Iba1 immunofluorescence images of the ipsilateral dorsal horns from naïve young (YN) and middle-aged (MN) rats rendered in 3D.

<p>Upper Panels: individual optical sections (1.25 μm intervals through 25 μm thick sections) were acquired with a LSCM and 40x 1.4 NA objective lens and then recombined and rendered as three-dimensional images. Lower Panels: Two-photon immunofluorescence 3D renderings of Iba1 positive dorsal horn microglia from young and middle-aged naïve (YN and MN). Image stacks (0.44 μm optical sections through 25 μm thick specimens) were obtained using a two-photon laser scanning confocal microscope and 100 X 1.4 NA objective lens. Each stack set was recombined to create the 3D rendering. A single plane of the 3D image is shown for each. Scale bar = 25 μm. Rotatable 3D images are also available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.s003" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.s004" target="_blank">S2</a> Files.</p

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

<div><p>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 aged-matched 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 middle-aged 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 T-cell 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.</p></div

Lumbar Spinal Cord Gene Expression Comparing Sham and CCI: Effects of Age.

<p>LSC ipsilateral hemisections were removed on day 7. Expression levels in lumbar spinal cord were normalized to the geomean of Hprt1 and Pplb expression and the ratios multiplied by 100 and presented as the mean ratios +/- SD. Two-way ANOVA results are shown with contrasts and associated p values. Numbers of animals (n) assayed per group are shown. Bolded/italicized genes indicate specific contrast differences p < 0.05</p><p>* indicates difference trending to significance. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.t002" target="_blank">Table 2</a>.</p><p>Lumbar Spinal Cord Gene Expression Comparing Sham and CCI: Effects of Age.</p

Gene Expression Profile of Young and Middle-Aged Sham and CCI LSC.

<p>LSC ipsilateral hemisections were removed on day 7. Expression levels in LSC samples were normalized to the geomean of Hprt1 and Pplb expression and the ratios multiplied by 100 and presented as the mean ratios +/- SD. Two-way ANOVA results are shown with contrasts and associated p values. Numbers of animals (n) assayed per group are shown. Bolded/italicized genes indicate specific contrast differences p < 0.05</p><p>* indicates difference trending to significance. YS = young sham, YCCI = young CCI, MS = middle-aged sham, MCCI = middle-aged CCI.</p><p>Gene Expression Profile of Young and Middle-Aged Sham and CCI LSC.</p

Gene expression Profile of Naïve Young and Middle-Aged DRG.

<p>Results are expressed as mean +/-SD and numbers of animals assayed per group are indicated. Bolded/italicized genes indicate significant differences. Further statistical results and corrections for multiple comparisons are in Additional File 1. YN = young naïve, MN = middle-aged naïve.</p><p>Gene expression Profile of Naïve Young and Middle-Aged DRG.</p

Gene Expression Profile of Young Sham and CCI DRG at Day 7.

<p>Gene Expression Profile of Young Sham and CCI DRG at Day 7.</p

Representative Iba1 immunofluorescence images of young and middle aged of lumbar spinal cord dorsal horns from post-CCI day 7 animals, ipsilateral or contralateral to injury.

<p>Immunofluorescence confocal images of the dorsal horns stained with Iba1 antibody were combined with corresponding transmitted light images. Young (YCCI) and middle-aged (MCCI) dorsal horns ipsilateral (IPSL) to injury are compared to the contralateral (CL) sides. Scale bars = 100 μm.</p

Neuroimmune gene expression profile in lumbar spinal cords of naïve young and middle-aged rats.

<p>Expression levels were normalized to the geomean of Hprt1 and Pplb expression and the ratios multiplied by 100. Results are presented as means +/- SD. Significance of differences between YN v MN * p < 0.0015.</p

Iba1 immunofluorescence of the ipsilateral dorsal horns from post-CCI day 7 young (YCCI) and middle-aged (MCCI) rats rendered in 3D.

<p>Left Panels: optical sections (1.25 μm intervals through 25 μm thick sections) were acquired with a LSCM and 40x 1.4 NA objective lens and then recombined and rendered as three-dimensional images. Middle and Right Panels: image stacks (0.44 μm optical sections through 25 μm thick specimens) were obtained using a two-photon laser scanning confocal microscope and 100 X 1.4 NA objective lens. Each stack set was recombined to create the 3D rendering. A single plane of the 3D image is shown for each. Scale bar = 25 μm.</p

Summary of multiplex assay results and immunofluorescence imaging of the LSC.

<p>LSC = lumbar spinal cord; MN = middle-aged naïve; YN = young naïve; MS = middle-aged sham; YS = young sham; MCCI = middle-aged CCI; YCCI = young CCI. + sign indicates ratios significantly > 1 but <u><</u> 2-fold; ++ > 2-fold but < 3; +++ > 3-fold;–sign indicates ratios significantly < 1; and ~ sign indicates ratio ~ 1. Density of microglia refers to the dorsal horn, and for CCI, the ipsilateral side 7 days post-injury.</p><p>Summary of multiplex assay results and immunofluorescence imaging of the LSC.</p