were housed in a 14 h light/10 h dark cycle at a constant temperature (23 ± 2°C) with food and water ad libitum. For mice euthanized at 7DPI, daily postoperative care was performed to include physical examination and documentation of each animal's condition for the first 3DPI.

Remote ischemic conditioning (RIC)

One hour prior to diffuse brain injury or sham injury, mice were randomized to RIC or no RIC groups. All mice were anesthetized for 45 s with 5% isoflurane. RIC mice had an orthodontic rubber band placed on the left hind limb using a hemorrhoidal ligator. Absence of blood flow was indicated by limb discoloration and the occluding band was left in place for a 5-min ischemic period while mice were allowed to ambulate in the holding chamber. After 5 min of ischemia with the band, the band was cut off using surgical suture scissors allowing a 5-min reperfusion period. This process was repeated four consecutive times for a total of 4×5 min ischemia with 5-min reperfusions between each session (23). Mice in the no RIC group received equivalent anesthesia (45 s every 10 min for four rounds) with no band applied to the limb. This model does not require direct surgical clamping of the femoral artery and mimics the use of a blood pressure cuff in clinical treatment (29).

Midline fluid percussion injury (mFPI)

Mice were subjected to mFPI consistent with methods previously described (4, 30, 31). Mice were anesthetized using 5% isoflurane in 100% oxygen for 3 min. The animal was placed in a stereotaxic frame with continuously delivered isoflurane at 2.5% via nose cone. Body temperature was maintained using a Deltaphase isothermal heating pad (Braintree Scientific Inc, Braintree, Mass). A midline incision was made exposing the skull and a 3 mm midline craniectomy was performed between bregma and lambda using a circular trephining tool. A modified Luer lock hub was glued directly over the exposed intact dura and reinforced with cyanoacrylate gel and methyl-methacrylate (Hygenic Corp, Akron, Ohio). The hub was filled with saline to ensure a complete seal. A cap made from a modified syringe tip was placed in the hub to prevent debris and air exposure overnight. Animals were placed in a heated recovery cage and monitored until ambulatory before they were returned to their home cage (approximately 10 min).

After 24 h, animals were re-anesthetized with 5% isoflurane delivered for 2 min to prepare for TBI induction. The cap was removed, and the hub was visually inspected for debris, an intact sinus, and no dura damage. The hub was then refilled with normal saline to create a continuous port of fluid from the injury device to the intact dura. The mouse was attached to the fluid percussion device (Custom Design and Fabrication, Virginia Commonwealth University, Richmond, Va) using extension tubing (Baxter, #2C5643). A toe pinch response was observed before the pendulum on the injury device was dropped from a predetermined height. This caused a fluid pressure pulse directly onto the dura to induce a diffuse brain injury. Hubs were removed immediately after sham or brain injury and mice were placed on their side on a heating pad. Apnea and seizure-like activity were recorded. The brain was inspected for uniform herniation, bleeding, formation of hematoma, and dura damage. Animals were monitored for the return of the righting reflex as a metric of injury severity (32). The righting reflex time was defined as the total time from the initial impact until the animal spontaneously righted itself from a lateral position. Brain-injured mice in this study had a righting reflex time of 4 to 9 min; six animals were excluded for not meeting these criteria. Sham mice were treated in the same manner without delivery of the injury. Sham animals spontaneously righted within 20 s, no shams were excluded for not righting within this time.

Blood, broncho-alveolar lavage (BAL) fluid, and tissue collection

Blood was collected from the submandibular vein into BD microtainer MAP microtubes coated with ethylene diamine tetraacetic acid. Mice were then injected with Euthasol (0.002 mL/g; Patterson veterinary, code: 07-805-9296). A BAL and left lung lobectomies were done to collect specimens for protein analysis. BAL fluid was collected via standard methods. Briefly, a midline incision at the neck was made and the trachea isolated by blunt dissection. The trachea was cannulated using a 22-gauge catheter that was secured in place using cotton suture. 1.5 mL of saline was delivered to the lungs via syringe and recovered via aspiration, this was repeated for collection of the total 1.5 mL volume administered. The left lobes of the lung were collected and flash frozen in liquid nitrogen for protein analysis. Mice were then transcardially perfused with phosphate-buffered saline (PBS) flush followed by 4% paraformaldehyde for 5 min. The right lobes of the lung were collected for histological analysis.

Immunohistochemistry

All samples used for immunohistochemistry were post-fixed in 4% paraformaldehyde in PBS for 24 h. Lungs were then switched into PBS with 0.5% sodium azide until processing. Directly before paraffin processing, lungs were circumferentially transected into multiple blocks perpendicular to the long axis of the organ, then placed into its own cassette. Lungs were dehydrated and infiltrated with paraffin (Surgipath Tissue Infiltration Medium) overnight. After processing, lungs were embedded in paraffin in separate tissue molds (Thermo Scientific Stainless Steel; 24 mm × 24 mm × 5 mm). Lungs were cut at 7 μm thickness and mounted on slides. Sections were deparaffinized via xylene washes (three times, 5 min) then rehydrated in sequential baths of 100% ethanol (two times, 10 min), 95% ethanol (two times, 2 min), 75% ethanol (two times, 2 min), 50% ethanol (5 min), and deionized water (two submersions each). Hematoxylin and eosin (H&E) were used to stain lung sections to visualize structures as previously described (33). Slides were dehydrated and cover slipped. Slides were imaged using the Keyence BZ-X800E digital microscope. Images were scored at ×10magnification by an expert blinded to the experimental conditions for inclusion in a weighted lung histology score (17). Briefly, three regions of each lung were given a score from 0 to 2 (two being the most severe) on presentation of five pathologies associated with ALI: (A) presence of neutrophils in alveolar space, (B) presence of neutrophils in interstitial space, (C) debris filling the airspace, (D) hyaline membrane deposition, and (E) septal wall thickening. The individual five scores were averaged among the three regions. Averaged scores were then weighted based on previous studies on TBI-induced lung injury (34). Weighted scores were calculated as follows:WeightedLungInjuredScore=((20×A)+(14×B)+(7×C)+(7×D)+(2×E))÷100

BAL cell counts

The BAL fluid was centrifuged, and the resulting cellular pellet was resuspended in PBS. A sample was used to estimate total cell counts using the BioRad TC10 automated cell counter. Remaining BAL fluid from each animal was transferred to slides via a Thermo Scientific cytospin 4. Slides were then stained with JorVet quick dip stain (Jorgenson labs: J0322) using standard protocols. Slides were then cover slipped, scored, and imaged. A student microscope was used by an experimenter blind to the conditions of the study to score the type of immune cells in the first 300 immune cells counted using a manual counter. The total neutrophils and monocytes out of the 300 cells are reported along with the neutrophil to monocyte ratio. Representative images were taken at ×40 magnification using the Keyence BZ-X800E microscope.

Western blots

Frozen samples were homogenized, cell lysates were sonicated, and the supernatant was collected. Total protein concentration was determined through a DC Protein Assay (Bio-Rad, Hercules, Calif). Samples were run in 4% to 12% gradient Bolt Bis-Tris Plus gels (Invitrogen, Carlsbad, Calif) at 100 V for 1.5 h then transferred to polyvinylidene difluoride membranes following transfer box manufacturer's directions (Bio-Rad, Hercules, Calif). Membranes were stained with Ponceau S (Boston BioProducts), blocked with 5% (weight/volume) bovine serum albumin (BSA) or milk (depending on optimized blocking buffer for primary antibody used) in Tris-buffered saline with Tween (TBST) for 1 h, then incubated with primary antibodies (Irisin: 1:1,000, NBP2-59680, Novus Biologicals; S1PR1: 1:1,000, 140952, Abcam, Cambridge, United Kingdom; S1PR3: 1:10,000, 108370, Abcam) in 5% BSA or milk in TBST overnight at 4°C. The membranes were washed and incubated in secondary antibody (donkey antirabbit: 1:5,000, SA1-200; Thermo Fisher, Waltham, Mass). Membranes were then incubated in horseradish peroxidase-linked secondary antibody in 5% BSA or milk at room temperature for 1 h before washes and incubation in enhanced chemiluminescence detection system. Blots were stripped and reprobed for all primary antibodies. The target band intensity was quantified using GeneTools software (Syngene). The target proteins were normalized by Ponceau S staining. Uneven light distribution while imaging was corrected by a background subtraction. Briefly, a representative area for each sample lane was chosen as the background by a researcher who was blinded to the conditions of the study. The intensity of this selected area was then subtracted from the whole lane intensity to produce the loading control volume for normalization calculations.

Myokine plate

Blood was collected and centrifuged for plasma within 2 h of submandibular vein collection. Plasma myokine concentrations were measured using commercially available Milliplex MAP mouse myokine magnetic bead panel (MMYOMAG-74K, Millipore Sigma). Manufacturer's instructions were followed, and all the samples were run in duplicate. Luminex magnetic beads containing fluorescent dye bonded to antibodies against the myokines irisin/FNDC5, myostatin, oncostatin M, follistatin- like 1, osteocrin/musclin, interleukin (IL)-6, and fractalkine were measured on a Bio-Plex 200 system (Bio-Rad, Hercules, Calif). Duplicate samples were averaged and analyzed among groups. Plasma samples from both cohorts were used for this analysis with a maximum of 38 animals allowed per plate. Therefore 8 sham mice, 11 TBI mice, 9 sham RIC mice, and 10 TBI RIC mice were used in this analysis.

Sphingosine-1-phosphate (S1P)

Circulating S1P was quantified by mass spectrometry at the Mass Spectrometry Laboratory at National Jewish health (Denver, Colorado; https://sites.google.com/site/lipidomicsandmetabolomicsatnjh/). Briefly, S1P in the mouse plasma was extracted and quantified using a liquid chromatography–tandem mass spectrometry, as previously described (35). The instrumentation used was an API4000 Q-trap hybrid triple-quadruple linear ion-trap mass spectrometer (Applied Biosystems, Foster City, Calif). S1P was analyzed as bis-acetylated derivatives. Repeated blood draws at baseline, 1 h postinjury, 1DPI, and 7DPI were taken from all animals from the 7DPI cohort (Fig. 1A), but only 16 were used for S1P measurement: 3 sham, 5 TBI, 3 sham RIC, and 5 TBI RIC.

F1
Fig. 1:
A schematic of the study design. (A) Briefly, mice assigned to the 7DPI cohort were bled using the submandibular vein at 2 days before injury as a baseline. Male CD1 mice were subjected to either diffuse TBI by midline fluid percussion or sham injury and randomly assigned among four groups: sham, TBI, sham RIC, or TBI RIC; RIC was performed 1 h prior to TBI. Mice were euthanized at 1-h postinjury or 7DPI and lung tissue for histology and protein quantification, bronchoalveolar lavage (BAL) fluid, and blood were collected. (B) Righting reflex times were recorded at the time of injury. Mice included in the study had righting reflex times between 4 and 8 min, and sham animals spontaneously righted within 20 s. (C) A Kaplan–Meier survival curve shows mortality between TBI and TBI RIC mice within 1 h of TBI. (D) Histological representation of normal, non-pathological lung in CD1 mice. (E) Histological representation of lung from a CD1 mouse that died of TBI-induced pulmonary edema. Scale bar 50 μm for all micrographs. TBI indicates traumatic brain injury; RIC, remote ischemic conditioning; 7DPI, 7 days postinjury.

Statistical analysis

Results were analyzed using GraphPad Prism 6 (La Jolla, Calif) and are shown as mean ± SEM, with statistical significance assigned at the 95% confidence level (α < 0.05). Righting reflex times were analyzed using a two-tailed unpaired t test. A Kaplan–Meier survival curve was used to analyze mortality in TBI and TBI RIC. Western blot, lung histology scores, BAL cell counts, and myokines were analyzed using a two-way analysis of variance (ANOVA) followed by Tukey multiple comparisons test. S1P time course was analyzed using a repeated measure two-way ANOVA followed by Dunnet multiple comparisons test. Grubb test was used to determine outliers and significant outliers were excluded. One sham was excluded from IL-6 statistical analysis, one sham RIC was excluded from osteocrin analysis, and one TBI RIC was excluded from irisin analysis. Resulting test values are included in the Results section where main effects of TBI and RIC and interactions are reported. Significant differences (P < 0.05) between groups after post hoc testing are indicated by an asterisk between groups. Significant main effects are distinguished by asterisks above TBI groups (for a main effect of TBI) and RIC groups (for a main effect of RIC) when post hoc testing between individual groups was not significant.

RESULTS

Righting reflex time of diffuse brain-injured mice was the same regardless of RIC

In this study, ALI was investigated following a diffuse brain injury by mFPI in the mouse and righting reflex time indicated injury severity. We previously reported on the suppression of the righting reflex in mice following mFPI as an injury-induced deficit and indicator of injury severity (31). The average righting reflex for this study was 315.5 s ± 20.82 for TBI mice and 317.6 s ± 21.73 for TBI RIC mice. Sham animals spontaneously righted within 20 s. There were no differences in the righting reflex times between the TBI mice groups (t(29) = 0.072, P = 0.944; Fig. 1B). There was a 24% mortality in TBI mice and 20% mortality in TBI RIC mice due to mFPI. However, there was no significant difference in survival rates between either group (χ2 = 0.0001, P = 0.99; Fig. 1C). Furthermore, all mice that died, died within 10 min of injury. In TBI mice that died, a lung injury was confirmed by pulmonary edema, leukocyte infiltration, and alveolar disruption. Histological representative images of lungs from a normal (non-TBI) mouse (Fig. 1D) and a TBI mouse (Fig. 1E) have been included.

RIC reduced TBI-induced lung injury

Sham RIC mice had intact alveoli sacs and blood vessels, with some neutrophil mobilization at 1 h after sham injury (Fig. 2, A1–A4) compared with sham mice without RIC that had intact alveoli sacs and blood vessels and no appreciable neutrophil mobilization at 1 h after sham injury (Fig. 2, B1–B4). However, TBI mice lungs showed fluid-filled alveoli sacs and neutrophil infiltration at 1 h postinjury (Fig. 2, C1–C4). TBI RIC mice lungs had some fluid-filled alveoli sacs and neutrophil mobilization at 1 h postinjury (Fig. 2, D1–D4). Overall, TBI mice had distinct ALI pathophysiology. Quantification of the histology captured the observations, where TBI mice had significantly higher lung histology scores (F(1, 20) = 9.298, P = 0.006), there was no difference between RIC mice (F(1, 20) = 0.597, P = 0.449), and no significant interaction between RIC and TBI (F(1, 20) = 2.352, P = 0.141; Fig. 2E).

F2
Fig. 2:
Representative micrographs of lungs from mice at 1 h after sham injury or brain injury.

RIC intervention prevented TBI-increased neutrophils in BAL fluid

BAL cells were isolated and stained with Diff-Quik (Fig. 3A). Automated cell counts from BAL fluid showed that TBI mice had the highest average cell numbers, though there were no significant differences in cell number among any groups either due to TBI (F(1, 20) = 3.378, P = 0.081) or RIC (F(1, 20) = 1.834, P = 0.191) and no significant interaction between TBI and RIC (F(1, 20) = 3.116, P = 0.093; Fig. 3B). A researcher-blind to the experimental groups scored the immune cells from the BAL slides to distinguish, monocytes, neutrophils, and lymphocytes. There were no significant differences in the number of monocytes in BAL fluid among any groups due to TBI (F(1, 20) = 1.637, P = 0.215) or RIC (F(1, 20) = 0.254, P = 0.620) and no significant interaction between TBI and RIC (F(1, 20) = 0.757, P = 0.395; Fig. 3C). TBI mice had significantly more neutrophils compared with sham mice in their BAL fluid (F(1, 20) = 19.070, P = 0.0003). Furthermore, TBI RIC mice had significantly fewer neutrophils than TBI mice (F(1, 20) = 7.618, P = 0.012) with a significant interaction between RIC and TBI (F(1, 20) = 9.592, P = 0.006; Fig. 3D). In addition, the ratio between neutrophils and monocytes was significantly higher in TBI mice compared with sham mice (F(1, 20) = 10.710, P = 0.004). RIC-treated mice had significantly lower neutrophil to monocyte ratios when compared with TBI mice (F(1, 20) = 5.515, P = 0.029) with a significant interaction of RIC and TBI (F(1, 20) = 6.337, P = 0.021; Fig. 3E). These data suggest that TBI-induced neutrophil infiltration, a key feature of ALI, is reduced by RIC intervention.

F3
Fig. 3:
Representative images of BAL fluid from sham, TBI, sham RIC, and TBI RIC mice at 1 h.

RIC protected against the TBI-associated changes in levels of S1PR3 and irisin in lung tissue

S1PR isoforms and irisin protein levels were analyzed by western blot in lung tissue and normalized to total protein (Ponceau S) at 1 h postinjury (Fig. 4A). TBI mice had lower S1PR1 levels compared with shams (F(1, 20) = 6.030, P = 0.023), regardless of RIC (F(1, 20) = 0.022, P = 0.880) but no significant interaction between RIC and TBI (F(1, 20) = 0.180, P = 0.676; Fig. 4B). TBI mice had significantly more S1PR3 levels in their lungs compared with sham mice (F(1, 20) = 9.298, P = 0.006). However, TBI RIC mice were not significantly different from either sham group (F(1, 20) = 0.597, P = 0.449), and there was no significant interaction between TBI and RIC (F(1, 20) = 2.352, P = 0.141; Fig. 4C). Irisin is cleaved from the protein FNDC5 (∼143kDa) into products of ∼15kDa and ∼27kDa molecular weights. TBI mice had significantly more FNDC5 in their lung compared with sham (F(1, 20) = 11.180, P = 0.003) and TBI RIC mice (F(1, 20) = 1.011, P = 0.327) with a significant interaction between TBI and RIC (F(1, 20) = 11.580, P = 0.003; Fig. 4D). Furthermore, TBI mice had significantly more irisin (27kDa) in their lungs compared with sham mice (F(1, 20) = 9.344, P = 0.006), and TBI RIC mice were not significantly different from either sham group (F(1, 20) = 1.426, P = 0.246), with no significant interaction between TBI and RIC (F(1, 20) = 3.931, P = 0.061; Fig. 4E). TBI mice had significantly more irisin (15kDa) in their lungs compared with sham mice (F(1, 20) = 15.040, P = 0.001), but TBI RIC mice did not have significantly different irisin (15kDa) levels compared with sham groups (F(1, 20) = 0.100, P = 0.755), with no significant interaction between TBI and RIC (F(1, 20) = 3.416, P = 0.079; Fig. 4F).

F4
Fig. 4:
Representative western blots of lung tissue at 1 h from sham, TBI, sham RIC, and TBI RIC mice for S1PR1, S1PR3, FNDC5, irisin (27 kDa), and irisin (15kDa) with Ponceau S as the loading control.

TBI-induced lung injury remained at 7DPI

After 7DPI, sham RIC mice had intact alveoli sacs and blood vessels with some neutrophils present (Fig. 5, A1–A4) as compared with normal lung histology in sham mice (Fig. 5, B1–B4). Lungs from TBI mice had fluid-filled alveoli sacs and neutrophil infiltration still present at 7DPI (Fig. 5, C1–C4). Lungs from TBI RIC mice had some neutrophils still present at 7DPI, but no obvious filled alveoli (Fig. 5, D1–D4). When quantified with the weighted lung histology score, there was a significant interaction between TBI and RIC (F(1, 21) = 5.348, P = 0.0310), without main effects of TBI (F(1, 21) = 0.001, P = 0.970) or RIC (F(1, 21) = 2.034, P = 0.169; Fig. 5E).

F5
Fig. 5:
Representative micrographs of lungs from mice at 7DPI.

TBI-induced increase in BAL neutrophils resolved by 7DPI

BAL fluid was stained to visualize immune cells; monocytes and neutrophils were counted at 7DPI (Fig. 6A). Automated cell counts of BAL fluid showed no significant differences in cell numbers among any groups either due to either TBI (F(1, 21) = 0.934, P = 0.345) or RIC (F(1, 21) = 0.323, P = 0.576) and no significant interaction between TBI and RIC (F(1, 21) = 0.582, P = 0.454; Fig. 6B). There were no significant differences in the number of monocytes in BAL fluid among any groups due to TBI (F(1, 21) = 1.197, P = 0.286) or RIC (F(1, 21) = 0.115, P = 0.738) and no significant interaction between TBI and RIC (F(1, 21) = 0.916, P = 0.350; Fig. 6C). Furthermore, there were no significant differences in neutrophil cell numbers in BAL fluid among any groups due to TBI (F(1, 21) = 2.144, P = 0.158) or RIC (F(1, 21) = 2.885, P = 0.104) and no significant interaction between TBI and RIC (F(1, 21) = 0.003, P = 0.954; Fig. 6D). The ratio between neutrophils and monocytes was not different in BAL among any groups due to TBI (F(1, 21) = 1.23, P = 0.280) or RIC (F(1, 21) = 3.376, P = 0.080) and no significant interaction between TBI and RIC (F(1, 21) = 0.001, P = 0.973, Fig. 6E).

F6
Fig. 6:
TBI-induced increase in neutrophils resolved by 7DPI.

S1PR3 is upregulated in TBI mice at 7DPI

S1PR and irisin proteins were quantified in lung tissue and normalized to Ponceau S at 7DPI (Fig. 7A). There were no differences in S1PR1 among any of the groups due to TBI (F(1, 22) = 3.402, P = 0.079) or RIC (F(1, 22) = 0.132, P = 0.719) and no significant interaction between TBI and RIC (F(1, 22) = 0.780, P = 0.402; Fig. 7B). TBI mice had significantly more S1PR3 expression in their lungs compared with sham mice (F(1, 22) = 4.570, P = 0.044), regardless of RIC (F(1, 22) = 0.011, P = 0.919) and there was no significant interaction between TBI and RIC (F(1, 22) = 4.198, P = 0.052; Fig. 7C). There were no differences in FNDC5 levels among any of the groups due to TBI (F(1, 22) = 1.430, P = 0.245) or RIC (F(1, 22) = 0.487, P = 0.492) and no significant interaction between TBI and RIC (F(1, 22) = 0.921, P = 0.348; Fig. 7D). There were no differences in irisin (27 kDa) levels among any of the groups due to TBI (F(1, 22) = 2.699, P = 0.115) or RIC (F(1, 22) = 0.046, P = 0.8316) and no significant interaction between TBI and RIC (F(1, 22) = 1.844, P = 0.188; Fig. 7E). There were no differences in irisin (15 kDa) levels among any of the groups due to TBI (F(1, 22) = 2.911, P = 0.102) or RIC (F(1, 22) = 0.424, P = 0.522) and no significant interaction between TBI and RIC (F(1, 22) = 2.601, P = 0.121; Fig. 7F).

F7
Fig. 7:
Representative western blots of lung tissue at 7DPI from sham, TBI, sham RIC, and TBI RIC mice for S1PR1, S1PR3, FNDC5, irisin (27 kDa), and irisin (15 kDa) with Ponceau S as the loading control.

Neither TBI nor RIC changed irisin or S1P levels in plasma

Plasma irisin was measured at 1-h postinjury by multiplex immunoassay in both cohorts and was not significantly different among any groups due to TBI (F(1, 33) = 0.316, P = 0.578) or RIC (F(1, 33) = 0.573, P = 0.454) and there was no significant interaction between TBI and RIC (F(1, 33) = 1.274, P = 0.267; Fig. 8A). In the plasma S1P time course, there were no significant differences among any groups at any timepoint, though there was an overall decrease of S1P over time (F(1.806, 21.67) = 13.15, P = 0.0003). Also, TBI RIC mice had lower S1P levels at 1-h postinjury and 7DPI compared with baseline levels (F(12, 36) = 2.379, P = 0.022, 1-h postinjury: post hoc: q = 0.016, 7DPI: q = 0.033; Fig. 8B). Other myokines (myostatin, oncostatin, follistatin-like 1, osteocrin/musclin, IL-6, fractalkine) measured were not significantly different among groups at 1-h postinjury (Supplemental Figure 1, https://links.lww.com/SHK/B98).

F8
Fig. 8:
No significant differences in plasma irisin and S1P at 1-h postinjury, but a significant difference of S1P at 7DPI in TBI RIC mice compared with baseline levels.

DISCUSSION

ALI is a critical condition that can lead to death or reduced quality of life with very few treatments or preventive options. This study provides evidence that a single-occurrence TBI can lead directly to lung damage within an hour of injury and can persist up to 7DPI. Interventions specifically for TBI-induced lung injury are necessary to reduce secondary complications from injury and increase quality of life in TBI patients. Thus, we evaluated the therapeutic efficacy of RIC to mitigate TBI-induced ALI. The results provide evidence that RlC attenuated the development of ALI. Furthermore, the resolution of ALI at 7DPI reinforces the acute effect of TBI on the lungs and justifies further investigations of early postinjury time points to improve survival. Though RIC did not protect against TBI mortality, it did protect against the TBI-associated lung pathology observed in the alveolar sacs and with neutrophil infiltration in BAL. In previous studies, TBI disrupted alveolar capillary membranes in the lung, increased neutrophil infiltration in the lung, and increased pulmonary edema with the first 4 h after controlled cortical impact (6). Our current study provided further evidence that TBI-induced lung injury can be found as early as 1-h post-TBI with increased neutrophil populations compared with shams present in the lungs. Therefore, targeting inflammation to reduce TBI-induced ALI is a viable treatment option. RIC has been shown to modulate inflammation in ischemia/reperfusion lung injury (20, 36, 37). This current study provides further evidence that RIC can be an effective intervention for TBI-induced ALI as RIC animals had significantly fewer neutrophils in BAL fluid. However, further studies on the mechanism of TBI-induced ALI and how RIC intervention can modulate inflammation are needed. Data in this current study suggest that S1P-associated pathways may be involved with the effectiveness of RIC on TBI-induced ALI.

In this study, we found a TBI-associated decrease in S1PR1 in the lung at 1-h postinjury regardless of RIC, which was resolved by 7DPI. This is the first study to show significant changes of S1PR1 in lung tissue after TBI. Previous studies of TBI and S1PR1 have focused on brain tissue expression (38, 39). In a rat cortical impact model, S1PR1 expression increased in the hippocampus, peaking at 7DPI (39). S1PR1 is also associated with worsened outcome of TBI, where introduction of S1PR1 agonist reduced TBI-induced brain lesions, T-cell infiltration, reactive astrocytes, and multiple cytokines (39). Furthermore, treatments used to reduce ALI-associated increased vascular permeability may work by preserving S1PR1 (40). Though S1PR1 agonists can improve TBI-induced neuroinflammation and inflammation in models of lung injury, whether S1PR1 is involved in TBI-induced lung inflammation is unknown. S1PR3 in plasma is a biomarker for ALI in both humans and mice (14). Though there was no RIC effect on S1PR1 found in this study at 1-h postinjury, S1PR3 levels in lung were significantly higher only in TBI groups compared with shams; there was no significant difference in S1PR3 expression between TBI RIC and sham mice. And so, RIC prevented TBI-induced increases of S1PR3, but not TBI-associated decreases in S1PR1. Mechanisms of RIC may be specific to S1PR3 rather than S1PR1. In previous studies, higher plasma S1PR3 concentrations were found in patients with pulmonary edema compared with healthy controls and were associated with increased sepsis and ALI mortality (14). Though little is known about S1PR3 in TBI, mouse models of cerebral ischemia have confirmed that S1PR3 contributes to brain injury, where S1PR3 agonists reduced lesion size, neuroinflammation, and neurodegeneration (41). Furthermore, in a clinical study where RIC was administered after subarachnoid hemorrhage, S1PR3 was differentially expressed in whole blood collected after RIC compared with blood taken from the same patients before RIC was administered (42). However, the exact abundance or directionally of this differential expression was not reported and only 10 out of 13 patients saw improved clinical functional outcomes or no change, while three patients had worsened outcome over time (42). The previous reports, coupled with the current data, suggest that TBI-induced pathophysiology may include S1PR1 and S1PR3, such that RIC may effect only S1PR3 expression. And so, more studies on TBI-induced S1PR1 and S1PR3 expression in the lung will be necessary to determine the exact mechanism.

S1PR3 is associated with neuroprotective and anti-inflammatory myokine expression. In this study, there was no significant difference in plasma irisin/FNDC5 expression at 1-h postinjury between any groups; however, irisin/FNDC5 expression was more in lungs from TBI mice compared with shams. Thus, irisin may be sequestered in the lung after TBI. Irisin can ameliorate detrimental outcomes associated with both TBI and ALI (20, 43, 44). Irisin pretreatment reduced lipopolysaccharide (LPS)-induced ALI in a mouse model (43). This protection is associated with the preservation of mitochondria in the lung (20). In a mouse model of stroke, irisin was lower in plasma and muscle compared with shams (45). However, the concentration in lung and brain was not measured (45). Here, no differences in plasma irisin were observed; however, levels were trending to be different in TBI groups compared with sham. Other studies have suggested irisin may travel through the blood stream to the site of injury, though future studies will be necessary to determine if that occurs after TBI (20). Further, irisin levels did not change in TBI RIC groups but irisin/FNDC5 levels were significantly lower in TBI RIC mice compared with TBI mice. Previous studies suggested that RIC effectiveness is due to irisin release (20); however, in the current study, RIC did not change irisin levels in the plasma or lungs. Our data suggest that irisin is involved in TBI pathophysiology and RIC efficacy, though the exact mechanism is still unknown.

Though this study provides evidence for the potential use of RIC in TBI-induced ALI, more evidence will be necessary before clinical application. Frist, this study was limited by the selected timepoints, both in terms of the pre-injury RIC application and the protracted outcomes at 1 h and 7 days. Further, the mechanistic and therapeutic efficacy investigations of S1P and irisin though rational, lent limited support for group differences. Possibly, the half-life of S1P in plasma (approximately 15 min) was contraindicated with the study timeline (46). Though there was no increase in plasma irisin, irisin remains a potential therapeutic mechanism or pharmacodynamic measure, as levels were significantly increased in the lung after TBI. Sequestration of irisin in the lung may indicate injury and/or RIC response. Therefore, future studies to evaluate exogenous irisin as a therapeutic intervention for TBI-induced ALI are warranted. Furthermore, we hypothesized that RIC would reduce inflammation. Complementary pharmacological compounds can investigate the extent to which inflammatory pathways drive RIC treatment efficacy in TBI-induced ALI. For example, Enoxaparin, a blocker of extracellular vesicle -mediated inflammasome signaling, reduced TBI-induced ALI in a controlled cortical impact model of TBI in the mouse (6). RIC and enoxaparin may be synergistic in achieving therapeutic efficacy for TBI-induced ALI. Lastly, both pre- and post-conditioning reduce inflammation in a mouse model of sepsis (37), which justifies investigations into postinjury RIC for TBI-ALI. Further studies will add evidentiary support to implement RIC interventions in TBI-induced ALI.

The primary objective of this study was to determine the therapeutic efficacy of RIC to reduce TBI-induced ALI. Since TBI can occur to an individual of any age or sex, studying the effect of RIC on TBI-induced ALI in different populations will be necessary. This study focused on one sex (male) to reduce variability and sex differences because females and males respond differently to both TBI and RIC (47–50). Further studies will determine whether TBI induces ALI in females, and the therapeutic efficacy of RIC intervention. And, as with all preclinical investigation, additional models and strains will complete the profile of TBI-ALI and opportunities for RIC treatment (32, 51). A study comparing strains of mice in the development of LPS-induced ALI determined that CD-1 mice were most susceptible to developing ALI within 4 and 24 h compared with A/J, Balb/c, DBA/2J, C57BL/6J, DBA/1J, NMRI, and C3H/HeN mice (51). Furthermore, CD-1 mice have a more robust neuroinflammatory response to LPS injections than C57BL/6 mice (32). Therefore, CD-1 mice were justified in this initial study on TBI-ALI. With the ultimate goal to develop targeted pharmacotherapy, the results should be reproduced in the C57BL/6 strain. A selective S1PR3 inhibitor or S1PR3 transgenic mouse (tissue-specific knockout in lung tissues) can then be used to advance mechanistic studies.

CONCLUSIONS AND FUTURE DIRECTIONS

TBI-induced ALI is a major health concern that can complicate TBI recovery and reduce quality of life. Acute critical care interventions are necessary to alleviate ALI and reduce risk for long-term deficits after TBI. The results of our study confirm that a single occurrence, isolated TBI can lead to ALI, and that RIC is a viable intervention for TBI-induced ALI. Additional studies are necessary to determine risk factors for the development of TBI-induced ALI and whether RIC is an effective intervention for both sexes, all ages, and in the clinic. These studies provide rationale for continued research on TBI-induced ALI, specifically with mechanisms that focus on peripheral inflammation.

Acknowledgments

The authors thank Nabia Kheshtchin-Kamel, Laura Cox, Stefany Gonzalez, and Yao Zang for assistance in data acquisition. The authors thank Yerin Hur for assistance in data and image acquisition and microscope training. The authors thank Katherine R. Giordano for assistance with running the myokine plate and maintaining the Bio-plex 200 used in this study. The authors thank Conor Young and Luisa M. Rojas for assistance in H&E staining.

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Keywords:

Acute lung injury; diffuse brain injury; irisin; mice; myokine; remote ischemic preconditioning; sphingosine-1-phosphate

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