Loss of Corneal Sensory Nerve Fibers in SIV-Infected Macaques: An Alternate Approach to Investigate HIV-Induced PNS Damage

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.^1,2 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.^3,4 Although HIV-induced PN (HIV-PN) is not life threatening, this debilitating disorder greatly compromises patient quality of life.¹ 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).^5–7 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.^8,9 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.¹⁰ 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).^11–15Although 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.^16,17 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.¹⁸ 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.