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Microglia and astrocyte activation in the spinal cord of lame horses
Accepted Manuscript Microglia and astrocyte activation in the spinal cord of lame horses Constanza S. Meneses, Heine Y. Müller, Daniel E. Herzberg, Benjamin Uberti, Marianne P. Werner, Hedie A. Bustamante PII:
S1467-2987(17)30353-7
DOI:
10.1016/j.vaa.2017.10.001
Reference:
VAA 209
To appear in:
Veterinary Anaesthesia and Analgesia
Received Date: 21 April 2017 Revised Date:
6 October 2017
Accepted Date: 10 October 2017
Please cite this article as: RRH: Microglia and astrocytes in lame horses This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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RESEARCH PAPER Running head (Author): CS Meneses et al. Running head (short title): Microglia and astrocytes in lame horses
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Microglia and astrocyte activation in the spinal cord of lame horses
Constanza S Menesesa, Heine Y Müllerb, Daniel E Herzbergb, Benjamin Ubertib, Marianne P Wernerc & Hedie A Bustamanteb
Graduate School, Faculty of Veterinary Sciences, Universidade Austral de Chile, Valdivia, Chile
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a
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Veterinary Clinical Sciences Department, Faculty of Veterinary Sciences, Universidade Austral
c
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de Chile, Valdivia, Chile
Animal Science Department, Faculty of Veterinary Sciences, Universidade Austral de Chile,
Valdivia, Chile
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Correspondence: Hedie A Bustamante, Institute of Veterinary Clinical Sciences, Faculty of Veterinary Sciences, Universidade Austral de Chile, Independencia 631, Valdivia 5090000, Chile.
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Abstract
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E-mail: [email protected]
Objective To determine the microglial and astrocyte response to painful lameness in horses. Study design Ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) expression, cell density and morphology were determined through immunofluorescence within the dorsal horn of equine spinal cord. Animals Five adult horses with acute or chronic unilateral lameness, previously scheduled for euthanasia. 1
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Methods Musculoskeletal lameness was evaluated in five horses through visual evaluation according to clinical guidelines. Spinal cord samples were obtained immediately after euthanasia, and distal limb lesions were confirmed through dissection and radiography. Iba-1 immunostaining
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was used for detection and characterization of dorsal horn microglia. GFAP was used for
immunostaining of dorsal horn astrocytes. Iba-1 and GFAP labeled cells were quantified in the dorsal horn, and intensity of fluorescence was compared between the ipsi- and contralateral
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dorsal horn to the affected limb, and between dorsal horn segments of all horses.
Results Iba-1 expression was higher in the ipsilateral dorsal horn of the affected limb in contrast
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to the contralateral side dorsal horn. GFAP markers did not demonstrate increased astrocytic activity on the dorsal horn ipsilateral side to the distal limb lesion of affected horses. Horses with acute lameness predominantly had a spherical shape microglial phenotype, while cells from chronic lameness cases had variable morphology. Astrocytes evidenced small somas and large
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processes in both acute and chronic lameness, with higher GFAP localization in the main branches. Like rodents, the localization of microglia and astrocytes in horses was mainly situated within laminae I, II and III.
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Conclusions and clinical relevance Iba-1 and GFAP are functional and morphological markers
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of spinal microglial cells and astrocytes in horses with lameness.
Keywords astrocyte, horses, lameness, microglia.
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Introduction In horses, pain is usually the first and only sign of disease, becoming a valuable clinical parameter for veterinarian practitioners (Price et al. 2002). Furthermore, severe or chronic cases
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of pain are often refractory to conventional anti-inflammatory analgesia, and euthanasia is
required (Jones et al. 2007). Osteoarthritis, laminitis and navicular disease cause chronic pain in horses and their treatment is considered one of the greatest challenges in equine clinical practice
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(Driessen 2010). Nevertheless, few studies have focused on the importance of chronic pain in
neuropathic pain in this species.
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horses (Jones et al. 2007), and no studies have identified major factors in the modulation of
Recently, the role of glial cells in the central nervous system (CNS) of rodents in the maintenance of pain has been described, making glial cells potentially a novel therapeutic target (Narita et al. 2006). Glial cells are capable of enhancing pain and dynamically modulating
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neurons (Temburni & Jakob 2001; Watkins et al. 2001). Spinal and peripheral nerve lesions result in physiological activation of glial cells in the ipsilateral dorsal horn of the affected limb (Mika et al. 2013). Once activated, these cells undergo morphological and functional modifications that
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trigger the release of algesic mediators capable of changing the neuron-glia interaction and enhance pain sensitivity (Ji et al. 2013). Of all glial cells described, spinal cord microglia and
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astrocytes have been the main focus for research. These cells generate a significant neuronal depolarization that makes dorsal horn neurons more hyperexcitable, resulting in evoked and spontaneous activity in dorsal horn neurons and contributing to the development of pathologic pain (Gwak et al. 2012).
The recognition of function and morphology of these cells has been described using specific biomarkers. Ionized calcium binding adaptor molecule 1 (Iba-1) is specifically expressed both in vitro and in vivo by microglia (Ito et al. 1998). After trauma, ischemia or nerve 3
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inflammation, spinal microglia become the first responders, increasing Iba-1 expression inducing functional and phenotypic modifications (Raghavendra et al. 2003; Faustino et al. 2011). These modifications increase the convergent nociceptive inputs to spinal dorsal horn neurons, and
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consequently the development of mechanical allodynia and thermal hyperalgesia (Yamamoto et al. 2015). These morphological changes reflect the active state of these cells. Normally, resting microglial cells have a small body from which short extensions arise around spinal neurons.
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Following long-term nociceptive input, they adopt an amoeboid shape and phagocytic functions (Hansson 2006). Microglial morphology is described as flexible and dynamic, with different
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morphologies that can markedly change and represent transient functional states (Hanisch & Kettenmann 2007).
Glial fibrillary acidic protein (GFAP) is one of the most commonly used markers for identification of astrocytes. GFAP modulates the shape and motility of astrocytes (Eng et al.
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2000) and is the major component of intermediate filaments of mature astrocytes (Nolte et al. 2001). After microglial activation, astrocytes overreact to nerve damage, and their activation is characterized by an increase in GFAP expression, enlargement of astrocytic processes (Gosselin
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et al. 2010) and cell swelling/hypertrophy. These changes vary qualitatively and quantitatively depending on the nature of the injury (Hansson et al. 2000; Allansson et al. 2001).
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Although murine models of pain have allowed the elucidation of factors that mediate
chronic pain, there is still an insufficient understanding of chronic pain and its consequences in other mammals (Whiteside et al. 2008). In horses, microglial and astrocytic responses to acute or chronic painful conditions have not been previously described. Therefore, the purpose of this study was to determine the microglial and astrocyte responses to lameness in horses, thus providing new data about central gliosis response in another mammalian species. Iba-1 and GFAP
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expression, cell density and morphology within the dorsal horn were compared between the ipsiand contralateral side of the affected limb in horses with unilateral acute or chronic lameness.
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Materials and methods
The experimental protocol was approved by the Ethics Committee of Animal Research of
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the Universidad Austral de Chile (no. 001/2017).
Animals
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Study subjects were selected prospectively, either from Universidad Austral de Chile teaching hospital herd (two horses) or from a commercial slaughterhouse (eight horses). Inclusion criteria were animals aged > 2 years of age, without sex, breed or size restriction. Researchers were not involved in the decision for euthanasia or slaughter. Euthanasia was performed by
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intravenous general anesthesia and intrathecal lidocaine injection at the atlantooccipital foramen for teaching herd horses or by mechanical stunning and exsanguination for the slaughterhouse
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horses, according to national regulations.
Lameness assessment and post-mortem radiography and dissection
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Selection criteria for horses included visible unilateral lameness, evaluated clinically by
an experienced equine clinician (CSM). Teaching herd horses were evaluated at the University teaching hospital, and slaughterhouse horses were evaluated visually in the holding pens. Horses with grade 2–5 lameness, according to the American Association of Equine Practitioners grading system (Keegan et al. 2010), were selected. Exclusion criteria included the presence of visibly acute wounds or visibly neurological gait alteration (central or peripheral ataxia). After euthanasia, affected limbs and the contralateral limbs distal to the proximal metacarpal or 5
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metatarsal epiphysis were collected from all selected horses. Six radiographic views (dorsopalmar and lateromedial of the hoof, dorsopalmar, lateromedial, dorsolateral-palmaromedial oblique and dorsomedial-palmarolateral oblique of the metacarpophalangeal /metatarsophalangeal joint) were
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obtained from the selected limbs using standard equipment (Diagnostic X-Ray, Orange 8016 HF; EcoRay Co Ltda., Korea). Digital images were stored on hard drives and interpreted by an
experienced equine clinician (CSM). For confirmation of radiological diagnosis, limbs were
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anatomically dissected (including examination of soft tissues, bones, joints and hooves). Lame horses were classified as either acute (less than 6 months duration) or chronic (more than 6
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months duration) based on teaching hospital records and information provided by owners of the horses obtained from the slaughterhouse. Spinal cord samples from these horses were included for immunohistochemical assays.
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Sampling of spinal cord tissues
Spinal cord tissue was collected immediately after euthanasia following longitudinal section of spinal vertebrae. Cervical spinal segments (C2-C7) were harvested from horses with
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thoracic limb lameness, or lumbar spinal segments (L1-L6) from horses with pelvic limb lameness. Samples were sectioned transversely and then stored in individual jars containing
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Bouin fixative [75 mL of saturated aqueous picric acid (1.2% w/v), 25 mL of formalin (40% w/v formaldehyde) and 5 mL of glacial acetic acid]. The cranial and caudal aspects of the spinal cord segments were marked using 18 gauge needles. Spinal cord sections of horses with confirmed limb lesions after radiographic and dissection examination (n = 5) were then further processed. The spinal cord sides were identified as either ipsi- or contralateral to the site of distal limb lesions in each horse.
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Histology and immunohistochemistry Spinal cord segments remained for 48 hours in Bouin fixative and were then separated into 2 mm thick sections. Sections were first dehydrated using a 10% graded ethanol (Sigma-
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Aldrich, Chile) series (70% to 100% for 1 hour each, plus an extra hour in 100% concentration), then with a mixture of 100% ethanol and 100% butanol (Sigma-Aldrich) in a 1:1 ratio for 1 hour, and finally a dehydration process with pure butanol for 2 hours. These segments were paraffin-
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embedded for 4 hours (divided into four separate sessions, 1 hour each at 60 °C), to be later sliced into 6 µm sagittal sections using a manual rotatory microtome (Leica Biosystem RM2235,
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Germany), and mounted in xylene embedded slides. Each histological section was dewaxed using pure xylene xylol for 10 minutes each, rehydrated in graded series of alcohols (100% to 70%, 5 minutes each) and washed with distilled water. Sections were then treated with sodium citrate buffer (10 mM sodium citrate, pH 6.0) and microwaved three times for 4 minutes each. All
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sections were blocked overnight with Terminal buffer controller (TCT) buffer [carrageenan 0.7%, Triton X-100 0.5% in Tris-buffered saline (TBS), pH 7.6] at 4 °C. Tissue sections were then rinsed 3 times for 10 minutes in TBS and incubated with the primary antibody overnight at room
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temperature. After incubation, tissue sections were washed 3 times for 10 minutes in TBS and incubated with the secondary antibody for 1 hour. Primary antibody was omitted for negative
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controls.
A polyclonal goat antibody raised against GFAP (1:50; Santa Cruz Biotechnology, CA,
USA) was used to detect spinal astrocytes. Detection of microglial cells was performed using a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of Iba-1 (1:50; Santa Cruz Biotechnology). Iba-1 primary antibody was conjugated with an Alexa Fluor, labeled with 488 (1:500; Invitrogen, CA, USA) goat anti-rabbit secondary antibody for 1 hour at room temperature. GFAP primary antibody was conjugated with an Alexa Fluor 488 (1:500; 7
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Invitrogen) donkey anti-goat secondary antibody for 1 hour at room temperature. After immunolabeling, sections were counterstained with DAPI (4’, 6-diamidino-2-phenylindole;
(Fluorescence Mounting Medium; DAKO, Agilent, CA, USA).
Image collection and analysis
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1:5000; Invitrogen) for 20 minutes at room temperature, washed with TBS and mounted
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Stained spinal cord sections were viewed using 10×, 20×, 40× and 63× oil objective lens and examined using an epifluorescence microscope (Eclipse E200; Nikon Instruments, NY,
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USA). Images were captured with a digital camera (Basler Scout scA780-541C; Basler AG, Germany) and collected using the Pylon Viewer 4 software (Basler AG). At 10× magnification, the entire spinal cord section was viewed in order to select those horses with spinal cord sections that had both dorsal horns completely defined. Owing to the large size of the horse spinal cord,
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several photos (each 2592×1944 pixels) were obtained at 10× magnification from 20 transverse sections and reconstructed using Adobe Illustrator CC (Adobe System Inc, CA, USA). Iba-1 and GFAP staining intensity was measured after converting images to black and white using the ‘split
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channels’ option of Image J software (https://imagej.nih.gov/ij/,1997-2016). Fluorescence intensity was quantified using the method described by Göbel et al. (2007).
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To accomplish this, 20 square areas (each 40×40 pixels) were randomly selected from segments in the ipsi- and contralateral dorsal horn sections of each horse. The dorsal horn segments were defined as 1) posterior dorsal horn (including laminae I, II and III), 2) intermediate dorsal horn (including lamina IV), and 3) ventral dorsal horn (including laminae V and IV). To eliminate the effect of background fluorescence, the mean of 60 minimum fluorescence intensity measurements was subtracted from each fluorescence measurement in each area.
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Additionally, the total number of Iba-1 and GFAP stained cells within the dorsal horn was evaluated at 10× magnification using 30 areas (each 260×240 pixels) randomly selected in each of the previously defined dorsal horn segments.
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Astrocyte and microglial morphology were evaluated and compared across the whole dorsal horn segments of each horse. To accomplish this, 100 Iba-1 stained microglia and 50
GFAP stained astrocytes were evaluated. Morphological criteria for evaluation included cell
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shape and presence of cellular processes.
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Statistical analyses
Following background correction, the least square mean intensity of fluorescence and standard error was measured for each marker within the ipsi- and contralateral dorsal horn, and between dorsal horn segments. Relative intensity of fluorescence (RIF) data is presented as
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relative expression compared with the contralateral side and the ventral dorsal horn segment which were used as controls. Normality of the data was evaluated using Shapiro Wilk test, and variance homogeneity by means of the Fligner-Killeen test. Mean intensity of fluorescence
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between ipsi- and contralateral dorsal horn was compared using the non-parametric Wilcoxon rank sum test. Mean intensity of fluorescence between dorsal horn segments and cell count was
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compared using the non-parametric Kruskal Wallis rank sum test with Dunn´s post hoc test (R Statistical Software; R Core Team, Austria). Differences were considered significant when p ≤ 0.05.
Results Of the 10 selected horses, 5 horses with pelvic limb lameness met the inclusion criteria and tissues were submitted for immunohistochemical studies (Table 1). Three horses were 9
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classified as chronic lameness: two with chronic osteoarthritis and evidence of metatarsophalangeal degenerative joint disease (horses 1 and 2), and one horse with chronic laminitis and rotation of the third phalanx (horse 3). Two horses were classified as acute lameness
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(horses 4 and 5), with a subsolar hematoma and dorsal metatarsal periosteal hematoma, respectively.
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Microglial response
Microglial response was quantified by measuring the RIF displayed by Iba-1 in the dorsal
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horn of horses, ipsilateral and contralateral to the affected limb. The sum averages of RIF in all horses within the ipsilateral side was significantly increased compared with the contralateral side (p = 0.0002) (Fig. 1a). Differences in the RIF between both the ipsi- and contralateral were found in three horses, two with chronic lameness (horses 1 and 2), and one with acute lameness (horse
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4) (Fig. 1b). This difference was easily observed at 10× magnification (Figs 2b & 2c). The larger population of microglia was found in the posterior dorsal horn (p = 0.00001; Figs 1c & 2a). The average number of cells per segment in all horses (both acute and chronically lame) in a 260×240
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pixels area was: posterior dorsal horn 10.5 ± 0.4 cells, intermediate dorsal horn 7.9 ± 0.5, and ventral dorsal horn 5.4 ± 0.3 (Fig. 1c). The immunohistochemical labeling of Iba-1 differentiated
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a cellular phenotype with intense staining of the cell soma and minimal or non-staining of the cellular processes. Horses with acute lameness predominantly had a spherical shape phenotype (Fig. 2d–f), while cells from horses with chronic lameness had variable morphology, ranging from spherical or amoeboid to elongated cell types (Fig. 2g–i), with minimal staining of the initial portion of the cellular process and an uneven distribution and interposed areas of little or no microglial activation.
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Astrocyte response Astrocyte response was determined using GFAP. For GFAP, the RIF was not significantly
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higher in the ipsilateral side in the total of dorsal horn sections (p = 0.7149; Fig. 3a), but
significant differences between ipsi- and contralateral sides were found in two horses with
chronic lameness (horses 2 and 3; Fig. 3b). Despite the fact that marked GFAP fluorescence was
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found (Figs 4a & 4b), it was not possible to quantify GFAP positive cell density through cell count at 10× magnification. However, the RIF differences identified that the posterior dorsal horn
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had a significant major population of astrocytes compared with the intermediate and ventral dorsal horn segments (p = 0.00001; Fig. 3c). Cells from all horses showed a uniform morphological pattern with GFAP labeling, small somas and long processes, on both ipsilateral
Discussion
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and contralateral sides (Fig. 4c-e).
Peripheral inflammatory or neuropathic lesions that lead to pain induce an activation of
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spinal cord microglia and astrocytes (Zhang et al. 2008; Chen et al. 2012; Yamamoto et al. 2015). After nerve damage or inflammation, these cells respond quickly, increasing in number and
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altering their morphological and functional pattern to maintain states of central sensitivity for pathologically prolonged time periods (Watkins et al. 2003; Clark et al. 2007). Therefore, a higher activity of these cells plays a key role in maintaining long-term pathological pain sensations (Raghavendra et al. 2003; Chen et al. 2012). The main goal of this research was to demonstrate the response of microglia and astrocytes in the dorsal horn of the spinal cord to painful conditions in horses, and to highlight differences in cell activity, density and morphology between the ipsi-
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and contralateral side of lameness. To achieve this, two recognized glial markers were used: Iba-1 for microglial cells and GFAP for astrocytes. This study demonstrates functional and morphological changes in microglial cells of lame
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horses. Morphologically, microglial cells have been shown to react rapidly to changes in CNS homeostasis, and change their normal ramified phenotype to a phagocytic functional shape
represented by an amoeboid cell aspect (Ahmed et al. 2007). Although we did not find the typical
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resting microglia with ramified morphology, differences in microglial morphology were observed between cells from horses with acute versus chronic lameness. Microglia in horses with acute
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lameness had a predominantly spherical shape, while in horses with chronic lameness, a more varied morphology was found. This diversity could be closely associated with pain sustained over time, and may indicate that cells could have been transitioning into phagocytic phenotypes, surveillance states, or even a post-activated shape, as Hanisch & Kettenmann et al. (2007)
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previously described. These changes may be directly associated with the increase of RIF in the ipsilateral dorsal horns, confirming that after injury, microglial cells become activated maintaining their activity for long periods of time (Fu et al. 1999). Additionally, cases of chronic
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or severe lameness showed significantly higher RIF in the dorsal horn segments on the ipsilateral side of the limb lesion than on the contralateral side. These microglial results highlight that severe
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or chronic lesions can produce a pronounced increase of Iba-1 expression which, in turn, suggests that the plasticity of the microglial cells could signal an association between greater levels of activity and exacerbated painful signs. This was previously demonstrated in murine models (Raghavendra et al. 2003). Moreover, it has been previously described that after peripheral inflammation and nerve injury in rodents, Iba-1 positive cells are mainly situated in the first dorsal horn laminae (Kim et al. 2002). Similarly, in this study, a higher number of Iba-1 microglial cells in the posterior dorsal horn of lame horses is described. Iba-1 was therefore an 12
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effective marker in defining microglial functionality, morphology and organization in the spinal cord dorsal horn of horses. Horse astrocytes were successfully immunostained with GFAP. However, differentiation
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of cell functionality using the GFAP marker in the ipsi- and contralateral side of affected horses was not possible owing to the small sample size. Furthermore, several studies have shown that GFAP immunoreactivity can vary considerably, including variations in immunostaining
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technique, incubation time, and individual physiological modifications (Martin et al. 1997; Eng et al. 2000; Sullivan et al. 2010). GFAP-labeled astrocytes demonstrated similar shapes on both ipsi-
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and contralateral sides of the spinal cord with small somas and long processes. These findings are similar to that described in murine models (Song et al. 2016). It is difficult to precisely define the entire morphology of these cells, and therefore counting this cell type was much more challenging than microglia cells. According to Bushong et al. (2002), GFAP immunostaining only
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reveals the structure of astrocyte primary branches, which represent 15% of the total volume of
studies.
Conclusions
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the astrocyte, thus morphological findings cannot be associated to functionality without further
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These results confirm the use of Iba-1 and GFAP as functional and morphological
biomarkers of spinal microglial cells and astrocytes in horses with lameness. Microglia had significantly higher Iba-1 expression, on the ipsilateral side of the affected limb. Increased astrocyte activity on the ipsilateral dorsal horn was not proven through GFAP labeling, but a numerical increase in fluorescence is reported, with significant increases in two horses with chronic lameness. Both cells were mainly found in the more superficial laminae. Microglial morphology was relatively homogeneous in cases of acute lameness, with a preponderance of 13
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activated cell states. By contrast, microglial morphology was heterogeneous in cases of chronic lameness, including different functional cell types. Astrocytes, in both acute and chronic lameness, have small somas and large processes, with more GFAP expression in the main
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astrocytes branches. The sample size of this work does not allow us to accurately define glial differences between acute and chronic lesion; thus, more immunohistochemical analyses of these marker proteins are necessary to associate a temporality to the glial response, and to obtain an
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overall picture of microglial and astrocyte activation in lame horses. These findings are relevant for the field of pain physiopathology in veterinary medicine, confirming previous findings using
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murine models.
Acknowledgements
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This work was supported by the Direction of Research and Development (DID), N° S-2014-26, Universidad Austral de Chile, Valdivia, Chile. The authors thank Genaro Alvial (Research Assistant at the Faculty of Medicine, Universidade Austral de Chile) for his technical support in
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immunohistochemical analysis.
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Authors’ contributions
CSM: collected all tissue samples, performed all laboratory analysis, interpreted the data and drafted the manuscript; HYM: performed statistical analysis; DEH, MPW and HAB: designed and coordinated the study, analyzed and interpreted the data and critically revised the manuscript; BU: participated in specimen collection and critically revised the manuscript. All authors approved the final version of the manuscript.
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Conflict of interest statement
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Authors declare no conflict of interest.
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References Ahmed Z, Shaw G, Sharma VP et al. (2007) Actin-binding proteins coronin-1a and IBA-1 are effective microglial markers for immunohistochemistry. J Histochem Cytochem 55, 687–700.
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Allansson L, Khatibi S, Olsson T, Hansson E (2001) Acute ethanol exposure induces [Ca2+]i transients, cell swelling and transformation of actin cytoskeleton in astroglial primary cultures. J Neurochem 76, 472–479.
SC
Bushong EA, Martone MA, Jones YZ, Ellisman MH (2002) Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J Neurosci 22, 183–192.
M AN U
Chen MJ, Kress B, Han X et al. (2012) Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury. Glia 60, 1660–1670.
Clark AK, Gentry C, Bradbury EJ et al. (2007) Role of spinal microglia in rat models of
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peripheral nerve injury and inflammation. Eur J Pain11, 223–230. Driessen B, Bauquier SH, Zarucco L (2010) Neuropathic pain management in chronic laminitis. Vet Clin North Am Equine Pract 26, 315–337.
EP
Eng LF, Ghirnikar RS, Lee YL (2000) Glial fibrillary acidic protein: GFAP-thirty-one years (1969–2000). Neurochem Res 25, 1439–1451.
AC C
Faustino JV, Wang X, Johnson CE et al. (2011) Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke. J Neurosci 31, 12992–13001. Fu KY, Light AR, Matsushima GK, Maixner W (1999) Microglial reactions after subcutaneous formalin injection into the rat hind paw. Brain Res 825, 59–67. Göbel W, Kampa BM, Helmchen F (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4, 73–79.
16
ACCEPTED MANUSCRIPT
Gosselin RD, Suter M, Ji RR, Decosterd I (2010) Glial cells and chronic pain. Neuroscientist 16, 519–531. Gwak YS, Kang J, Unabia GC, Hulsebosch CE (2012) Spatial and temporal activation of spinal
Exp Neurol 234, 362–372.
RI PT
glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats.
Hanisch UK, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the
SC
normal and pathologic brain. Nat Neurosci 10, 1387–1394.
Hansson E, Muyderman H, Leonova J et al. (2000) Astroglia and glutamate in physiology and
M AN U
pathology: aspects on glutamate transport, glutamate-induced cell swelling and gap-junction communication. Neurochem Int 37, 317–329.
Hansson E (2006) Could chronic pain and spread of pain sensation be induced and maintained by glial activation? Acta Physiol 187, 321–327.
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Ito D, Imai Y, Ohsawa K et al. (1998) Microglia-specific localisation of a novel calcium binding protein, Iba1. Mol Brain Res 57, 1–9.
Ji RR, Berta T, Nedergaard M (2013) Glia and pain: is chronic pain a gliopathy? Pain 154
EP
(Suppl), S10–S28.
Jones E, Viñuela-Fernandez I, Eager RA et al. (2007) Neuropathic changes in equine laminitis
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pain. Pain 132, 321–331.
Keegan KG, Dent EV, Wilson DA et al. (2010) Repeatability of subjective evaluation of lameness in horses. Equine Vet J 42, 92–97. Kim SY, Bae JC, Kim JY et al. (2002) Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury. Neuroreport 13, 2483– 2486.
17
ACCEPTED MANUSCRIPT
Martin LJ, Brambrink AM, Lehmann C et al. (1997) Hypoxia–ischemia causes abnormalities in glutamate transporters and death of astroglia and neurons in newborn striatum. Ann Neurol 42, 335–348.
neuropathic pain. Eur J Pharmacol 716, 106–119.
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Mika J, Zychowska M, Popiolek-Barczyk K et al. (2013) Importance of glial activation in
Narita M, Yoshida T, Nakajima M et al. (2006) Direct evidence for spinal cord microglia in the
SC
development of a neuropathic pain-like state in mice. J Neurochem 97, 1337–1348.
Nolte C, Matyash M, Pivneva T et al. (2001) GFAP promoter-controlled EGFP-expressing
M AN U
transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33, 72–86.
Price J, Marques JM, Welsh EM, Waran NK (2002) Pilot epidemiological study of attitudes towards pain in horses. Vet Rec 151, 570–575.
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Raghavendra V, Tanga F, DeLeo JA (2003) Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther 306, 624–630.
EP
Song ZP, Xiong BR, Guan XH et al. (2016) Minocycline attenuates bone cancer pain in rats by inhibiting NF-kB in spinal astrocytes. Acta Pharmacol Sin 37, 753–762.
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Sullivan SM, Björkman ST, Miller SM et al. (2010) Morphological changes in white matter astrocytes in response to hypoxia/ischemia in the neonatal pig. Brain Res 1319, 164–174. Temburni MK, Jacob MH (2001) New functions for glia in the brain. Proc Natl Acad Sci 98, 3631–3632.
Watkins LR, Milligan ED, Maier SF (2001) Spinal cord glia: new players in pain. Pain 93, 201– 205.
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Watkins LR, Milligan ED, Maier SF (2003) Glial proinflammatory cytokines mediate exaggerated pain states: implications for clinical pain. Adv Exp Med Biol 521, 1–21. Whiteside GT, Adedoyin A, Leventhal L (2008) Predictive validity of animal pain models? A
humans. Neuropharmacology 54, 767–775.
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comparison of the pharmacokinetic–pharmacodynamic relationship for pain drugs in rats and
Yamamoto Y, Terayama R, Kishimoto N et al. (2015) Activated microglia contribute to
neuropathic pain. Neurochem Res 40, 1000–1012.
SC
convergent nociceptive inputs to spinal dorsal horn neurons and the development of
M AN U
Zhang F, Vadakkan KI, Kim SS et al. (2008) Selective activation of microglia in spinal cord but
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not higher cortical regions following nerve injury in adult mouse. Mol pain 4, 15–31.
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Table 1 Description of the lesions and lameness of five horses Horse
Lesion
Duration
Lameness grade (0–5)*
Osteoarthritis
Chronic
2
2
Osteoarthritis
Chronic
2
3
Laminitis
Chronic
4
4
Subsolar hematoma
Acute
4
5
Periosteal hematoma of a metatarsus
Acute
2
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*Keegan et al. 2010
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1
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Figure legends Figure 1 Relative intensity of fluorescence (RIF) indicating microglial activity using the glial marker Iba-1 in the dorsal horns of five horses with unilateral lameness. (a) Averages of RIF in all
posterior, intermediate and ventral dorsal horn segments.
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horses in ipsi- and contralateral sides; (b) RIF in individual horses; (c) RIF and cell counts in the
Data are presented as least square mean ± standard error of the mean. *Significant difference
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between ipsi- and contralateral dorsal horns. †RIF significantly different from intermediate and ventral dorsal horn segments (p < 0.05). ‡Cell count significantly different from posterior and
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ventral dorsal horn segments (p < 0.05). §Cell count significantly lower than posterior and intermediate dorsal horn segments (p < 0.05).
Sample size, 260 × 240 pixels. AU, arbitrary units
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Figure 2 Immunostaining against Iba-1 in dorsal horns of lame horses (Iba-1, green; DAPI, blue). (a) Microglial cells positive to Iba-1 labeling, mainly distributed at the posterior dorsal horn level, 10× magnification; (b) ipsilateral and (c) contralateral sections of dorsal horn in a horse with
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chronic laminitis, fluorescence significantly higher in (b) than (c), 10× magnification); (d–f) microglial cells with spherical shapes in horses with acute lameness; (g–i) more varied
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morphology, varying from spherical or amoeboid shape to elongated cell types, identified from horses with chronic lameness, 63× magnification.
Figure 3 Relative intensity of fluorescence (RIF) indicating astrocyte response using the glial marker GFAP in the dorsal horns of five horses with unilateral lameness. (a) Averages of RIF in all horses in ipsi- and contralateral sides; (b) RIF in individual horses; (c) RIF in the dorsal horn segments. 21
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Data are presented as least square mean ± standard error of the mean. *Significant difference between ipsi- and contralateral dorsal horns. †Significantly different from intermediate and ventral dorsal horn segments (p < 0.05). ‡Significantly different from intermediate dorsal horn
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segment (p < 0.05). AU, arbitrary units
Figure 4 Immunostaining against GFAP in dorsal horns of lame horses (GFAP, green; DAPI,
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blue). (a, b) Areas of reactive astrogliosis in lame horses were evident, 10× magnification; (c–e) uniform morphological pattern in astrocytes with GFAP labeling observed in all horses, showing
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cells with small somas and long processes, 63× magnification.
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S1467-2987(17)30353-7
DOI:
10.1016/j.vaa.2017.10.001
Reference:
VAA 209
To appear in:
Veterinary Anaesthesia and Analgesia
Received Date: 21 April 2017 Revised Date:
6 October 2017
Accepted Date: 10 October 2017
Please cite this article as: RRH: Microglia and astrocytes in lame horses This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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RESEARCH PAPER Running head (Author): CS Meneses et al. Running head (short title): Microglia and astrocytes in lame horses
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Microglia and astrocyte activation in the spinal cord of lame horses
Constanza S Menesesa, Heine Y Müllerb, Daniel E Herzbergb, Benjamin Ubertib, Marianne P Wernerc & Hedie A Bustamanteb
Graduate School, Faculty of Veterinary Sciences, Universidade Austral de Chile, Valdivia, Chile
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a
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Veterinary Clinical Sciences Department, Faculty of Veterinary Sciences, Universidade Austral
c
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de Chile, Valdivia, Chile
Animal Science Department, Faculty of Veterinary Sciences, Universidade Austral de Chile,
Valdivia, Chile
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Correspondence: Hedie A Bustamante, Institute of Veterinary Clinical Sciences, Faculty of Veterinary Sciences, Universidade Austral de Chile, Independencia 631, Valdivia 5090000, Chile.
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Abstract
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E-mail: [email protected]
Objective To determine the microglial and astrocyte response to painful lameness in horses. Study design Ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) expression, cell density and morphology were determined through immunofluorescence within the dorsal horn of equine spinal cord. Animals Five adult horses with acute or chronic unilateral lameness, previously scheduled for euthanasia. 1
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Methods Musculoskeletal lameness was evaluated in five horses through visual evaluation according to clinical guidelines. Spinal cord samples were obtained immediately after euthanasia, and distal limb lesions were confirmed through dissection and radiography. Iba-1 immunostaining
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was used for detection and characterization of dorsal horn microglia. GFAP was used for
immunostaining of dorsal horn astrocytes. Iba-1 and GFAP labeled cells were quantified in the dorsal horn, and intensity of fluorescence was compared between the ipsi- and contralateral
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dorsal horn to the affected limb, and between dorsal horn segments of all horses.
Results Iba-1 expression was higher in the ipsilateral dorsal horn of the affected limb in contrast
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to the contralateral side dorsal horn. GFAP markers did not demonstrate increased astrocytic activity on the dorsal horn ipsilateral side to the distal limb lesion of affected horses. Horses with acute lameness predominantly had a spherical shape microglial phenotype, while cells from chronic lameness cases had variable morphology. Astrocytes evidenced small somas and large
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processes in both acute and chronic lameness, with higher GFAP localization in the main branches. Like rodents, the localization of microglia and astrocytes in horses was mainly situated within laminae I, II and III.
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Conclusions and clinical relevance Iba-1 and GFAP are functional and morphological markers
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of spinal microglial cells and astrocytes in horses with lameness.
Keywords astrocyte, horses, lameness, microglia.
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Introduction In horses, pain is usually the first and only sign of disease, becoming a valuable clinical parameter for veterinarian practitioners (Price et al. 2002). Furthermore, severe or chronic cases
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of pain are often refractory to conventional anti-inflammatory analgesia, and euthanasia is
required (Jones et al. 2007). Osteoarthritis, laminitis and navicular disease cause chronic pain in horses and their treatment is considered one of the greatest challenges in equine clinical practice
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(Driessen 2010). Nevertheless, few studies have focused on the importance of chronic pain in
neuropathic pain in this species.
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horses (Jones et al. 2007), and no studies have identified major factors in the modulation of
Recently, the role of glial cells in the central nervous system (CNS) of rodents in the maintenance of pain has been described, making glial cells potentially a novel therapeutic target (Narita et al. 2006). Glial cells are capable of enhancing pain and dynamically modulating
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neurons (Temburni & Jakob 2001; Watkins et al. 2001). Spinal and peripheral nerve lesions result in physiological activation of glial cells in the ipsilateral dorsal horn of the affected limb (Mika et al. 2013). Once activated, these cells undergo morphological and functional modifications that
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trigger the release of algesic mediators capable of changing the neuron-glia interaction and enhance pain sensitivity (Ji et al. 2013). Of all glial cells described, spinal cord microglia and
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astrocytes have been the main focus for research. These cells generate a significant neuronal depolarization that makes dorsal horn neurons more hyperexcitable, resulting in evoked and spontaneous activity in dorsal horn neurons and contributing to the development of pathologic pain (Gwak et al. 2012).
The recognition of function and morphology of these cells has been described using specific biomarkers. Ionized calcium binding adaptor molecule 1 (Iba-1) is specifically expressed both in vitro and in vivo by microglia (Ito et al. 1998). After trauma, ischemia or nerve 3
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inflammation, spinal microglia become the first responders, increasing Iba-1 expression inducing functional and phenotypic modifications (Raghavendra et al. 2003; Faustino et al. 2011). These modifications increase the convergent nociceptive inputs to spinal dorsal horn neurons, and
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consequently the development of mechanical allodynia and thermal hyperalgesia (Yamamoto et al. 2015). These morphological changes reflect the active state of these cells. Normally, resting microglial cells have a small body from which short extensions arise around spinal neurons.
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Following long-term nociceptive input, they adopt an amoeboid shape and phagocytic functions (Hansson 2006). Microglial morphology is described as flexible and dynamic, with different
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morphologies that can markedly change and represent transient functional states (Hanisch & Kettenmann 2007).
Glial fibrillary acidic protein (GFAP) is one of the most commonly used markers for identification of astrocytes. GFAP modulates the shape and motility of astrocytes (Eng et al.
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2000) and is the major component of intermediate filaments of mature astrocytes (Nolte et al. 2001). After microglial activation, astrocytes overreact to nerve damage, and their activation is characterized by an increase in GFAP expression, enlargement of astrocytic processes (Gosselin
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et al. 2010) and cell swelling/hypertrophy. These changes vary qualitatively and quantitatively depending on the nature of the injury (Hansson et al. 2000; Allansson et al. 2001).
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Although murine models of pain have allowed the elucidation of factors that mediate
chronic pain, there is still an insufficient understanding of chronic pain and its consequences in other mammals (Whiteside et al. 2008). In horses, microglial and astrocytic responses to acute or chronic painful conditions have not been previously described. Therefore, the purpose of this study was to determine the microglial and astrocyte responses to lameness in horses, thus providing new data about central gliosis response in another mammalian species. Iba-1 and GFAP
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expression, cell density and morphology within the dorsal horn were compared between the ipsiand contralateral side of the affected limb in horses with unilateral acute or chronic lameness.
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Materials and methods
The experimental protocol was approved by the Ethics Committee of Animal Research of
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the Universidad Austral de Chile (no. 001/2017).
Animals
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Study subjects were selected prospectively, either from Universidad Austral de Chile teaching hospital herd (two horses) or from a commercial slaughterhouse (eight horses). Inclusion criteria were animals aged > 2 years of age, without sex, breed or size restriction. Researchers were not involved in the decision for euthanasia or slaughter. Euthanasia was performed by
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intravenous general anesthesia and intrathecal lidocaine injection at the atlantooccipital foramen for teaching herd horses or by mechanical stunning and exsanguination for the slaughterhouse
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horses, according to national regulations.
Lameness assessment and post-mortem radiography and dissection
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Selection criteria for horses included visible unilateral lameness, evaluated clinically by
an experienced equine clinician (CSM). Teaching herd horses were evaluated at the University teaching hospital, and slaughterhouse horses were evaluated visually in the holding pens. Horses with grade 2–5 lameness, according to the American Association of Equine Practitioners grading system (Keegan et al. 2010), were selected. Exclusion criteria included the presence of visibly acute wounds or visibly neurological gait alteration (central or peripheral ataxia). After euthanasia, affected limbs and the contralateral limbs distal to the proximal metacarpal or 5
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metatarsal epiphysis were collected from all selected horses. Six radiographic views (dorsopalmar and lateromedial of the hoof, dorsopalmar, lateromedial, dorsolateral-palmaromedial oblique and dorsomedial-palmarolateral oblique of the metacarpophalangeal /metatarsophalangeal joint) were
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obtained from the selected limbs using standard equipment (Diagnostic X-Ray, Orange 8016 HF; EcoRay Co Ltda., Korea). Digital images were stored on hard drives and interpreted by an
experienced equine clinician (CSM). For confirmation of radiological diagnosis, limbs were
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anatomically dissected (including examination of soft tissues, bones, joints and hooves). Lame horses were classified as either acute (less than 6 months duration) or chronic (more than 6
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months duration) based on teaching hospital records and information provided by owners of the horses obtained from the slaughterhouse. Spinal cord samples from these horses were included for immunohistochemical assays.
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Sampling of spinal cord tissues
Spinal cord tissue was collected immediately after euthanasia following longitudinal section of spinal vertebrae. Cervical spinal segments (C2-C7) were harvested from horses with
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thoracic limb lameness, or lumbar spinal segments (L1-L6) from horses with pelvic limb lameness. Samples were sectioned transversely and then stored in individual jars containing
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Bouin fixative [75 mL of saturated aqueous picric acid (1.2% w/v), 25 mL of formalin (40% w/v formaldehyde) and 5 mL of glacial acetic acid]. The cranial and caudal aspects of the spinal cord segments were marked using 18 gauge needles. Spinal cord sections of horses with confirmed limb lesions after radiographic and dissection examination (n = 5) were then further processed. The spinal cord sides were identified as either ipsi- or contralateral to the site of distal limb lesions in each horse.
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Histology and immunohistochemistry Spinal cord segments remained for 48 hours in Bouin fixative and were then separated into 2 mm thick sections. Sections were first dehydrated using a 10% graded ethanol (Sigma-
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Aldrich, Chile) series (70% to 100% for 1 hour each, plus an extra hour in 100% concentration), then with a mixture of 100% ethanol and 100% butanol (Sigma-Aldrich) in a 1:1 ratio for 1 hour, and finally a dehydration process with pure butanol for 2 hours. These segments were paraffin-
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embedded for 4 hours (divided into four separate sessions, 1 hour each at 60 °C), to be later sliced into 6 µm sagittal sections using a manual rotatory microtome (Leica Biosystem RM2235,
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Germany), and mounted in xylene embedded slides. Each histological section was dewaxed using pure xylene xylol for 10 minutes each, rehydrated in graded series of alcohols (100% to 70%, 5 minutes each) and washed with distilled water. Sections were then treated with sodium citrate buffer (10 mM sodium citrate, pH 6.0) and microwaved three times for 4 minutes each. All
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sections were blocked overnight with Terminal buffer controller (TCT) buffer [carrageenan 0.7%, Triton X-100 0.5% in Tris-buffered saline (TBS), pH 7.6] at 4 °C. Tissue sections were then rinsed 3 times for 10 minutes in TBS and incubated with the primary antibody overnight at room
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temperature. After incubation, tissue sections were washed 3 times for 10 minutes in TBS and incubated with the secondary antibody for 1 hour. Primary antibody was omitted for negative
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controls.
A polyclonal goat antibody raised against GFAP (1:50; Santa Cruz Biotechnology, CA,
USA) was used to detect spinal astrocytes. Detection of microglial cells was performed using a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of Iba-1 (1:50; Santa Cruz Biotechnology). Iba-1 primary antibody was conjugated with an Alexa Fluor, labeled with 488 (1:500; Invitrogen, CA, USA) goat anti-rabbit secondary antibody for 1 hour at room temperature. GFAP primary antibody was conjugated with an Alexa Fluor 488 (1:500; 7
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Invitrogen) donkey anti-goat secondary antibody for 1 hour at room temperature. After immunolabeling, sections were counterstained with DAPI (4’, 6-diamidino-2-phenylindole;
(Fluorescence Mounting Medium; DAKO, Agilent, CA, USA).
Image collection and analysis
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1:5000; Invitrogen) for 20 minutes at room temperature, washed with TBS and mounted
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Stained spinal cord sections were viewed using 10×, 20×, 40× and 63× oil objective lens and examined using an epifluorescence microscope (Eclipse E200; Nikon Instruments, NY,
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USA). Images were captured with a digital camera (Basler Scout scA780-541C; Basler AG, Germany) and collected using the Pylon Viewer 4 software (Basler AG). At 10× magnification, the entire spinal cord section was viewed in order to select those horses with spinal cord sections that had both dorsal horns completely defined. Owing to the large size of the horse spinal cord,
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several photos (each 2592×1944 pixels) were obtained at 10× magnification from 20 transverse sections and reconstructed using Adobe Illustrator CC (Adobe System Inc, CA, USA). Iba-1 and GFAP staining intensity was measured after converting images to black and white using the ‘split
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channels’ option of Image J software (https://imagej.nih.gov/ij/,1997-2016). Fluorescence intensity was quantified using the method described by Göbel et al. (2007).
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To accomplish this, 20 square areas (each 40×40 pixels) were randomly selected from segments in the ipsi- and contralateral dorsal horn sections of each horse. The dorsal horn segments were defined as 1) posterior dorsal horn (including laminae I, II and III), 2) intermediate dorsal horn (including lamina IV), and 3) ventral dorsal horn (including laminae V and IV). To eliminate the effect of background fluorescence, the mean of 60 minimum fluorescence intensity measurements was subtracted from each fluorescence measurement in each area.
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Additionally, the total number of Iba-1 and GFAP stained cells within the dorsal horn was evaluated at 10× magnification using 30 areas (each 260×240 pixels) randomly selected in each of the previously defined dorsal horn segments.
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Astrocyte and microglial morphology were evaluated and compared across the whole dorsal horn segments of each horse. To accomplish this, 100 Iba-1 stained microglia and 50
GFAP stained astrocytes were evaluated. Morphological criteria for evaluation included cell
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shape and presence of cellular processes.
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Statistical analyses
Following background correction, the least square mean intensity of fluorescence and standard error was measured for each marker within the ipsi- and contralateral dorsal horn, and between dorsal horn segments. Relative intensity of fluorescence (RIF) data is presented as
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relative expression compared with the contralateral side and the ventral dorsal horn segment which were used as controls. Normality of the data was evaluated using Shapiro Wilk test, and variance homogeneity by means of the Fligner-Killeen test. Mean intensity of fluorescence
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between ipsi- and contralateral dorsal horn was compared using the non-parametric Wilcoxon rank sum test. Mean intensity of fluorescence between dorsal horn segments and cell count was
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compared using the non-parametric Kruskal Wallis rank sum test with Dunn´s post hoc test (R Statistical Software; R Core Team, Austria). Differences were considered significant when p ≤ 0.05.
Results Of the 10 selected horses, 5 horses with pelvic limb lameness met the inclusion criteria and tissues were submitted for immunohistochemical studies (Table 1). Three horses were 9
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classified as chronic lameness: two with chronic osteoarthritis and evidence of metatarsophalangeal degenerative joint disease (horses 1 and 2), and one horse with chronic laminitis and rotation of the third phalanx (horse 3). Two horses were classified as acute lameness
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(horses 4 and 5), with a subsolar hematoma and dorsal metatarsal periosteal hematoma, respectively.
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Microglial response
Microglial response was quantified by measuring the RIF displayed by Iba-1 in the dorsal
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horn of horses, ipsilateral and contralateral to the affected limb. The sum averages of RIF in all horses within the ipsilateral side was significantly increased compared with the contralateral side (p = 0.0002) (Fig. 1a). Differences in the RIF between both the ipsi- and contralateral were found in three horses, two with chronic lameness (horses 1 and 2), and one with acute lameness (horse
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4) (Fig. 1b). This difference was easily observed at 10× magnification (Figs 2b & 2c). The larger population of microglia was found in the posterior dorsal horn (p = 0.00001; Figs 1c & 2a). The average number of cells per segment in all horses (both acute and chronically lame) in a 260×240
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pixels area was: posterior dorsal horn 10.5 ± 0.4 cells, intermediate dorsal horn 7.9 ± 0.5, and ventral dorsal horn 5.4 ± 0.3 (Fig. 1c). The immunohistochemical labeling of Iba-1 differentiated
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a cellular phenotype with intense staining of the cell soma and minimal or non-staining of the cellular processes. Horses with acute lameness predominantly had a spherical shape phenotype (Fig. 2d–f), while cells from horses with chronic lameness had variable morphology, ranging from spherical or amoeboid to elongated cell types (Fig. 2g–i), with minimal staining of the initial portion of the cellular process and an uneven distribution and interposed areas of little or no microglial activation.
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Astrocyte response Astrocyte response was determined using GFAP. For GFAP, the RIF was not significantly
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higher in the ipsilateral side in the total of dorsal horn sections (p = 0.7149; Fig. 3a), but
significant differences between ipsi- and contralateral sides were found in two horses with
chronic lameness (horses 2 and 3; Fig. 3b). Despite the fact that marked GFAP fluorescence was
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found (Figs 4a & 4b), it was not possible to quantify GFAP positive cell density through cell count at 10× magnification. However, the RIF differences identified that the posterior dorsal horn
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had a significant major population of astrocytes compared with the intermediate and ventral dorsal horn segments (p = 0.00001; Fig. 3c). Cells from all horses showed a uniform morphological pattern with GFAP labeling, small somas and long processes, on both ipsilateral
Discussion
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and contralateral sides (Fig. 4c-e).
Peripheral inflammatory or neuropathic lesions that lead to pain induce an activation of
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spinal cord microglia and astrocytes (Zhang et al. 2008; Chen et al. 2012; Yamamoto et al. 2015). After nerve damage or inflammation, these cells respond quickly, increasing in number and
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altering their morphological and functional pattern to maintain states of central sensitivity for pathologically prolonged time periods (Watkins et al. 2003; Clark et al. 2007). Therefore, a higher activity of these cells plays a key role in maintaining long-term pathological pain sensations (Raghavendra et al. 2003; Chen et al. 2012). The main goal of this research was to demonstrate the response of microglia and astrocytes in the dorsal horn of the spinal cord to painful conditions in horses, and to highlight differences in cell activity, density and morphology between the ipsi-
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and contralateral side of lameness. To achieve this, two recognized glial markers were used: Iba-1 for microglial cells and GFAP for astrocytes. This study demonstrates functional and morphological changes in microglial cells of lame
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horses. Morphologically, microglial cells have been shown to react rapidly to changes in CNS homeostasis, and change their normal ramified phenotype to a phagocytic functional shape
represented by an amoeboid cell aspect (Ahmed et al. 2007). Although we did not find the typical
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resting microglia with ramified morphology, differences in microglial morphology were observed between cells from horses with acute versus chronic lameness. Microglia in horses with acute
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lameness had a predominantly spherical shape, while in horses with chronic lameness, a more varied morphology was found. This diversity could be closely associated with pain sustained over time, and may indicate that cells could have been transitioning into phagocytic phenotypes, surveillance states, or even a post-activated shape, as Hanisch & Kettenmann et al. (2007)
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previously described. These changes may be directly associated with the increase of RIF in the ipsilateral dorsal horns, confirming that after injury, microglial cells become activated maintaining their activity for long periods of time (Fu et al. 1999). Additionally, cases of chronic
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or severe lameness showed significantly higher RIF in the dorsal horn segments on the ipsilateral side of the limb lesion than on the contralateral side. These microglial results highlight that severe
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or chronic lesions can produce a pronounced increase of Iba-1 expression which, in turn, suggests that the plasticity of the microglial cells could signal an association between greater levels of activity and exacerbated painful signs. This was previously demonstrated in murine models (Raghavendra et al. 2003). Moreover, it has been previously described that after peripheral inflammation and nerve injury in rodents, Iba-1 positive cells are mainly situated in the first dorsal horn laminae (Kim et al. 2002). Similarly, in this study, a higher number of Iba-1 microglial cells in the posterior dorsal horn of lame horses is described. Iba-1 was therefore an 12
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effective marker in defining microglial functionality, morphology and organization in the spinal cord dorsal horn of horses. Horse astrocytes were successfully immunostained with GFAP. However, differentiation
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of cell functionality using the GFAP marker in the ipsi- and contralateral side of affected horses was not possible owing to the small sample size. Furthermore, several studies have shown that GFAP immunoreactivity can vary considerably, including variations in immunostaining
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technique, incubation time, and individual physiological modifications (Martin et al. 1997; Eng et al. 2000; Sullivan et al. 2010). GFAP-labeled astrocytes demonstrated similar shapes on both ipsi-
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and contralateral sides of the spinal cord with small somas and long processes. These findings are similar to that described in murine models (Song et al. 2016). It is difficult to precisely define the entire morphology of these cells, and therefore counting this cell type was much more challenging than microglia cells. According to Bushong et al. (2002), GFAP immunostaining only
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reveals the structure of astrocyte primary branches, which represent 15% of the total volume of
studies.
Conclusions
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the astrocyte, thus morphological findings cannot be associated to functionality without further
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These results confirm the use of Iba-1 and GFAP as functional and morphological
biomarkers of spinal microglial cells and astrocytes in horses with lameness. Microglia had significantly higher Iba-1 expression, on the ipsilateral side of the affected limb. Increased astrocyte activity on the ipsilateral dorsal horn was not proven through GFAP labeling, but a numerical increase in fluorescence is reported, with significant increases in two horses with chronic lameness. Both cells were mainly found in the more superficial laminae. Microglial morphology was relatively homogeneous in cases of acute lameness, with a preponderance of 13
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activated cell states. By contrast, microglial morphology was heterogeneous in cases of chronic lameness, including different functional cell types. Astrocytes, in both acute and chronic lameness, have small somas and large processes, with more GFAP expression in the main
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astrocytes branches. The sample size of this work does not allow us to accurately define glial differences between acute and chronic lesion; thus, more immunohistochemical analyses of these marker proteins are necessary to associate a temporality to the glial response, and to obtain an
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overall picture of microglial and astrocyte activation in lame horses. These findings are relevant for the field of pain physiopathology in veterinary medicine, confirming previous findings using
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murine models.
Acknowledgements
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This work was supported by the Direction of Research and Development (DID), N° S-2014-26, Universidad Austral de Chile, Valdivia, Chile. The authors thank Genaro Alvial (Research Assistant at the Faculty of Medicine, Universidade Austral de Chile) for his technical support in
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immunohistochemical analysis.
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Authors’ contributions
CSM: collected all tissue samples, performed all laboratory analysis, interpreted the data and drafted the manuscript; HYM: performed statistical analysis; DEH, MPW and HAB: designed and coordinated the study, analyzed and interpreted the data and critically revised the manuscript; BU: participated in specimen collection and critically revised the manuscript. All authors approved the final version of the manuscript.
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Conflict of interest statement
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Authors declare no conflict of interest.
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References Ahmed Z, Shaw G, Sharma VP et al. (2007) Actin-binding proteins coronin-1a and IBA-1 are effective microglial markers for immunohistochemistry. J Histochem Cytochem 55, 687–700.
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Allansson L, Khatibi S, Olsson T, Hansson E (2001) Acute ethanol exposure induces [Ca2+]i transients, cell swelling and transformation of actin cytoskeleton in astroglial primary cultures. J Neurochem 76, 472–479.
SC
Bushong EA, Martone MA, Jones YZ, Ellisman MH (2002) Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J Neurosci 22, 183–192.
M AN U
Chen MJ, Kress B, Han X et al. (2012) Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury. Glia 60, 1660–1670.
Clark AK, Gentry C, Bradbury EJ et al. (2007) Role of spinal microglia in rat models of
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peripheral nerve injury and inflammation. Eur J Pain11, 223–230. Driessen B, Bauquier SH, Zarucco L (2010) Neuropathic pain management in chronic laminitis. Vet Clin North Am Equine Pract 26, 315–337.
EP
Eng LF, Ghirnikar RS, Lee YL (2000) Glial fibrillary acidic protein: GFAP-thirty-one years (1969–2000). Neurochem Res 25, 1439–1451.
AC C
Faustino JV, Wang X, Johnson CE et al. (2011) Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke. J Neurosci 31, 12992–13001. Fu KY, Light AR, Matsushima GK, Maixner W (1999) Microglial reactions after subcutaneous formalin injection into the rat hind paw. Brain Res 825, 59–67. Göbel W, Kampa BM, Helmchen F (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4, 73–79.
16
ACCEPTED MANUSCRIPT
Gosselin RD, Suter M, Ji RR, Decosterd I (2010) Glial cells and chronic pain. Neuroscientist 16, 519–531. Gwak YS, Kang J, Unabia GC, Hulsebosch CE (2012) Spatial and temporal activation of spinal
Exp Neurol 234, 362–372.
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glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats.
Hanisch UK, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the
SC
normal and pathologic brain. Nat Neurosci 10, 1387–1394.
Hansson E, Muyderman H, Leonova J et al. (2000) Astroglia and glutamate in physiology and
M AN U
pathology: aspects on glutamate transport, glutamate-induced cell swelling and gap-junction communication. Neurochem Int 37, 317–329.
Hansson E (2006) Could chronic pain and spread of pain sensation be induced and maintained by glial activation? Acta Physiol 187, 321–327.
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Ito D, Imai Y, Ohsawa K et al. (1998) Microglia-specific localisation of a novel calcium binding protein, Iba1. Mol Brain Res 57, 1–9.
Ji RR, Berta T, Nedergaard M (2013) Glia and pain: is chronic pain a gliopathy? Pain 154
EP
(Suppl), S10–S28.
Jones E, Viñuela-Fernandez I, Eager RA et al. (2007) Neuropathic changes in equine laminitis
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pain. Pain 132, 321–331.
Keegan KG, Dent EV, Wilson DA et al. (2010) Repeatability of subjective evaluation of lameness in horses. Equine Vet J 42, 92–97. Kim SY, Bae JC, Kim JY et al. (2002) Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury. Neuroreport 13, 2483– 2486.
17
ACCEPTED MANUSCRIPT
Martin LJ, Brambrink AM, Lehmann C et al. (1997) Hypoxia–ischemia causes abnormalities in glutamate transporters and death of astroglia and neurons in newborn striatum. Ann Neurol 42, 335–348.
neuropathic pain. Eur J Pharmacol 716, 106–119.
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Mika J, Zychowska M, Popiolek-Barczyk K et al. (2013) Importance of glial activation in
Narita M, Yoshida T, Nakajima M et al. (2006) Direct evidence for spinal cord microglia in the
SC
development of a neuropathic pain-like state in mice. J Neurochem 97, 1337–1348.
Nolte C, Matyash M, Pivneva T et al. (2001) GFAP promoter-controlled EGFP-expressing
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transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33, 72–86.
Price J, Marques JM, Welsh EM, Waran NK (2002) Pilot epidemiological study of attitudes towards pain in horses. Vet Rec 151, 570–575.
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Raghavendra V, Tanga F, DeLeo JA (2003) Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther 306, 624–630.
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Song ZP, Xiong BR, Guan XH et al. (2016) Minocycline attenuates bone cancer pain in rats by inhibiting NF-kB in spinal astrocytes. Acta Pharmacol Sin 37, 753–762.
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Sullivan SM, Björkman ST, Miller SM et al. (2010) Morphological changes in white matter astrocytes in response to hypoxia/ischemia in the neonatal pig. Brain Res 1319, 164–174. Temburni MK, Jacob MH (2001) New functions for glia in the brain. Proc Natl Acad Sci 98, 3631–3632.
Watkins LR, Milligan ED, Maier SF (2001) Spinal cord glia: new players in pain. Pain 93, 201– 205.
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Watkins LR, Milligan ED, Maier SF (2003) Glial proinflammatory cytokines mediate exaggerated pain states: implications for clinical pain. Adv Exp Med Biol 521, 1–21. Whiteside GT, Adedoyin A, Leventhal L (2008) Predictive validity of animal pain models? A
humans. Neuropharmacology 54, 767–775.
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comparison of the pharmacokinetic–pharmacodynamic relationship for pain drugs in rats and
Yamamoto Y, Terayama R, Kishimoto N et al. (2015) Activated microglia contribute to
neuropathic pain. Neurochem Res 40, 1000–1012.
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convergent nociceptive inputs to spinal dorsal horn neurons and the development of
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not higher cortical regions following nerve injury in adult mouse. Mol pain 4, 15–31.
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Table 1 Description of the lesions and lameness of five horses Horse
Lesion
Duration
Lameness grade (0–5)*
Osteoarthritis
Chronic
2
2
Osteoarthritis
Chronic
2
3
Laminitis
Chronic
4
4
Subsolar hematoma
Acute
4
5
Periosteal hematoma of a metatarsus
Acute
2
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*Keegan et al. 2010
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1
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Figure legends Figure 1 Relative intensity of fluorescence (RIF) indicating microglial activity using the glial marker Iba-1 in the dorsal horns of five horses with unilateral lameness. (a) Averages of RIF in all
posterior, intermediate and ventral dorsal horn segments.
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horses in ipsi- and contralateral sides; (b) RIF in individual horses; (c) RIF and cell counts in the
Data are presented as least square mean ± standard error of the mean. *Significant difference
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between ipsi- and contralateral dorsal horns. †RIF significantly different from intermediate and ventral dorsal horn segments (p < 0.05). ‡Cell count significantly different from posterior and
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ventral dorsal horn segments (p < 0.05). §Cell count significantly lower than posterior and intermediate dorsal horn segments (p < 0.05).
Sample size, 260 × 240 pixels. AU, arbitrary units
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Figure 2 Immunostaining against Iba-1 in dorsal horns of lame horses (Iba-1, green; DAPI, blue). (a) Microglial cells positive to Iba-1 labeling, mainly distributed at the posterior dorsal horn level, 10× magnification; (b) ipsilateral and (c) contralateral sections of dorsal horn in a horse with
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chronic laminitis, fluorescence significantly higher in (b) than (c), 10× magnification); (d–f) microglial cells with spherical shapes in horses with acute lameness; (g–i) more varied
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morphology, varying from spherical or amoeboid shape to elongated cell types, identified from horses with chronic lameness, 63× magnification.
Figure 3 Relative intensity of fluorescence (RIF) indicating astrocyte response using the glial marker GFAP in the dorsal horns of five horses with unilateral lameness. (a) Averages of RIF in all horses in ipsi- and contralateral sides; (b) RIF in individual horses; (c) RIF in the dorsal horn segments. 21
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Data are presented as least square mean ± standard error of the mean. *Significant difference between ipsi- and contralateral dorsal horns. †Significantly different from intermediate and ventral dorsal horn segments (p < 0.05). ‡Significantly different from intermediate dorsal horn
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segment (p < 0.05). AU, arbitrary units
Figure 4 Immunostaining against GFAP in dorsal horns of lame horses (GFAP, green; DAPI,
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blue). (a, b) Areas of reactive astrogliosis in lame horses were evident, 10× magnification; (c–e) uniform morphological pattern in astrocytes with GFAP labeling observed in all horses, showing
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cells with small somas and long processes, 63× magnification.
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