Jessica K. Alexander

Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord

Macrophages dominate sites of CNS injury in which they promote both injury and repair. These divergent effects may be caused by distinct macrophage subsets, i.e., “classically activated” proinflammatory (M1) or “alternatively activated” anti-inflammatory (M2) cells. Here, we show that an M1 macrophage response is rapidly induced and then maintained at sites of traumatic spinal cord injury and that this response overwhelms a comparatively smaller and transient M2 macrophage response. The high M1/M2 macrophage ratio has significant implications for CNS repair. Indeed, we present novel data showing that only M1 macrophages are neurotoxic and M2 macrophages promote a regenerative growth response in adult sensory axons, even in the context of inhibitory substrates that dominate sites of CNS injury (e.g., proteoglycans and myelin). Together, these data suggest that polarizing the differentiation of resident microglia and infiltrating blood monocytes toward an M2 or “alternatively” activated macrophage phenotype could promote CNS repair while limiting secondary inflammatory-mediated injury.

Beta-adrenergic Modulation of Cognitive Flexibility during Stress

Stress-induced activation of the locus ceruleus-norepinephrine (LC-NE) system produces significant cognitive and behavioral effects, including enhanced arousal and attention. Improvements in discrimination task performance and memory have been attributed to this stress response. In contrast, for other cognitive functions that require cognitive flexibility, increased activity of the LC-NE system may produce deleterious effects. The aim of the present study was to determine the effect of pharmacological modulation of the LC-NE system on stress-induced impairments in cognitive flexibility performance in healthy individuals. Cognitive performance, plus psychological and physiological parameters for 16 adults without any history of anxiety disorders, was assessed during four test sessions: stress and no-stress, with each condition tested after administration of propranolol and placebo. The Trier Social Stress Test, a public-speaking and mental arithmetic stressor, was presented to participants for the stress sessions, whereas a similar, but nonstressful, control task (reading, counting) was utilized for the no-stress sessions. Tests of cognitive flexibility included lexical-semantic and associative problem-solving tasks (anagrams, Compound Remote Associates Test). Visuo-spatial memory and motor processing speed tests served as control tasks. Results indicate that (1) stress impaired performance on cognitive flexibility tasks, but not control tasks; (2) compared to placebo, cognitive flexibility improved during stress with propranolol. Therefore, psychological stress, such as public speaking, negatively impacts performance on tasks requiring cognitive flexibility in normal individuals, and this effect is reversed by beta-adrenergic antagonism. This may provide support for the hypothesis that stress-related impairments in cognitive flexibility are related to the noradrenergic system.

2006 Curt P. Richter award winner: Social influences on stress responses and health.

Both positive and negative social interactions can modulate the hypothalamic-pituitary-adrenal (HPA) axis and influence recovery from injuries and illnesses, such as wounds, stroke, and cardiac arrest. Stress exacerbates neuronal death following stroke and cardiac arrest, and delays cutaneous wound healing, via a common mechanism involving stress-induced increases in corticosterone, acting on glucocorticoid receptors. In contrast, hamsters and mice that form social bonds are buffered against stress and heal cutaneous wounds more quickly than socially isolated animals, presumably because the physical contact experienced by the pairs releases oxytocin, which in turn suppresses the HPA axis and facilitates wound healing. Social housing also decreases stroke-induced neuronal death and improves functional recovery, but the mechanism appears to involve suppressing the inflammatory response that accompanies stroke, rather than alterations in HPA axis activity. An interaction between the HPA axis and immune system determines stroke outcome in neonatally manipulated mice that exhibit life-long dampening of the HPA axis. Taken together, these studies provide support for the detrimental effects of stress and identify potential mechanisms underlying the well-documented clinical observation that social support positively influences human health.

Neuroinflammation in spinal cord injury: therapeutic targets for neuroprotection and regeneration.

Traumatic spinal cord injury triggers a complex local inflammatory reaction capable of enhancing repair and exacerbating pathology. The composition and effector potential of the post-injury cellular and molecular immune cascade changes as a function of time and distance from the lesion. Production along this time-space continuum of cytokines, proteases, and growth factors establishes dynamic environments that lead to the death, damage, repair or growth of affected neurons and glia. Microenvironmental cues, therefore, generated by the cells therein, may determine these distinct fates of repair versus pathology. To harness repair, it is necessary to manipulate the assembly and phenotype of cells that comprise the neuroinflammatory response to injury. Here, the potential of the neuroinflammatory response to cause outcomes such as pain, regeneration, and functional recovery is reviewed.

Stress Exacerbates Neuropathic Pain via Glucocorticoid and NMDA Receptor Activation

There is growing recognition that psychological stress influences pain. Hormones that comprise the physiological response to stress (e.g., corticosterone; CORT) may interact with effectors of neuropathic pain. To test this hypothesis, mice received a spared nerve injury (SNI) after exposure to 60 min restraint stress. In stressed mice, allodynia was consistently increased. The mechanism(s) underlying the exacerbated pain response involves CORT acting via glucocorticoid receptors (GRs); RU486, a GR antagonist, prevented the stress-induced increase in allodynia whereas exogenous administration of CORT to non-stressed mice reproduced the allodynic response caused by stress. Since nerve injury-induced microglial activation has been implicated in the onset and propagation of neuropathic pain, we evaluated cellular and molecular indices of microglial activation in the context of stress. Activation of dorsal horn microglia was accelerated by stress; however, this effect was transient and was not associated with the onset or maintenance of a pro-inflammatory phenotype. Stress-enhanced allodynia was associated with increased dorsal horn extracellular signal-regulated kinase phosphorylation (pERK). ERK activation could indicate a stress-mediated increase in glutamatergic signaling, therefore mice were treated prior to SNI and stress with memantine, an N-methyl-D-aspartate receptor (NMDAR) antagonist. Memantine prevented stress-induced enhancement of allodynia after SNI. These data suggest that the hormonal responses elicited by stress exacerbate neuropathic pain through enhanced central sensitization. Moreover, drugs that inhibit glucocorticoids (GCs) and/or NMDAR signaling could ameliorate pain syndromes caused by stress.

Macrophage Migration Inhibitory Factor Potentiates Autoimmune-Mediated Neuroinflammation

Macrophage migration inhibitory factor (MIF) is a multipotent cytokine that is associated with clinical worsening and relapses in multiple sclerosis (MS) patients. The mechanism through which MIF promotes MS progression remains undefined. In this study, we identify a critical role for MIF in regulating CNS effector mechanisms necessary for the development of inflammatory pathology in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Despite the ability to generate pathogenic myelin-specific immune responses peripherally, MIF-deficient mice have reduced EAE severity and exhibit less CNS inflammatory pathology, with a greater percentage of resting microglia and fewer infiltrating inflammatory macrophages. We demonstrate that MIF is essential for promoting microglial activation and production of the innate soluble mediators IL-1β, IL-6, TNF-α, and inducible NO synthase. We propose a novel role for MIF in inducing microglial C/EBP-β, a transcription factor shown to regulate myeloid cell function and play an important role in neuroinflammation. Intraspinal stereotaxic microinjection of MIF resulted in upregulation of inflammatory mediators in microglia, which was sufficient to restore EAE-mediated inflammatory pathology in MIF-deficient mice. To further implicate a role for MIF, we show that MIF is highly expressed in human active MS lesions. Thus, these results illustrate the ability of MIF to influence the CNS cellular and molecular inflammatory milieu during EAE and point to the therapeutic potential of targeting MIF in MS.

Macrophage migration inhibitory factor (MIF) is essential for inflammatory and neuropathic pain and enhances pain in response to stress.

Stress and glucocorticoids exacerbate pain via undefined mechanisms. Macrophage migration inhibitory factor (MIF) is a constitutively expressed protein that is secreted to maintain immune function when glucocorticoids are elevated by trauma or stress. Here we show that MIF is essential for the development of neuropathic and inflammatory pain, and for stress-induced enhancement of neuropathic pain. Mif null mutant mice fail to develop pain-like behaviors in response to inflammatory stimuli or nerve injury. Pharmacological inhibition of MIF attenuates pain-like behaviors caused by nerve injury and prevents sensitization of these behaviors by stress. Conversely, injection of recombinant MIF into naïve mice produces dose-dependent mechanical sensitivity that is exacerbated by stress. MIF elicits pro-inflammatory signaling in microglia and activates sensory neurons, mechanisms that underlie pain. These data implicate MIF as a key regulator of pain and provide a mechanism whereby stressors exacerbate pain. MIF inhibitors warrant clinical investigation for the treatment of chronic pain.

SEMI-AUTOMATED SHOLL ANALYSIS FOR QUANTIFYING CHANGES IN GROWTH AND DIFFERENTIATION OF NEURONS AND GLIA

There is a need to develop therapies that promote growth or remyelination of mammalian CNS axons. Although the feasibility of pre-clinical treatment strategies should be tested in animal models, in vitro assays are usually faster and less expensive. As a result, in vitro models are ideal for screening large numbers of potential therapeutics prior to use in more complex in vivo systems. In 1953, Sholl introduced a technique that is a reliable and sensitive method for quantifying indices of neurite outgrowth. However, application of the technique is limited because it is labor-intensive. Several methods have been developed to reduce the analysis time for the Sholl technique; but these methods require extensive pre-processing of digital images, they introduce user bias or they have not been compared to manual analysis to ensure accuracy. Here we describe a new, semi-automated Sholl technique for quantifying neuronal and glial process morphology. Using MetaMorph, we developed an unbiased analysis protocol that can be performed approximately 3x faster than manual quantification with a comparable level of accuracy regardless of cell morphology. The laborious image processing typical of most computer-aided analysis is avoided by embedding image correction functions into the automated portion of the analysis. The sensitivity and validity of the technique was confirmed by quantifying neuron growth treated with growth factors or oligodendroglial maturation in the presence or absence of thyroid hormone. Thus, this technique provides a rapid and sensitive method for quantifying changes in cell morphology and screening for treatment effects in multiple cell types in vitro.

The Effect of Auditory Stressors on Cognitive Flexibility

Stress is known to activate the noradrenergic system which may have a modulatory influence on cognitive flexibility. We investigated whether an auditory stressor would thus affect performance on cognitive flexibility. A task utilizing cognitive flexibility and two memory tasks were presented in both stressful and non-stressful condition. In the stressful condition performance was impaired for the cognitive flexibility task but not for the memory tasks, arguing against the stressor serving as a general distracter. These findings suggest that stress caused by an auditory stressor may negatively impact performance on more complex tasks requiring a rapid search of the semantic and lexical associative networks.

Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs

Abstract Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice (Mus musculus) basal GC receptor (Nr3c1, also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity.