Loss of Corneal Sensory Nerve Fibers in SIV-Infected Macaques

Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third of 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, corneal nerve alterations have not been characterized in HIV. Here, to determine whether SIV infection leads to corneal nerve fiber loss, we immunostained corneas for the nerve fiber marker βIII tubulin. We developed and applied both manual and automated methods to measure nerves in the corneal subbasal plexus. These counting methods independently indicated significantly lower subbasal corneal nerve fiber density among SIV-infected animals that rapidly progressed to AIDS compared with slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA and CD68-positive macrophages and expression of glial fibrillary acidic protein by glial satellite cells in the trigeminal ganglia, the location of the neuronal cell bodies of corneal sensory nerve fibers. In addition, corneal nerve fiber density was directly correlated with epidermal nerve fiber length. These findings indicate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and to set the stage for introducing noninvasive techniques to measure corneal nerve fiber density in HIV clinical settings. Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third of 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, corneal nerve alterations have not been characterized in HIV. Here, to determine whether SIV infection leads to corneal nerve fiber loss, we immunostained corneas for the nerve fiber marker βIII tubulin. We developed and applied both manual and automated methods to measure nerves in the corneal subbasal plexus. These counting methods independently indicated significantly lower subbasal corneal nerve fiber density among SIV-infected animals that rapidly progressed to AIDS compared with slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA and CD68-positive macrophages and expression of glial fibrillary acidic protein by glial satellite cells in the trigeminal ganglia, the location of the neuronal cell bodies of corneal sensory nerve fibers. In addition, corneal nerve fiber density was directly correlated with epidermal nerve fiber length. These findings indicate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and to set the stage for introducing noninvasive techniques to measure corneal nerve fiber density in HIV clinical settings. Peripheral neuropathy (PN) is the most frequent neurological complication caused by HIV-1, affecting more than one-third of infected persons, including patients receiving combination antiretroviral therapy.1McArthur J.C. Brew B.J. Nath A. Neurological complications of HIV infection.Lancet Neurol. 2005; 4: 543-555Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 2Ellis R.J. Rosario D. Clifford D.B. McArthur J.C. Simpson D. Alexander T. Gelman B.B. Vaida F. Collier A. Marra C.M. Ances B. Atkinson J.H. Dworkin R.H. Morgello S. Grant I. CHARTER Study GroupContinued high prevalence and adverse clinical impact of human immunodeficiency virus-associated sensory neuropathy in the era of combination antiretroviral therapy: the CHARTER Study.Arch Neurol. 2010; 67: 552-558Crossref PubMed Scopus (251) Google Scholar The typical clinical presentation is known as distal sensory polyneuropathy, a length-dependent neuropathy that is characterized by bilateral aching, painful numbness or burning, and is most pronounced in the lower extremities.3Cornblath D.R. McArthur J.C. Predominantly sensory neuropathy in patients with AIDS and AIDS-related complex.Neurology. 1988; 38: 794-796Crossref PubMed Google Scholar, 4Pardo C.A. McArthur J.C. Griffin J.W. HIV neuropathy: insights in the pathology of HIV peripheral nerve disease.J Peripher Nerv Syst. 2001; 6: 21-27Crossref PubMed Scopus (209) Google Scholar Although HIV-induced PN (HIV-PN) is not life threatening, this debilitating disorder greatly compromises patient quality of life.1McArthur J.C. Brew B.J. Nath A. Neurological complications of HIV infection.Lancet Neurol. 2005; 4: 543-555Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar Currently, skin biopsy is the accepted standard for measuring the loss of small, unmyelinated C fibers in the epidermis, one of the earliest detectable signs of damage to the peripheral nervous system (PNS).5Polydefkis M. Yiannoutsos C.T. Cohen B.A. Hollander H. Schifitto G. Clifford D.B. Simpson D.M. Katzenstein D. Shriver S. Hauer P. Brown A. Haidich A.B. Moo L. McArthur J.C. Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy.Neurology. 2002; 58: 115-119Crossref PubMed Scopus (254) Google Scholar, 6Sumner C.J. Sheth S. Griffin J.W. Cornblath D.R. Polydefkis M. The spectrum of neuropathy in diabetes and impaired glucose tolerance.Neurology. 2003; 60: 108-111Crossref PubMed Scopus (601) Google Scholar, 7Smith A.G. Howard J.R. Kroll R. Ramachandran P. Hauer P. Singleton J.R. McArthur J. The reliability of skin biopsy with measurement of intraepidermal nerve fiber density.J Neurol Sci. 2005; 228: 65-69Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar However, skin biopsy is an invasive procedure, and longitudinal assessment requires repeated surgical biopsies. Electrophysiological testing to measure properties of peripheral nerve conduction is not considered a viable alternative because current methods lack the sensitivity required to detect damage to small, unmyelinated fibers, especially in early stages of disease.8Gonzalez-Duarte A. Cikurel K. Simpson D.M. Managing HIV peripheral neuropathy.Curr HIV/AIDS Rep. 2007; 4: 114-118Crossref PubMed Scopus (34) Google Scholar, 9Gonzalez-Duarte A. Robinson-Papp J. Simpson D.M. Diagnosis and management of HIV-associated neuropathy.Neurol Clin. 2008; 26 (x): 821-832Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar For these reasons, new, sensitive, noninvasive methods of assessing small fiber nerve damage are urgently needed to detect and monitor PN in persons infected with HIV. Like HIV, small sensory nerve fiber loss is common in patients with diabetes mellitus and results in a clinical syndrome that closely resembles HIV distal sensory polyneuropathy.10Boulton A.J. Management of Diabetic Peripheral Neuropathy.Clin Diabetes. 2005; 23: 9-15Crossref Scopus (238) Google Scholar Of interest, studies have documented the utility of measuring changes in corneal sensory innervation to track diabetic neuropathy as an alternative to measuring epidermal nerve fiber (ENF) density via skin biopsy. In particular, decreases in the nerve density in the corneal subbasal plexus (SBP) have been reported in both isolated corneal whole mount studies and by noninvasive in vivo corneal confocal microscopy (CCM).11Müller L.J. Marfurt C.F. Kruse F. Tervo T.M. Corneal nerves: structure, contents and function.Exp Eye Res. 2003; 76: 521-542Crossref PubMed Scopus (901) Google Scholar, 12Malik R.A. Kallinikos P. Abbott C.A. van Schie C.H. Morgan P. Efron N. Boulton A.J. Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients.Diabetologia. 2003; 46: 683-688Crossref PubMed Scopus (411) Google Scholar, 13Kallinikos P. Berhanu M. O'Donnell C. Boulton A.J. Efron N. Malik R.A. Corneal nerve tortuosity in diabetic patients with neuropathy.Invest Ophthalmol Vis Sci. 2004; 45: 418-422Crossref PubMed Scopus (236) Google Scholar, 14Midena E. Brugin E. Ghirlando A. Sommavilla M. Avogaro A. Corneal diabetic neuropathy: a confocal microscopy study.J Refract Surg. 2006; 22: S1047-S1052PubMed Google Scholar, 15He J. Bazan H.E.P. Mapping the nerve architecture of diabetic human corneas.Ophthalmology. 2012; 119: 956-964Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Although corneal nerve assessments have found increasing value as a surrogate to evaluate ENFs in diabetic PN, the use of corneal alterations in tracking HIV-induced neuropathy has yet to be explored. To study the pathogenesis of HIV-induced PNS disease, our group developed a SIV-infected macaque model that closely recapitulates key PNS alterations seen in patients with HIV with PN. These changes include macrophage infiltration, SIV replication, and neuronal loss in sensory ganglia, including the trigeminal ganglia, which house the cell bodies of sensory neurons that innervate the cornea.16Laast V.A. Pardo C.A. Tarwater P.M. Queen S.E. Reinhart T.A. Ghosh M. Adams R.J. Zink M.C. Mankowski J.L. Pathogenesis of simian immunodeficiency virus-induced alterations in macaque trigeminal ganglia.J Neuropathol Exp Neurol. 2007; 66: 26-34Crossref PubMed Scopus (33) Google Scholar, 17Laast V.A. Shim B. Johanek L.M. Dorsey J.L. Hauer P.E. Tarwater P.M. Adams R.J. Pardo C.A. McArthur J.C. Ringkamp M. Mankowski J.L. Macrophage-mediated dorsal root ganglion damage precedes altered nerve conduction in SIV-infected macaques.Am J Pathol. 2011; 179: 2337-2345Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar In this report, we aimed to determine whether SIV infection leads to decreases in corneal nerve fiber (CNF) density, whether changes in corneal nerves correspond with the extent of SIV replication and the severity of cellular immune responses in the trigeminal ganglia, and whether CNF density correlates with ENF length, thereby setting the stage for follow-up CCM investigation. To achieve optimal immunostaining of the corneal SBP, we modified an immunohistochemical staining method developed by Marfurt et al.18Marfurt C.F. Cox J. Deek S. Dvorscak L. Anatomy of the human corneal innervation.Exp Eye Res. 2010; 90: 478-492Crossref PubMed Scopus (345) Google Scholar Because conventional image analysis of the corneal nerve is labor intensive and requires trained observers, we developed two novel counting methods: a relatively simple manual counting method of nerve fibers and an automated method of nerve detection and counting to facilitate efficient, objective assessment of nerve fiber density. Male juvenile pigtailed macaques (Macaca nemestrina) were inoculated intravenously with either a combination of the infectious clone SIV/17E-Fr and the viral swarm SIV/DeltaB670 (n = 7) or with the infectious clone SIV/17E-Fr (n = 4).19Clements J.E. Mankowski J.L. Gama L. Zink M.C. The accelerated simian immunodeficiency virus macaque model of human immunodeficiency virus-associated neurological disease: from mechanism to treatment.J Neurovirol. 2008; 14: 309-317Crossref PubMed Scopus (69) Google Scholar Serology was negative for SIV, simian T-cell leukemia virus, and simian type D retrovirus for all macaques before inoculation. For control purposes, corneas also were obtained from three were in that are by the for and of were a macaque and with were by the and and were in with set in the of and the for the and of of To evaluate SIV RNA RNA was from trigeminal or was by with the use of to SIV Tarwater P.M. D. Queen S.E. Adams R.J. Mankowski J.L. is with viral in simian immunodeficiency 2012; PubMed Scopus Google Scholar an biopsy was used to the from the were in and for before in were three in and in a biopsy was used to the corneal which was immunostained as a whole To whole were for in and in Corneal were three for in that and also immunostaining was by for in in that before a with a the neuronal marker βIII Corneal were in by for and detection with the for to corneas were and were in in in and with For of from and one from the of the were with a a were of to for the of the the of the a The of in for image were in and to a image of the subbasal nerve plexus for The were used in the assessment of nerve fiber density. Corneal nerves were with to three image to the longitudinal subbasal nerve fibers. The of nerves was and by the length. For the of nerves of the three was and the of corneal nerves was from the for To nerve in the a was developed to nerve fibers in to the manual counting was applied to the of P. Malik J. and Scopus Google Scholar The of the nerves was with the use of Google Scholar To the of the the image was in which nerves the the were to the most of the as of the of image in the The of nerves was by image the most and counting the in the with a nerve The nerve was from the of the image for of the was of trigeminal were by for in changes of and in a of were for by with before were with in was with were in the clone and glial fibrillary acidic protein for by and for with detection with the were and with The of immunostaining for the macrophage marker and satellite cell marker was by image analysis as V.A. Shim B. Johanek L.M. Dorsey J.L. Hauer P.E. Tarwater P.M. Adams R.J. Pardo C.A. McArthur J.C. Ringkamp M. Mankowski J.L. Macrophage-mediated dorsal root ganglion damage precedes altered nerve conduction in SIV-infected macaques.Am J Pathol. 2011; 179: 2337-2345Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar For trigeminal were with a a were with The by the in the trigeminal ganglion was for To measure ENF density in the skin of the of the were obtained from the skin were for to in with and to in as A. Hauer P. Cornblath D.R. Griffin J.W. McArthur J.C. nerve fiber density in patients with painful sensory neuropathy.Neurology. PubMed Scopus Google Scholar were a of a and immunostained for a marker as A. Hauer P. C. Cornblath D.R. Griffin J.W. McArthur J.C. innervation in sensory by skin 45: PubMed Scopus Google Scholar, M. C. A. G. L. of and in human Neurol. 2003; PubMed Scopus Google Scholar ENF was with by the by et McArthur J.C. Polydefkis M. Dorsey J.L. O'Donnell R. Hauer P. Adams R.J. Mankowski J.L. of a potential for Neurovirol. 2012; PubMed Scopus Google Scholar, O'Donnell R. Hauer P. McArthur J.C. Polydefkis M. repair in with diabetes 2011; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar and McArthur J.C. Polydefkis M. Dorsey J.L. O'Donnell R. Hauer P. Adams R.J. Mankowski J.L. of a potential for Neurovirol. 2012; PubMed Scopus Google Scholar, O'Donnell R. Hauer P. McArthur J.C. Polydefkis M. repair in with diabetes 2011; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar immunostained from one biopsy from were The of was as the epidermal that from the to the and was a objective a The ENF was with a The of the was with a of nerve fibers the were The results were as the of nerve fibers. were obtained with of O'Donnell R. Hauer P. McArthur J.C. 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Pardo C.A. Tarwater P.M. Queen S.E. Reinhart T.A. Ghosh M. Adams R.J. Zink M.C. Mankowski J.L. Pathogenesis of simian immunodeficiency virus-induced alterations in macaque trigeminal ganglia.J Neuropathol Exp Neurol. 2007; 66: 26-34Crossref PubMed Scopus (33) Google Scholar, 17Laast V.A. Shim B. Johanek L.M. Dorsey J.L. Hauer P.E. Tarwater P.M. Adams R.J. Pardo C.A. McArthur J.C. Ringkamp M. Mankowski J.L. Macrophage-mediated dorsal root ganglion damage precedes altered nerve conduction in SIV-infected macaques.Am J Pathol. 2011; 179: 2337-2345Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar To whether SIV replication in the trigeminal SIV RNA levels were by SIV RNA in the trigeminal among the rapid progressors was significantly SIV RNA = = compared with the slow progressors with to SIV RNA levels SIV RNA = In the sensory of key cellular immune including macrophages and satellite are in to SIV V.A. Pardo C.A. Tarwater P.M. Queen S.E. Reinhart T.A. Ghosh M. Adams R.J. Zink M.C. Mankowski J.L. Pathogenesis of simian immunodeficiency virus-induced alterations in macaque trigeminal ganglia.J Neuropathol Exp Neurol. 2007; 66: 26-34Crossref PubMed Scopus (33) Google Scholar, 17Laast V.A. Shim B. Johanek L.M. Dorsey J.L. Hauer P.E. Tarwater P.M. Adams R.J. Pardo C.A. McArthur J.C. Ringkamp M. Mankowski J.L. Macrophage-mediated dorsal root ganglion damage precedes altered nerve conduction in SIV-infected macaques.Am J Pathol. 2011; 179: 2337-2345Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar To determine whether were in cellular immune responses the rapid and slow trigeminal were for the macrophage marker and the satellite cell marker The of and immunostaining was by image J.L. Queen S.E. 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Macrophage-mediated dorsal root ganglion damage precedes altered nerve conduction in SIV-infected macaques.Am J Pathol. 2011; 179: 2337-2345Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar manual and automated CNF were correlated with ENF = = for manual = = for automated and Because the is the most in the corneal nerve assessment is for damage to small sensory nerve fibers compared with including measurement of intraepidermal nerve fibers in the L.J. Marfurt C.F. Kruse F. Tervo T.M. Corneal nerves: structure, contents and function.Exp Eye Res. 2003; 76: 521-542Crossref PubMed Scopus (901) Google Scholar, C. M. M. Kallinikos P. S. J. A. Boulton A.J. Efron N. Malik R.A. of small fiber damage in human diabetic 2007; PubMed Scopus Google Scholar The potential for early detection of nerve with the and of in vivo has to increased use of corneal nerve assessments to monitor and evaluate a of peripheral neuropathies, diabetic R.A. Kallinikos P. Abbott C.A. van Schie C.H. Morgan P. Efron N. Boulton A.J. Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients.Diabetologia. 2003; 46: 683-688Crossref PubMed Scopus (411) Google Scholar, 14Midena E. Brugin E. Ghirlando A. Sommavilla M. Avogaro A. Corneal diabetic neuropathy: a confocal microscopy study.J Refract Surg. 2006; 22: S1047-S1052PubMed Google Scholar, M. A. R. H. D. Efron N. Boulton A.J. Malik R.A. Corneal confocal microscopy: a novel to detect nerve fibre damage in small fibre Neurol. 2010; PubMed Scopus Google Scholar, M. A. L. M. S. Efron N. Malik R.A. Corneal confocal microscopy: a novel noninvasive to diagnose neuropathy in patients with PubMed Scopus Google Scholar, M. C. Abbott C. Kallinikos P. A. J. Morgan P. Efron N. Boulton Malik R.A. Corneal confocal microscopy: novel noninvasive to diagnose and the severity of human diabetic 2010; PubMed Scopus Google Scholar the high prevalence of as as the clinical and diabetic PN, alterations in corneal nerves among persons infected with HIV have not yet been explored. the use of an model that closely resembles HIV infection, this study indicated loss of in the corneal subbasal nerve plexus. CNF closely with of ENF length. CNF loss also was with viral replication and cellular immune in the trigeminal ganglia, the location of the neuronal cell bodies of the corneal sensory nerve fibers. The and SIV replication in the trigeminal and CNF loss that damage to neurons in the trigeminal the of neurons to sensory nerve fiber these findings indicate that emerging noninvasive techniques to measure CNF alterations as in vivo CCM be clinical to for and monitor of PN in patients infected with HIV. of current corneal nerve assessments include the for manual image analysis and in the of nerve fibers, the for trained automated of in vivo confocal microscopy of the human corneal subbasal nerve Ophthalmol Vis Sci. 2012; PubMed Scopus Google Scholar, analysis of in vivo confocal microscopy a 58: Full Text Full Text PDF PubMed Scopus Google Scholar To these we developed a immunostaining and image To measure in the SBP, we developed and two applied methods for nerve in the a manual method that all nerve fibers a and an automated method that a to and nerve fibers. Although both methods viable methods of the of an automated method of thereby counting and counting methods decreased CNF density in the rapid the the rapid and slow was most pronounced with the use of the automated method of nerve our method of immunostaining that of all as as the in the sensitivity of nerve fiber detection the the use of the manual nerve fiber is of or to counting In the automated is to nerve with detection to the nerve to current in vivo CCM of the nerve fibers also J. Bazan H.E.P. Mapping the nerve architecture of diabetic human corneas.Ophthalmology. 2012; 119: 956-964Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, M. P. Malik R.A. of corneal confocal 2008; Google Scholar The in nerve fiber that the density of nerve the severity of CNF damage in In this to analysis and to we CNF density a to have high and and T. Morgan P. H. G. J. M. Malik R.A. of in vivo corneal confocal microscopy to corneal nerve PubMed Scopus Google Scholar However, as fiber length, density, and tortuosity of subbasal corneal nerves also have been to changes of PN disease, clinical are or and skin ENF R.A. Kallinikos P. Abbott C.A. van Schie C.H. Morgan P. Efron N. Boulton A.J. Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients.Diabetologia. 2003; 46: 683-688Crossref PubMed Scopus (411) Google Scholar, 13Kallinikos P. Berhanu M. O'Donnell C. Boulton A.J. Efron N. Malik R.A. Corneal nerve tortuosity in diabetic patients with neuropathy.Invest Ophthalmol Vis Sci. 2004; 45: 418-422Crossref PubMed Scopus (236) Google Scholar, 14Midena E. Brugin E. Ghirlando A. Sommavilla M. Avogaro A. Corneal diabetic neuropathy: a confocal microscopy study.J Refract Surg. 2006; 22: S1047-S1052PubMed Google Scholar, 15He J. Bazan H.E.P. Mapping the nerve architecture of diabetic human corneas.Ophthalmology. 2012; 119: 956-964Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, C. M. M. Kallinikos P. S. J. A. Boulton A.J. Efron N. Malik R.A. of small fiber damage in human diabetic 2007; PubMed Scopus Google Scholar, K. N. D. A. Malik R.A. Efron N. of corneal confocal microscopy for assessing diabetic neuropathy: findings of the Exp 2012; PubMed Scopus Google Scholar studies that use corneal nerve assessment as a surrogate for also include of these to our of CNF damage as as repair the of in this study we were to of because of the immunohistochemical staining method of nerve that corneal whole However, the to noninvasive and methods of nerve detection as in vivo CCM study of PN by early detection of damage and of nerve fiber and assessment of in the in vivo CCM for use in tracking HIV-induced PNS damage in patients be of great value in and early PNS damage of skin biopsies. 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