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).
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).
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.
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.
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.
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.
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.
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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