Changes in plasma alpha-1 acid glycoprotein following hemorrhagic trauma: Possible role in dose differences of ALM drug therapy in rat and pig resuscitation

Introduction: The binding of drugs to plasma proteins is an important consideration in drug development. We have reported that the dose of adenosine, lidocaine, and magnesium (ALM) fluid therapy for resuscitation from hemorrhagic shock is nearly 3-times higher for pigs than rats. Since lidocaine strongly binds to serum alpha-1-acid glycoprotein (AGP), the aim of the study was to investigate the effect of hemorrhagic shock on levels of AGP in rats and pigs. Materials and Methods: Healthy adult male Sprague–Dawley rats and female crossbred pigs (n = 33 each) underwent tail vein and peripheral ear vein blood sampling, respectively, to collect plasma for AGP measurements. Rats (n = 17) and pigs (n = 16) underwent surgical instrumentation and uncontrolled hemorrhage via liver resection, and were treated with 3% NaCl ± ALM IV bolus followed 60 min later by 4 h 0.9% NaCl ± ALM IV drip. Rats were monitored for 72 h with blood samples taken post-surgery, and at 5.25, 24, and 72 h. Pigs were monitored for 6 h with blood samples taken post-surgery, and at 60 min and 6 h. Plasma AGP was measured with rat- and pig-specific enzyme-linked immunosorbent assay kits. Results: Baseline AGP levels in rats were 3.91 μg/mL and significantly 83-fold lower than in pigs (325 μg/mL). Surgical instrumentation was associated with ~10-fold increases in AGP in rats and a 21% fall in pigs. AGP levels remained elevated in rats after hemorrhage and resuscitation (28–29 μg/mL). In contrast, no significant differences in plasma AGP were found in ALM- or Saline-treated pigs over the monitoring period. Conclusions: We conclude that the trauma of surgery alone was associated with significant increases in AGP in rats, compared to a contrasting decrease in pigs. Higher levels of plasma AGP in pigs prior to hemorrhagic shock is consistent with the higher ALM doses required to resuscitate pigs compared with rats

Anterior Cruciate Ligament Reconstruction Surgery: Creating a Permissive Healing Phenotype in Military Personnel and Civilians for Faster Recovery

Introduction: Anterior cruciate ligament (ACL) rupture in military personnel and civilians can be a devastating injury. A service member is 10 times more likely to suffer an ACL injury than their civilian counterparts, and despite successful surgical stabilization, 4%-35% will develop arthrofibrosis, over 50% will not return to full active duty, and up to 50% will develop post-traumatic osteoarthritis (PTOA) within 15 years. Equally concerning, woman are 2 to 8 times more likely to experience ACL injuries than men, which represents a major knowledge gap. Materials and Methods: A comprehensive literature search was performed in December 2021 using structured search terms related to prevalence, risk factors, disease progression, and treatment of ACL injury and reconstruction. The literature search was conducted independently by two researchers using PubMed, Cochrane, and Embase databases, with inclusion of articles with military, civilian, and sex relevance, and exclusion of most papers with a publication date greater than 10 years. The resources used for the review reflect the most current data, knowledge, and recommendations associated with research and clinical findings from reliable international sources. Results: Currently, there is no effective system-based drug therapy that creates a “permissive environment” to reduce synovial and cartilage stress after ACL injury and reconstruction and prevent secondary complications. We argue that progress in this area has been hampered by researchers and clinicians failing to recognize that (1) an ACL injury is a system’s failure that affects the whole joint, (2) the early molecular events define and perpetuate different injury phenotypes, (3) male and female responses may be different and have a molecular basis, (4) the female phenotype continues to be under-represented in basic and clinical research, and (5) the variable outcomes may be perpetuated by the trauma of surgery itself. The early molecular events after ACL injury are characterized by an overexpression of joint inflammation, immune dysfunction, and trauma-induced synovial stress. We are developing an upstream adenosine, lidocaine, and magnesium therapy to blunt these early molecular events and expedite healing with less arthrofibrosis and early PTOA complications. Conclusions: ACL injuries continue to be a major concern among military personnel and civilians and represent a significant loss in command readiness and quality of life. The lack of predictability in outcomes after ACL repair or reconstruction underscores the need for new joint protection therapies. The male–female disparity requires urgent investigation

Why are bleeding trauma patients still dying? Towards a systems hypothesis of trauma

Over the years, many explanations have been put forward to explain early and late deaths following hemorrhagic trauma. Most include single-event, sequential contributions from sympathetic hyperactivity, endotheliopathy, trauma-induced coagulopathy (TIC), hyperinflammation, immune dysfunction, ATP deficit and multiple organ failure (MOF). We view early and late deaths as a systems failure, not as a series of manifestations that occur over time. The traditional approach appears to be a by-product of last century’s highly reductionist, single-nodal thinking, which also extends to patient management, drug treatment and drug design. Current practices appear to focus more on alleviating symptoms rather than addressing the underlying problem. In this review, we discuss the importance of the system, and focus on the brain’s “privilege” status to control secondary injury processes. Loss of status from blood brain barrier damage may be responsible for poor outcomes. We present a unified Systems Hypothesis Of Trauma (SHOT) which involves: 1) CNS-cardiovascular coupling, 2) Endothelial-glycocalyx health, and 3) Mitochondrial integrity. If central control of cardiovascular coupling is maintained, we hypothesize that the endothelium will be protected, mitochondrial energetics will be maintained, and immune dysregulation, inflammation, TIC and MOF will be minimized. Another overlooked contributor to early and late deaths following hemorrhagic trauma is from the trauma of emergent surgery itself. This adds further stress to central control of secondary injury processes. New point-of-care drug therapies are required to switch the body’s genomic and proteomic programs from an injury phenotype to a survival phenotype. Currently, no drug therapy exists that targets the whole system following major trauma

Immune dysfunction following severe trauma: A systems failure from the central nervous system to mitochondria

When a traumatic injury exceeds the body’s internal tolerances, the innate immune and inflammatory systems are rapidly activated, and if not contained early, increase morbidity and mortality. Early deaths after hospital admission are mostly from central nervous system (CNS) trauma, hemorrhage and circulatory collapse (30%), and later deaths from hyperinflammation, immunosuppression, infection, sepsis, acute respiratory distress, and multiple organ failure (20%). The molecular drivers of secondary injury include damage associated molecular patterns (DAMPs), pathogen associated molecular patterns (PAMPs) and other immune-modifying agents that activate the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic stress response. Despite a number of drugs targeting specific anti-inflammatory and immune pathways showing promise in animal models, the majority have failed to translate. Reasons for failure include difficulty to replicate the heterogeneity of humans, poorly designed trials, inappropriate use of specific pathogen-free (SPF) animals, ignoring sex-specific differences, and the flawed practice of single-nodal targeting. Systems interconnectedness is a major overlooked factor. We argue that if the CNS is protected early after major trauma and control of cardiovascular function is maintained, the endothelial-glycocalyx will be protected, sufficient oxygen will be delivered, mitochondrial energetics will be maintained, inflammation will be resolved and immune dysfunction will be minimized. The current challenge is to develop new systems-based drugs that target the CNS coupling of whole-body function

Pathophysiology of severe burn injuries: new therapeutic opportunities from a systems perspective

Severe burn injury elicits a profound stress response with the potential for high morbidity and mortality. If polytrauma is present, patient outcomes appear to be worse. Sex-based comparisons indicate females have worse outcomes than males. There are few effective drug therapies to treat burn shock and secondary injury progression. The lack of effective drugs appears to arise from the current treat-as-you-go approach rather than a more integrated systems approach. In this review, we present a brief history of burns research and discuss its pathophysiology from a systems’ perspective. The severe burn injury phenotype appears to develop from a rapid and relentless barrage of damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs) and neural afferent signals, which leads to a state of hyperinflammation, immune dysfunction, coagulopathy, hypermetabolism and intense pain. We propose that if the central nervous system (CNS) control of cardiovascular function and endothelial-glycocalyx-mitochondrial coupling can be restored early, these secondary injury processes may be minimized. The therapeutic goal is to switch the injury phenotype to a healing phenotype by reducing fluid leak and maintaining tissue O2 perfusion. Currently, no systems-based therapies exist to treat severe burns. We have been developing a small-volume fluid therapy comprising adenosine, lidocaine and magnesium (ALM) to treat hemorrhagic shock, traumatic brain injury and sepsis. Our early studies indicate that the ALM therapy holds some promise in supporting cardiovascular and pulmonary functions following severe burns. Future research will investigate the ability of ALM therapy to treat severe burns with polytrauma and sex disparities, and potential translation to humans

Adenosine, lidocaine and Mg2+ update: teaching old drugs new tricks

If a trauma (or infection) exceeds the body’s evolutionary design limits, a stress response is activated to quickly restore homeostasis. However, when the injury severity score is high, death is often imminent. The goal of this review is to provide an update on the effect of small-volume adenosine, lidocaine and Mg2+ (ALM) therapy on increasing survival and blunting secondary injury after non-compressible hemorrhagic shock and other trauma and infective/endotoxemic states. Two standout features of ALM therapy are: (1) resuscitation occurs at permissive hypotensive blood pressures (MAPs 50–60 mmHg), and (2) the drug confers neuroprotection at these low pressures. The therapy appears to reset the body’s baroreflex to produce a high-flow, hypotensive, vasodilatory state with maintained tissue O2 delivery. Whole body ALM protection appears to be afforded by NO synthesis-dependent pathways and shifting central nervous system (CNS) control from sympathetic to parasympathetic dominance, resulting in improved cardiovascular function, reduced immune activation and inflammation, correction of coagulopathy, restoration of endothelial glycocalyx, and reduced energy demand and mitochondrial oxidative stress. Recently, independent studies have shown ALM may also be useful for stroke, muscle trauma, and as an adjunct to Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA). Ongoing studies have further shown ALM may have utility for burn polytrauma, damage control surgery and orthopedic surgery. Lastly, we discuss the clinical applications of ALM fluid therapy for prehospital and military far-forward use for non-compressible hemorrhage and traumatic brain injury (TBI)

Conventional and specific-pathogen free rats respond differently to anesthesia and surgical trauma

Specific-pathogen free (SPF) animals were introduced in the 1960s to minimize disease and infection as variables in biomedical research. Our aim was to examine differences in physiological response in rat colonies bred and housed in a conventional versus SPF facility, and implications for research. Sprague-Dawley rats were anesthetized and catheterized for blood and pressure monitoring, and electrocardiogram (ECG) leads implanted. Hematology was assessed, and coagulation profile using rotational thromboelastometry. Health screening was outsourced to Cerberus Sciences. SPF rats had significantly lower pulse pressure (38% decrease), arrhythmias and prolonged QTc (27% increase) compared to conventional rats. No arrhythmias were found in conventional rats. SPF rats had significantly higher white cell, monocyte, neutrophil and lymphocyte counts, and were hyperfibrinolytic, indicated by EXTEM maximum lysis >15%. Independent assessment revealed similar pathogen exclusion between colonies, with the exception of Proteus in SPF animals. Returning to a conventional facility restored normal host physiology. We conclude that SPF animals displayed an abnormal hemodynamic, hematological and hemostatic phenotype in response to anesthesia and surgery, and provide a number of recommendations to help standardize research outcomes and translation

Specific pathogen-free (SPF) animal status as a variable in biomedical research: have we come full circle?

In this commentary, we discuss the pros and cons of using specific pathogen-free (SPF) animals in biomedical research, and present individual cases where altering the gut microbiome has dramatically changed the animal's basic physiology, immune/inflammatory functions and susceptibility to infection and disease. We argue that SPF manipulation of the microbiome-host relationship has itself become a confounding variable in biomedical research, which could have major implications to human translation

Living in a hostile world: inflammation, new drug development, and coronavirus

We present a brief history of the immune response and show that Metchnikoff’s theory of inflammation and phagocytotic defense was largely ignored in the 20th century. For decades, the immune response was believed to be triggered centrally, until Lafferty and Cunningham proposed the initiating signal came from the tissues. This shift opened the way for Janeway’s pattern recognition receptor theory, and Matzinger’s danger model. All models failed to appreciate that without inflammation, there can be no immune response. The situation changed in the 1990s when cytokine biology was rapidly advancing, and the immune system’s role expanded from host defense, to the maintenance of host health. An inflammatory environment, produced by immune cells themselves, was now recognized as mandatory for their attack, removal and repair functions after an infection or injury. We explore the cellular programs of the immune response, and the role played by cytokines and other mediators to tailor the right response, at the right time. Normally, the immune response is robust, self-limiting and restorative. However, when the antigen load or trauma exceeds the body’s internal tolerances, as witnessed in some COVID-19 patients, excessive inflammation can lead to increased sympathetic outflows, cardiac dysfunction, coagulopathy, endothelial and metabolic dysfunction, multiple organ failure and death. Currently, there are few drug therapies to reduce excessive inflammation and immune dysfunction. We have been developing an intravenous (IV) fluid therapy comprising adenosine, lidocaine and Mg2+ (ALM) that confers a survival advantage by preventing excessive inflammation initiated by sepsis, endotoxemia and sterile trauma. The multi-pronged protection appears to be unique and may provide a tool to examine the intersection points in the immune response to infection or injury, and possible ways to prevent secondary tissue damage, such as that reported in patients with COVID-19

Adenosine, Lidocaine and Magnesium (ALM) therapy modulates early sex-specific inflammatory and immune responses following experimental anterior cruciate ligament rupture and reconstruction

Background: Early dysregulation of local and systemic inflammatory and immune responses is implicated in the pathogenesis of fibrotic and degenerative complications after anterior cruciate ligament reconstruction (ACLR) surgery. In other surgical trauma models, ALM therapy has been shown to blunt inflammation, leading to a more permissive healing environment in injured tissues. The purpose of this study was to evaluate sex-specific effects of surgery and perioperative ALM therapy on leukocyte mobilization and activation, and systemic and joint tissue inflammation in a rat model of ACL rupture and reconstruction. Methods: Adult male and female Sprague–Dawley rats were randomly divided into ALM (male, n = 15; female, n = 14) or Saline control (male, n = 13; female, n = 14) treatment groups. Three days after non-invasive ACL rupture, ACLR surgery was performed on the injured knee. Animals received a 1 h perioperative IV ALM or saline drip, and a 0.1 ml IA bolus of ALM or saline, and were monitored to 120 h postoperative. Hematology, leukocyte immunophenotyping, plasma and synovial inflammatory mediator concentrations, and joint tissue histopathology and gene expression of inflammatory markers were assessed. Results: Following ACLR surgery, plasma concentrations of inflammatory cytokines IL-6, TNF-α and IL-1β peaked later and at a higher magnitude in females compared to males, with ALM dampening this systemic inflammatory response. At 1 h postoperative, ALM boosted circulating B cell numbers in males and females, and decreased neutrophil activation in females. By 72 h, numbers of circulating T cells with immunoregulatory potential were increased in all ALM-treated animals compared to Saline controls, and corresponded to a significant reduction in synovial TNF-α concentrations within the operated knees. Sex-specific treatment differences were found in inflammatory and immune profiles in the synovial fluid and joint tissues. Inflammatory cell infiltration and gene expression of markers of inflammation (Nfκb, Nlrp3), cytoprotective responses (Nrf2), and angiogenesis (Vegf ) were increased in joint synovial tissue from ALM-treated males, compared to controls. In females, ALM treatment was associated with increased mononuclear cell recruitment, and expression of M2 macrophage marker (Arg1) in joint synovial tissue