Loss of Corneal Sensory Nerve Fibers in SIV-Infected Macaques

• DOI: 10.1016/j.ajpath.2014.02.009
• Publication Date: 2014-05-12T00:15:14Z
• Authors: Jamie L. Dorsey; Lisa M. Mangus; Jonathan D. Oakley; Sarah E. Beck; Kathleen M. Kelly; Suzanne E. Queen; Kelly A. Metcalf ...; Robert J. Adams; Carl F. Marfurt; Joseph L. Mankowski
• Keywords: Nerve fiber; Sensory nerve

Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third infected patients, including patients treated with antiretroviral therapy. Although emerging noninvasive techniques for corneal nerve assessments are increasingly being used to diagnose and monitor peripheral neuropathies, alterations have not been characterized in HIV. Here, determine whether SIV infection leads fiber loss, we immunostained corneas marker βIII tubulin. We developed applied both manual automated methods measure nerves subbasal plexus. These counting independently indicated significantly lower density among SIV-infected animals that rapidly progressed AIDS compared slow progressors. Concomitant decreased density, rapid progressors had increased levels RNA CD68-positive macrophages expression glial fibrillary acidic protein by satellite cells trigeminal ganglia, location neuronal cell bodies sensory fibers. In addition, was directly correlated epidermal length. findings indicate assessment has great potential HIV-induced set stage introducing clinical settings. (PN) caused HIV-1, persons, receiving combination therapy.1McArthur J.C. Brew B.J. Nath A. Neurological complications infection.Lancet Neurol. 2005; 4: 543-555Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 2Ellis R.J. Rosario D. Clifford D.B. McArthur Simpson Alexander T. Gelman B.B. Vaida F. Collier Marra C.M. Ances B. Atkinson J.H. Dworkin R.H. Morgello S. Grant I. CHARTER Study GroupContinued high prevalence adverse impact human immunodeficiency virus-associated era therapy: Study.Arch 2010; 67: 552-558Crossref (251) Scholar The typical presentation known as distal polyneuropathy, a length-dependent bilateral aching, painful numbness or burning, pronounced extremities.3Cornblath D.R. Predominantly AIDS-related complex.Neurology. 1988; 38: 794-796Crossref 4Pardo C.A. Griffin J.W. neuropathy: insights pathology disease.J Peripher Nerv Syst. 2001; 6: 21-27Crossref (209) PN (HIV-PN) life threatening, this debilitating disorder greatly compromises patient quality life.1McArthur Currently, skin biopsy accepted standard measuring loss small, unmyelinated C fibers epidermis, one earliest detectable signs damage nervous system (PNS).5Polydefkis M. Yiannoutsos C.T. Cohen B.A. Hollander H. Schifitto G. D.M. Katzenstein Shriver Hauer P. Brown Haidich A.B. Moo L. Reduced intraepidermal HIV-associated neuropathy.Neurology. 2002; 58: 115-119Crossref (254) 6Sumner C.J. Sheth Cornblath Polydefkis spectrum diabetes impaired glucose tolerance.Neurology. 2003; 60: 108-111Crossref (601) 7Smith A.G. Howard J.R. Kroll R. Ramachandran Singleton J. reliability measurement density.J Neurol Sci. 228: 65-69Abstract (115) However, an invasive procedure, longitudinal requires repeated surgical biopsies. Electrophysiological testing properties conduction considered viable alternative because current lack sensitivity required detect fibers, especially early stages disease.8Gonzalez-Duarte Cikurel K. Managing neuropathy.Curr HIV/AIDS Rep. 2007; 114-118Crossref (34) 9Gonzalez-Duarte Robinson-Papp Diagnosis management neuropathy.Neurol Clin. 2008; 26 (x): 821-832Abstract (54) For these reasons, new, sensitive, assessing small urgently needed persons Like HIV, common mellitus results syndrome closely resembles polyneuropathy.10Boulton A.J. Management Diabetic Neuropathy.Clin Diabetes. 23: 9-15Crossref (238) Of interest, studies documented utility changes innervation track diabetic (ENF) via biopsy. particular, decreases plexus (SBP) reported isolated whole mount vivo confocal microscopy (CCM).11Müller L.J. Marfurt C.F. Kruse Tervo T.M. Corneal nerves: structure, contents function.Exp Eye Res. 76: 521-542Crossref (901) 12Malik R.A. Kallinikos Abbott van Schie C.H. Morgan Efron N. Boulton microscopy: non-invasive surrogate fibre repair patients.Diabetologia. 46: 683-688Crossref (411) 13Kallinikos Berhanu O'Donnell C. Malik tortuosity neuropathy.Invest Ophthalmol Vis 2004; 45: 418-422Crossref (236) 14Midena E. Brugin Ghirlando Sommavilla Avogaro study.J Refract Surg. 2006; 22: S1047-S1052PubMed 15He Bazan H.E.P. Mapping architecture corneas.Ophthalmology. 2012; 119: 956-964Abstract (63) found increasing value evaluate ENFs PN, use tracking yet be explored. To study pathogenesis PNS disease, our group macaque model recapitulates key seen PN. include macrophage infiltration, replication, which house neurons innervate cornea.16Laast V.A. Pardo Tarwater P.M. Queen S.E. Reinhart T.A. Ghosh Adams Zink M.C. Mankowski J.L. Pathogenesis simian virus-induced ganglia.J Neuropathol Exp 66: 26-34Crossref (33) 17Laast Shim Johanek L.M. Dorsey P.E. Ringkamp Macrophage-mediated dorsal root ganglion precedes altered macaques.Am J Pathol. 2011; 179: 2337-2345Abstract (41) report, aimed (CNF) correspond extent replication severity cellular immune responses CNF correlates ENF length, thereby setting follow-up CCM investigation. achieve optimal immunostaining SBP, modified immunohistochemical staining method et al.18Marfurt Cox Deek Dvorscak Anatomy innervation.Exp 90: 478-492Crossref (345) Because conventional image analysis labor intensive trained observers, two novel methods: relatively simple detection facilitate efficient, objective density. Male juvenile pigtailed macaques (Macaca nemestrina) were inoculated intravenously either infectious clone SIV/17E-Fr viral swarm SIV/DeltaB670 (n = 7) 4).19Clements J.E. Gama accelerated virus disease: from mechanism treatment.J Neurovirol. 14: 309-317Crossref (69) Serology negative SIV, T-cell leukemia virus, type D retrovirus all before inoculation. control purposes, also obtained three additional uninfected macaques. Animals housed Johns Hopkins University facilities fully accredited Association Assessment Accreditation Laboratory Animal Care International. Macaques fed commercial diet (Harlan, Indianapolis, IN), given water ad libitum, provided environmental enrichment daily. All procedures approved Institutional Use Committee conducted accordance guidelines forth Welfare Regulations (US Department Agriculture) Guide (Office Welfare). levels, extracted frozen ganglia plasma. Viral load measured quantitative RT-PCR primers quantitate gag.20Kelly K.M. Karper J.M. Bedja Tunin R.S. Kass D.A. Diastolic dysfunction associated myocardial virus-infected macaques.AIDS. 26: 815-823Crossref (22) After euthanasia, 8-mm punch remove central cornea eye. Corneas immediately placed 0.1 mol/L PBS stored at 4°C 8 hours preservation 10% neutral-buffered formalin. then fixed overnight 4°C, rinsed times PBS, cryoprotected 20% glycerol/0.1 Sørensen's phosphate buffer. Before immunostaining, 5-mm button, free-floating mount. enhance permeability, mounts incubated 48 37°C 0.01% hyaluronidase (type IV-S; Sigma-Aldrich, St. Louis, MO) 0.1% EDTA (Sigma-Aldrich) (pH 5.3). buttons washed 15 minutes each contained 0.3% Triton X-100 between subsequent step. Nonspecific labeling blocked incubation room temperature 2 blocking serum (1% bovine albumin X-100) 4-day mouse monoclonal antibody against tubulin (1:500; Promega Corporation, Madison, WI). secondary (biotinylated goat anti-mouse IgG, 1:200; Vector Laboratories, Burlingame, CA) followed avidin-biotin-horseradish peroxidase complex (Vectastain Elite ABC Reagent; Laboratories) diaminobenzidine chromogen after 10 minutes. washing, mounted on chrome alum-gelatin–coated slides air-dried overnight. Tissue distilled water, dehydrated graded alcohols, cleared HistoClear (National Diagnostics, Atlanta, GA), coverslipped Permount (Fisher Scientific, Pittsburgh, PA). cornea, images five ×200 fields (one field quadrant cornea) SBP captured Retiga 2000R digital camera (QImaging, Surrey, BC, Canada) Nikon E600 microscope (Nikon, Tokyo, Japan). Several serial Z-stack taken accommodate curvature thus bringing entire into single focused plane. subregions focus selected Adobe Photoshop CS4 software version 11.0 (Adobe Systems, San Jose, autoblended assemble cohesive field. blended subsequently counted Stereo Investigator 9 (MicroBrightField Inc, Colchester, VT) draw transect lines across oriented perpendicular number crossing divided total image, transects averaged, final mean per millimeter derived animal. automate counts custom algorithm specifically quantify emulate method. First, anisotropic diffusion filtering reduce noise while preserving structure image.21Perona Scale-space edge-detection using diffusion.IEEE Trans Pattern Anal Mach Intell. 1990; 12: 629-639Crossref (10066) linear enhanced various scales Frangi's Hessian-based filter.22Frangi A.F. Niessen W.J. Vincken K.L. Viergever M.A. Multiscale vessel enhancement filtering.Lect Notes Comput Sc. 1998; 1496: 130-137Crossref exploit piecewise linearity nerves, subdivided smaller regions exhibited same orientation. Next, subimages reoriented find best (ie, prominent) orientation defined minimal entropy distribution data resulting 1D profile. visible determined summing along prominent peaks profile, peak representing fiber. count cumulatively sum images. IHC performed HistoChoice-fixed (HistoChoice Fixative; Amresco, Solon, OH) paraffin-embedded sections ganglia. deparaffinized heating 30 60°C, Diagnostics), rehydrated series alcohol. Slides pretreated antigen retrieval 0.01 sodium citrate 6.0) immunostaining. Endogenous peroxidases 3% hydrogen peroxide methanol, nonspecific dilute block (Vector Laboratories). Sections appropriate primary dilution [CD68, 1:4000, KP1, (GFAP), 1:80,000; Dako, Carpinteria, CA] 1 hour, biotinylated anti-mouse/rabbit washed, cleared, Scientific). amount CD68 GFAP digitized described.17Laast ganglion, 20 nonoverlapping magnification (QImaging) (Nikon). Binarized analyzed iVision imaging 4.0.14 (BioVision Technologies, Exton, area occupied immunopositive pixels calculated plantar footpad surface hind limb, 3-mm diameter samples necropsy identical site. Footpad 12 24 2% paraformaldehyde/lysine/periodate fixative 0.08 buffer, transferred cryoprotective buffer (20% glycerol buffer) until processed previously described.23Holland N.R. Stocks Intraepidermal 1997; 48: 708-711Crossref (271) Cryoprotected sectioned thickness 50 μm sliding microtome PGP9.5, pan axonal (1:2000; Chemicon, Temecula, CA), described.24McCarthy B.G. Hsieh S.T. Macko Cutaneous neuropathies: evaluation biopsy.Neurology. 1995; 1848-1855Crossref (405) 25Nolano Provitera V. Crisci Stancanelli Wendelschafer-Crabb Kennedy W.R. Santoro Quantification myelinated endings mechanoreceptors skin.Ann 54: 197-205Crossref (153) PGP9.5 length quantified unbiased stereology methodology space ball probe (MBF Bioscience, Williston, described Ebenezer al26Ebenezer G.J. SIV-induced impairment neurovascular repair: role VEGF.J 18: 222-230Crossref (13) 27Ebenezer Cimino N.P. Impaired subjects following experimental intracutaneous axotomy.Brain. 134: 1853-1863Crossref (74) 28Mouton P.R. Gokhale A.M. Ward N.L. West M.J. Stereological estimation spherical probes.J Microsc. 206: 54-64Crossref (157) Mouton.26Ebenezer Three 50-μm animal measured. interest region extended epidermal/dermal junction stratum corneum drawn under 4×/0.2 Plan Apo Eclipse light microscope. 60×/1.40 oil objective. radius hemisphere guard zone μm. Only within epidermis counted. expressed measurements DAT files Investigator.27Ebenezer Seven dual-inoculated SIV/17E-Fr, clone, SIV/DeltaB670, immunosuppressive swarm. An four only molecular SIV/17E-Fr. disease progression, plasma loads (PVLs) acute through terminal time points. During similar PVLs 14 days inoculation 107 109 copies/mL (Figure 1A). Among animals, point remained high, sacrifice (84 inoculation), severely diminished CD4+ T-lymphocyte (median PVL 1.18 × copies/mL, median T cells/μL 276). contrast, singly much continued 1.89 103 copies/mL) maintained higher 989; P 0.0061) did Given differences groups, versus classified progressors, respectively, comparisons. induced 5 mm 2A). Visual inspection progressor 2, B–E). CNFs, manually 3, A–D). Manual showed strong, positive correlation (P 0.0004, r 0.90), indicating excellent agreement 3E). Both progressors; decline robust 0.024 counts; 0.0061 counts) F G). uniform sample individual animals. Neither nor different > 0.05). This expected finding, approximately 50% do evidence damage.Figure 3Manual RP SP A: Image represents acquired CNF. B: assessment, (blue lines) trunks (denoted red circles) transect, average calculated. C: (Voxeleron) identify CNFs. Applying filters generated output depicted would detected algorithm. D: steps Each first 16 rotated vertical (top panel). pixel summed axis (middle panel), generating profile represented denoted asterisk top (bottom Lower excluded thresholding. E: A strong methods. F: statistically significant decrease CNFs assessment. G: Similarly, RPs. Lines represent values (E–G). (G), Mann-Whitney test; 0.90, Spearman rank (G). Original (A–C). RP, SP, progressors.View Large Figure ViewerDownload Hi-res Download (PPT) Previous shown end points.16Laast investigate differed RT-PCR. elevated 1076 copies/μg RNA; 0.024) 1B) low undetectable 3 RNA). macaques, responders, cells, activated response infection.16Laast there progresso