The Role of Substance P in Pulmonary Clearance of Bacteria in Comparative Injury Models
2016; Elsevier BV; Volume: 186; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2016.08.014
ISSN1525-2191
AutoresTerry Hsieh, Max Vaickus, Thor D. Stein, Bethany L. Lussier, Jiyoun Kim, David M. Stepien, Elizabeth R. Duffy, Evan L. Chiswick, Daniel G. Remick,
Tópico(s)Sepsis Diagnosis and Treatment
ResumoNeural input to the immune system can alter its ability to clear pathogens effectively. Patients suffering mild traumatic brain injury (mTBI) have shown reduced rates of pneumonia and a murine model replicated these findings, with better overall survival of TBI mice compared with sham-injured mice. To further investigate the mechanism of improved host response in TBI mice, this study developed and characterized a mild tail trauma model of similar severity to mild TBI. Both mild tail trauma and TBI induced similar systemic changes that normalized within 48 hours, including release of substance P. Examination of tissues showed that injuries are limited to the target tissue (ie, tail in tail trauma, brain in mTBI). Pneumonia challenge showed that mild TBI mice showed improved immune responses, characterized by the following: i) increased survival, ii) increased pulmonary neutrophil recruitment, iii) increased bacterial clearance, and iv) increased phagocytic cell killing of bacteria compared with tail trauma. Administration of a neurokinin-1–receptor antagonist to block substance P signaling eliminated the improved survival of mTBI mice. Neurokinin-1–receptor antagonism did not alter pneumonia mortality in tail trauma mice. These data show that immune benefits of trauma are specific to mTBI and that tail trauma is an appropriate control for future studies aimed at elucidating the mechanisms of improved innate immune responses in mTBI mice. Neural input to the immune system can alter its ability to clear pathogens effectively. Patients suffering mild traumatic brain injury (mTBI) have shown reduced rates of pneumonia and a murine model replicated these findings, with better overall survival of TBI mice compared with sham-injured mice. To further investigate the mechanism of improved host response in TBI mice, this study developed and characterized a mild tail trauma model of similar severity to mild TBI. Both mild tail trauma and TBI induced similar systemic changes that normalized within 48 hours, including release of substance P. Examination of tissues showed that injuries are limited to the target tissue (ie, tail in tail trauma, brain in mTBI). Pneumonia challenge showed that mild TBI mice showed improved immune responses, characterized by the following: i) increased survival, ii) increased pulmonary neutrophil recruitment, iii) increased bacterial clearance, and iv) increased phagocytic cell killing of bacteria compared with tail trauma. Administration of a neurokinin-1–receptor antagonist to block substance P signaling eliminated the improved survival of mTBI mice. Neurokinin-1–receptor antagonism did not alter pneumonia mortality in tail trauma mice. These data show that immune benefits of trauma are specific to mTBI and that tail trauma is an appropriate control for future studies aimed at elucidating the mechanisms of improved innate immune responses in mTBI mice. Neural input exerts significant control on the ability of the immune system to clear pathogens effectively. Early studies have shown that stress has a profound detrimental effect on the immune response.1Stein M. Keller S.E. Schleifer S.J. Stress and immunomodulation: the role of depression and neuroendocrine function.J Immunol. 1985; 135: 827s-833sPubMed Google Scholar Psychologic or physiologic stress can result in dysregulation of these pathways, such as chronic activation, which ultimately leads to immunosuppression.2Padgett D.A. Glaser R. How stress influences the immune response.Trends Immunol. 2003; 24: 444-448Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar Activation of the vagus nerve also has been shown to induce powerful anti-inflammatory effects through the α7 nicotinic acetylcholine receptor. In severe traumatic brain injury (TBI) patients, it has been proposed that hyperactivity of the vagus depresses immune responses through strong dampening of proinflammatory mediator production.3Hazeldine J. Lord J.M. Belli A. Traumatic brain injury and peripheral immune suppression: primer and prospectus.Front Neurol. 2015; 6: 235Crossref PubMed Scopus (87) Google Scholar In contrast to these studies showing that the neuroimmune axis decreases immune responses, our previous work documented that head trauma patients showed significantly reduced rates of pneumonia compared with blunt trauma patients.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar A murine model of mild traumatic brain injury (mTBI) was able to reproduce these findings with enhanced resistance to bacterial pneumonia compared with sham injury mice.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar mTBI mice showed improved survival, augmented pulmonary neutrophil recruitment, and reduced bacterial burdens compared with sham-injured mice. These findings show that neuroimmune modulation can show beneficial effects by improving immune function. Further investigation into the mechanisms by which mTBI augments the innate immune response could offer valuable insight into fighting infections in today's increasingly antibiotic-resistant world (CDC, http://www.cdc.gov/drugresistance, last accessed April 20 2016). An important question raised by the previous study was whether any mild trauma would augment host resistance to bacterial pathogens.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar To address this issue, a model of peripheral injury was needed for comparison. In our previous clinical study, blunt non–head trauma patients were used as controls, but our murine studies used sham-injured mice.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar Traumatic injury models have been shown to have drastic effects on immunity. For example, a pulmonary contusion model showed enhanced neutrophil recruitment and expression of inflammatory mediators in response to an intratracheal lipopolysaccharide challenge.5Hoth J.J. Wells J.D. Hiltbold E.M. McCall C.E. Yoza B.K. Mechanism of neutrophil recruitment to the lung after pulmonary contusion.Shock. 2011; 35: 604-609Crossref PubMed Scopus (42) Google Scholar However, unlike our mTBI model, these neutrophils ultimately were deleterious and caused lung injury. Fracture and pseudofracture models replicate a common clinical injury and can have powerful immunosuppressive effects.6Li H. Itagaki K. Sandler N. Gallo D. Galenkamp A. Kaczmarek E. Livingston D.H. Zeng Y. Lee Y.T. Tang I.T. Isal B. Otterbein L. Hauser C.J. Mitochondrial damage-associated molecular patterns from fractures suppress pulmonary immune responses via formyl peptide receptors 1 and 2.J Trauma Acute Care Surg. 2015; 78 (discussion 279–281): 272-279Crossref PubMed Scopus (28) Google Scholar, 7Napolitano L.M. Koruda M.J. Meyer A.A. Baker C.C. The impact of femur fracture with associated soft tissue injury on immune function and intestinal permeability.Shock. 1996; 5: 202-207Crossref PubMed Scopus (65) Google Scholar These disparate findings show the wide spectrum of immune alterations and the necessity for proper controls in studying traumatic sequelae. Prior publications have raised the concern that any mild trauma could alter the host immune response and that the protective effects seen in our mTBI model are not specific to head trauma. Thus, we sought to develop an injury model that induces similar hematologic, physiologic, and inflammatory changes without compromising the lung, our primary organ of interest. A model of blunt tail trauma was studied to test the hypothesis that only TBI will augment pulmonary antibacterial defenses. The current study also showed that specific blockade of substance P (SP) signaling decreased survival to a pulmonary pathogen only in mTBI mice, confirming the mechanism of the improved survival.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar The significance of this work is highlighted further by a 2015 report showing that TBI patients had increased levels of SP.8Lorente L. Martin M.M. Almeida T. Hernandez M. Ramos L. Argueso M. Caceres J.J. Sole-Violan J. Jimenez A. Serum substance P levels are associated with severity and mortality in patients with severe traumatic brain injury.Crit Care. 2015; 19: 192Crossref PubMed Scopus (34) Google Scholar The mechanism of improved survival occurs through enhanced phagocytic cell clearance of bacteria. Female ICR mice (Harlan Sprague Dawley, Indianapolis, IN) were used for all experiments. All mice were 8 to 10 weeks old, weighed between 24 and 30 g, and were acclimated for at least 3 days before experiments. Animals were housed in a temperature- and humidity-controlled facility with a 12-hour light/dark cycle. Food and water were provided ad libitum. The Institutional Animal Care and Use Committee at Boston University approved all experiments. The neurokinin-1 receptor (NK1R) is the preferred receptor for SP.9Douglas S.D. Leeman S.E. Neurokinin-1 receptor: functional significance in the immune system in reference to selected infections and inflammation.Ann N Y Acad Sci. 2011; 1217: 83-95Crossref PubMed Scopus (125) Google Scholar The NK1R antagonist, aprepitant (50 mg/kg; Merck, Kenilworth, NJ; 2% carboxymethylcellulose; Sigma, St. Louis, MO) was given 2 hours before trauma and afterward at 12-hours intervals by oral gavage (p.o.) until pneumonia instillation. Vehicle mice were administered 2% carboxymethylcellulose p.o. mTBI was performed as previously described.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar Mice were evaluated 2 hours after injury using our Modified Mouse Coma Scale (MMCS) (Table 1) by two blinded evaluators (M.H.V. and B.L.L.) and only mice meeting the criteria for mild TBI (MMCS > 13) were included. Of the mTBI mice, >95% had a MMCS > 13. To induce mild tail trauma (TT), mice were anesthetized using isoflurane and placed in a prone position on the surface of the mTBI apparatus. The 170 g steel rod was released from a height of 8.5 cm onto a spot 2.0 cm from the base of the tail. This height was chosen based on pilot studies in which it induced neutrophilia at 4 hours similar to the mTBI mice. Immediately after impact the mouse was removed and given a subcutaneous injection of 0.05 mg/kg buprenorphine in 1 mL of normal saline. The mTBI mice also received the same dose of buprenorphine. Mice were placed on a warming pad until they were able to right themselves. Mice were selected randomly to undergo TBI, tail, or sham injury. Sham injury mice were anesthetized, placed in the apparatus, but no trauma was induced, although they were given the same dose of buprenorphine as the mTBI mice to keep the groups equivalent. Pneumonia was induced 48 hours after trauma by administration of 0.5 to 1 × 108 colony forming units (CFU) of Pseudomonas aeruginosa (Boston 41501; ATCC, Manassas, VA) in 50 μL of Hank's balanced salt solution by intratracheal instillation as previously described.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar The majority of the current data do not include a sham-injured group because the study attempts to determine if any trauma will augment survival to a pathogen challenge. Not including the sham-injured group complies with the three R ethics of use of animals in science (replacement, reduction, and refinement), specifically the reduction component.10Fenwick N. Griffin G. Gauthier C. The welfare of animals used in science: how the "Three Rs" ethic guides improvements.Can Vet J. 2009; 50: 523-530PubMed Google ScholarTable 1Modified Mouse Coma ScaleHuman Glasgow Coma ScaleModified Mouse Coma ScaleEye openingSpontaneous4PostureMoving about cage4To command3Hunched3To pain2Dazed sternal recumbence2None1Prone1Verbal responseOriented5Response to soundTurns to investigate5Confused, disoriented4Moves away4Inappropriate words3Startle3Incomprehensible sounds2Wincing (no body movement)2None1No response1Motor responseObeys commands6Tail lift responseExtension and reaching6Localizes pain5Extension without reaching5Withdraws4Lifts head flexion4Abnormal flexion to pain3Flexion no head lift3Abnormal extension to pain2Deviates to one side2None1No response1Best total score1515 Open table in a new tab Blood was collected (20 μL) from the facial vein at serial time points into 180 μL of phosphate-buffered saline–EDTA (50:1) and centrifuged for 5 minutes at 1000 × g.11Craciun F.L. Schuller E.R. Remick D.G. Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival.J Immunol. 2010; 185: 6930-6938Crossref PubMed Scopus (94) Google Scholar Plasma later was removed for later analysis while the pellets were resuspended in Hemavet diluent and used to obtain a complete blood count with differential using a Hemavet 950 instrument (Drew Scientific, Miami Lakes, FL). Physiologic measurements (heart rate, O2 saturation, and pulse distension) were obtained by cervical collar telemetry (MouseOx; Starr Lifesciences, Oakmont, PA). Respiratory measurements (respiratory rate, time of inspiration/expiration, and tidal volume) also were measured using unrestrained whole-body plethysmography (Buxco Research Systems, Wilmington, NC). Initial readings were taken on the day before trauma and then at 4 hours, 24 hours, and 48 hours after injury. Mice were sacrificed under ketamine/xylazine anesthesia. Bronchoalveolar lavage (BAL) was performed 4 hours after instillation of Pseudomonas with 5 mL of warm Hank's balanced salt solution–EDTA (50:1) in 500-μL aliquots. Of the first 1 mL, 100 μL was taken to determine the bacterial burden by serial dilutions on 5% sheep's blood agar plates. Cell pellets were combined and counted using a Beckman-Coulter particle counter (Coulter Electronics, Danvers, MA). A cell differential was performed by counting 300 cells spun using a Cytospin (Shandon, Waltham, MA) and slides were stained with Diff-Quick (SIEMENS Healthcare Diagnostics, Newark, DE). Mice were sacrificed at 30 minutes after mTBI to collect blood and BAL fluid. SP concentrations in samples were measured by enzyme-linked immunosorbent assay using a commercial kit (Substance P Enzyme-Linked Immunosorbent Assay Kit; Cayman Chemical Co, Ann Arbor, MI). Neutrophils (2 × 105) in whole blood were obtained via retroorbital exsanguination or neutrophils (5 × 105) were isolated using a PerColl gradient from bone marrow. Hemavet analysis was performed on whole blood for normalization of neutrophil number. Cytospins of bone marrow were performed to ensure efficient separation. Samples were incubated at a multiplicity of infection of 5 with Pseudomonas for 1 hour at 37°C in a final volume of 200 μL. Two control samples of bacteria only were incubated to provide a control. After 1 hour, cells were lysed in H2O followed by serial dilutions in Hank's balanced salt solution onto 5% sheep's blood agar plates. The microbicidal capacity was calculated as the percentage of killing = (CFUbac−CFUsample)/(CFUbac) × 100. During the development of the phagocyte killing assay, neutrophils from sham-injured animals were first studied. Because the bacterial killing by sham-injured animals was similar to TT animals the results were pooled to increase the power of the study. To quantify the extent of plasma leakage from the vasculature, 1% body weight of a 1% Evans blue solution was injected i.p. 1 hour before trauma. Mice were sacrificed 48 hours after trauma using ketamine/xylazine anesthesia, then perfused with phosphate-buffered saline–heparin (10 U/mL) followed by 10% formalin. Whole brains and a 1.5-cm long section of the tail encompassing the injury site were collected and stored in 10% formalin. Two-millimeter (brain) or 3-mm (tail) slices were imaged using an Odyssey near-infrared scanner (Li-Cor, Lincoln, NE) to measure Evans' blue extravasation from the vasculature and penetration into the tissue. After Li-Cor imaging, tissue slices then were embedded in paraffin blocks for sectioning, hematoxylin and eosin staining, and histologic analysis. Glass slides were scanned on an iScanCoreo (Ventana, Tucson, AZ). Statistical analyses were performed using Prism software version 5.0 (GraphPad, La Jolla, CA). Hematologic, physiological, and pulmonary data were analyzed by two-way analysis of variance, with Bonferroni post-tests where appropriate. SP BAL levels were below the detection limit in naive mice and a Fisher exact test was used to compare the levels with the injured groups. Survival data were analyzed by log-rank tests. Comparisons between mTBI and TT after pneumonia challenge and microbial killing were made using a two-tailed t-test. To test the hypothesis that only mTBI primes the immune system, we developed a model of peripheral injury with a similar level of injury as our mTBI. TT caused by a weight being dropped on the tail was chosen because it avoids several potential confounders. First, our studies focused on the immune response to pneumonia and TT would not compromise pulmonary or diaphragmatic function.5Hoth J.J. Wells J.D. Hiltbold E.M. McCall C.E. Yoza B.K. Mechanism of neutrophil recruitment to the lung after pulmonary contusion.Shock. 2011; 35: 604-609Crossref PubMed Scopus (42) Google Scholar, 12Nemzek-Hamlin J.A. Hwang H. Hampel J.A. Yu B. Raghavendran K. Development of a murine model of blunt hepatic trauma.Comp Med. 2014; 63: 398-408Google Scholar Second, this model closely mimicked our closed-head mTBI model that avoided skin manipulation and potential damage to organs implicated in mounting immune responses.4Yang S. Stepien D. Hanseman D. Robinson B. Goodman M.D. Pritts T.A. Caldwell C.C. Remick D.G. Lentsch A.B. Substance P mediates reduced pneumonia rates after traumatic brain injury.Crit Care Med. 2014; 42: 2092-2100Crossref PubMed Scopus (15) Google Scholar Third, as in our mTBI model, we avoided bone fractures that are known to be immunosuppressive.6Li H. Itagaki K. Sandler N. Gallo D. Galenkamp A. Kaczmarek E. Livingston D.H. Zeng Y. Lee Y.T. Tang I.T. Isal B. Otterbein L. Hauser C.J. Mitochondrial damage-associated molecular patterns from fractures suppress pulmonary immune responses via formyl peptide receptors 1 and 2.J Trauma Acute Care Surg. 2015; 78 (discussion 279–281): 272-279Crossref PubMed Scopus (28) Google Scholar, 7Napolitano L.M. Koruda M.J. Meyer A.A. Baker C.C. The impact of femur fracture with associated soft tissue injury on immune function and intestinal permeability.Shock. 1996; 5: 202-207Crossref PubMed Scopus (65) Google Scholar Because virtually any trauma induces an inflammatory response, including neutrophilia related to the severity of the injury, we first analyzed hematologic changes. Both the mTBI and TT groups showed similar early neutrophilia 4 hours after injury (Figure 1A), indicating that both models induced a detectable level of trauma. Concomitantly, the lymphocyte concentration in both groups decreased in an expected fashion (Figure 1B). The total white blood cell count did not differ significantly (Figure 1C). There were no significant differences between trauma groups at the time points analyzed by two-way analysis of variance. Comparable increases in neutrophils and decreases in lymphocytes suggest that neither model induced a higher level of trauma. The normalization of hematologic alterations by 48 hours suggests that differences in circulating immune cell numbers does not contribute to enhanced immune responses. Additional experiments sought to characterize changes after injury to determine whether physiologic differences were induced by the trauma and if they persisted up to the time of the pneumonia challenge. Physiological measurements of heart rate, pulse distension (a measure of local arterial blood flow proportional to cardiac output), and O2 saturation were measured using cervical collar telemetry. The telemetry data allowed repeated measurements on the same experimental subject to see the onset and resolution over the 48 hours after injury. There were no significant differences between TT and mTBI mice in heart rate during the 48 hours after injury (Figure 2A). Although pulse distension (Figure 2B) was significantly higher in TT mice, and O2 saturation (Figure 2C) was lower at 4 hours after trauma, these normalized by 24 hours. Because we would be inducing a pulmonary infection, additional lung studies were performed with an alternative technology. Respiratory parameters were measured by unrestrained whole-body plethysmography.13Vaickus L.J. Bouchard J. Kim J. Natarajan S. Remick D.G. Assessing pulmonary pathology by detailed examination of respiratory function.Am J Pathol. 2010; 177: 1861-1869Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Tidal volume remained essentially unchanged in both trauma models (Figure 3A). Both trauma models had a significant decrease in respiratory rate (Figure 3B) as a result of increased times of inspiration (Figure 3C) and expiration (Figure 3D), with greater changes in the mTBI group. These results show that the relatively minor, early physiologic, and respiratory differences between models at 4 hours resolved by 24 hours. The systemic measurements support that both models showed similar although not identical responses through the first 48 hours after trauma. Pilot studies have shown that both BAL and plasma levels of SP peaked within 30 minutes after either TT or mTBI (data not shown). A group of naive mice were studied to determine systemic as well as local levels of SP. SP was not detectable in the BAL of naive mice, but did increase after either TT or mTBI (Figure 4). Similarly, the plasma SP level was increased in both trauma groups, and the naive mice had low levels as previously reported (ie, <20 pg/mL).14Wang Y. Wang D.H. Role of substance P in renal injury during DOCA-salt hypertension.Endocrinology. 2012; 153: 5972-5979Crossref PubMed Scopus (12) Google Scholar, 15Amadesi S. Reni C. Katare R. Meloni M. Oikawa A. Beltrami A.P. Avolio E. Cesselli D. Fortunato O. Spinetti G. Ascione R. Cangiano E. Valgimigli M. Hunt S.P. Emanueli C. Madeddu P. Role for substance p-based nociceptive signaling in progenitor cell activation and angiogenesis during ischemia in mice and in human subjects.Circulation. 2012; 125 (S1–S19): 1774-1786Crossref PubMed Scopus (85) Google Scholar These data indicate that either mTBI or TT was sufficient to induce the release of SP into both the BAL and plasma. Figure 1, Figure 2, Figure 3, and 4 show that both trauma models produce a similar initial inflammatory response. Our data indicate similar systemic responses to trauma but additional experiments were performed to quantify the severity of neurologic injury in our mice. The Glasgow Coma Scale is the most commonly used neurologic injury scale for adults for traumatic head injuries.16Teasdale G. Maas A. Lecky F. Manley G. Stocchetti N. Murray G. The Glasgow Coma Scale at 40 years: standing the test of time.Lancet Neurol. 2014; 13: 844-854Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar We adapted the Glasgow Coma Scale criteria to mice (MMCS) by observing their spontaneous behavior, response to auditory stimuli, and motor reflex responses similar to a clinical evaluation. The MMCS allowed us to assess the severity of neurologic deficits quickly and accurately (Table 1), as well as limit interobserver variability. To mimic the clinical scenario of a patient presenting to the emergency department for evaluation after head injury, two blinded investigators (M.H.V. and B.L.L.) first scored the mice 2 hours after trauma. This time point was chosen to recapitulate the common delay between injury and clinical evaluation, as well as the superior performance of the 2-hour evaluation as a predictor of outcome.17Lesko M.M. Jenks T. O'Brien S.J. Childs C. Bouamra O. Woodford M. Lecky F. Comparing model performance for survival prediction using total Glasgow Coma Scale and its components in traumatic brain injury.J Neurotrauma. 2013; 30: 17-22Crossref PubMed Scopus (22) Google Scholar These same mice were also evaluated at 24 and 48 hours after trauma to longitudinally track any deficits in neurologic function. The same Glasgow Coma Scale score for TBI severity was used in our MMCS, as follows: mild was indicated by a score of 13 to 15, moderate was indicated by a score of 9 to 13, and severe was indicated by a score of <8. Our mTBI model reproducibly generated scores in the mild range and TT mice showed no deficits at all (Figure 5). The results show that our TBI model consistently was in the mild category and the MMCS can be used as a robust tool for assessing head trauma severity in mice. To characterize the location of injury after trauma, we performed detailed analyses of tail and brain tissue from both groups at 48 hours after injury (ie, immediately before pulmonary pathogen challenge). Evans' blue dye was injected intraperitoneally before the trauma to allow us to visualize and quantify the injury. Evans' blue is a fluorescent dye that binds to serum albumin with high affinity and is unable to cross the intact blood brain barrier. These properties allowed us to identify the area of injury, notably extravasation from the vasculature into tissue spaces, by its blue coloration, as well as quantify the signal intensity using the Li-Cor Odyssey near-infrared scanner. Evans' blue fluorescence can be detected at 700 nm using the Odyssey near-infrared scanner. mTBI mice showed an intense signal in the area of injury that reflected the histologic damage described later (Figure 6A). TT brains had a slight signal owing to the blood vessels in the brain (Figure 6A). Accordingly, TT tails reflect intense signal throughout the sections whereas mTBI tail signal was limited to the vasculature (Figure 6D). Brains of mTBI mice showed focal lesions with acute necrosis and hemorrhage generally involving the anterior and ventral portions of the brain. The ventral pallidum and anterior commissure were the most common structures damaged in the brains. No lesions were identified in any of the brains of TT mice (Figure 6B). Tails of TT mice reflect the damage seen grossly with areas of extravasated red blood cells in the tissue space, and infiltration of neutrophils (Figure 6E). None of these changes were observed in the tails of the mTBI mice. For both tissues, the positive dye signal appears to be larger than the extravasation seen microscopically, probably owing to the increased sensitivity to damage using Evans' blue dye compared with hematoxylin and eosin–stained sections.18Hamer P.W. McGeachie J.M. Davies M.J. Grounds M.D. Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.J Anat. 2002; 200: 69-79Crossref PubMed Scopus (221) Google Scholar Quantification of the dye intensity shows that the injury was isolated to the targeted tissue (Figure 6, C and F). The combination of the near-infrared quantification and histologic observations indicated the localization of trauma between these models. Because both trauma groups induced similar physiologic and hematologic responses that normalized by 24 to 48 hours, we proceeded to study whether only mTBI induced pulmonary immune priming to help eradicate pathogens. Each trauma group was challenged 48 hours after injury with 0.5 to 1.0 × 108 CFU of Pseudomonas intratracheally using a well-described nonsurgical model.19Kim J. Merry A.C. Nemzek J.A. Bolgos G.L. Siddiqui J. Remick D.G. Eotaxin represents the principal eosinophil chemoattractant in a novel murine asthma model induced by house dust containing cockroach allergens.J Immunol. 2001; 167: 2808-2815Crossref PubMed Scopus (52) Google Scholar, 20Bouchard J.C. Kim J. Beal D.R. Vaickus L.J. Craciun F.L. Remick D.G. Acute oral ethanol exposure triggers asthma in cockroach allergen-sensitized mice.Am J Pathol. 2012; 181: 845-857Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar mTBI mice showed significantly improved 7-day survival compared with TT mice (76.5% versus 40%) (Figure 7A). In fact, TT mice survived at a rate similar to sham mice, and thus in future experiments the sham group was combined or omitted following Animal Research: Reporting of in Vivo Experiments guidelines to reduce the number of mice used.10Fenwick N. Griffin G. Gauthier C. The welfare of animals used in science: how the "Three Rs" ethic guides improvements.Can Vet J. 2009; 50: 523-530PubMed Google Scholar TT or mTBI mice were challenged with Pseudomonas 48 hours after trauma and BAL was performed 4 hours after infection. An enhanced
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