JAMA Ophthalmology

Wax, Martin B.; Tezel, Gülgün; Edward, P. Deepak

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.8.993pmid: 9715678

ObjectiveTo study the histopathological changes of eyes from a patient with normal-pressure glaucoma whose clinical and laboratory findings were well documented.MethodsPostmortem histopathological findings in a patient with normal-pressure glaucoma who had monoclonal gammopathy and serum immunoreactivity to retinal proteins were examined in comparison with those of an age-matched control subject. Clinicopathological correlations to laboratory findings were examined.ResultsClinical and histopathological findings of the patient, including cavernous degeneration of optic nerve and characteristic optic nerve cupping, were similar to those in patients with glaucoma who had high intraocular pressure. In addition, we found immunoglobulin G and immonuglobulin A deposition in the ganglion cells, inner and outer nuclear layers of the retina, and evidence of apoptotic retinal cell death using terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling technique.ConclusionsSerum antibodies to retinal proteins and retinal immunoglobulin deposition constitute novel findings in a patient with normal-pressure glaucoma and may contribute to better understanding of the mechanisms underlying glaucomatous optic neuropathy in this disorder.GLAUCOMATOUS optic neuropathy is characterized by loss of retinal ganglion cells and their axons, excavated appearance of optic nerve head, and progressive loss of visual field sensitivity. Although clinical studies have shown the role of several risk factors in glaucomatous optic neuropathy, including high intraocular pressure (IOP),about 20% to 25% of glaucomatous optic neuropathy develops in patients with normal IOP.Despite several histopathological reports of postmortem human eyes from patients with primary open-angle glaucoma,and despite experimental studies using glaucoma models in which IOP is elevated,it is not clear whether the pathological findings of glaucomatous eyes with normal IOP are similar to those seen in glaucomatous eyes with high IOP. We herein present the clinical and postmortem histopathological findings in a patient with normal-pressure glaucoma, including evidence of immunoglobulin deposition in the retina and apoptotic retinal cell death using terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique.MATERIALS AND METHODSCLINICAL FINDINGSA 74-year-old, white woman received a diagnosis of normal-pressure glaucoma and was followed up for 5 years. The initial diagnosis was based on the presence of open iridocorneal angles, no evidence of IOP greater than 20 mm Hg without antiglaucoma treatment, progressive glaucomatous changes in visual fields and optic disc cupping, and absence of alternative causes of optic neuropathy. Alternative causes of optic neuropathy (ie, meningeal disease, infection, inflammation, ischemia, demyelinization, or compressive lesions) were excluded using neuro-ophthalmological examination with magnetic resonance imaging. She had coronary artery disease and family history of glaucoma. During the last 2 years of follow-up, she received topical β-blocker treatment in an effort to lower IOP from the middle to low teens and to retard progressive glaucomatous optic neuropathy.During the initial diurnal IOP measurements (3 times between 6 AM and 5 PM and 3 times between 5 PM and 6 AM) and during regular visits every 3 to 6 months at which measurements were obtained using applanation tonometry, IOP readings never exceeded 20 mm Hg. A visual field analyzer, 30-2 program (Humphrey Instruments, San Leandro, Calif) was used for visual field examinations. Initial visual field defects were characterized by bilateral nasal steps and dense paracentral scotoma in the left eye close to fixation. These defects progressed compared with baseline values based on the glaucoma change probability analysis. In Figure 1, the last stereoscopic optic disc photographs of the patient taken 1 year before the date of death and first and last results of visual field testing (5-year interval) are shown. The patient had bilateral, large optic disc cups (larger in left than right eye) that further enlarged during follow-up. During regular optic disc examinations, recurrent optic disc hemorrhages were observed in both eyes. The patient had also bilateral advanced parapapillary chorioretinal atrophy consisting of the α and β zones.Results of indocyanin green angiography and early stages of fluorescein angiography showed nonperfusion areas of the choriocapillaris in the parapapillary region. Fundus fluorescein angiography demonstrated a window defect corresponding to zone α of parapapillary atrophy, in which there are pigmentary and structural changes of the retinal pigment epithelium. One of the nonperfusion areas that was located adjacent to the inferotemporal optic disc border corresponded to the more advanced zone of parapapillary atrophy (zone β). In the later stages of the fluorescein angiography, fluorescein diffusion was seen in the previously nonperfused area of the more advanced zone of parapapillary atrophy extending to optic disc (Figure 2).Figure 1. Clinical findings of glaucomatous optic neuropathy in a patient with normal-pressure glaucoma. Stereoscopic optic disc photographs of the right (A and B) and left (C and D) eyes; initial 30° visual field findings of the right (E) and left (G) eyes; and progression of visual field defects during 5-year follow-up of the right (F) and left (H) eyes are seen. Fixation losses, false positives, false negatives, pupil size, and fluctuations are as follows: E, 5/28, 0/17, 2/17, 5.5 mm, and 1.62 dB, respectively; F, 9/33, 0/20, 3/19, 5.0 mm, and 2.13 dB, respectively; G, 0/27, 0/18, 2/16, 5.0 mm, and 3.04 dB, respectively; and H, 8/24, 0/19, 1/14, 5.0 mm, and 1.68 dB, respectively.Figure 2. Parapapillary atrophy in a patient with normal-pressure glaucoma. A and B, Indocyanin green angiography of the right eye showing the peripapapillary ischemic areas. Fundus fluorescein angiography of the same eye shows a window defect in zone α of parapapillary atrophy and a filling defect in zone β that is adjacent to inferotemporal optic disc border (C). D, Fluorescein diffused into zone β of parapapillary atrophy and optic disc in the late stages. E, Histopathological appearance of the parapapillary area with severe damage of retinal pigment epithelium and adjacent photoreceptors and choriocapillaris (arrow) (hematoxylin-eosin; original magnification ×100).Laboratory studies revealed the presence of abnormal humoral autoimmunity, as demonstrated by an IgA-λ paraproteinemia in addition to anticardiolipin antibodies. To study the possible presence of antiretinal antibodies in serum, we performed immunoblotting using retinal substrates as previously described.Results of Western blot analysis and enzyme-linked immunosorbent assay showed the presence and high titers of circulating antibodies against several retinal proteins, including rhodopsin and heat shock proteins (hsp). Antibodies to retinal hsp included those directed to human and bacterial hsp60, hsp27, and αA-crystallin (Figure 3).Figure 3. Results of Western blot analysis of a patient with normal-pressure glaucoma. Each lane contains patient serum (dilution, 1:1000) against bovine retinal supernatant (BRS), bovine retinal membrane (BRM) (15 µg/lane), purified αA- and αB-crystallin, heat shock protein (hsp)27, bacterial (B) and human (H) hsp60, and rhodopsin (3 µg/lane), as labeled. Secondary antibody (goat anti–human IgG) dilution is 1:2000.METHODSPostmortem eyes of our patient with normal-pressure glaucoma and, for comparisons, of a 72-year-old, white female donor with no history of ocular or neurological disease were obtained. All eyes were enucleated within 4 hours of death and processed within 12 hours. All eyes were fixed in 10% formalin, processed, and embedded in paraffin. Eyes were sectioned in the coronal plane from the distal end of the optic nerve to the equator. Serial sections, 4 µm thick, were prepared. Some of the sections were stained with hematoxylin-eosin and examined under a light microscope. Some of the sections were used to identify the apoptotic cells or for immunohistochemical analysis.Identification of the apoptotic cells was performed using TUNEL technique, an in situ end-labeling technique for apoptotic cells that couples 2 major approaches: morphological examination and DNA fragmentation.It is a sensitive and specific technique that allows precise and rapid identification and quantification of the cell population involved in apoptotic death. Using an in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany), deparaffinized sections were incubated with a mixture of fluorescein-labeled nucleotides and terminal deoxynucleotidyl transferase (TdT) from calf thymus for 1 hour. The TdT catalyzes the polymerization of labeled nucleotides to free 3‘-hydroxyl terminals of DNA fragments. A fluorescence microscope (Olympus, Tokyo, Japan) was used to visualize the apoptotic cells at the end of this period. Sections incubated with fluorescein-labeled nucleotide mixture without TdT served as a negative control. Sections previously treated with DNAse I (1 mg/mL) to induce breaks in the DNA strands served as a positive control.The sections were also examined using Alcian blue to identify mucopolysaccharides, Masson trichrome to outline the areas of gliosis, Luxol fast blue to delineate the myelin sheaths of the optic nerve, phosphotungstic acid–hematoxylin to identify fibrin deposits in blood vessels, and Congo red to examine perivascular amyloid deposits.We also performed immunohistochemical analysis to investigate the immunoglobulin deposition in the retina and optic nerve using antibodies against human IgG and IgA. For immunostaining, deparaffinized sections were incubated with proteinase K (20 µg/mL) for 20 minutes at room temperature. The samples were then treated with 3% bovine serum albumin at 37oC for 30 minutes to block nonspecific binding sites. After several washes, they were incubated at 37oC for 1 hour with fluorescein-conjugated monoclonal antibodies against human IgG or IgA (dilution, 1:16) (Sigma Chemical Company, St Louis, Mo). At the end of the incubation time, the sections were washed several times and examined using the fluorescence microscope. Age-matched healthy control eyes and antibodies against mouse immunoglobulins were used as negative controls.RESULTSThe histopathological changes of the optic nerve head and retina were more severe in the left eye of the patient with normal-pressure glaucoma than those seen in the right eye that were correlated with the clinical appearance of the optic discs. Both optic nerve heads exhibited remarkable cupping characterized by disarrangement, compression, and backward bowing of lamina cribrosa. The number of axons passing through the nerve head were decreased, and there were compact bundles of extracellular matrix. However, in the normal eyes, the lamina cribrosa displayed a regular horizontal arrangement. In both eyes with normal-pressure glaucoma, there were empty spaces in the optic nerve suggestive of Schnabel cavernous degeneration that stained with Alcian blue (Figure 4). In the parapapillary area, the retinal pigment epithelium, choriocapillaris, and photoreceptors were atrophic (Figure 2).Figure 4. Optic nerve head in normal-pressure glaucoma. A, Optic disc cupping, posterior bowing of the lamina cribrosa (arrowheads), and Schnabel cavernous degeneration in the prelaminar (small arrows) and postlaminar (large arrows) areas of the right optic nerve head (Masson trichrome; original magnification ×25). B, Extensive Schnabel cavernous degeneration in the postlaminar area of the left optic nerve (S). Posterior bowing of the lamina cribrosa (arrowheads) is seen (hematoxylin-eosin; original magnification ×10). C, Cavernous areas in the left optic nerve (Alcian blue; original magnification ×25).Examination of serial hematoxylin-eosin–stained sections of the retina from the patient with normal-pressure glaucoma revealed a significant loss of retinal ganglion cells and their axons compared with that of the control eyes. In addition, the thickness of the inner nuclear layer (3-4 cell layers) appeared to be diminished compared with that of control eyes (8-9 cell layers) (Figure 5, A). Examination of the retinal sections revealed occasional retinal cells with nuclear or cytoplasmic condensation, pyknotic nuclei, or apoptotic bodies. The TUNEL technique showed brightly fluorescein-stained nuclei representing fragmented DNA and nuclear chromatin condensation (Figure 5, B and C). The TUNEL-positive cells were found mostly in the ganglion cell layer of the retina; however, a few cells were found in the inner and outer nuclear layers. The TUNEL-positive ganglion cells were sparsely distributed, corresponding to 0.1% of the total number of ganglion cells in each section. The TUNEL-positive cells were virtually absent in the age-matched control eyes.Figure 5. Evidence of apoptosis in normal-pressure glaucoma. A, Loss of ganglion cells and their axons in the midperipheral retina of the patient with normal-pressure glaucoma. Arrowhead shows a ganglion cell with pyknotic nucleus and intact cytoplasm; arrows show a ganglion cell with condensed nucleus and shrunken cytoplasm (hematoxylin-eosin; original magnification ×100). B, Cells positively labeled using terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique (arrowheads) in the ganglion cells and inner nuclear layers of the retina sectioned from the eyes with normal-pressure glaucoma (original magnification ×40). C, Cells positively labeled using TUNEL technique (arrow) in the ganglion cells layer of the retina (original magnification ×100).The inner retina and optic nerve head demonstrated a decrease in the number and volume of capillaries compared with the control eyes, especially in some areas that exhibited significant loss of ganglion cells and their axons. However, patent capillaries were still present, and normal red blood cells were seen in those vessels. On the right posterior laminar area of the optic nerve, scattered structures resembling the vascular lumen without endothelial cells (Figure 6) and adjacent areas of axonal swelling and microglial infiltration were noted. However, no amyloid or fibrin deposition was demonstrated along vessel walls, and the general loss of capillaries appeared proportionate to the loss of neural tissue in the retina and optic nerve head. The filling defects seen during angiography appeared to correlate with these findings.Figure 6. The appearance of vasculature in the patient with normal-pressure glaucoma. A, Diminished but intact retinal vasculature (hematoxylin-eosin; original magnification ×100). B, In the postlaminar area of the optic nerve, focal axonal swelling and adjacent, partly occluded vessel devoid of endothelial cells are seen (hematoxylin-eosin; original magnification ×100).Immunostaining was observed with anti–human IgG and IgA antibodies in the retina and optic nerve head of the eyes with normal-pressure glaucoma, which demonstrates the presence of serum immunoglobulins in these tissues. Immunostaining was noticeable in ganglion cells and in the inner and outer nuclear layers of the retina. Control antibody did not stain these tissues, and the control eyes did not exhibit immunostaining with any of the antibodies used (Figure 7).Figure 7. Immunostaining of the retina from the patient with normal-pressure glaucoma with fluorescein-conjugated monoclonal antibodies to human immunoglobulins. Immunostaining with antibodies to human IgA (A) or IgG (C) is visible in the ganglion cells (g) and outer (on) and inner nuclear (in) layers of the retina sectioned from the patient. Fluorescein staining is not seen in the age-matched control eyes with antibodies to human IgA (B) or IgG (D) except normal autofluorescence.COMMENTWe observed a disarrangement of the lamina cribrosa in our patient with normal IOP and glaucoma, similar to that described in patients with primary open-angle glaucoma.Similarly to our findings, Iwatahas reported histopathological changes of the optic nerve head in normal-pressure glaucoma that were characterized by the disarrangement and backward bowing of the lamina cribrosa and loss of nerve fibers without evidence of vascular abnormality. Furthermore, histopathological optic nerve head changes correlated with the clinical appearance of the optic nerve head that is comparable in glaucoma with high and with normal IOP.It seems, then, that there are complex mechanisms related to individual anatomical, vascular, or other differences in the susceptibility to damage that result in similar changes of optic nerve head in glaucoma with high and normal IOP.Several previous studies suggest the importance of the structural support of the lamina cribrosa and its role in optic nerve fiber damage resulting from distortion of the cribriform plates by elevated IOP. In postmortem glaucomatous eyes with high IOP and in experimental glaucoma models, there are dramatic changes in the lamina cribrosa, eg, disarrangement and remodeling.However, it is not known whether these changes play a causal role in neural damage or whether they occur as a result of the rearrange