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Inflammation and retinal axons regeneration

Upon injury inflammation is one of the first responses that are triggered. Modulating the inflammatory response may have important consequences to enhance the regenerative capabilities in different systems. Thus, too little or too much inflammation can inhibit or alter processes such us wound healing or cell proliferation that are required for proper repair and regeneration. As previously mentioned in this blog, the failure of mammalian CNS to regenerate depends in great measure on the inhibitory action of myelin and the glial scar. This is also true for the axons of the retinal ganglion cells (RGC), that after optic nerve injury normally die by apoptosis. Previous works have shown that the JAK/STAT3 and PI3K/Akt signalling pathways can enhance the regenerative response of RGC axons. Similar enhancement can be seen after inflammation stimulation, that does not only promote axonal regrowth but also plays a neuroprotective function as it increases the survival rate of RGCs upon injury.

In a recent paper from the laboratory of Dietmar Fischer the authors show how interleukin-6 is an important mediator of RGCs regeneration upon inflammatory stimulation (http://www.nature.com/cddis/journal/v4/n4/abs/cddis2013126a.html) in rats. IL-6 is a cytokine that is lowly expressed in the CNS but strongly upregulated after ischemia, trauma or axotomy in the peripheral nervous system. Here, the authors first show that IL-6 is upregulated in the retina after optic nerve crush or inflammatory stimulation. In retinal cell cultures IL-6 increases neurite growth not only in permissive substrates but also in inhibitory substrates as CNS myelin extracts. Moreover, the presence of a IL-6 antibody in the medium blocks this IL-6-stimulated neurite outgrowth. Also, IL-6 exerts a neuroprotective role on the cultured RGCs, although this effect is lower compared to those exerted by other neuroprotective and axon-promoting factors such as CNTF (astrocyte-derived ciliary neurotrophic factor). Consistent with this function of IL-6, the authors show how a IL-6 receptor is expressed in retinal cells as well as in Müller cells. When cells are cultured with an antibody against IL-6 receptor, the growth-promoting effect of IL-6 is significantly reduced. Also, when this receptor is activated by another cytokine it induces neurite growth at a similar level as IL-6 does. These results indicate that the stimulation of the IL-6 receptor is sufficient to promote neurite growth.

The effects of IL-6 on RGCs regeneration is mediated through the activation of the JAK/STAT3 and PI3K/Akt pathways. Thus, IL-6 promotes the phosphorilation of STAT3 and, on the other side, drug-mediated inhibition of the of these two signalling pathways blocks the regeneration response induced by the treatment with IL-6. Finally, the authors also check the effects of IL-6 in vivo. To do that they injected IL-6 protein after optic nerve crush and saw the activation of the JAK/STAT3 pathway as well as the upregulation of some genes associated to regeneration such as Sprr1a, galanin and gap-43. Retinal cell cultures from rats that went through optic nerve crush and posterior IL-6 injections show a significant increase in neurite outgrowth, suggesting that IL-6 transforms those RGCs into a regenerative state.

In summary, IL-6 is an important element for optic nerve regeneration mediated by inflammatory stimulation. Further studies will be necessary to see up to what extend this system can become a therapeutical target to promote optic nerve regeneration in mammals.

 

MMP1 and digit regeneration in mice

In some previous posts I have commented on the role that matrix metalloproteinases (MMPs) play during regeneration. Among others, one important aspect in which MMPs play an important function is in allowing a type of wound healing that reduces the presence of a fibrotic scar that may block the regenerative response. In fact, a scarless healing is very important for creating this regeneration-permissive environment.  Although adult mammals do not really regenerate well, at the fetal stage this is quite different (at least for certain tissues and structures). Thus, fetal wounds in the skin heal without any fibrotic scar and, consequently, a normal skin architecture and function can be regenerated. However, the adult skin when wounded elicits the formation of a fibrotic scar that blocks regeneration. Among the factors that appear to control the transition from a scarless healing to a fibrotic scar response we may find the development of the immune system, the ratio between different isoforms of TGF-b expressed, or the presence of PDGF (platelet-derived growth factor).

MMPs are another important factor in the differences between how mammalian fetal and adult skin respond to wounding. A recent paper from the laboratory of Yong Li shows how the regeneration of soft tissues can be enhanced by MMP1 during digit regeneration in mice (http://www.ncbi.nlm.nih.gov/pubmed/23527099). During wound healing, MMPs are required to promote ECM (extracellular matrix) degradation that is necessary also to favour the migration of cells into the injury site. In this paper, the authors analyse the effects of applying exogenous MMP1 into adult mice digits amputated through the middle phalanx bones. In terms of the final size of the digits regenerated in control and MMP1-treated amputated digits the authors did not find any significant difference. However, whereas the wounds were almost completely closed by day 10 after MMP1 treatment, control wounds needed more time to heal. This indicates that MMP1 has a positive impact on the healing of soft tissues in those amputated digits. Next, the authors show how this faster healing correlates with an improved revascularization after MMP1 treatment. In addition, and as innervation is important in many regenerating contexts, such as limb regeneration, the authors checked here the amount of NCAM present in the peripheral nerve fibers and neuromuscular junctions. They also found that NCAM increases in those MMP1-treated amputated digits.

As said above, one of the factors that block regeneration in may cases is the excess of fibrotic connective tissue deposited in the ECM space of wounded tissues. It is known that MMP1 digest, for example, collagens types I and III that contribute to the fibrotic scar. This collagenase activity of MMP1 is repressed by TGF-b1 and, in fact, TGF-b1 is more abundant in the adult skin than in the fetal one. Also, knocking down TGF-b1 reduces scar formation. Here, the authors show how MMP1 treatment reduces significantly the deposition of collagen in the ECM, and therefore the fibrotic scar. Finally, the authors use Sca-1 (Stem cell antigen-1) that labels hematopoietic and other types of progenitor cells and found an enrichment in the number of positive cells in the MMP1-treated amputated digits.

In summary, the authors show, by using different markers, that MMP1 promoted a faster wound closure, the regeneration of soft tissues and a decrease in scar tissue formation. However, no beneficial action of MMP1 is observed for the bones or the final size of the regenerated digit. Future work should help to characterize better the targets of MMP1 and try to find factors to enhance the regeneration of the skeletal tissues.

Spinal cord regeneration in vertebrates

Patients of neurodegenerative diseases such as Alzheimer and Parkinson as well as people that suffer spinal cord injuries would surely benefit if we would manage to enhance somehow the very limited regenerative capacities of our central nervous system. But whereas mammals show almost no regeneration potential in their CNS, other vertebrates such as amphibians and zebrafish are capable of regenerating their spinal cord. Also, invertebrates such as planarians can regenerate a complete CNS de novo from any small piece of their bodies. So, how do amphibians and fish do to regenerate their spinal cord? What prevents mammals from doing the same? Because of the great potential benefits of enhancing mammalian CNS regeneration a lot of research is being done in this field; however, the results are not being really very promising yet, at least as fast as demanded by the society.

In a recent review from the laboratory of Karen Echeverri the authors discuss the current state-of-the-art of vertebrate CNS regeneration (http://www.ncbi.nlm.nih.gov/pubmed/23581406). When comparing the regenerative response of mammals to that from amphibians and fish important aspects that need to be considered include the cellular response to injuries and the formation and role of the glial scar. In zebrafish, upon injury, glial cells amplify and migrate to the lesion where they elongate creating a glial bridge that axons use as a scaffold to grow across the lesion. A very interesting observation in zebrafish is that the re-wiring of the regenerated CNS does not need to be one hundred per cent accurate (in terms of re-establishing the original connections) to yield a functional recovery.

Amphibians, both anurans (tailless amphibians) and urodeles (tailed amphibians) regenerate the spinal cord during tail regeneration. In Xenopus it has been described a population of putative neural stem cells positive for Sox-2 that, upon injury, proliferate and migrate forming a substrate for axonal regeneration. In urodeles, also, fibroblasts and glial cells migrate to create a substrate for axonal regeneration. Thus, in all these models once the spinal cord is injured certain cell types are able to create a regeneration-permissive environment.  Another thing that is shared by amphibians and zebrafish refers to the upregulation of signalling pathways such as Wnt, BMP and FGF during spinal cord regeneration.

In contrast, CNS regeneration in mammals is inhibited by both extrinsic and intrinsic factors. One of the most important inhibitors is the glial scar that acts as a physical and chemical barrier to regeneration. In zebrafish and amphibians, however, no inhibitory glial scar is formed. Recently it is emerging the view that in mammals the glial scar although inhibiting axonal regeneration may have an important role as a protective agent during early stages after injury. In this sense it becomes interesting to further analyse whether the inhibitory action of the glial scar on regeneration is an unavoidable trade-off of its beneficial role on stabilizing the injury site preventing further damage. If this is true maybe it means that there is a kind of evolutionary constraint that really works against the possibility of enhancing CNS regeneration in mammals. Another field to develop is the study of the inhibitory action on regeneration by myelin components such us Nogo, myelin-associated protein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp). Different studies have shown contradictory results in terms of enhancing the regenerative capabilities after inhibiting these factors. Therefore more data and comparative analyses are needed from animals that can and cannot regenerate their CNS.

In summary, and as it has been pointed out in previous posts on this blog, it is important not only to understand as deeply as possible how regeneration takes place at both cellular and molecular level, but also to try to determine why the regeneration potential has been lost in certain lineages. In this sense spinal cord regeneration provides with a very attractive model in which direct comparisons between regenerating and non-regenerating models (phylogenetically close, such as amphibians, fish and mammals) can be beneficial for the field as it may help, for example, to determine what really makes that some species are able to create a regeneration-permissive environment whereas others, in which similar cell types and molecular pathways are present and even upregulated after injury, cannot do it.

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