Glaucoma, Inflammation and Oxidative Stress: An Attempt to Unify Recent News

This article is my attempt to unify some of what has been proposed about the mechanism underlying retinal ganglion cell (RGC) loss in glaucoma. I am going to quickly dive right into the biochemical details of the process as I understand it, and then I will conclude this article with supplementary material meant to familiarize readers with many of the technical terms. Some of you may want to read this supplementary material first if you are not familar with things like glial cells. (And please ask for further clarifications in the comments to this article.)

Glaucoma affects more than seventy million people worldwide. Glaucoma is a progressive optic neuropathy characterized by a specific pattern of optic nerve head and visual field damage which result from loss of retinal ganglion cells (RGCs) by apoptosis [17]. Growing evidence obtained from clinical and experimental studies over the past decade strongly suggests the involvement of the immune system in glaucoma [9]. There are several mechanisms by which inflammatory immune system responses have been shown to be directly responsible for retinal ganglion cell apoptosis. All the proposed immune system responses involve reactive oxygen species (ROS). Reactive oxygen species regulate the immune system response in general and are directly involved in the specific responses that lead to retinal ganglion cell apoptosis [9]. ROS is a term that includes oxygen free radicals as well as other atomic and molecular oxygen species that can participate in free radical chain reactions. A free radical is an atom or molecule with at least one unpaired electron. In this article I will group reactive oxygen species under the term free radicals for simplicity. The biological damage caused by free radicals is termed oxidative stress. Inflammation and oxidative stress are two topics at the forefront of glaucoma research and I in this article I want to consolidate some of what I understand about those important topics into one unified mechanism of optic nerve damage in glaucoma. In that sense you could call this article a mashup.

At present it is unclear which of either free radical damage or inflammatory immune reactions comes earlier in the sequence of events that ultimately lead to retinal ganglion cell apoptosis. This is a bit like the chicken-and-egg problem. However, elevation of free radical reactions and inflammatory immune responses go hand-in-hand and the process tends to feed on itself in a cycle that can be hard to break. (In another article, I will discuss some factors that may precede both inflammation and oxidative stress.) In this article, I will outline a linear sequence of events for simplicity so I ask you to keep in mind that the in vivo situation is not linear. Because free radical reactions are the more fundamental process, I will start my proposed sequence of events with them.

Outside of glaucoma research, it has been shown that free radicals can directly induce neuronal death by a protease and phosphatase-gated mechanism distinct from apoptosis [13]. Although we can speculate that free radicals can also directly induce retinal ganglion cell death by a non-apoptotic mechanism, most of the current glaucoma research is focusing on apoptotic mechanisms of retinal ganglion cell death. Here as well, we find that free radicals can induce retinal ganglion cell death and I will outline the proposed apoptotic mechanism below.

As we know, not all forms of glaucoma involve ocular hypertension. I propose that both primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) ultimately involve free radical-mediated mechanisms of optic nerve loss.

In primary open angle glaucoma patients, free radicals may damage the trabecular meshwork (TM). This has been shown to lead to elevated intraocular pressure [12]. Incidently, physical stresses and the resulting damage - which is not signficantly different from the effects of ocular hypertension -- have been shown to generate free radicals in biological systems. In my view, elevated intraocular pressure may start with free radical damage, but ocular hypertension then turns into a source of free radicals in a vicious cycle.

In normal tension glaucoma patients, the trabecular meshwork may be more resistant to free radical damage or the optic nerve may be more susceptible to free radical damage at lower concentrations of free radicals, but I know of no reason to think that the apoptotic mechanisms of retinal ganglion cell death differ between primary open angle glaucoma and normal tension glaucoma. Therefore, I believe the follow sequence of events may be common to many types of glaucoma.

The process of apoptotic retinal ganglion cell death starts with exposure of glial cells to elevated concentrations of free radicals [2]. As a result of this, we find major histocompatibility complex (MHC) class II molecules become upregulated [2]. This occurs predominantly on microglia. The microglia exposed to free radicals become activated. T-cells in the eye become activated by both glial cells and directly by free radicals [9].

In cases of glaucoma with elevated intraocular pressure it has been shown that calcineurin is cleaved which turns it into its activated form. Calcineurin also plays an essential role in T-cell activation (and in coupling Ca++ ion signals to cellular responses in general). (Calcineurin was previously known as protein phasphatase 2B.)

Tumor necrosis factor alpha (TNF-alpha) is produced by T-cells activated by either glial cells or by free radicals. In addition, calcineurin mediates TNF-alpha production in both T-cells and B-cells. Calcineurin triggers inflammatory processes in astrocytes (macroglia) and is thought to contribute to several neurodegenerative diseases other than glaucoma.

Therefore, any of these three classes of items - activated glial cells, calcineurin and free radicals -- either together or separately, can lead to increased concentrations of TNF-alpha in the eye.

In the apoptotic mechanisms of retinal ganglion cell death, tumor necrosis factor receptor 2 (TNFR2) is required. This is interesting because TNFR2 activates the Akt signaling pathway and promotes cell survival [2]. TNFR2 suppresses oxidative stress in microglial cells. This finding infers that retinal ganglion cell death is mediated by other cells rather than caused directly by TNF-alpha (more on this in the next paragraph). TNFR2 may also be important for cytotoxic lymphocyte recruitment although this hasn't yet been demonstrated in the eye (as far as I know).

Oligodendrocyte loss follows next in the sequence of events. Oligodendrocytes primarily express TNFR2 which promotes survival, so TNF-alpha is not likely directly cytotoxic to oligodendrocytes [2]. It is the activated microglia that cause the loss of oligodendrocytes.

After some delay (several weeks in the mouse model of glaucoma), retinal ganglion cells apoptosis follows the oligodendrocyte loss. This apoptosis has been shown to be connected with "Bad" protein dephosphorylation (which is proapoptotic) as well as with the release of mitochondrial cytochrome C.

Here is a condensed outline of what I described above:

1. ROS damage various tissues in the eye. In some people the TM is susceptible to damage and OH results [12], which further increases ROS. In the case of NTG, I speculate that ROS alone leads to the next steps.
2. ROS can directly induce neuronal death by a protease and phosphatase-gated mechanism distinct from apoptosis [13].
3. ROS can induce neuronal death (and specifically RGC death) by inflammatory immune reactions involving TNF-alpha as follows:
4. Glial cells exposed to ROS [2].
   
   1. MHC class II upregulated (predominantly on microglia)
   2. Microglia activated
5. T-cells activated (by both glial cells and by ROS) [9]
6. T-cells may also be activated by a calcineurin-mediated mechanism [15].
7. TNF-alpha produced by the activated T-cells
8. TNFR2 required. (TNFR2 activates the Akt signaling pathway and promotes cell survival) [2].
9. TNFR2 suppresses oxidative stress in microglia. TNFR2 may also be important for cytotoxic lymphocyte recruitment although this hasn't yet been demonstrated in the eye (as far as I know) [2].
10. Oligodendrocyte degradation/loss [2]. (Oligodendrocytes primarily express TNFR2 which promotes survival, so TNF-alpha is not likely directly cytotoxic to oligodendrocytes [2]).
11. RGC apoptosis follows oligodendrocyte loss.
    
    1. RGC apoptosis following elevated IOP has been shown to be connected with Bad protein dephosphorylation (which is proapoptotic) as well as with the release of mitochondrial cytochrome C.

Obviously, there are a lot of gaps in the above chain of events. However, I hope to continue elaborating on this in future blog posts.

Supplement

Inflammation is commonly defined as a defensive reaction involving the immune system [18]. Inflammation is necessary for our survival. However, many of our most common diseases involve a chronic inflammatory component that results from inappropriate activation of the innate immune system. For example, cardiovascular disease involves chronic inflammation in the arterial wall.

Free radical and lipid peroxide generation are crucial to the entire sequence of events in inflammation [5]. Webster's Dictionary would point out that inflammation is a process that involves setting on fire, kindling, arousing to strong emotion, making more violent, and to intensify. Given that free radical reactions are involved in fire as well as in the biology of living organisms, this definition seems appropriate.

The release of cytokines such as tumor necrosis factor alpha (TNF-alpha) or interleukin-6 is integral to the ultimate acquired inflammatory immune response. TNF-alpha is a very potent inflammatory cytokine that promotes and promulgates inflammation [19].

Inflammation involves the massive release of free radicals into the extracellular area. Nerve tissue, because of its high content of membranes rich in polyunsaturated fatty acids such as neuronal myelin sheaths, is particularly prone to free radical damage [5].

Glial cells are a type of cell within the nervous system (and eye) that provide metabolic and structural support for neurons while not being involved in the transmission of electrical and chemical (or soliton) signals. They help maintain homeostatis, form myelin and provide physical support as well as nutrition to the nerve cells. Glial cells are sometimes called neuroglia or simply glia.

Microglia are a type of immune system cell found in the central nervous system and in the eye. They are small glial cells. Microglia are normally inactive. However, once activated they proliferate and migrate to the site of injury. Their job is to engulf dead cells and other debris. They are specifically involved in digesting dead neurons. Knowing this we can see how dangerous it is to activate microglial cells around the area of the optic nerve. Activated microglial cells are also associated with dying nerve cells in Alzheimer's disease, for example.

As I mentioned above, calcineurin activation has been shown to lead to retinal ganglion cell loss via a mechanism involving astrocytes. Astrocytes are one of the larger glial cells types. Astrocytes are also called astroglia, Deiters' cell or macroglia. They get their name because they are star-shaped. Astrocytes are the largest and most numerous type of neuroglial cell within the central nervous system. The astrocyte projections form part of the blood-brain barrier.

References (fortunately or unfortunately, my reference list includes some citations which will not be referenced until a future blog post)

1. Guthauser, U., J. Flammer, and F. Mahler. "The relationship between digital and ocular vasospasm." Graefe's Archive for Clinical and Experimental Ophthalmology 226, no. 3 (May 30, 1988): 224-226. http://dx.doi.org/10.1007/BF02181185 (accessed April 18, 2007).

2. Nakazawa, Toru, Chifuyu Nakazawa, Akihisa Matsubara, Kousuke Noda, Toshio Hisatomi, Haicheng She, et al. "Tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma." The Journal of neuroscience : the official journal of the Society for Neuroscience 26, no. 49 (December 6, 2006): 12633-41.

3. Epel, Elissa S, Elizabeth H Blackburn, Jue Lin, Firdaus S Dhabhar, Nancy E Adler, Jason D Morrow, et al. "Accelerated telomere shortening in response to life stress." Proceedings of the National Academy of Sciences of the United States of America 101, no. 49 (December 7, 2004): 17312-5.

4. Takagi, Hitoshi. "[Aging and retinal vascular diseases]." Nippon Ganka Gakkai zasshi 111, no. 3 (March 2007): 207-30; discussion 231. [Article in Japanese]

5. Levine, S. A., and P. M. Kidd. Antioxidant Adaptation: Its Role in Free Radical Pathology. Biocurrents Division, Allergy Research Group, 1985. [Book]

6. Sies, Helmut, Wilhelm Stahl, and Alex Sevanian. "Nutritional, Dietary and Postprandial Oxidative Stress." J. Nutr. 135, no. 5 (May 1, 2005): 969-972. http://jn.nutrition.org/cgi/content/abstract/135/5/969 (accessed April 18, 2007).

7. Monemi, Sharareh, George Spaeth, Alexander DaSilva, Samuel Popinchalk, Elena Ilitchev, Jeffrey Liebmann, et al. "Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1." Human molecular genetics 14, no. 6 (March 15, 2005): 725-33.

8. Fuse, Nobuo, Kana Takahashi, Hiroshi Akiyama, Toru Nakazawa, Motohiko Seimiya, Soichiro Kuwahara, et al. "Molecular genetic analysis of optineurin gene for primary open-angle and normal tension glaucoma in the Japanese population." Journal of glaucoma 13, no. 4 (August 2004): 299-303.

9. Tezel, Gülgün, Xiangjun Yang, Cheng Luo, Yong Peng, Sheher L Sun, and Deming Sun. "Mechanisms of immune system activation in glaucoma: oxidative stress-stimulated antigen presentation by the retina and optic nerve head glia." Investigative ophthalmology & visual science 48, no. 2 (February 2007): 705-14.

10. Personal note: in my case I have orthostatic hypotension. This is exacerbated by adrenal fatigue and other lifestyle factors. In my own experience, I can reduce or eliminate my orthostatic hypotension through my lifestyle choices; I can also make it worse by poor choices.

11. Singh, A. "Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen." Canadian Journal of Physiology and Pharmacology 60 (November 1982): 1330-1345.

12. Saccà, Sergio Claudio, Alberto Izzotti, Pietro Rossi, and Carlo Traverso. "Glaucomatous outflow pathway and oxidative stress." Experimental eye research 84, no. 3 (March 2007): 389-99.

13. Sée, V, and J P Loeffler. "Oxidative stress induces neuronal death by recruiting a protease and phosphatase-gated mechanism." The Journal of biological chemistry 276, no. 37 (September 14, 2001): 35049-59.

14. Klee, C B, H Ren, and X Wang. "Regulation of the calmodulin-stimulated protein phosphatase, calcineurin." The Journal of biological chemistry 273, no. 22 (May 29, 1998): 13367-70.

15. Huang, Wei, John B Fileta, Adam Dobberfuhl, Theodoros Filippopolous, Yan Guo, Gina Kwon, et al. "Calcineurin cleavage is triggered by elevated intraocular pressure, and calcineurin inhibition blocks retinal ganglion cell death in experimental glaucoma." Proceedings of the National Academy of Sciences of the United States of America 102, no. 34 (August 23, 2005): 12242-7.

16. Norris, Christopher M, Inga Kadish, Eric M Blalock, Kuey-Chu Chen, Veronique Thibault, Nada M Porter, et al. "Calcineurin triggers reactive/inflammatory processes in astrocytes and is upregulated in aging and Alzheimer's models." The Journal of neuroscience : the official journal of the Society for Neuroscience 25, no. 18 (May 4, 2005): 4649-58.

17. Ritch, R. Natural Compounds: Evidence for a protective role in eye disease. Can J Ophthalmol 2007;42:prepublication

18. Benaroyo L. How do we define inflammation? Schweiz Rundsch Med Prax. 1994 Nov 29;83(48):1343-7.

19. Plutzky, J. Inflammation and the Metabolic Syndrome. http://www.medscape.com/viewarticle/440972_8