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Glaucoma Dialogue IGR 11-2

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Comment by Takuji Kurimoto on:

23848 Axonal regeneration induced by repetitive electrical stimulation of crushed optic nerve in adult rats, Tagami Y; Kurimoto T; Miyoshi T et al., Japanese Journal of Ophthalmology, 2009; 53: 257-266

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We were very happy to learn that our paper was chosen for thesection of Glaucoma Dialogue, and greatly appreciate the positivecomments from Jeff Goldberg, Len Levin, Keith Martin and BarbaraLorber, James Morgan, Manuel Vidal Sanz, and Makoto Aihara. Hereare our answers to their comments.

1. We completely agree about the mechanisms. Current flow may directly stimulate or injure M�ller cells, which may then leadto an up-regulation of different molecules including IGF-1.Another possible mechanism is an inflammatory reaction.Unfortunately, we did not check the inflammatory reaction afterrepetitive TES by histological methods. Several investigators haveshown that activated macrophages promote RGC survival andaxonal regeneration after an optic nerve crush. We also plan toinvestigate whether TES activates immunological reactions.
2. The optic nerve crush model using forceps is widely used althoughthere is a limitation to this method because the degree of injurymay vary depending on the degree and length of time of thepressure on the optic nerve. We have shown that the uptakeof Fluorogold applied to the superior colliculus into the RGCswas completely disrupted by crushing the optic nerve with forcepsand morphological changes were observed at the injurysite (Neuroreport 17: 1525-1529). We counted the number ofCTB-labeled axons in more than seven sections of each opticnerve. Thus, counting the number of CTB-labeled axons shouldgive a reasonable estimate of axonal regeneration. However, weshould further evaluate the regenerated axons in cross sectionsand GAP43 staining.
   Actually, TES has a potential of stimulating the brain stemby the trigeminal nerves that innervate cornea. Previous studieshave shown that TES increased the blood flow in the middlecerebral artery of rats. Further experiments are required todemonstrate how TES affects the blood flow in the retina andbrain.
3. As suggested by the reviewer, TES may promote just sproutingof RGC axons. So, we are investigating whether combining TESwith different neurotrophic factors will enhance axonal growth. Ithas been already been shown that TrkB, BDNF receptors, arerecruited onto the cell surface by the elevation of neural activityor cAMP in vitro. We have hypothesized that the combinationof TES with BDNF will enhance axonal regeneration in vivo.
4. We greatly appreciate the reviewer pointing out our mistake in Figure 3. We have corrected Figure 3 as an erratum of the figure.As the reviewer commented, the survival effect of repetitiveTES may be mediated by a different pathway than IGF-1.We are currently investigating what intracellular signals contributeto the survival effects.
5. Our data may not be conclusive that TES affects axonal regeneration.However, comparing our data with others, TES probablyinduces various effects on the retina. We would like to emphasizethat repetitive TES, which is noninvasive method, has apossibility of initiating axonal growth in the naive optic nerve.
6. Our findings suggest that IGF-1 is mainly associated with theaxonal regeneration induced by repetitive TES. However, theywere not conclusive because we did not demonstrate changesin the expression of IGF-1 by other methods, e.g., western blots,ELISA, or quantitative PCR. Thus, we need to further experimentsto confirm the relationship between IGF-1 and axonalregeneration. As the reviewer commented, the electrical inhibitionof RGC by TTX injection should be an important experimentto prove that active potentials induced by TES is critical for axonalregeneration in vivo. Unfortunately, we did not try this experimentbecause we were concerned that TTX may have variouseffects on the retina in addition to that on the RGC, and it hasalready been proven that electrical activities is critical for axongrowth of RGC in vitro. However, we plan to investigate the relationshipbetween electrical activity and axon growth in vivo.

In conclusion, we strongly believe that the electrical activity ofthe RGC is a critical factor for axon growth in adult mammals.As suggested by several of the reviewers, TES may promote not onlyaxonal sprouting in vivo, but also has a high potential for enhancingthe axonal regeneration when combined with neurotrophic factorsor counteracting of axon-growth inhibitory factors.

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