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Immunohistochemical Evidence of Inducible Nitric Oxide Synthase and Nitrotyrosine in a Case of Clinically Isolated Optic Neuritis

Tsoi, Veda L; Hill, Kenneth E BS; Carlson, Noel G PhD; Warner, Judith E. A MD; Rose, John W MD

Journal of Neuro-Ophthalmology: June 2006 - Volume 26 - Issue 2 - p 87-94
doi: 10.1097/01.wno.0000223266.48447.1b
Original Contribution
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Background: Optic neuritis (ON) is a demyelinating inflammation of the optic nerve that may occur as an isolated disease or related to multiple sclerosis (MS). There is little evidence of whether the immunohistochemistry of ON resembles that of typical cerebral MS lesions.

Methods: Pathologic optic nerves were obtained from a patient who died of causes unrelated to ON after clinical recovery from clinically isolated ON. Normal control optic nerves were obtained from an eye bank. Normal and pathologic tissues were probed with antibodies to pathologic proteins including myelin basic protein (MBP) fragment, the inducible form of nitric oxide synthase (iNOS), macrophage markers CD14 and CD64, nitrotyrosine, and cyclooxygenase (COX-2). We also examined MBP, the oligodendrocyte marker cyclic nucleotide phosphodiesterase (CNPase), and glial fibrillary acidic protein.

Results: In the affected pathologic nerve, iNOS-positive macrophages/microglia, iNOS-positive astrocytes, COX-2, and nitrotyrosine were observed. iNOS and COX-2 were occasionally observed in the unaffected nerve.

Decreased expression of MBP and CNPase was seen in the pathologic optic nerves, along with evidence of gliosis and ongoing myelin degradation indicated by the presence of MBP fragment.

Conclusions: The immunohistochemistry of clinically isolated optic neuritis, as judged by this single case, resembles that of cerebral lesions of MS in showing abnormally high levels of iNOS and nitrotyrosine as well as other mediators of immune damage.

Neurovirology Research Laboratory (VLT, KEH, NGC, JWR), Geriatric Research Education and Clinical Center (GRECC) (NGC), and Neuro-ophthalmology (JW), Veterans Affairs Salt Lake City Health Care System, Salt Lake City, Utah; Neurobiology and Anatomy (NGC), Neurology (KEH, NGC, JWR), Brain Institute (NGC, JWR), and Neuro-Ophthalmology (JW), Moran Eye Center, University of Utah, Salt Lake City, Utah.

Address correspondence to John W. Rose, MD, Neurovirology Research Laboratory (151B), VASLCHCS, 500 Foothill Drive, Salt Lake City, UT 84148; E-mail: jrose@genetics.utah.edu

Supported in part by an unrestricted grant from Research to Prevent Blindness, Inc., New York, NY, to the Department of Ophthalmology & Visual Sciences, University of Utah (JEAW).

Optic neuritis (ON) is characterized by inflammation and demyelination of the optic nerve. Clinical symptoms include a loss of visual acuity, scotomas, periocular pain, and Uhthoff phenomenon. ON is often the first clinical manifestation of multiple sclerosis (MS) in 15%-20% of individuals ultimately diagnosed with clinically definite MS (CDMS). ON eventually occurs in 50%-75% of patients with MS (1,2).

Previous studies of ON have focused on predicting the likelihood of development of CDMS after a first demyelinating event such as ON. These studies demonstrate that the risk of development of CDMS after ON increases if the patient has an abnormal MRI scan showing at least one T2 lesion that also enhances on T1 imaging after gadolinium administration (3,4). Additional studies have shown that an increase in anti-myelin antibodies to myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG) in the cerebrospinal fluid of patients with clinically isolated syndromes (CIS) may also be used to predict the likelihood of development of MS (5-7). Prior pathologic investigations on ON have described the inflammation and demyelination in the optic nerves, but few studies have detailed the immunohistochemistry of ON.

The mechanisms leading to the death and damage of oligodendrocytes and the loss of myelin from immune and inflammatory-mediated responses is not yet fully understood. However, several factors have been implicated in this process. One possible mediator is nitric oxide (NO), which is synthesized by the inducible form of the enzyme nitric oxide synthase (iNOS) (8-10). iNOS has been found in multiple cell types, including reactive astrocytes and inflammatory monocytes/macrophages (11-13). NO can combine with superoxide to generate peroxynitrite (ONOO-), a molecule capable of damaging cells, membranes, and DNA (14). Peroxynitrite-mediated damage is indicated by the presence of nitrotyrosine (15,16). COX-2 has also been implicated in the pathogenesis of MS and has been found closely associated with iNOS activity in MS lesions (17). Myelin degradation products, MBP fragments, and lipids have also shown a close association with iNOS (12). To study how the pathology of ON may parallel that seen in MS lesions, we have used immunofluorescent confocal analysis to examine whether similar pathologic markers are present in the optic nerves of a patient with CIS recovering from an episode of ON.

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METHODS

Tissue

For this study, normal optic nerves were obtained from the Utah Lion's Eye Bank at the Moran Eye Center, University of Utah. ON-affected optic nerves were obtained from a patient with CIS treated at the University of Utah who died of causes not related to the disease (see patient history). Research using these tissues was performed in compliance with Department of Health and Human Services federal regulation 45CFR46. Pathologic tissues were formalin-fixed and paraffin-embedded. Normal optic nerves were paraformaldehyde-fixed and paraffin-embedded. Normal optic nerves were dissected in both longitudinal and cross sections. ON nerve tissue was fixed only in cross section. Tissues were sliced in 20 μm thick sections and mounted on glass slides. Tissue obtained from the patient with CIS included the clinically affected left and clinically unaffected right optic nerves.

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Patient History

The patient presented with left periocular pain and loss of vision in February 1997. Visual acuity was 20/15 in the right eye and 20/400 in the left eye. He had a l.2 log unit left relative afferent pupillary defect (APD) with impaired color vision (0/13 Ishihara plates). There was no uveitis. Mild optic nerve swelling was observed in the left eye. Humphrey visual fields showed a large central scotoma in the left eye and was normal in the right eye. MRI performed at another facility revealed a high T2 signal in the anterior portion of the left optic nerve. Contrast enhancement of the left optic nerve on T1 images was suspected, but interpretation was limited by technical factors. No imaging abnormality was detected in the right optic nerve. In addition, the T2-weighted MRI revealed five high signal cerebral lesions, including one periventricular ovoid lesion. The patient was initially treated with intravenous methylprednisolone, with recovery of visual acuity from 20/400 to 20/20 within one month. The central scotoma and APD resolved completely, but left optic disc pallor remained. The patient was then placed on treatment with interferon beta-1a. In subsequent MRI scans, the optic nerves did not enhance and were normal in appearance on T2-weighted and T1-weighted scans. The patient died of accidental causes ten months later, and the optic nerves were obtained at autopsy.

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Immunofluorescent Assay

Immunofluorescent assays were performed to identify the presence of various inflammatory signals and demyelination in both normal and MS optic nerves (Table 1). Primary antibodies used included goat anti-COX 2 (Alpha Diagnostic International, San Antonio, TX), rabbit anti-myelin basic protein (Accurate Chemical and Scientific Corporation, Westbury, NY), goat anti-myelin oligodendrocyte glycoprotein (Santa Cruz Biotechnology Inc., Santa Cruz, CA), mouse antibody to human cyclic nucleotide phosphodiesterase (CNPase) (Sigma-Aldrich Corp., St. Louis, MO), mouse anti-MBP fragment, mouse anti-CD64, mouse anti-CD14 (Research Diagnostics Inc., Benecia, CA), rabbit anti-nitrotyrosine, rabbit anti-inducible nitric oxide synthase, and rat anti-glial fibrillary acidic protein (GFAP) (Calbiochem, San Diego, CA). Primary antibodies were diluted in ranges from 1:40 to 1:1000, according to manufacturer specifications. Secondary fluorochrome antibodies were Cy5-conjugated anti-mouse, anti-rat, and anti-goat, FITC-conjugated anti-rabbit and anti-mouse (Jackson Immunoresearch Laboratories, West Grove, PA). Secondary antibodies were used in concentrations ranging from 1:800 to 1:2000. The fluorochrome antibodies were cross-adsorbed to immunoglobulins from other species to ensure specificity. Phosphate buffered saline (PBS) with 0.02% Tween-20 was used in all dilutions. Tissues were permeabilized with PBS with 0.2% Triton X-100 and blocked with Image-iT Fx signal enhancer solution (Molecular Probes, Eugene, OR). The combined primary antibodies were added to each section and incubated overnight in a humidified chamber at 4°C. After washing in PBS, conjugated secondary antibodies were then applied for one hour at room temperature. Propidium iodide (PI) (15 μM) was added for an additional hour. Sections were mounted with ProLong anti-fade mounting medium (Molecular Probes, Eugene, OR.)

TABLE 1

TABLE 1

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Microscopy

Personal Confocal Microscopy PCM-2000 (Nikon, Melville, NY) with Argon, green and red HeNe lasers was used to acquire the image. The fluorochromes were resolved into three different image channels. The FITC label was detected with the Argon laser at 488 nm, Cy5 with the red HeNe laser at 633 nm, and PI with the green HeNe laser at 605/32 nm. Tissues were individually scanned for each respective fluorochrome. Simple Personal Confocal Image Program (PCI, Compix, Cranberry Township, PA), a multifocus (z-focus) program, was used to create a stereopsis image. The three separate images were then merged to create the final triple-colored image. The PI was converted to blue during the merging process, allowing for comprehensive visualization of labeling throughout the tissue. Images were stored as digital files, and final figures were created with the use of Adobe Photoshop 7.0.

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RESULTS

Immunohistochemistry of Normal Optic Nerves

Antibodies that recognize normal myelin and cellular antigens including anti-CNPase specific for oligodendrocytes, anti-MBP specific for myelin basic protein, and anti-GFAP specific for astrocytes were used to demonstrate the structure of the nerves. Fluorescent labeling of the normal optic nerves with anti-MBP showed complete myelination extending throughout the nerve (Fig. 1A). CNPase expression was similarly abundant, showing no apparent signs of damage or death of oligodendrocytes. In the normal optic nerve, iNOS and nitrotyrosine were infrequently seen and usually not observed (Fig. 1, B and C, respectively). Subsequent probing for MBP fragments, CD64+ and CD14+ macrophages/microglia, and COX-2 revealed no detectable labeling of the tissue (data not shown). The results from labeling normal and pathologic optic nerves are summarized in Table 2.

FIG. 1

FIG. 1

TABLE 2

TABLE 2

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Expression of Myelin Basic Protein, Cyclic Nucleotide Phosphodiesterase, and Glial Fibrillary Acidic Protein in the Pathologic Optic Nerves

Compared with the normal optic nerves, there was a sharp contrast in patterns of MBP, GFAP, and CNPase expression in the pathologic optic nerves. The clinically unaffected optic nerve showed only one small area of demyelination (Fig. 2A). However, there was a marked reduction in both CNPase and MBP expression in the affected nerve. In both normal and pathologic tissues, the pattern of CNPase distribution mirrored that of MBP (Fig. 2, C and D). In the clinically affected optic nerve, the majority of myelin and oligodendrocytes was concentrated in the center of the nerve, whereas demyelinating activity occurred mostly on the periphery of the nerve. GFAP was inversely expressed in the affected pathologic optic nerve; intense labeling of reactive astrocytes was present in demyelinated areas compared with more uniform and diminished expression in the myelinated areas (Fig. 2E).

FIG. 2

FIG. 2

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Expression of Myelin Oligodendrocyte Glycoprotein

After establishing that the pathologic optic nerve showed a decrease in myelin expression, we searched for other characteristics associated with inflammation and demyelination in ON. The anti-MOG antibody used in this investigation binds to a region near the carboxy terminus of MOG. This antigen is generally detected only when damaged myelin is present. The MOG c-terminal antigen was seen only in the affected optic nerve and was restricted to isolated cells located toward the interior of nerve fascicles (Fig. 2B).

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Active Demyelination

We then searched for evidence of recent demyelination by assessing whether MBP fragments were present in the pathologic optic nerves. Detection of MBP fragments would be indicative of an ongoing demyelinating process. MBP fragments detected by a monoclonal antibody specific for an epitope found on recently digested MBP were present in both the unaffected (Fig. 3A, see *) and affected optic nerves (Fig. 3B, see arrow); however, MBP fragments were more prominent on the affected optic nerve. MBP fragments were predominantly found in areas exhibiting extensive demyelination, generally near the periphery of areas still rich in myelin.

FIG. 3

FIG. 3

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Inducible Nitric Oxide Synthase Expression in Reactive Astrocytes and Macrophages/Microglia

We assessed whether iNOS, a major inflammatory enzyme associated with demyelinating lesions, was expressed in ON (11-13). Cellular expression was observed predominantly in the affected nerve, although small amounts were seen in the unaffected nerve. The majority of iNOS was associated with inflammatory infiltrates in the nerve septa toward the periphery of the nerve as well as in the adventitia of the central retinal arteries. Most of the iNOS was expressed in or near macrophage and microglia subtypes co-labeled with either anti-CD14 (Fig. 4A), a specific marker for a type of macrophages/microglia that phagocytose apoptotic cells, or anti-CD64, a specific marker for the high-affinity Fcγ receptor (18,19). CD64+ macrophages/microglia localized to cell clusters in the septa in iNOS positive regions (Fig. 4B, see arrow). In these areas, CD64+ macrophages/microglia sometimes expressed iNOS.

FIG. 4

FIG. 4

CD14+ macrophages/microglia were found adjacent to iNOS-positive cells (Fig. 4A, see arrow). CD14+ cells were most numerous in the septa on the perimeter of the myelinated area of the affected optic nerve. They were also observed in the tissue surrounding the central retinal artery. Little or no expression of CD14+ cells was seen toward the interior of the nerve in the myelinated regions. In regions of overlap, iNOS and CD14 were generally co-expressed within the same cells. Neither CD14+ nor CD64+ cells were detected in the unaffected nerve (not shown).

In addition to expression of iNOS in inflammatory infiltrates, occasional isolated reactive astrocytes in the affected optic nerve also showed considerable expression iNOS (Fig. 4C, see arrow). Nearby non-GFAP-positive cells also expressed iNOS (Fig. 4C, see *). These iNOS-positive reactive astrocytes were found near the outer edge of the myelinated areas in both the septa and in parenchymal tissue. Additionally, a few iNOS-positive astrocytes and other cells were also detected toward the interior of the nerve in the myelinated area.

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Expression of Nitrotyrosine and COX-2

After establishing the presence of iNOS in the diseased nerves, we determined whether nitrotyrosine and COX-2 were present and associated with iNOS, as seen in MS plaques. The degree of peroxynitrite-mediated damage was assessed by determining the formation of nitrotyrosine. Nitrotyrosine was present among inflammatory infiltrates in the adventitia of the central retinal vessels and in the septa between fascicles (Fig. 4D). Nitrotyrosine was largely not detected in GFAP-positive cells (Fig. 4D, see *), nor was it found in the unaffected nerve.

COX-2 was rarely detected in either the unaffected (Fig. 4E) or affected (Fig. 4F) pathologic optic nerves. However, when present, COX-2 was located in large inflammatory cells either on the periphery of the nerves, where most of the myelin damage occurred, or in the nerve septa.

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DISCUSSION

In this study, we have demonstrated antigen detection by MBP, GFAP, and CNPase antibodies specific for normal cellular and myelin components in the control optic nerves. Positive labeling of these three antibodies provided a structural image of the nerve consistent with normal optic nerve histology. Additional probing for pathologic markers associated with MS lesions (anti-iNOS, anti-COX-2, anti-nitrotyrosine, and anti-MBP fragment) was negative, demonstrating that normal nerves lack these indicators.

Histological analysis of the affected optic nerve from a patient with CIS recovering from ON showed a pathology similar to that of chronic active MS plaques, including localized loss of myelin proteins, evidence of myelin breakdown, and presence of iNOS and nitrotyrosine (12). The formalin- and paraformaldehyde-fixed tissues used in this study allowed for better preservation of tissue structure and reduced non-specific staining of artifacts and lipids common to frozen tissues. Our ability to detect antigens in these tissues illustrates the potential to analyze archival tissues for future investigations of ON pathology.

iNOS was detected in the affected and unaffected optic nerves of the patient with CIS but not in the normal control nerves. iNOS has been found in astrocytes as well as activated macrophages/microglia in both acute and chronic active MS lesions (11-13). Although iNOS immunoreactivity in the optic nerves was not as intense as had been previously observed in MS lesions, it was prominent in or adjacent to CD14+ and CD64+ macrophages/microglia. These inflammatory infiltrates were most frequently observed on the edges of the nerve exhibiting active demyelination, implicating iNOS in the inflammation and demyelination in ON.

Our patient was in clinical remission after methylprednisolone treatment and had undergone interferon therapy for ten months at the time of his accidental death. Treatment with interferon beta-1a after a first episode of ON has been shown to significantly decrease the risk of development of CDMS and has led to slower disease progression among patients who have CDMS (20-24). Clinical remission as well as ongoing therapy may have contributed to less intense iNOS staining in the ON compared with the chronic active plaques from MS patients that we previously studied (12).

Additionally, some isolated reactive astrocytes in damaged areas expressed iNOS. This observation parallels our previous studies on MS plaques (12). However, a few scattered iNOS-positive astrocytes were also detected in areas showing no apparent active or past demyelination. It has been suggested that NO derived from these distal astrocytes, which are observed in mice with experimental allergic encephalomyelitis (EAE) and in MS brain sections, may actually serve a protective role in hindering the spread of inflammation (13,25). This idea is supported by the results obtained in some experiments with EAE animals, in which NO inhibition actually aggravated the symptoms of the disease (26-29). Our results suggest that iNOS expressed by astrocytes may have opposing effects in ON, although the net effect of iNOS is probably deleterious.

Significantly less COX-2 was observed in the optic nerves than had been previously seen in chronic active MS lesions (17). In addition to the goat anti-COX-2 used to obtain the final results, the tissues were tested with multiple antibodies to COX-2, including mouse and rabbit anti-COX-2, which demonstrated limited COX-2 presence, possibly suggesting that distinct pathologic features of ON may not induce increased COX-2 expression. Alternatively, in MS brain tissue, COX-2 was mostly found in CD64-positive macrophages/microglia on the active edges of MS plaques (17). Although CD64-positive cells were observed in ON, they were not detected as frequently as in MS lesions. Since activation of CD64, the high-affinity Fcγ1 receptor, is linked to COX-2 expression in monocytic cells, a reduced number of activated CD64-positive cells may also contribute to the rarity of COX-2 detection (30).

Additionally, interferon therapy could have suppressed COX-2 expression in our patient. However, when detected, COX-2 was found in inflammatory infiltrates in areas commonly expressing iNOS. Like iNOS and MBP fragments, COX-2 was occasionally detected in the unaffected nerve. However, it was entirely undetected in the normal control nerves, indicating a low level of pathology in the unaffected eye.

Peroxynitrite has been strongly implicated as a mediator of myelin and tissue damage very early in the progression of demyelinating disease (31). This role is supported by evidence that uric acid and other inhibitors of peroxynitrite formation have led to improvements in clinical symptoms in experimental models of MS (32,33). We identified peroxynitrite-mediated damage by the presence of nitrotyrosine, a stable marker. Nitrotyrosine was commonly found among inflammatory infiltrates in iNOS-positive regions only in the affected optic nerve, which is consistent with previous observations of co-localization of nitrotyrosine and iNOS (11-13). Although nitrotyrosine was one of the more prevalent markers detected, immunoreactivity for nitrotyrosine in the affected optic nerve was not as intense as has been observed in acute or chronic active MS lesions. However, because the patient was in a period of recovery and treatment, our findings parallel previous observations indicating that nitrotyrosine is less frequently detected in the remitting disease phase in the mouse model of EAE, even when inflammation and some clinical symptoms are still present (31).

Although the patient was undergoing treatment of ON and showed clinical recovery, evidence of ongoing demyelinating and inflammatory activity continued in the affected eye. MBP fragments, which are only present for approximately seven days after phagocytosis of myelin, were frequently observed in the affected nerve and occasionally in the unaffected nerve, signifying active demyelination (34). MBP fragments were also observed in chronic active MS lesions (12). The detection of MOG in the affected nerve, as well as the reduced expression of MBP and oligodendrocytes in both pathologic optic nerves, demonstrates extensive myelin and oligodendrocyte damage. The optic nerves from patients with neuromyelitis optica have shown similar signs of demyelination but without evidence of active demyelination and with some signs of partial remyelination (35). In these examples, there was intense labeling of GFAP in astrocytes in demyelinated areas, consistent with reactive astrogliosis in response to central nervous system and optic nerve injury (36-38). Astrogliosis in these areas may lead to astrocytic scar formation, preventing remyelination of axons (36,39).

We have demonstrated that the affected optic nerve from a patient with CIS has a pathology similar to MS lesions. The unaffected and affected nerves showed a decrease in oligodendrocytes, mild to extensive demyelination, astrogliosis, and ongoing demyelination. Pathologic indicators were less prevalent in the unaffected nerve, although it did show some signs of disease, including occasional expression of iNOS and COX-2. Nitrotyrosine and the macrophage markers CD14 and CD64, as well as iNOS and COX-2, were also detected in the affected optic nerve. Overall, these findings indicate the need for better imaging techniques to detect disease progression after a first demyelinating event and suggest possible treatments for ON. Diffusion tensor imaging, magnetization transfer imaging, and proton MR spectroscopy may be more sensitive than conventional MRI and may allow pathologic events seen in the postmortem optic nerve including myelin and axonal loss, gliosis, and inflammatory activity to be noted, opening possibilities for novel therapy.

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Acknowledgments

We greatly appreciate the donation of normal optic nerve samples from the Utah Lions Eye Bank. MS samples were provided by the Rocky Mountain Multiple Sclerosis Center and the UCLA Brain Bank. This work was supported by grants from the National Multiple Sclerosis Society (to JWR and NGC), VA Merit Review (JWR and NGC), and the Cumming Foundation.

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