C. Amy Tovar

Deficient CX3CR1 Signaling Promotes Recovery after Mouse Spinal Cord Injury by Limiting the Recruitment and Activation of Ly6Clo/iNOS+ Macrophages

Macrophages exert divergent effects in the injured CNS, causing either neurotoxicity or regeneration. The mechanisms regulating these divergent functions are not understood but can be attributed to the recruitment of distinct macrophage subsets and the activation of specific intracellular signaling pathways. Here, we show that impaired signaling via the chemokine receptor CX3CR1 promotes recovery after traumatic spinal cord injury (SCI) in mice. Deficient CX3CR1 signaling in intraspinal microglia and monocyte-derived macrophages (MDMs) attenuates their ability to synthesize and release inflammatory cytokines and oxidative metabolites. Also, impaired CX3CR1 signaling abrogates the recruitment or maturation of MDMs with presumed neurotoxic effects after SCI. Indeed, in wild-type mice, Ly6Clo/iNOS+/MHCII+/CD11c− MDMs dominate the lesion site, whereas CCR2+/Ly6Chi/MHCII−/CD11c+ monocytes predominate in the injured spinal cord of CX3CR1-deficient mice. Replacement of wild-type MDMs with those unable to signal via CX3CR1 resulted in anatomical and functional improvements after SCI. Thus, blockade of CX3CR1 signaling represents a selective anti-inflammatory therapy that is able to promote neuroprotection, in part by reducing inflammatory signaling in microglia and MDMs and recruitment of a novel monocyte subset.

Kinetics of glucocorticoid response to restraint stress and/or experimental influenza viral infection in two inbred strains of mice

The murine model of influenza viral infection was used to evaluate the effects of restraint stress on pathogenesis and survival in inbred strains of mice. We recently reported that restraint stress was associated with an enhanced probability of survival in one strain of inbred mouse, DBA/2, and not in another, C57BL/6. Those studies suggested that the protective mechanism(s) of stress on mortality in the DBA/2 mice might be attributable to elevated levels of circulating glucocorticoids. Therefore, daily levels of plasma glucocorticoids were measured during influenza viral infection in both these strains. The present studies demonstrated that influenza infection itself is perceived as a stressor in both C57BL/6 and DBA/2 mice as evidenced by elevated plasma glucocorticoid levels within 48 h of infection. However, augmentation of glucocorticoid levels was not seen in the DBA/2 mice that were also subjected to restraint stress during the course of infection. Thus, corticosterone levels alone did not account for the enhanced survival seen in this group of animals.

An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat.

Changes in sensory function including chronic pain and allodynia are common sequelae of spinal cord injury (SCI) in humans. The present study documents the extent and time course of mechanical allodynia and cold hyperalgesia after contusion SCI in the rat using stimulation with graded von Frey filaments (4.97–50.45 g force) and ice probes. Fore- and hind-paw withdrawal thresholds to plantar skin stimulation were determined in rats with a range of SCI severities (10-g weight dropped from 6.25, 12.5, or 25 mm using the MASCIS injury device); animals with 25-mm injuries most consistently showed decreased hind-paw withdrawal thresholds to touch and cold, which developed over several weeks after surgery. Stimulation of the torso with graded von Frey hairs was performed at specified locations on the back and sides from the neck to the haunch. Suprasegmental responses (orientation, vocalization, or escape) to mechanical stimulation of these sites were elicited infrequently in the laminectomy control rats and only during the first 3 weeks after surgery, whereas in 25-mm SCI rats, such responses were obtained for the entire 10 weeks of the study. These data suggest that rats with contusion SCI may exhibit sensory alterations relevant to human spinal cord injuries.

Restraint stress differentially affects the pathogenesis of an experimental influenza viral infection in three inbred strains of mice

Genetic variation in the response to stress may play a critical role in susceptibility to inflammatory diseases and development of the immune response. Experimental influenza viral infection was used to study the effects of restraint stress (RST) on pathogenesis and development of the immune response. Three inbred strains of mice (C57BL/6, DBA/2, and C3H/HeN) were infected with influenza A/PR8 and subjected to repetitive cycles of RST during development of the immune response. RST diminished cellular immune and inflammatory responses in all three strains; yet only the DBA/2 strain demonstrated RST-associated reduction in influenza viral-induced mortality.

The effect of restraint stress on the kinetics, magnitude, and isotype of the humoral immune response to influenza virus infection

The stress of physical restraint has been shown to modulate the cellular immune response during a viral infection. We have studied the effects of stress on the humoral immune response during infection by influenza virus. Restraint stress altered the kinetics of the antibody response; seroconversion in the IgG and IgA isotypes was delayed in virus-infected C57BL/6 mice subjected to repeated cycles of physical restraint. However, the magnitude and isotype of the mature antibody response were unaffected during the plateau phase; no significant differences were observed between restrained/infected and nonrestrained/infected mice. Thus, the time during infection at which the antibody response was measured was a significant variable in the study of stress-induced alterations of the hosts response to a replicating viral antigen. While restraint stress did not significantly affect the magnitude or class of the humoral response, it did alter the kinetics of response.

STRESS-INDUCED CHANGES ATTRIBUTABLE TO THE SYMPATHETIC NERVOUS SYSTEM DURING EXPERIMENTAL INFLUENZA VIRAL INFECTION IN DBA/2 INBRED MOUSE STRAIN

The murine model of influenza viral infection was used to evaluate the effects of restraint stress on pathogenesis and survival in the DBA/2 inbred strain of mice. Restraint stress has been associated with an enhanced probability of survival during influenza infection in this strain of mouse. Previous studies suggested that the protective mechanism(s) of stress on mortality might be attributable to elevated levels of circulating glucocorticoids. Subsequent work demonstrated that corticosterone levels alone could not account for the enhanced survival seen in the DBA/2 mice. The present studies examined the role of catecholamines in behavioral stress during influenza infection. It appears that glucocorticoids may play a primary role in trafficking and restriction of inflammation, while catecholamines may play role in limiting activation of virus-specific effector cells. The studies presented here suggest that the interplay between these two physiological response mechanisms needs to be coordinated to optimize development of the immune response to an infection.

Topological Data Analysis for Discovery in Preclinical Spinal Cord Injury and Traumatic Brain Injury

Data-driven discovery in complex neurological disorders has potential to extract meaningful syndromic knowledge from large, heterogeneous data sets to enhance potential for precision medicine. Here we describe the application of topological data analysis (TDA) for data-driven discovery in preclinical traumatic brain injury (TBI) and spinal cord injury (SCI) data sets mined from the Visualized Syndromic Information and Outcomes for Neurotrauma-SCI (VISION-SCI) repository. Through direct visualization of inter-related histopathological, functional and health outcomes, TDA detected novel patterns across the syndromic network, uncovering interactions between SCI and co-occurring TBI, as well as detrimental drug effects in unpublished multicentre preclinical drug trial data in SCI. TDA also revealed that perioperative hypertension predicted long-term recovery better than any tested drug after thoracic SCI in rats. TDA-based data-driven discovery has great potential application for decision-support for basic research and clinical problems such as outcome assessment, neurocritical care, treatment planning and rapid, precision-diagnosis.

Developmental stage of oligodendrocytes determines their response to activated microglia in vitro

BackgroundOligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.MethodsOPCs and microglia were isolated from rat cortex. OPCs were induced to differentiate into oligodendrocytes with thyroid hormone in defined medium. For selected experiments, microglia were added to OPC or oligodendrocyte cultures. Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFα ELISA. Cell survival was assessed with immunocytochemistry and cell counts. OPC proliferation and oligodendrocyte apoptosis were also assessed.ResultsOPCs and oligodendrocytes displayed phenotypes representative of immature and mature oligodendrocytes, respectively. Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes. Activated microglia also underwent cell death themselves.ConclusionActivated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival. This may be of importance because activated microglia are present in several disease states where both OPCs and mature oligodendrocytes are also reacting to injury. Activated microglia may simultaneously have deleterious and helpful effects on different cells after central nervous system injury.

Hypertonic saline attenuates cord swelling and edema in experimental spinal cord injury: A study utilizing magnetic resonance imaging

Objective: To use magnetic resonance imaging (MRI) to characterize secondary injury immediately after spinal cord injury (SCI), and to show the effect of hypertonic saline on MRI indices of swelling, edema, and hemorrhage within the cord. Design: A prospective, randomized, placebo-controlled study. Setting: Research laboratory. Subjects: Twelve adult Long-Evans female rats. Interventions: Rats underwent a unilateral 12.5 mm SCI at vertebral level C5. Animals were administered 0.9% NaCl (n = 6) or 5% NaCl (n = 6) at 1.4 mL/kg intravenously every hour starting 30 minutes after SCI. Immediately after SCI, rats were placed in a 4.7T Bruker MRI system and images were obtained continuously for 8 hours using a home-built transmitter/receiver 3 cm Helmholtz coil. Rats were killed 8 hours after SCI. Measurements and Main Results: Quantification of cord swelling and volumes of hypointense and hyperintense signal within the lesion were determined from MRI. At 36 minutes after SCI, significant swelling of the spinal cord at the lesion center and extending rostrally and caudally was demonstrated by MRI. Also, at this time point, a hypointense core was identified on T1, PD, and T2 weighted images. Over time this hypointense core reduced in size and in some animals was no longer visible by 8 hours after SCI, although histopathology demonstrated presence of red blood cells. A prominent ring of T2-weighted image hyperintensity, characteristic of edema, surrounded the hypointense core. At the lesion center, this rim of edema occupied the entire unilateral injured cord and in all animals extended to the contralateral side. Administration of HS resulted in increased serum [Na], attenuation of cord swelling, and decreased volume of hypointense core and edema at the last time points. Conclusions: We were able to use MRI to detect rapid and acute changes in the evolution of tissue pathophysiology, and show potentially beneficial effects of hypertonic saline in acute cervical SCI.

Induction of endogenous tumor necrosis factor-α: suppression of centrally stimulated gastric motility

Gastric stasis is frequently seen in conjunction with critical infectious illness, chronic inflammatory disorders, radiation sickness, and carcinogenesis. These conditions are associated with elevated circulating levels of the cytokine tumor necrosis factor-α (TNF-α). The present studies examined the relationship between endogenously produced TNF-α and the central neural mechanisms that augment gastric motility. Systemic lipopolysaccharide (LPS) was employed to induce TNF-α production in thiobutabarbital-anesthetized rats. Sixty minutes after intravenous LPS injection, gastric motility could not be stimulated by a potent centrally acting gastrokinetic stimulant, thyrotropin-releasing hormone (TRH). This failure to elicit gastric motility via central mechanisms coincided with high circulating levels of TNF-α. However, intravenous injections of bethanecol, a peripherally acting cholinergic agonist with direct gastrokinetic effects, were still able to elicit normal increases in gastric motility in the presence of TNF-α and LPS. Therefore, the inability to stimulate gastric motility via central TRH could not be attributed to the direct inhibitory effects of either LPS or TNF-α on the stomach. If the production of endogenous TNF-α was suppressed via the use of urethan as the anesthetic agent, then intravenous injections of LPS were no longer effective in suppressing gastric motility. Thus these effects on gastric motility are not directly attributable to LPS nor are they due to direct effects on the gastric smooth muscle. Our previous study demonstrated that microinjection of femtomole quantities of TNF-α in the brain stem dorsal vagal complex (DVC) can modulate gastric motility. This central TNF-α effect on gastric motility was dose dependent and required an intact vagal efferent pathway. The results from these two studies suggest that systemically produced TNF-α may gain access to the DVC to modulate gastric function.