Neuroimmune and Neuropathic Responses of Spinal Cord and Dorsal Root Ganglia in Middle Age

RESEARCH ARTICLE Neuroimmune and Neuropathic Responses of Spinal Cord and Dorsal Root Ganglia in Middle Age William Galbavy, Martin Kaczocha, Michelino Puopolo, Lixin Liu, Mario J. Rebecchi* Department of Anesthesiology, Stony Brook University, Stony Brook, New York, United States of America * Abstract OPEN ACCESS Citation: Galbavy W, Kaczocha M, Puopolo M, Liu L, Rebecchi MJ (2015) Neuroimmune and Neuropathic Responses of Spinal Cord and Dorsal Root Ganglia in Middle Age. PLoS ONE 10(8): e0134394. doi:10.1371/journal.pone.0134394 Editor: Simon Beggs, Toronto University, CANADA Received: March 31, 2015 Accepted: July 8, 2015 Published: August 4, 2015 Copyright: © 2015 Galbavy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Prior studies of aging and neuropathic injury have focused on senescent animals compared to young adults, while changes in middle age, particularly in the dorsal root ganglia (DRG), have remained largely unexplored. 14 neuroimmune mRNA markers, previously associated with peripheral nerve injury, were measured in multiplex assays of lumbar spinal cord (LSC), and DRG from young and middle-aged (3, 17 month) naïve rats, or from rats subjected to chronic constriction injury (CCI) of the sciatic nerve (after 7 days), or from agedmatched sham controls. Results showed that CD2, CD3e, CD68, CD45, TNF-α, IL6, CCL2, ATF3 and TGFβ1 mRNA levels were substantially elevated in LSC from naïve middle-aged animals compared to young adults. Similarly, LSC samples from older sham animals showed increased levels of T-cell and microglial/macrophage markers. CCI induced further increases in CCL2, and IL6, and elevated ATF3 mRNA levels in LSC of young and middleaged adults. Immunofluorescence images of dorsal horn microglia from middle-aged naïve or sham rats were typically hypertrophic with mostly thickened, de-ramified processes, similar to microglia following CCI. Unlike the spinal cord, marker expression profiles in naïve DRG were unchanged across age (except increased ATF3); whereas, levels of GFAP protein, localized to satellite glia, were highly elevated in middle age, but independent of nerve injury. Most neuroimmune markers were elevated in DRG following CCI in young adults, yet middle-aged animals showed little response to injury. No age-related changes in nociception (heat, cold, mechanical) were observed in naïve adults, or at days 3 or 7 post-CCI. The patterns of marker expression and microglial morphologies in healthy middle age are consistent with development of a para-inflammatory state involving microglial activation and Tcell marker elevation in the dorsal horn, and neuronal stress and satellite cell activation in the DRG. These changes, however, did not affect the establishment of neuropathic pain. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was entirely supported by internal funds of the Department of Anesthesiology, Stony Brook University School of Medicine. Introduction Competing Interests: The authors have declared that no competing interests exist. Normal healthy aging is associated with neuroimmune changes that have been referred to as “inflammaging”, an elevation of inflammatory tone with age that may contribute to the aging PLOS ONE | DOI:10.1371/journal.pone.0134394 August 4, 2015 1 / 27 Aging and Neuropathic Pain Response process itself, as well as enhance susceptibility to neurodegeneration [1–5]. As a result, an incipient or para-inflammatory state is thought to develop that predisposes the senescent CNS to deleterious neurotoxic responses following injury or infection or stress. A large body of evidence now supports this idea. For example, multiple inflammatory markers increase with age in various brain regions of healthy rats, mice, and primates [5–7], particularly the pro-inflammatory cytokines interleukin 1β (IL1β), tumor necrosis factor α (TNFα) and interleukin 6 (IL6), as well as microglial activation markers, Cd11b (Ox42, C3A receptor) and MHCII (major histocompatibility complex II), and the astrogliosis marker, glial fibrillary acidic protein (GFAP); moreover, challenging the senescent CNS with lipopolysaccharide (LPS) or with mechanical injury induces exaggerated neuroinflammatory responses, exacerbates decline, and delays functional recovery [6–9]. Furthermore, neuroimmune profiles of healthy aged and diseased brains suggest that early para-inflammatory changes, particularly activation of microglia [10], may contribute to neurodegenerative disorders, such as Alzheimer’s dementia [11, 12] and Parkinson’s disease [13]. In contrast to the extensive work on the aging mammalian brain, relatively few reports have examined inflammatory markers in the aging spinal cord in healthy or nerve-injured subjects. Early work showed some differences in the numbers of Ox42+ (CD11b) microglia in lumbar spinal cord (LSC) from healthy young and middle-aged adults, whereas senescent adults had greater numbers and staining intensities of activated microglia [14]. Similarly, sections of spinal cords and brainstems from healthy senescent rats showed increased CD11b and ED1 (CD68) immunoreactivity in microglia, and GFAP in astrocytes compared to young adults [15]. Many of these CD68-positive microglia were hypertrophic with short stout processes, many were localized to the white matter, and these were found at higher levels in senescent animals with severe sensorimotor deficits. While it has been reported that sciatic nerve injury increases the numbers of CD11b-positive microglia in both young and middle-aged LSC, this increase was attenuated in senescent animals [14, 16]. In canine spinal cord, increased numbers of Iba1-positive microglia with “activated” morphology have been found in lumbar and cervical cords of older (10–12 years) compared to young adults (1–2 years)[17]. Taken together, these studies demonstrate age-related changes in spinal cord microglia and astrocytes that are consistent with inflammaging and that could lead to exaggerated responses and/or to delayed recovery following nerve injury. Indeed, increased sensitivities to noxious heat [18–20], and increased mechanical allodynia [21] and hyperalgesia [20] have been found in older neuropathic animals. Contrary to these reports, however, reduced mechanical allodynia and decreased ongoing pain have been reported in older rats following spinal nerve ligation [22]. Nonetheless, these evoked response differences were modest, and their interpretation could be complicated by age-related changes in sensory thresholds. On the other hand, substantial delays in pain resolution have been consistently reported in senescent animals following nerve injury [23–25]. Peripheral nerve injury provokes a rapid innate immune re (...truncated)