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Veterinary Immunology and Immunopathology 148 (2012) 348– 352

Contents lists available at SciVerse ScienceDirect

Veterinary Immunology and Immunopathology

j o ur nal ho me p age: w ww.elsev ier .com/ locate /vet imm

hort communication

nvolvement of the choroid plexus in the inflammatory response aftercute spinal cord injury in dogs: An immunohistochemical study

arah A. Moorea,∗, Michael J. Oglesbeeb

College of Veterinary Medicine, The Ohio State University, Department of Veterinary Clinical Sciences, Columbus, OH, USACollege of Veterinary Medicine, The Ohio State University, Department of Veterinary Biosciences, Columbus, OH, USA

r t i c l e i n f o

rticle history:eceived 29 May 2012eceived in revised form 3 July 2012ccepted 5 July 2012

eywords:aninepinal cord injurynflammationhoroid plexuseat shock protein 70

a b s t r a c t

The choroid plexus (CP) is increasingly recognized as an important contributor to centralnervous system (CNS) inflammation by recruitment of inflammatory cells and release ofinflammatory cytokines. Here we investigate the role of the CP epithelium (CPE) as a sourceof three pro-inflammatory molecules of potential importance in inflammation after acutespinal cord injury (SCI): IL-1�, TNF-�, and hsp70. Immunohistochemical (IHC) staining forthese three proteins was performed on 4th ventricular CPE from 4 dogs euthanized 12–48 hafter spontaneous acute SCI, and from 4 neurologically normal dogs euthanized for otherreasons. IHC staining was quantified using Aperio ImageScope software. IHC staining in theCPE of dogs with acute SCI was 2.2, 1.6 and 1.5 times higher than that of normal dogs, forIL-1�, TNF-�, and hsp70, respectively. Increases were statistically significant (p < 0.1) forIL-1� and TNF-�, and closely approached significance for hsp70. These findings indicate

that the CPE could serve as an important source of these inflammatory mediators after SCI.There was also an inverse correlation between IL-1� and hsp70 staining and duration ofclinical signs in acute SCI, suggesting that increased expression of these proteins by the CPEmay be of particular importance in the immediate-early inflammatory response after acuteSCI.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Spinal cord injury (SCI) is accompanied by primarynflammatory changes in the central nervous system (CNS).his inflammation not only serves various purposes suchs removal of cellular debris, but also causes secondaryamage which has a negative impact on functional recov-ry (Wang et al., 2004; Ankeny et al., 2009; Peng et al.,009; Olby, 2010). This secondary damage occurs over

ays, weeks and even months after the initial insult, buthe exact mechanisms and mediators of this detrimentalnflammatory response have yet to be fully defined.

∗ Corresponding author. Tel.: +1 614 292 3551; fax: +1 614 292 0895.E-mail addresses: moore.2204@osu.edu, sarah.moore@cvm.osu.edu

S.A. Moore).

165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.vetimm.2012.07.001

The choroid plexus (CP) is a specialized CNS tissuelocated within the ventricular system of the brain. It ishighly vascular and composed of a single sheets of cuboidalepithelial cells. Traditionally, roles ascribed to the CPinclude production of cerebrospinal fluid (CSF) and contri-bution to the blood–CSF barrier; however, recent studieshave implicated the CP as a potentially important media-tor of inflammatory responses within the CNS. For example,the CP has been shown to play an important role in theautoimmune inflammation associated with experimentalallergic encephalitis (EAE) and its human counterpart, mul-tiple sclerosis (MS) (Englehardt et al., 2001; Vercellinoet al., 2008). Because the CP forms an important inter-

face between the CSF and the peripheral vascular system,the potential for the CP to serve as both a key entrypoint for leukocytes into the CNS, as well as a sourceof inflammatory mediators during various disease states

nology a

S.A. Moore, M.J. Oglesbee / Veterinary Immu

seems obvious, albeit previously underappreciated. Indeed,choroid plexus epithelial cells (CPE) have recently beenshown to be a source of pro-inflammatory cytokines, suchas IL-6, IL-8, TNF-�, and IL-1�, released into the CSF asso-ciated with peripheral systemic inflammation (Marqueset al., 2009; Mitchell et al., 2009). Increases in IL-1� andTNF-� mRNA and protein expression in the CPE have alsobeen recently documented in a rodent model of trau-matic brain injury (Szmydynger-Chodobska et al., 2009),demonstrating the potential importance of the CPE in theinflammatory response to traumatic CNS injury.

The objective of the present study was to character-ize the immunohistochemical (IHC) changes occurring inthe CPE after spontaneously occurring acute SCI in dogs. Inparticular, we examined expression of IL-1�, TNF-�, andhsp70. These three proteins have been previously impli-cated as markers or mediators of a robust inflammatoryresponse of the spinal cord following acute SCI, spinalcord ischemia-reperfusion (IR) injury and meningomyeli-tis (Sharma et al., 2006; Awad et al., 2008; Hecker et al.,2008; Peng et al., 2009; Moore et al., 2012). Particularlyrelated to SCI, extracellular release of hsp70, IL-1�, andTNF-� may represent important contributors to inflam-mation, and likely inhibit recovery; IL-1� by exerting adirect detrimental effect (Allan et al., 2005), and TNF-� andhsp70 by serving as danger associated molecular patterns(DAMPs) and TLR agonists that stimulate further inflam-matory cytokine release (Calderwood et al., 2007).

2. Materials and methods

2.1. Tissue samples and histology

Immunohistochemical (IHC) staining for IL-1�, TNF-� and hsp70 were investigated in the CPE of 4 dogswith acute SCI secondary to intervertebral disc extrusion(IVDE). These dogs represented submissions to the OhioState University College of Veterinary Medicine diagnosticpathology service, Columbus, OH. Clinical injury severityat the time of euthanasia was quantified using a previ-ously published scale ranging from 1 to 5, with a score of5 indicating a dog with paraplegia and absence of noci-ception (Sharp and Wheeler, 2005). All dogs with SCIwere euthanized between 12 and 48 h of onset of clinicalsigns. Complete necropsies were performed on each dogand brain and spinal cord tissue were processed for rou-tine histologic evaluation. Tissues were formalin-fixed andparaffin-embedded. Routine hematoxylin and eosin (H&E)was used to evaluate representative sections of brain andspinal cord, including sites of spinal cord compression andthe choroid plexus of the 4th ventricle. Tissue from 4 dogsfree of neurologic disease and euthanized and submittedfor necropsy for other reasons were used as controls.

2.2. Immunohistochemistry (IHC)

Sections of fourth ventricular CP from both SCI dogs

and normal controls underwent IHC staining for IL-1� andTNF-�, and hsp70. Specificity, source, and dilution of theantibodies are listed in Table 1. After deparaffinization, sec-tions were treated with 3% hydrogen peroxide for 10 min.

nd Immunopathology 148 (2012) 348– 352 349

Species-specific biotin conjugated second step antibodieswere added at a working dilution of 1:100–1:200. Perox-idase labeling was visualized with 3,3-diaminobenzidine.Sections were counterstained with hematoxylin. Appro-priate isotype controls were used to eliminate nonspecificstaining as a reason for IHC positivity. A section of spinalcord from the lesion epicenter of each dog with acute SCIwas also stained with each antibody using an identicalmethod, allowing comparison of proximate versus distalinflammatory responses.

2.3. Evaluation of IHC images

Digital quantitative analysis of IHC staining was per-formed. Individual slides were digitized using the AperioScanScope (Aperio Technologies, Vista, CA, USA), anddigital images were analyzed using Aperio ImageScopesoftware. For each tissue examined, a region of interest(ROI) containing only CPE was manually defined by a sin-gle investigator (SAM) using 20× magnification on theImageScope software. A pre-programmed algorithm foranalysis of IHC staining consisted of the following: huevalue of 0.1 (consistent with recognition of brown pixels),hue width of 0.5 and color saturation of 0.04. These param-eters allowed for consistent identification of brown pixels(positive immunoperoxidase signal) and consistent exclu-sion of pixels containing other colors. Positive pixel countswere performed, and a mean positivity was reported foreach ROI. Mean positivity was calculated as the number ofpositive pixels identified by the algorithm, divided by thenumber of total pixels in the ROI. Possible values rangedfrom 0 to 1.0. A single slide containing 4th ventricular CPEwas evaluated from each dog, and three regions of CPE wererandomly chosen for evaluation on each slide. The threevalues obtained for each available tissue section were aver-aged to produce a single value for mean positivity from eachdog examined. The same process was repeated for spinalcord tissue from each dog with acute SCI. ROIs for spinalcord included the ependymal lining of the central canal,spinal cord gray matter, and spinal cord white matter.

2.4. Statistical analysis

For each of the IHC stains, mean positivity for sectionsof CPE from the group of SCI dogs was compared to thatof the normal controls and to spinal cord tissue usingan unpaired t-test. Correlations between various clinicalparameters and degree of IHC staining in the SCI dogs werealso examined using a Pearson correlation. p < 0.1 was con-sidered statistically significant. All values are presented asmean ± standard error of the mean (SEM).

3. Results and discussion

The group of normal dogs consisted of 4 animals withno clinical or histopathologic evidence of neurologic dis-ease. One dog died from a pulmonary thromboembolism

identified at necropsy, one was euthanized due to severepneumonia, another due to pancreatitis, and one doghad no apparent cause of death. Mean positivity for theCPE of normal dogs was 0.284 ± 0.09, 0.423 ± 0.10, and

350 S.A. Moore, M.J. Oglesbee / Veterinary Immunology and Immunopathology 148 (2012) 348– 352

Table 1List of antibodies used, including name, specificity, source, and dilution.

Antibody Specificity Source Dilution

0StCw

adei2g

FCs

Rabbit polyclonal IL-1�

Rabbit polyclonal TNF-�

Mouse polyclonal hsp70/hsp72

.302 ± 0.07 for IL-1�, TNF-� and hsp70, respectively.taining patterns for all three proteins were most consis-ent with a primarily cytoplasmic distribution within thePE, although a small amount of apparent nuclear stainingas also present.

The group of dogs with SCI consisted of 4 dogs withcute IVDE affecting the T3-L3 spinal cord segment. Theuration of clinical signs ranged from 12 to 48 h prior touthanasia, with a mean duration of 28.5 h. Severity of

njury ranged from grade 3 to grade 5 (Sharp and Wheeler,005) with a mean injury severity of 4 (equating to paraple-ia with nociception intact). All sections of CPE appeared

ig. 1. IHC staining results at 40× magnification for the CP of dogs with acute sp and E). IL-1� (A and B), hsp70 (C and D) and TNF-� (E and F) staining were alignificance only for IL-1� and TNF-� (p < 0.1).

Abbiotec, San Diego, CA 1:100Abbiotec, San Diego, CA 1:50Enzo Life Sciences, Farmingdale, NY 1:50

histologically normal when evaluated via H&E staining.Spinal cord sections from these dogs contained variabledegrees of hemorrhage, necrosis, and inflammatory cellinfiltrates, consistent with acute SCI. Acute disc herniationwas confirmed at necropsy in each case.

Mean positivity for the CPE of dogs with SCI was0.630 ± 0.01, 0.660 ± 0.05, and 0.407 ± 0.08 for IL-1�, TNF-� and hsp70, respectively. When comparing SCI dogs tonormal control dogs, significant increases is IHC staining

of the CPE were identified for IL-1� (p = 0.014), and TNF-� (p = 0.038). CPE staining for hsp70 was also higher indogs with SCI. This relationship did not achieve statistical

inal cord injury (SCI – B, D and F) compared to normal control dogs (A,l increased in dogs with acute SCI. This relationship achieved statistical

S.A. Moore, M.J. Oglesbee / Veterinary Immunology a

Fig. 2. Mean positivity for IL-1�, TNF-� and hsp70 (±SEM) in the CP ofdogs with acute spinal cord injury (SCI) compared to normal control dogs.Statistically significant differences (p < 0.1) are indicated with an asterisk.

Table 2Mean positivity of IHC staining for IL-1�, TNF-� and hsp70 in the CPE of the4th ventricle and the “fold” increase of this value compared to ependymalcells, gray matter and white matter located in the lesion epicenter in thespinal cord of dogs with acute SCI.

IL-1� TNF-� hsp70

Mean positivity of CPE 0.630 0.660 0.406Fold increase compared to:

Ependymal cells 2.2 2.7 0.6Gray matter 3.2 2.9 5.3White matter 6.5 6.2 12.4

significance, but approached it (p = 0.176) (Figs. 1 and 2).There was not a significant correlation between injuryseverity and degree of IL-1�, TNF-� or hsp70 staining inthe SCI group; however, there was an inverse correlationbetween duration of clinical signs and degree of IL-1�,TNF-� and hsp70 staining. This relationship was statisti-cally significant for hsp70 (r = −0.90, p = 0.054), and IL-1�(r = −0.88, p = 0.059), but not for TNF-�.

To assess the relative importance of the CPE as asource of IL-1�, TNF-� and hsp70 in the injured CNS, sec-tions of spinal cord located at the lesion epicenter werealso evaluated for the dogs with acute SCI. Differences inmean positivity between the CPE and regions of spinalcord evaluated are detailed in Table 2. IHC staining ofthe CPE was significantly higher than for the ependymallining of the central canal, spinal cord gray matter andwhite matter at the lesion epicenter for IL-1� (p = 0.023,0.003, <0.001, respectively), and for TNF-� (p = 0.008, 0.004,<0.001, respectively). This was also the case for hsp70 stain-ing in the spinal cord gray matter (p = 0.02) and whitematter (p = 0.013); however, there was a significant increasein hsp70 staining in the ependymal cells lining the cen-tral canal of the spinal cord when compared to the CPE

(p = 0.065).

Our results demonstrate significant increases in IL-1�,TNF-�, and possibly hsp70 staining in the CPE of dogswith acute SCI compared to that of normal control dogs.

nd Immunopathology 148 (2012) 348– 352 351

Additionally, these responses appear to be significantlymore robust that those appreciated within the gray andwhite matter of the spinal cord lesion epicenter. To theauthors’ knowledge, this is the first study demonstratinga potential role of the CP in the inflammatory responsefollowing acute SCI in the dog. IL-1�, TNF-� and hsp70have all been previously implicated as potential mediatorsof CNS inflammation in acute SCI and immune-mediatedinflammation (Wang et al., 2004; Sharma et al., 2006; Penget al., 2009; Moore et al., 2012), and the pathologic sig-nificance of each of these molecules after SCI has beenstudied to various degrees. IL-1� and TNF-� responseshave been documented in various pathologic conditionsof the canine spinal cord, including chemically inducedinflammation and ageing (Horais et al., 2003; Chung et al.,2010). Additionally, laboratory models of SCI have repeat-edly demonstrated IL-1� and TNF-� responses within theCNS, and improved outcome with attenuation of theseresponses (Wang et al., 2004; Peng et al., 2009; Sawaiet al., 2010). Hsp70 responses have been less thoroughlyevaluated; however, the inducible form of hsp70 is nor-mally near the lower limit of detection by ELISA in theCSF of health dogs, and rises in conditions of cellularischemia or inflammation (Awad et al., 2008; Moore et al.,2012).

An inducible hsp70 response has been documented inrodent models of SCI, and attenuation of this responseappears to reduce tissue pathology and improve outcome;however, the sources of hsp70 in the CNS, as well asits role in the inflammatory sequel to acute SCI requirefurther investigation (Sharma et al., 1995, 2006). Eval-uation of extracellular hsp70 release in various otherdisease models implicates this protein as an importantinflammatory mediator and contributor to tissue dam-age. Apparent mechanisms of action include interactingwith Toll-like receptors (TLR), inducing pro-inflammatorycytokine expression via the NF-KB pathway, up-regulatingmacrophage-mediated phagocytosis, and enhancing apo-ptosis (Clemons and Anderson, 2006; Kovalchin et al., 2006;Yokota et al., 2010).

Given the results of the current study, it appears thatthe CPE could serve as a potential source for extracellularrelease of IL-1�, TNF-�, and hsp70 after acute SCI in dogs.For hsp70, ependymal cells lining the central canal mayalso serve as an important source, a phenomenon previ-ously suggested in a canine model of IR injury of the spinalcord (Awad et al., 2008). In the present study, CSF concen-trations of each protein could not be evaluated as definitiveproof of extracellular release because CSF samples were notavailable from any of the dogs included in this study.

Given the inverse correlation between hsp70 and IL-1� staining and the duration of clinical signs in SCI, thesetwo molecules, in particular, may play an important rolein the immediate-early inflammatory response after SCI indogs. Extracellular release of pro-inflammatory cytokinessuch as IL-1� and hsp70 from either the CPE or ependy-mal cells into the CSF would allow for dissemination of

inflammatory signals throughout the neuroaxis. Ultimatelythis could contribute to the chronic pro-inflammatory stateknown to exist in the CNS long after the acute traumaticinsult of SCI has passed. Given these findings, the role of the

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52 S.A. Moore, M.J. Oglesbee / Veterinary Immu

horoid plexus in the inflammatory response after canineCI warrants further investigation.

onflict of interest statement

The authors have no financial or personal relationshipshat could inappropriately influence or bias the content ofhe paper.

cknowledgments

The project described was supported by the Gray Ladyoundation. The content is solely the responsibility of theuthors.

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