Urokinase-Type Plasminogen Activator Receptor (uPAR) in Inflammation and Disease: A Unique Inflammatory Pathway Activator.

The urokinase-type plasminogen activator receptor (uPAR) is a unique protease binding receptor, now recognized as a key regulator of inflammation. Initially, uPA/uPAR was considered thrombolytic (clot-dissolving); however, recent studies have demonstrated its predominant immunomodulatory functions in inflammation and cancer. The uPA/uPAR complex has a multifaceted central role in both normal physiological and also pathological responses. uPAR is expressed as a glycophosphatidylinositol (GPI)-linked receptor interacting with vitronectin, integrins, G protein-coupled receptors, and growth factor receptors within a large lipid raft. Through protein-to-protein interactions, cell surface uPAR modulates intracellular signaling, altering cellular adhesion and migration. The uPA/uPAR also modifies extracellular activity, activating plasminogen to form plasmin, which breaks down fibrin, dissolving clots and activating matrix metalloproteinases that lyse connective tissue, allowing immune and cancer cell invasion and releasing growth factors. uPAR is now recognized as a biomarker for inflammatory diseases and cancer; uPAR and soluble uPAR fragments (suPAR) are increased in viral sepsis (COVID-19), inflammatory bowel disease, and metastasis. Here, we provide a comprehensive overview of the structure, function, and current studies examining uPAR and suPAR as diagnostic markers and therapeutic targets. Understanding uPAR is central to developing diagnostic markers and the ongoing development of antibody, small-molecule, nanogel, and virus-derived immune-modulating treatments that target uPAR.

Viral SERPINS-A Family of Highly Potent Immune-Modulating Therapeutic Proteins.

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2-10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics.

Viral anti-inflammatory serpin reduces immuno-coagulopathic pathology in SARS-CoV-2 mouse models of infection.

SARS-CoV-2 acute respiratory distress syndrome (ARDS) induces uncontrolled lung inflammation and coagulopathy with high mortality. Anti-viral drugs and monoclonal antibodies reduce early COVID-19 severity, but treatments for late-stage immuno-thrombotic syndromes and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases. The myxoma virus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated thrombotic, thrombolytic, and complement proteases as a self-defense strategy to combat clearance. Serp-1 is effective in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 as a therapy for immuno-coagulopathic complications during ARDS. Treatment with PEGSerp-1 in two mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved outcomes. PEGSerp-1 significantly reduced M1 macrophages in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR), thrombotic proteases, and complement membrane attack complex (MAC). Sequential changes in gene expression for uPAR and serpins (complement and plasminogen inhibitors) were observed. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for severe viral ARDS, immuno-coagulopathic responses, and Long COVID.

The under-appreciated world of the serpin family of serine proteinase inhibitors.

In the practice of medicine, many fundamental biological pathways that require tight on/off control, such as inflammation and circulatory homeostasis, are regulated by serine proteinases, but we rarely consider the unique protease inhibitors that, in turn, regulate these proteases. The serpins are a family of proteins with a shared tertiary structure, whose members largely act as serine protease inhibitors, found in all forms of life, ranging from viruses, bacteria, and archaea to plants and animals. These proteins represent up to 2-10% of proteins in the human blood and are the third most common protein family.

Mouse Corneal Transplantation.

Corneal transplantation is the most common form of organ transplantation worldwide. Transplant survival depends on various factors, many of which are not fully understood. Due to the existence of many genetically defined strains, mouse models of corneal transplantation are most commonly used. Here, we describe a method for a mouse corneal transplantation.

Mouse Models of Renal Allograft Transplant Rejection: Methods to Investigate Chemokine-GAG Interaction and Therapeutic Blockade.

Chemokine-glycosaminoglycan (GAG) interactions direct immune cell activation and invasion, e.g., directing immune cells to sites of infection or injury, and are central to initiating immune responses. Acute innate and also adaptive or antibody-mediated immune cell responses both drive damage to kidney transplants. These immune responses are central to allograft rejection and transplant failure. While treatment for acute rejection has advanced greatly, ongoing or chronic immune damage from inflammation and antibody-mediated rejection remains a significant problem, leading to transplant loss. There are limited numbers of organs available for transplant, and preventing chronic graft damage will allow for longer graft stability and function, reducing the need for repeat transplantation. Chemokine-GAG interactions are the basis for initial immune responses, forming directional gradients that allow immune cells to traverse the vascular endothelium and enter engrafted organs. Targeting chemokine-GAG interactions thus has the potential to reduce immune damage to transplanted kidneys.Mouse models for renal transplant are available, but are complex and require extensive microsurgery expertise. Here we describe simplified subcapsular and subcutaneous renal allograft transplant models, for rapid assessment of the roles of chemokine-GAG interactions during allograft surgery and rejection. These models are described, together with treatment using a unique chemokine modulating protein (CMP) M-T7 that disrupts chemokine-GAG interactions.

Myxomavirus Serp-1 Protein Ameliorates Inflammation in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is an X-linked disease afflicting 1 in 3500 males that is characterized by muscle weakness and wasting during early childhood, and loss of ambulation and death by early adulthood. Chronic inflammation due to myofiber instability leads to fibrosis, which is a primary cause of loss of ambulation and cardiorespiratory insufficiency. Current standard of care focuses on reducing inflammation with corticosteroids, which have serious adverse effects. It is imperative to identify alternate immunosuppressants as treatments to reduce fibrosis and mortality. Serp-1, a Myxoma virus-derived 55 kDa secreted glycoprotein, has proven efficacy in a range of animal models of acute inflammation, and its safety and efficacy has been shown in a clinical trial. In this initial study, we examined whether pegylated Serp-1 (PEGSerp-1) treatment would ameliorate chronic inflammation in a mouse model for Duchenne muscular dystrophy. Our data revealed a significant reduction in diaphragm fibrosis and increased myofiber diameter, and significantly decreased pro-inflammatory M1 macrophage infiltration. The M2a macrophage and overall T cell populations showed no change. These data demonstrate that treatment with this new class of poxvirus-derived immune-modulating serpin has potential as a therapeutic approach designed to ameliorate DMD pathology and facilitate muscle regeneration.

RNA Virus Gene Signatures Detected in Patients With Cardiomyopathy After Chemotherapy; A Pilot Study.

Background: Viral infections are pervasive and leading causes of myocarditis. Immune-suppression after chemotherapy increases opportunistic infections, but the incidence of virus-induced myocarditis is unknown. Objective: An unbiased, blinded screening for RNA viruses was performed after chemotherapy with correlation to cardiac function. Methods: High-throughput sequencing of RNA isolated from blood samples was analyzed following chemotherapy for hematological malignancies (N = 28) and compared with left ventricular ejection fraction (LVEF). Results: On initial rigorous analysis, low levels of influenza orthomyxovirus and avian paramyxovirus sequences were detectable, but without significant correlation to LVEF (r = 0.208). A secondary broad data mining analysis for virus sequences, without filtering human sequences, detected significant correlations for paramyxovirus with LVEF after chemotherapy (r = 0.592, P < 0.0096). Correlations were similar for LVEF pre- and post- chemotherapy for orthomyxovirus (R = 0.483, P < 0.0421). Retrovirus detection also correlated with LVEF post (r = 0.453, p < 0.0591), but not pre-chemotherapy, but is suspect due to potential host contamination. Detectable phage and anellovirus had no correlation. Combined sequence reads (all viruses) demonstrated significant correlation (r = 0.621, P < 0.0078). Reduced LVEF was not associated with chemotherapy (P = NS). Conclusions: This is the first report of RNA virus screening in circulating blood and association with changes in cardiac function among patients post chemotherapy, using unbiased, blinded, high-throughput sequencing. Influenza orthomyxovirus, avian paramyxovirus and retrovirus sequences were detectable in patients with reduced LVEF. Further analysis for RNA virus infections in patients with cardiomyopathy after chemotherapy is warranted.

Systemic Delivery of mLIGHT-Armed Myxoma Virus Is Therapeutic for Later-Stage Syngeneic Murine Lung Metastatic Osteosarcoma.

Cancers that metastasize to the lungs represent a major challenge in both basic and clinical cancer research. Oncolytic viruses are newly emerging options but successful delivery and choice of appropriate therapeutic armings are two critical issues. Using an immunocompetent murine K7M2-luc lung metastases model, the efficacy of MYXV armed with murine LIGHT (TNFSF14/CD258) expressed under virus-specific early/late promoter was tested in an advanced later-stage disease K7M2-luc model. Results in this model show that mLIGHT-armed MYXV, delivered systemically using ex vivo pre-loaded PBMCs as carrier cells, reduced tumor burden and increased median survival time. In vitro, when comparing direct infection of K7M2-luc cancer cells with free MYXV vs. PBMC-loaded virus, vMyx-mLIGHT/PBMCs also demonstrated greater cytotoxic capacity against the K7M2 cancer cell targets. In vivo, systemically delivered vMyx-mLIGHT/PBMCs increased viral reporter transgene expression levels both in the periphery and in lung tumors compared to unarmed MYXV, in a tumor- and transgene-dependent fashion. We conclude that vMyx-mLIGHT, especially when delivered using PBMC carrier cells, represents a new potential therapeutic strategy for solid cancers that metastasize to the lung.

Serp-1 Promotes Corneal Wound Healing by Facilitating Re-epithelialization and Inhibiting Fibrosis and Angiogenesis.

Purpose: Chemical corneal injuries carry a high morbidity and commonly lead to visual impairment. Here, we investigate the role of Serp-1, a serine protease inhibitor, in corneal wound healing. Methods: An alkaline-induced corneal injury was induced in 14 mice. Following injury, five mice received daily topical saline application while nine mice received Serp-1 100 µL topically combined with a daily subcutaneous injection of 100 ng/gram body weight of Serp-1. Corneal damage was monitored daily through fluorescein staining and imaging. Cross sectional corneal H&E staining were obtained. CD31 was used as marker for neovascularization. Results: Serp-1 facilitates corneal wound healing by reducing fibrosis and neovascularization while mitigating inflammatory cell infiltration with no noticeable harm related to its application. Conclusions: Serp-1 effectively mitigates inflammation, decreases fibrosis, and reduce neovascularization in a murine model of corneal injury without affecting other organs. Translational Relavence: Our study provides preclinical data for topical application of Serp-1 to treat corneal wounds.

Distinct Coagulopathy With Myocardial Injury and Pulmonary Embolism in COVID-19.

In this article, we report a case of a 61-year-old male who was diagnosed with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), presenting with acute respiratory distress syndrome requiring intubation and hemodynamic support, marked D-Dimer and troponin I elevation, worsening ST-elevation myocardial infarction on repeat electrocardiograms, and a negative coronary angiogram ruling out a coronary artery thrombosis or occlusion. With worsening diffuse ST-segment elevation on electrocardiograms and reduced ejection fraction on echocardiography in the setting of systemic inflammation, fulminant myocarditis was highly suspected. Despite optimal medical treatment, the patient's condition deteriorated and was complicated by cardiac arrest that failed resuscitation. Although myocarditis was initially suspected, the autopsy revealed no evidence of myocarditis or pericarditis but did demonstrate multiple microscopic sites of myocardial ischemia together with thrombi in the left atrium and pulmonary vasculature. Additionally, scattered microscopic cardiomyocyte necrosis with pathological diagnosis of small vessel micro-thrombotic occlusions. These findings are potentially exacerbated by inflammation-induced coagulopathy, hypoxia, hypotension, and stress, that is, a multifactorial etiology. Further research and an improved understanding are needed to define the precise pathophysiology of the coagulopathic state causing widespread micro-thrombosis with subsequent myocardial and pulmonary injury.

Fibrinolytic Serine Proteases, Therapeutic Serpins and Inflammation: Fire Dancers and Firestorms.

The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex relationship of sequentially activated serine proteases and their regulatory inhibitors in the circulating blood. The proteases and inhibitors interact continuously throughout all branches of the cardiovascular system in the human body, representing one of the most abundant groups of proteins in the blood. There is an intricate interaction of the coagulation cascades with endothelial cell surface receptors lining the vascular tree, circulating immune cells, platelets and connective tissue encasing the arterial layers. Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic cascades initiate immune cell responses, representing a front line, "off-the-shelf" system for inducing inflammatory responses. These hemostatic pathways are one of the first response systems after injury with the fibrinolytic cascade being one of the earliest to evolve in primordial immune responses. An equally important contributor and parallel ancient component of these thrombotic and thrombolytic serine protease cascades are the serine protease inhibitors, termed serpins. Serpins are metastable suicide inhibitors with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory pathways throughout the body. Serpins are now known to also modulate the immune response, either via control of thrombotic and thrombolytic cascades or via direct effects on cellular phenotypes, among many other functions. Here we review the co-evolution of the thrombolytic cascade and the immune response in disease and in treatment. We will focus on the relevance of these recent advances in the context of the ongoing COVID-19 pandemic. SARS-CoV-2 is a "respiratory" coronavirus that causes extensive cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting in severe and potentially fatal coagulopathies.

Incidental Finding of a Large Right Atrial Thrombus in a Patient With Cerebral Lymphoma.

Right atrial (RA) masses are rare, challenging to diagnose, and potentially life-threatening with high mortality if untreated. We present a patient presenting with diffuse large B-cell lymphoma in the brain that was incidentally found to have a large RA mass. For a better definition of the RA mass, extensive workup using multimodality imaging including chest computed tomography, transthoracic echocardiography, transesophageal echocardiography, cardiac magnetic resonance imaging, and left heart catheterization was warranted. The imaging demonstrated a large RA mass extending through the tricuspid valve into the right ventricle and superior and inferior vena cava without a mobile component. The mass was then successfully resected, and further histology examination was performed to rule out lymphoma and rare subtypes of diffuse large B-cell lymphoma. The comprehensive workup proved the RA mass to be a calcified thrombus rather than a direct metastatic spread of lymphoma.

PEGylated Serp-1 Markedly Reduces Pristane-Induced Experimental Diffuse Alveolar Hemorrhage, Altering uPAR Distribution, and Macrophage Invasion.

Diffuse alveolar hemorrhage (DAH) is one of the most serious clinical complications of systemic lupus erythematosus (SLE). The prevalence of DAH is reported to range from 1 to 5%, but while DAH is considered a rare complication there is a reported 50-80% mortality. There is at present no proven effective treatment for DAH and the therapeutics that have been tested have significant side effects. There is a clear necessity to discover new drugs to improve outcomes in DAH. Serine protease inhibitors, serpins, regulate thrombotic and thrombolytic protease cascades. We are investigating a Myxomavirus derived immune modulating serpin, Serp-1, as a new class of immune modulating therapeutics for vasculopathy and lung hemorrhage. Serp-1 has proven efficacy in models of herpes virus-induced arterial inflammation (vasculitis) and lung hemorrhage and has also proved safe in a clinical trial in patients with unstable coronary syndromes and stent implant. Here, we examine Serp-1, both as a native secreted protein expressed by CHO cells and as a polyethylene glycol modified (PEGylated) variant (Serp-1m5), for potential therapy in DAH. DAH was induced by intraperitoneal (IP) injection of pristane in C57BL/6J (B6) mice. Mice were treated with 100 ng/g bodyweight of either Serp-1 as native 55 kDa secreted glycoprotein, or as Serp-1m5, or saline controls after inducing DAH. Treatments were repeated daily for 14 days (6 mice/group). Serp-1 partially and Serp-1m5 significantly reduced pristane-induced DAH when compared with saline as assessed by gross pathology and H&E staining (Serp-1, p = 0.2172; Serp-1m5, p = 0.0252). Both Serp-1m5 and Serp-1 treatment reduced perivascular inflammation and reduced M1 macrophage (Serp-1, p = 0.0350; Serp-1m5, p = 0.0053), hemosiderin-laden macrophage (Serp-1, p = 0.0370; Serp-1m5, p = 0.0424) invasion, and complement C5b/9 staining. Extracellular urokinase-type plasminogen activator receptor positive (uPAR+) clusters were significantly reduced (Serp-1, p = 0.0172; Serp-1m5, p = 0.0025). Serp-1m5 also increased intact uPAR+ alveoli in the lung (p = 0.0091). In conclusion, Serp-1m5 significantly reduces lung damage and hemorrhage in a pristane model of SLE DAH, providing a new potential therapeutic approach.

Serine Proteases and Chemokines in Neurotrauma: New Targets for Immune Modulating Therapeutics in Spinal Cord Injury.

Progressive neurological damage after brain or spinal cord trauma causes loss of motor function and treatment is very limited. Clotting and hemorrhage occur early after spinal cord (SCI) and traumatic brain injury (TBI), inducing aggressive immune cell activation and progressive neuronal damage. Thrombotic and thrombolytic proteases have direct effects on neurons and glia, both healing and also damaging bidirectional immune cell interactions. Serine proteases in the thrombolytic cascade, tissue- and urokinase-type plasminogen activators (tPA and uPA), as well as the clotting factor thrombin, have varied effects, increasing neuron and glial cell growth and migration (tPA), or conversely causing apoptosis (thrombin) and activating inflammatory cell responses. tPA and uPA activate plasmin and matrix metalloproteinases (MMPs) that break down connective tissue allowing immune cell invasion, promoting neurite outgrowth. Serine proteases also activate chemokines. Chemokines are small proteins that direct immune cell invasion but also mediate neuron and glial cell communication. We are investigating a new class of therapeutics, virus-derived immune modulators; One that targets coagulation pathway serine proteases and a second that inhibits chemokines. We have demonstrated that local infusion of these biologics after SCI reduces inflammation providing early improved motor function. Serp-1 is a Myxomavirus-derived serine protease inhibitor, a serpin, that inhibits both thrombotic and thrombolytic proteases. M-T7 is a virus-derived chemokine modulator. Here we review the roles of thrombotic and thrombolytic serine proteases and chemoattractant proteins, chemokines, as potential therapeutic targets for SCI. We discuss virus-derived immune modulators as treatments to reduce progressive inflammation and ongoing nerve damage after SCI.

The Role of Astrogliosis in Formation of the Syrinx in Spinal Cord Injury.

A massive localized trauma to the spinal cord results in complex pathologic events driven by necrosis and vascular damage which in turn leads to hemorrhage and edema. Severe, destructive and very protracted inflammatory response is characterized by infiltration by phagocytic macrophages of a site of injury which is converted into a cavity of injury (COI) surrounded by astroglial reaction mounted by the spinal cord. The tissue response to the spinal cord injury (SCI) has been poorly understood but the final outcome appears to be a mature syrinx filled with the cerebrospinal fluid with related neural tissue loss and permanent neurologic deficits. This paper reviews known pathologic mechanisms involved in the formation of the COI after SCI and discusses the integrative role of reactive astrogliosis in mechanisms involved in the removal of edema after the injury. A large proportion of edema fluid originating from the trauma and then from vasogenic edema related to persistent severe inflammation, may be moved into the COI in an active process involving astrogliosis and specifically over-expressed aquaporins.

Deriving Immune-Modulating Peptides from Viral Serine Protease Inhibitors (Serpins).

Viruses have devised highly effective approaches that modulate the host immune response, blocking immune responses that are designed to eradicate viral infections. Over millions of years of evolution, virus-derived immune-modulating proteins have become extraordinarily potent, in some cases working at picomolar concentrations when expressed into surrounding tissues and effectively blocking host defenses against viral invasion and replication. The marked efficiency of these immune-modulating proteins is postulated to be due to viral engineering of host immune modulators as well as design and development of new strategies (i.e., some derived from host proteins and some entirely unique). Two key characteristics of viral immune modulators confer both adaptive advantages and desirable functions for therapeutic translation. First, many virus-derived immune modulators have evolved structures that are not readily recognized or regulated by mammalian immune pathways, ensuring little to no neutralizing antibody responses or proteasome-mediated degradation. Second, these immune modulators tend to target early steps in central immune responses, producing a powerful downstream inhibitory "domino effect" which may alter cell activation and gene expression.We have proposed that peptide metabolites of these immune-modulating proteins can enhance and extend protein function. Active immunomodulating peptides have been derived from both mammalian and viral proteins. We previously demonstrated that peptides derived from computationally predicted cleavage sites in the reactive center loop (RCL) of a viral serine proteinase inhibitor (serpin ) from myxoma virus, Serp-1 , can modify immune response activation. We have also demonstrated modulation of host gut microbiota produced by Serp-1 and RCL-derived peptide , S7, in a vascular inflammation model. Of interest, generation of derived peptides that maintain therapeutic function from a serpin can act by a different mechanism. Whereas Serp-1 has canonical serpin-like function to inhibit serine proteases, S7 instead targets mammalian serpins. Here we describe the derivation of active Serp- RCL peptides and their testing in inflammatory vasculitis models.

Topical Application of Virus-Derived Immunomodulating Proteins and Peptides to Promote Wound Healing in Mouse Models.

Immune modulators play critical roles in the progression of wounds to normal or conversely delayed healing, through the regulation of normal tissue regrowth, scarring, inflammation, and growth factor expression. Many immune modulator recombinants are under active preclinical study or in clinical trial to promote improved acute or chronic wound healing and to reduce scarring. Viruses have evolved highly efficient immune modulators for the evasion of host-defensive immune responses that target and kill invasive viruses. Recent studies have proven that some of these virus-derived immune modulators can be used to promote wound healing with significantly improved speed and reduced scarring in rodent models. Mouse full-thickness excisional wound model is one of the most commonly used animal models used to study wound healing for its similarity to humans in the healing phases and associated cellular and molecular mechanisms. This chapter introduces this mouse dermal wound healing model in detail for application in studying viral immune modulators as new treatments to promote wound healing. Details of hydrogel, protein construction, and topical application methods for these therapeutic proteins are provided in this chapter.

Neurologic and Histologic Tests Used to Measure Neuroprotective Effectiveness of Virus-Derived Immune-Modulating Proteins.

Severe inflammatory disease initiated by neurotrauma and stroke is of primary concern in these intractable pathologies as noted in recent studies and understanding of the pathogenesis of spinal cord injury (SCI) in the rat model. Successful anti-inflammatory treatments should result in neuroprotection and limit the loss of neurological function to injury caused by the initial damage. Continuous subdural infusion offers direct access to the cavity of injury (COI) that forms after balloon crush SCI deep in the spinal cord. Some anti-inflammatory compounds are not likely capable of crossing the blood-spinal cord barrier. Subdural infusion of myxoma virus-derived Serp-1, an anti-thrombotic/anti-thrombolytic, and also of M-T7, a chemokine inhibitor, improved the locomotor scores and pain sensation scores as well as reduced the numbers of macrophages in the COI by 50 and 80%, respectively, while intraperitoneal infusion of either protein had little effect. Injection of a chitosan hydrogel loaded with Serp-1 into the dorsal spinal column crush also resulted in improved neurological deficits and in reduction of the size of the crush lesion 4 weeks after injury. While neurological scores in a simplified hind-end (HE) locomotor test together with a toe-pinch withdrawal test demonstrated improvement in all balloon crush injury and dorsal spinal crush injury rats, a severe inflammation is induced by the injury indicating additional damage to the spinal cord. Thus neurological function testing can be contradictory, rather than corresponding, to the pathogenesis of SCI. The count of macrophages in the COI offers a precise, reliable method of measuring the effectiveness of a neuroprotective treatment of SCI in preclinical studies.