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Neurologic and Histologic Tests Used to Measure Neuroprotective Effectiveness of Virus-Derived Immune-Modulating Proteins
Abstract
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.
... In the experiments using the subdural infusion of dexamethasone, M-T7 and Serp-1, the Basso Beattie Bresnahan (BBB) locomotor test used to evaluate SCI rats [63] by others was substituted by a simpler hind end locomotor test and supplemented by a hind end pinch withdrawal test developed by the author [22,23,64] to allow for a more detailed analysis of the neurologic function of SCI rats every day after the SCI. While both tests detected neurological improvements in rats treated with anti-inflammatory infusions in a 7 days long study [22], this was not the case in the follow-up study involving 56 days of treatment with Serp-1 despite the lowering effect of this treatment on the numbers of phagocytic macrophages in the COI [23]. ...
... While both tests detected neurological improvements in rats treated with anti-inflammatory infusions in a 7 days long study [22], this was not the case in the follow-up study involving 56 days of treatment with Serp-1 despite the lowering effect of this treatment on the numbers of phagocytic macrophages in the COI [23]. This observation suggests that scores in both neurological tests and perhaps the BBB test do not adequately represent the pathogenesis of the SCI where severe inflammation likely contributes to further destruction of the spinal cord [1] while the scores improve in untreated rats during the first 4 weeks post-SCI [23,64,65]. In another contradiction, while the phagocytic macrophage-rich inflammation persists beyond 4 weeks in untreated rats and in Serp-1 infused rats [1,23], the scores of both neurological tests stabilized during the week 4 post-SCI [23], an observation also made with the BBB test in treatment experiments lasting beyond 4 weeks [66][67][68]. ...
... In another contradiction, while the phagocytic macrophage-rich inflammation persists beyond 4 weeks in untreated rats and in Serp-1 infused rats [1,23], the scores of both neurological tests stabilized during the week 4 post-SCI [23], an observation also made with the BBB test in treatment experiments lasting beyond 4 weeks [66][67][68]. Thus the usefulness of the neurological test in pre-clinical studies on candidate neuroprotective compounds appears quite limited as it may not correspond to the pathogenesis of the SCI and cannot be used beyond week 4 while the effective treatments are expected to inhibit inflammation by lowering numbers of macrophages and the duration of treatment needs to last well beyond 4 weeks [23,64]. ...
The spinal cord injury (SCI) initiates an extraordinarily protracted disease with 3 phases; acute, inflammatory and resolution that are restricted to the cavity of injury (COI) or arachnoiditis by a unique CNS reaction against the severity of destructive inflammation. While the severity of inflammation involving the white matter is fueled by a potently immunogenic activity of damaged myelin, its sequestration in the COI and its continuity with the cerebrospinal fluid of the subdural space allows for anti-inflammatory therapeutics infused subdurally to inhibit phagocytic macrophage infiltration and thus provide neuroprotection. The role of astrogliosis in containing and ultimately in eliminating severe destructive inflammation post-trauma appears obvious but is not yet sufficiently understood to use in therapeutic neuroprotective and neuroregenerative strategies. An apparent anti-inflammatory activity of reactive astrocytes is paralleled by their active role in removing excess edema fluid in blood brain barrier damaged by inflammation. Recently elucidated pathogenesis of neurotrauma including SCI, traumatic brain injury (TBI) and of stroke, calls for the following principal therapeutic steps in its treatment leading to recovery of neurologic function: (1) inhibition and elimination of destructive inflammation from the COI with accompanying reduction of vasogenic edema, (2) insertion into the COI of a functional bridge supporting the crossing of regenerating axons, (3) enabling regeneration of axons to their original synaptic targets by a temporary safe removal of myelin in targeted areas of white matter, (4) in vivo, systematic monitoring of the consecutive therapeutic steps. The focus of this paper is on the therapeutic step 1.
Spinal cord injury (SCI) initiates a severe, destructive inflammation with pro-inflammatory, CD68+/CD163−, phagocytic macrophages infiltrating the area of necrosis and hemorrhage by day 3 and persisting for the next 16 weeks. Inhibition of macrophage infiltration of the site of necrosis that is converted into a cavity of injury (COI) during the first week post-SCI, should limit inflammatory damage, shorten its duration and result in neuroprotection. By sustained subdural infusion we administered Serp-1, a Myxoma virus-derived immunomodulatory protein previously shown to improve neurologic deficits and inhibit macrophage infiltration in the COI in rats with the balloon crush SCI. Firstly, in a 7 day long study, we determined that the optimal dose for macrophage inhibition was 0.2 mg/week. Then, we demonstrated that a continuous subdural infusion of Serp-1 for 8 weeks resulted in consistently accelerated lowering of pro-inflammatory macrophages in the COI and in their almost complete elimination similar to that previously observed at 16 weeks in untreated SCI rats. The macrophage count in the COI is a quantitative test directly related to the severity of destructive inflammation initiated by the SCI. This test has consistently demonstrated anti-inflammatory effect of Serp-1 interpreted as neuroprotection, the first and necessary step in a therapeutic strategy in neurotrauma.
Spinal cord injury (SCI) results in massive secondary damage characterized by a prolonged inflammation with phagocytic macrophage invasion and tissue destruction. In prior work, sustained subdural infusion of anti-inflammatory compounds reduced neurological deficits and reduced pro-inflammatory cell invasion at the site of injury leading to improved outcomes. We hypothesized that implantation of a hydrogel loaded with an immune modulating biologic drug, Serp-1, for sustained delivery after crush-induced SCI would have an effective anti-inflammatory and neuroprotective effect. Rats with dorsal column SCI crush injury, implanted with physical chitosan-collagen hydrogels (CCH) had severe granulomatous infiltration at the site of the dorsal column injury, which accumulated excess edema at 28 days post-surgery. More pronounced neuroprotective changes were observed with high dose (100 µg/50 µL) Serp-1 CCH implanted rats, but not with low dose (10 µg/50 µL) Serp-1 CCH. Rats treated with Serp-1 CCH implants also had improved motor function up to 20 days with recovery of neurological deficits attributed to inhibition of inflammation-associated tissue damage. In contrast, prolonged low dose Serp-1 infusion with chitosan did not improve recovery. Intralesional implantation of hydrogel for sustained delivery of the Serp-1 immune modulating biologic offers a neuroprotective treatment of acute SCI.
Spinal cord injury (SCI)-initiated inflammation was treated with anti-inflammatory reagents. We compared local spinal cord or intraperitoneal infusion of two Myxoma virus derived immune modulating proteins, Serp-1 and M-T7, with dexamethasone (DEX).
Hemorrhage and necrosis after SCI initiate a complex pathogenesis dominated by early, severe and highly destructive inflammatory macrophage infiltration. We examined sustained, 7-day, subdural infusion of either M-T7, a chemokine modulator or Serp-1, a plasminogen activator and factor inhibitor. Mature male rats had epidural balloon crush SCI and sustained subdural infusion of Serp-1, M-T7, DEX or saline for 7 days via the osmotic pump. A separate group of rats with SCI had intra-peritoneal infusion. Clinical evaluation included endpoint monitoring with body weight, hemorrhagic cystitis and bilateral toe pinch response. Sections of the spinal cord were analyzed histologically and macrophage numbers counted by standardized protocol in the cavity of injury (COI).
While the rats administered DEX demonstrated substantial body weight loss, dehydration and dermal atrophy consistent with steroid toxicity, rats infused with Serp-1 and M-T7 had no toxicity. Serp-1 improved withdrawal responses. Subdural infusion of Serp-1, M-T7 and DEX significantly reduced numbers of phagocytic, CD68-positive macrophages. With intraperitoneal infusion only M-T7 reduced macrophage counts, Serp-1 showed only a trend. Local infusion of highly active immune modulating proteins; Serp-1 and M-T7, targeting serine protease and chemokine pathways demonstrated excellent potential for neuroprotection after severe SCI in a rat model, without adverse side effects. Sustained subdural infusion offers an alternative route of administration for treatment of SCI.
Trauma in spinal cord injury often results in massive damage to the white matter and in damage to myelin that results in a severe phagocyte-rich infiltration apparently directed at removing immunologically toxic myelin debris. In the epidural balloon crush injury to the rat cranial thoracic spinal cord, the dorsal column was crushed, which at one week post-op resulted in its obliteration by a severe infiltration by a virtually pure population of macrophages that internalized all damaged myelin. A week-long subdural infusion of dexamethasone, a stable synthetic corticosteroid, resulted in remarkable inhibition of the macrophage infiltration of the crush cavity and in the lack of removal of myelin debris by phagocytosis. In this study we demonstrated that spinal cord injury results in a severe inflammatory response directed at massively damaged myelin, and we inhibited this response with a subdural infusion of a powerful anti-inflammatory drug, dexamethasone.
Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.
Binding of chemokines to glycosaminoglycans (GAGs) is classically described as initiating inflammatory cell migration and creating tissue chemokine gradients that direct local leukocyte chemotaxis into damaged or transplanted tissues. While chemokine-receptor binding has been extensively studied during allograft transplantation, effects of glycosaminoglycan (GAG) interactions with chemokines on transplant longevity are less well known. Here we examine the impact of interrupting chemokine-GAG interactions and chemokine-receptor interactions, both locally and systemically, on vascular disease in allografts.Analysis of GAG or CC chemokine receptor 2 (CCR2) deficiency were coupled with the infusion of viral chemokine modulating proteins (CMPs) in mouse aortic allograft transplants (n = 239 mice). Inflammatory cell invasion and neointimal hyperplasia were significantly reduced in N-deacetylase-N-sulfotransferase-1 (Ndst1(f/f)TekCre(+)) heparan sulfate (GAG)-deficient (Ndst1(-/-), p
Behavioral assessment after spinal cord contusion has long focused on open field locomotion using modifications of a rating scale developed by Tarlov and Klinger (1954). However, on-going modifications by several groups have made interlaboratory comparison of locomotor outcome measures difficult. The purpose of the present study was to develop an efficient, expanded, and unambiguous locomotor rating scale to standardize locomotor outcome measures across laboratories. Adult rats (n = 85) were contused at T7-9 cord level with an electromagnetic or weight drop device. Locomotor behavior was evaluated before injury, on the first or second postoperative day, and then for up to 10 weeks. Scoring categories and attributes were identified, operationally defined, and ranked based on the observed sequence of locomotor recovery patterns. These categories formed the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale. The data indicate that the BBB scale is a valid and predictive measure of locomotor recovery able to distinguish behavioral outcomes due to different injuries and to predict anatomical alterations at the lesion center. Interrater reliability tests indicate that examiners with widely varying behavioral testing experience can apply the scale consistently and obtain similar scores. The BBB Locomotor Rating Scale offers investigators a more discriminating measure of behavioral outcome to evaluate treatments after spinal cord injury.
As the systematic work on the pathogenesis of the white matter injury in the spinal cord models progresses, it becomes obvious that a severe and extraordinarily protracted, destructive inflammation follows the initial injury. Appropriate anti-inflammatory therapies of sufficient duration should not only inhibit but also lead to the elimination of this destructive inflammation, thus resulting in neuroprotection of the spinal cord tissue and a greater preservation of the neurologic function. While dexamethasone, a powerful, anti-inflammatory steroid analog administered continuously by subdural infusion for 7 days inhibited severe macrophage infiltration in the cavity of injury, the dose used was remarkably toxic. A 2-week-long infusion of lower doses of dexamethasone resulted in dose-dependent inhibition of macrophage infiltration and was better tolerated by the rats, but it became evident that a much longer duration of subdural administration of a powerful anti-inflammatory drug is required to eliminate myelin-rich, necrotic debris from the cavity and synthetic steroids such as dexamethasone, and methylprednisolone may be too toxic for this application. Therefore, nontoxic but powerful anti-inflammatory compounds are required for neuroprotective treatment of the spinal cord injury (SCI) and also brain trauma and stroke where the massive injury to the white matter occurs. Serpins have been associated with neurological damage. The mammalian serpin neuroserpin (SERPINI1) is reported to act in a protective manner after cerebrospinal infarction. The serine protease, tissue-type plasminogen activator (tPA), and the serpin plasminogen activator inhibitor (PAI-1, SERPINE1) are both upregulated at sites of central nervous system damage. In preliminary studies, subdural infusion of the myxomaviral serpin, Serp-1, resulted in the powerful inhibition of the macrophage infiltration of the cavity of injury, comparable to the inhibition by high dose of dexamethasone that has proven to be unduly toxic. Nontoxic, yet powerful neuroprotective, anti-inflammatory effects of Serp-1 may indicate this serpin protein as a potential attractive compound to treat SCI and similar syndromes involving massive injury to the white matter such as brain trauma and stroke. Novel methods of drug delivery, chronic subdural infusion, and novel analytic methods to measure the effectiveness of the neuroprotective serpin treatments are discussed in this chapter.
Current therapies to limit the neural tissue destruction following the spinal cord injury are not effective. Our recent studies indicate that the injury to the white matter of the spinal cord results in a severe inflammatory response where macrophages phagocytize damaged myelin and the fluid-filled cavity of injury extends in size with concurrent and irreversible destruction of the surrounding neural tissue over several months. We previously established that a high dose of 4. mg/rat of dexamethasone administered for 1 week via subdural infusion remarkably lowers the numbers of infiltrating macrophages leaving large amounts of un-phagocytized myelin debris and therefore inhibits the severity of inflammation and related tissue destruction. But this dose was potently toxic to the rats. In the present study the lower doses of dexamethasone, 0.125-2.0. mg, were administered via the subdural infusion for 2 weeks after an epidural balloon crush of the mid-thoracic spinal cord. The spinal cord cross-sections were analyzed histologically. Levels of dexamethasone used in the current study had no systemic toxic effect and limited phagocytosis of myelin debris by macrophages in the lesion cavity. The subdural infusion with 0.125-2.0. mg dexamethasone over 2 week period did not eliminate the inflammatory process indicating the need for a longer period of infusion to do so. However, this treatment has probably lead to inhibition of the tissue destruction by the severe, prolonged inflammatory process.
Cellular mechanisms of regeneration after the white matter injury are difficult to study because of severe, inflammatory response to massively damaged myelin. Myelin-lacking CNS of the adult Long Evans Shaker (LES) rat supplies a model where neuroregeneration can be studied conveniently. The crush site in the dorsal spinal column in LES rats implanted with the normal rat choroid plexus was studied under the light and electron microscopy at 5 time points 3-56 days post-op. While the crush injury in normal rats resulted in severe inflammation active beyond 8 weeks, the same injury in LES rats resulted in a brief inflammation that resolved before day 7 post-op. In a clear fluid-filled crush cavity, ependymal cells from the implanted choroid plexus encased multiple regenerating axons, apparently guided them across the crush cavity and participated in establishing of a zone of neuroregeneration, morphologically similar to the white matter, at the interface of the crush cavity and the surrounding tissue of the spinal cord. Axons that were not encased by implanted cells failed to cross the crush cavity and persisted as markedly swollen end bulbs filled with organelles. At 8 weeks post-op, a large proportion of axons in the zone of neuroregeneration became myelinated by Schwann cells, likely originating from dorsal nerve roots or by oligodendrocytes that formed thin sheaths with a major dense line and likely originated from the implanted choroid plexus. The LES rat can serve as a convenient model to study mechanisms of neuroregeneration including axonal regeneration in the adult CNS injury.
Vascular inflammation can lead to plaque instability and acute coronary syndromes (ACS). Viruses produce potent immunomodulating proteins that regulate key inflammatory pathways. A myxoma virus-derived serpin Serp-1 reduces inflammatory cell invasion and plaque growth in vascular injury models. Our objective was to evaluate the safety and efficacy of Serp-1 in patients with ACS undergoing percutaneous coronary intervention.
This double-blind pilot trial included 48 ACS patients undergoing percutaneous coronary intervention randomly assigned to Serp-1 at doses of 5 μg/kg (n=19) or 15 μg/kg (n=17) or to placebo (n=12). Serp-1 was given by intravenous bolus immediately before intervention and 24 and 48 hours later. Patients were assessed for safety (primary objective) and efficacy outcomes, including biomarker analysis. In-stent neointimal hyperplasia was evaluated by intravascular ultrasound at 6 months. Key safety outcomes including coagulation parameters and adverse events did not differ between Serp-1 and placebo groups. A dose-dependent reduction in troponin I levels was observed with Serp-1 at 8, 16, 24, and 54 hours (P<0.05) and in creatine kinase-MB levels at 8, 16, and 24 hours after dose (P<0.05). The composite of death, myocardial infarction, or coronary revascularization occurred in 2 of 12 patients with placebo, 5 of 19 in the low-dose group, and none of 17 patients with the high-dose (P=0.058). Intravascular ultrasound did not detect changes in neointimal hyperplasia among groups.
This is the first study of a viral serpin demonstrating its safety in ACS patients. The significant reduction in myocardial damage biomarkers supports further assessment of Serp-1 in ACS patients undergoing stent deployment.
URL: http://www.clinicaltrials.gov. Unique identifier: NCT00243308.
The immune system functions by maintaining a delicate balance between the activities of pro-inflammatory and anti-inflammatory pathways. Unbalanced activation of these pathways often leads to the development of serious inflammatory diseases. TNF (Tumor Necrosis Factor) is a key pro-inflammatory cytokine, which can cause several inflammatory diseases when inappropriately up-regulated. Inhibition of TNF activities by using modulatory recombinant proteins has become a successful therapeutic approach to control TNF activity levels but these anti-TNF reagents also have risks and certain limitations. Biological molecules with a different mode of action in regulating TNF biology might provide a clinically useful alternative to the current therapeutics or in some cases might be efficacious in combination with existinganti-TNF therapies. TNF is also a powerful host defense cytokine commonly induced in the host response against various invading pathogens. Many viral pathogens can block TNF function by encoding modulators of TNF, its receptors or downstream signaling pathways. Here, we review the known virus-encoded TNF inhibitors and evaluate their potential as alternative future anti-TNF therapies.
Chemokines are important for activation of a host of cellular immune and inflammatory responses including cell signaling, activation, and communication. M-T7, a myxoma virus protein, inhibits the activity of chemokines by direct binding to chemokines and/or with glycosaminoglycans (GAGs). To study the effects of this chemokine-modulating protein (CMP), we use a variety of in vitro and in vivo techniques to evaluate M-T7 inhibition of inflammatory cells. To quickly analyze the effects of M-T7, changes in cell adhesion and membrane fluidity are measured as well as cell migration in mouse ascites. For more physiological analyses, an aortic transplant model in rodents is used to assess change in inflammatory cell infiltrates and vascular plaque growth (rejection). Utilization of the combination of these in vitro and in vivo techniques allows for a more complete study of the chemokine-modulating activity of M-T7, and can be used to study other immune and inflammation-modulating proteins.
Protracted inflammation extends damage after spinal cord injury
- J M Kwiecien
- W Dabrowski
- B Dąbrowska-Bouta
An in vivo model of anti-inflammatory activity of subdural dexamethasone following the spinal cord injury
- J M Kwiecien
- B Jarosz
- W Oakden
- JM Kwiecien
Tissue reaction to acellular implants in the acute spinal cord injury in the dysmyelinated rat
- J M Kwiecien
- JM Kwiecien