Immune mapping of the peripheral part of the visual analyzer and optic nerve

Aim. To perform immune mapping of the peripheral part of visual analyzer and optic nerve in order to identify potential antigenic targets of autoimmune attack.

Methods. Eyes enucleated for terminal painful glaucoma (n = 30) were studied. Immunohistochemistry (IHC) was performed on paraffin-embedded sections of isolated retina and optic nerve using a broad panel of antibodies, i.e., monoclonal murine anti-MBP (myelin basic protein) antibodies, polyclonal rabbit anti-alpha fodrin antibodies, monoclonal murine anti-NSE2 (neuron-specific enolase) antibodies, monoclonal murine anti-GFAP (glial fibrillary acidic protein), and polyclonal rabbit anti-S100 antibodies. IHC reaction was visualized using Mouse and Rabbit Specific HRP / AEC Detection IHC Kit. IHC reaction without primary antibodies included was a negative control. IHC reaction was considered as follows: negative — no specific cellular staining or less than 10 % of cells are stained; mild — 10‑30 % of cells are stained (+); moderate — 30‑75 % of cells are stained (++); marked — more than 75 % of cells are stained (+++); overexpression — 100 % of cells intensively express markers. Additionally, staining intensity was considered as mild (+1), moderate (+2), strong (+3) and intense (+4).

Results. Immune mapping with a broad panel of monoclonal antibodies identified ocular structures which were stained with IHC markers. Retina was stained with almost all markers of neural differentiation (i.e., antibodies against NSE, GFAP, S100, and α-fodrin) excepting anti-MBP autoantibodies. IHC reaction intensity in retinal layers and structures varied and depended on markers. Moderate (2+) staining with antibodies against MBP, NSE, GFAP, and S100 and marked (3+) staining with antibodies against alpha-fodrin was detected in the cytoplasm of optic nerve glia.

Conclusion. Complete labelling of retina structures was performed. As a result, IHC profiles of retinal neurons, optic nerve axons, interneurons, and microglial cells were described. IHC profiles of retinal layers and optic nerve are useful markers which can be applied in serological diagnostics of various ocular disorders.

1. Chekhonin V. P., Lebedev S. V., Blinov D. V., Turina O. I., Semenova A. V., Lazarenko I. P., Petrov S. V., Ryabukhin I. L., Rogatkin S. O., Volodin N. N. [Pathogenetic role of the permeability disorder of the blood-brain barrier to neurospecific proteins with perinatal hypoxic-ischemic lesions of the central nervous system in newborns]. Patogeneticheskaya rol’ narusheniy pronitsaemosti gematoentsefalicheskogo bar’era dlya neyrospetsificheskikh belkov pri perinatal’nykh gipoksicheskkh narusheniyakh tsentral’noy nervnoy sistemy u novorozhdennykh. [Questions of gynecology, obstetrics and perinatology]. Voprosy ginekologii, akusherstva i perinatologii 2004; 3: 50‑61 (in Russ.).

2. Ilyenko L. I., Zubareva E. A., Kholodova I. N. [Modern approaches to diagnostics and treatment of hypoxic-ischemic CNS lesions in term infants of the first year of life]. Sovremennye podkhody k diagnostike i lecheniyu gipoksicheskikh-ishemicheskikh povrezhdeniy TsNS u detey pervogo goda zhizni. [Pediatrics]. Pediatriya. 2003; 2: 87‑92 (in Russ.).

3. Goncharova O. V., Bakanov M. I., Mutalov A. G., Greshilov A. A., Dzhumagaziev A. A., Yusupova E. S. [Modern biochemical criteria of diagnosis of perinatal hypoxic CNS lesions in infants]. Sovremennye biokhimicheskie kriterii diagnostiki perinatal’nykh gipoksicheskikh narusheniy TsNS. [Russian pediatric journal]. Rossiiskii pediatricheskii zhurnal 2007; 4: 13‑18 (in Russ.).

4. Anand N., Stead LG. Neuron-specific enolase as a marker for acute ischemic stroke: a systematic review. Cerebrovascular Disease. 2005; 20: 213‑219.

5. Zovein A., Flowers-Ziegler J., Thamotharan S., Shin D., Sankar R., Nguyen K., Gambhir S., Devaskar S. U. Postnatal hypoxic-ischemia brain injury alters mechanisms mediating neuronal glucose transport. Am J Physiol Regul Integr Comp Physiol. 2004; 286: 273‑282.

6. Yu-Wai-Man P., Griffiths P. G., Chinnery P. F. Mitochondrial optic neuropathies — disease mechanisms and therapeutic strategies. Prog Retin Eye Res. 2011; 30 (2): 81‑114.

7. Tezel G., Yang X., Luo C., Kain A., Powell D., Kuehn M. H., Kaplan H. J. Oxidative stress and the regulation of complement activation in human glaucoma. Invest Ophthalmol Vis Sci. 2010; 51 (10): 5071‑5082.

8. Bambrick L., Kristian T., Fiskum G., Bambrick L. Astrocyte mitochondrial mechanisms of ischemic brain injury and neuroprotection. Neurochemical Research. 2004; 29: 601‑608.

9. Lukyanova L. D. [Role of bioenergy disorders in the pathogenesis of hypoxia]. Rol’ bioenergeticheskikh narusheniy v patogeneze gipoksii. [Pathological physiology and experimental therapy]. Patologicheskaya fiziologiya i eksperimental’naya terapiya. 2004; 2: 2‑11 (in Russ.).

10. Kann O, Kovаcs R. Mitochondria and neuronal activity. American Journal of Physiology — Cell Physiology. 2007; 292: 641‑657.

11. Yang J., Tezel G., Patil R. V., Romano C., Wax M. B. Serum autoantibody against glutathione S-transferase in patients with glaucoma. Invest Ophthalmol Vis Sci. 2001; 42: 1273‑1276.

12. Kremmer S., Kreuzfelder E., Klein R., Bontke N., Henneberg-Quester K. B., Steuhl K. P. Antiphosphatidylserine antibodies are elevated in normal tension glaucoma. Clin Exp Immunol. 2001; 125: 211‑215.

13. Ikeda Y., Maruyama I., Nakazawa M., Ohguro H. Clinical significance of serum antibody against neuron-specific enolase in glaucoma patients. Jpn J Ophthalmol. 2002; 46: 13‑17.

14. Maruyama I., Ohguro H., Ikeda Y. Retinal ganglion cells recognized by serum autoantibody against gamma-enolase found in glaucoma patients. Invest Ophthalmol Vis Sci. 2000; 41: 1657‑1665.

15. Tezel G, Edward DP., Wax M. B. Serum autoantibodies to optic nerve head glycosaminoglycans in patients with glaucoma. Arch Ophthalmol. 1999; 117: 917‑924.

16. Romano C., Barrett D. A., Li Z., Pestronk A., Wax M. B. Anti-rhodopsin antibodies in sera from subjects with normal-pressure glaucoma. Invest Ophthalmol Vis Sci. 1995; 36: 1968‑1975.

17. Grus F. H., Joachim S. C., Bruns K., Lackner K. J., Pfeiffer N., Wax M. B. Serum autoantibodies to alpha-fodrin are present in glaucoma patients from Germany and the United States. Invest Ophthalmol Vis Sci. 2006; 47: 968‑976.

18. Tezel G., Li L. Y., Patil R. V., Wax M. B. TNF-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes. Invest Ophthalmol Vis Sci. 2001; 42: 1787‑1794.

19. Wax M. B., Tezel G. Immunoregulation of RGC fate in glaucoma. Experimental Eye Research. 2009; 88: 825‑830.

20. Surgucheva I., McMahan B., Ahmed F., Tomarev S., Wax M. B., Surguchov A. Synucleins in glaucoma: implication of gammasynuclein in glaucomatous alterations in the optic nerve. J Neurosci Res. 2002; 68: 97‑106.

21. Tezel G., Seigel G. M., Wax M. B. Autoantibodies to small heat shock proteins in glaucoma. Invest Ophthalmol Vis Sci. 1998; 39: 2277‑2287.

22. Tezel G., Hernandez R., Wax M. B. Immunostaining of heat shock proteins in the retina and optic nerve head of normal and glaucomatous eyes. Arch Ophthalmol. 2000; 118: 511‑518.

23. Wax M. B. The case for autoimmunity in glaucoma. Experimental Eye Research. 2011; 93 (2): 187‑190.

24. Junglas B., Kuespert S., Seleem A. A. Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork. Am J Pathol. 2012; 180 (6): 2386‑2403.

25. Pascale A., Drago F., Govoni S. Protecting the retinal neurons from glaucoma: Lowering ocular pressure is not enough. Pharmacological Research. 2012; 66 (1): 19‑32.