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    Originally posted by jsilver View Post
    It doesn't seem as if you are both on the same page to me....

    I thought we were headed in the right direction but, alas, we're as far apart as ever. The scar (or let's call it the lesion environment) is a potent barrier that needs strong measures to remove or over power it. It is absolutely not growing weaker and weaker just because Wise says so. Crabby person and Wise mention the Tuszynski paper as an example of axon regeneration past an established glial scar. Here's what they had to do to get a very small number of sensory axons past an established scar without physically removing it. (1) They fill the lesion with genetically altered neurotrophin expressing marrow stromal cells, then they (2) express the same powerful neurotrophin just beyond the scar using a virus and finally (3) they have to condition the sensory neurons with a prior crush lesion. At 6 weeks following injury using this approach they get several dozens of axons across the lesion and at 15 months post injury they get just a handful of sensory axons across a well established scar. Once past the scar the axons stop abruptly in the trophic oasis in the white matter on the other side of the lesion. No synapses, no functional recovery. I encourage you all to carefully read this paper ( it's downloadable from the Crabbyfish post above) and learn what it takes to get the smallest number of axons past an established scar. Does that sound weak to you? Just yesterday I saw a guardrail on the side of the road that had been smashed through by a fast moving vehicle. It was good example of the point I am trying to make. No barrier is absolute but would you suggest that steel guardrails are not potent barriers?
    Jerry,

    May I suggest that you press the button [Quote] on the lower right side of the post that you are answering. This includes the words and name of the person that you are answering. This reduces confusion of what you are saying versus the person that you are answering. You can put what you write either on top or below the quoted post, as well as edit what you want to quote. I also suggest that if you answer within the quotation that it is easier to read if you select your words and highlight them by changing the color of the text. All capitalized words indicate shouting on Internet and is considered poor manners. You also have be careful not to delete the {/quote} (curly brackets indicating square brackets) which is necessary to close the quotation. If you don't mind, I can edit your posts to clear up these display issues.

    I like your use of the term "lesion environment", instead of "glial scar". It accurately depicts the situation. Regarding the Lu, et al. (2012) paper that I had previously posted, the number of axons that are growing across the transection and transplantation site in the paper are quite numerous. Many thousands of axons are growing form the transplanted cells. But, as I have pointed out already, these axons come from the transplanted neural stem cells lines and may not behave like normal neurons.

    So, perhaps we should discuss another paper by Lu, et al. (2012) also from the Tuszynski lab. The reference and abstract of the paper is below. The study assesses the growth of reticulospinal axons across a transection site transplanted with mesenchymal stem cells and that has an area expressing BDNF on the other site. It is a beautiful study that shows that growing reticulospinal axons ignore the gliosis to grow into the injury site and can be enticed to grow out of the graft by expression of BDNF in the distal cord.

    Lu, P., Blesch, A., Graham, L., Wang, Y., Samara, R., Banos, K., et al. (2012). Motor axonal regeneration after partial and complete spinal cord transection. The Journal of Neuroscience : the Official Journal of the Society for Neuroscience, 32(24), 8208–8218. doi:10.1523/JNEUROSCI.0308-12.2012
    1Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, 2Veterans Administration Medical Center, San Diego, California 92161, 3Spinal Cord Injury Center, Heidelberg University Hospital, D-69120 Heidelberg, Germany, 4Department of Anesthesiology, University of California, Irvine, Irvine, California 92697, and 5University of Alberta, Centre for Neuroscience, Edmonton, Alberta T6G 2E1, Canada
    We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.

    Lu, et al. dorsally hemisected the spinal cord, placed a bone marrow stromal cell graft into the hemisetion site, labeled reticulospinal axons by injecting biotinylated dextran amine (BDA) into the brainstem, stimulated the axons to grow by injecting cAMP, and then transfected cells in the distal spinal cord to express brain-derived neurotrophic factor (BDNF). For the purposes of discussion, I attach Figure 2 from that article.

    Just to orient people, the figure shows photomicrographs of C5 hemisected spinal cords after combinatorial treatment with cAMP, a cell graft into the lesion site, and transected cells that express BDNF beyond the lesion site. Reticulospinal neurons (from brainstem) are labelled with BDA (red), host astrocyte GFAP in blue (showing the gliosis), and BDNF (a neutrophin) is green.

    I know that the figure legends gives the explanation but let me point out the salient findings in each panel. Panel A shows a low powered view where the lesion site (Les) is situated on the left, there is blue (gliosis) around the lesion site, and a flame-like gout of green towards the right.

    Panel B is a higher power view of the left side of the lesion site. The lesion site is filled with many red fibers. The spinal cord to the left of the lesion site clearly has axons that are heading into and through the gliosis (blue) to the lesion site. The dotted white line indicates the border of the lesion site. There are no glial cells on the lesion side.

    Panel C is a higher power view of the right side of the lesion site. You can see that some of the red (BDA-labelled) reticulospinal axons are leaving the lesion site and growing into the green (BDNF) expressing cells.

    Panel D is the same as Panel C except that the green color was removed, so that you can the large number of red reticulospinal axons that are growing out of the lesion site.

    Panel E is the a higher power view of the right (caudal) cord where you can see many red (BDA-labelled) reticulospinal axons growing beyond the area of BDNF expressing cells.

    Panel F-I are from another spinal cord where the graft (g) can be seen on the left and they have stained the cells not with fluorescence but stained DAB for BDNF light microscopy (dark black cells). The axonal growth seems to be where BDNF is expressed.

    This study indicates that
    1. Many thousands of reticulospinal axons grow into a hemisection site that had been grafted with mesenchymal stem cells.
    2. The reticulospinal axons clearly cross the area of gliosis into the mesenchymal graft and out of the graft into the distal spinal cord.
    3. The axonal growth seem to be associated with BDNF expressing cells. This is of interest because it suggests that axons don't like to leave the graft but can be induced to do so by BDNF in the host spinal cord.
    4. The regenerated axons make synapses with neurons in the distal spinal cord but the animals do not walk (although it seems to have reduced their spasticity).
    5. Finally, despite robust regeneration of reticulospinal axons (probably other axons as well) and connections of these axons with neurons below the injury site, the authors did not see improved motor function.


    The last point is of course discouraging. The authors have regenerated many axons in the spinal cord, showed that the cells have connected, but motor outcomes worsened. One possible reason is that the rats did not receive intensive locomotor training.

    Wise.
    Attached Files
    Last edited by Wise Young; 30 Jan 2013, 2:55 PM.

    Comment


      Here is the full paper Lu, et al. 2012 that I referred to in my last post.

      Comment


        You can fix my responses if you like. I haven't attempted to figure out the use of all the buttons. I didn't think there was much new info in the Lu paper you cite. Same techniques as before, except this time they added cAMP injections to the reticular formation rather than a conditioning type of lesion that they use to improve the intrinsic growth capacity of the DRGs. But now they were focusing on efferent projections with the hope of getting some crude locomotor recovery. Instead, they got a lot of spasticity and no improvements. Yes, again axons can cross a lesion if one simultaneously pushes them and pulls them forward. I don't know why you feel obligated to get dramatic and use the word "ignore" because clearly not all axons can bypass the lesion. Although clearly some do others are still stuck, and by the way where do you come up with "thousands" when the authors themselves report hundreds. Again this is an acute injury model. Why the animals develop spasticity is a matter of speculation but my guess is that directing several hundreds of axons to a single location might be causing a hyperinnervation of the cord.

        Comment


          Jerry,

          Is the intermittent hypoxia you did with the rats the same as, or similar to, intermittent hypoxic training?

          Best,

          Steven
          ...it's worse than we thought. it turns out the people at the white house are not secret muslims, they're nerds.

          Comment


            Jerry,

            Is the intermittent hypoxia you did with the rats the same as, or similar to, intermittent hypoxic training?

            Best,

            Steven
            Yes , this is basically the procedure we used but we did this for much less time per day and only for 5 days. The ch'ase really helped us to lower the amount of IH that we needed to give the animals.
            Last edited by Sue Pendleton; 30 Jan 2013, 10:20 PM. Reason: Quote fix.

            Comment


              Dr. silver,
              Hi, would you please go and read the new post on cure page Title " spinal research (ISRT): update" by fly_pelican_fly and see what you think of that research about CHAS . Thank you

              Comment


                Originally posted by Wise Young View Post
                Here is the full paper Lu, et al. 2012 that I referred to in my last post.
                I find this interesting from the paper:

                "Several lines of evidence support the observation that axons
                have regenerated in this study: (1) regenerating axons emerge
                from the midportions of the graft in the lesion site, where spared
                axons are unlikely to be located; (2) axons exhibit irregular trajectories,
                often with abrupt turns, rather than existing in tightly
                linear bundles typical of spared axons
                ;
                ..."(pg. 8216).

                Comment


                  Originally posted by kz View Post
                  Dr. silver,
                  Hi, would you please go and read the new post on cure page Title " spinal research (ISRT): update" by fly_pelican_fly and see what you think of that research about CHAS . Thank you
                  KZ, I believe Jerry is part of the Ch'ase+ISRT steering committee.

                  See: http://www.spinal-research.org/wp-co.../Programme.pdf

                  Comment


                    Dr. silver,
                    Hi, would you please go and read the new post on cure page Title " spinal research (ISRT): update" by fly_pelican_fly and see what you think of that research about CHAS . Thank you

                    I was extremely excited to read this. This is really wonderful news. I think they are finally going to do this.

                    Comment


                      Originally posted by NowhereMan
                      axons exhibit irregular trajectories,
                      often with abrupt turns, rather than existing in tightly
                      linear bundles typical of spared axons;

                      Yes, precisely!!! This type of haphazard axonal patterning is typical of truly re-growing axons. Everyone in the regeneration biology community, except Wise, understands the importance of this basic concept. This is exactly the argument I raised when I questioned the validity of Wise's DTI data and his conclusions that axons had regenerated in his patients. I hope he is paying close attention to this exchange between you and I.

                      from my post #124 "Truly regenerating axons NEVER grow in tightly aligned bundles. Regeneration in the adult is not like the fasciculated axon growth that occurs in the embryo. Indeed, the presence of aligned bundles of axons passing in the vicinity of a cord lesion is always suggestive of spared axons."
                      Last edited by Wise Young; 31 Jan 2013, 7:55 AM.

                      Comment


                        Originally posted by jsilver View Post
                        Yes, precisely!!! This type of haphazard axonal patterning is typical of truly re-growing axons. Everyone in the regeneration biology community, except Wise, understands the importance of this basic concept. This is exactly the argument I raised when I questioned the validity of Wise's DTI data and his conclusions that axons had regenerated in his patients. I hope he is paying close attention to this exchange between you and I.

                        from my post #124 "Truly regenerating axons NEVER grow in tightly aligned bundles. Regeneration in the adult is not like the fasciculated axon growth that occurs in the embryo. Indeed, the presence of aligned bundles of axons passing in the vicinity of a cord lesion is always suggestive of spared axons."
                        Jerry,

                        I edited the your post so that the quotes show...

                        Axons grow in bundles during development, a process called fasciculation, aided by expression of the L1 cellular adhesion molecule by axons. The fact that almost all the axons in the spinal cord are organized into tracts indicates that axons can grow in bundles. Likewise, axons in peripheral nerves grow in bundles. Just because axonal growth appears to be dispersed and haphazard in animal studies, it does not mean that bundled growth cannot occur under certain circumstances. In fact, such types of growth is desirable.

                        We did the DTI to assess the location of the white matter in the spinal cords. The results were surprising. *Every* one of five spinal cords of ASIA A subjects that we were able to image successfully with DTI, we saw a clear gap in the fiber tracts between the rostral and caudal spinal cord. In two of these patients, we saw the fiber tracts approach each other, overlap, and even bundles of fibers that extend long distances into the proximal and distal spinal cord.

                        In any case, we think that this is sufficiently promising to see if we can repeat this in a phase III randomized clinical trial in larger numbers of subjects. We are of course also still following the subjects for over one year in hopes that neurological scores correlate with these findings. Finally, we are currently cleaning up and analyzing the 6-12 month data from 20 subjects that were transplanted in Kunming with intensive locomotor training.

                        Wise.

                        Comment


                          Wise

                          "Just because axonal growth appears to be dispersed and haphazard in animal studies, it does not mean that bundled growth cannot occur under certain circumstances. In fact, such types of growth is desirable."

                          Sure, I agree that regenerating axons can be induced to cross a lesion in the cord within tight bundles, but this occurs only when they are given an aligned scaffold or tubular prosthesis or segments of peripheral nerves to guide them. However, when such axons exit the distal end of the bridge (and this rarely happens unless you lure them out of the other side with neurotrophins or add ch'ase or strongly stimulate their intrinsic growth capacity or some combination of the three) they then instantly begin to meander again. So the presence of a very long bundled thing extending long distances beyond a lesion in your patients will be interpreted by all those who do this for a living or read the relevant literature, as spared fibers and will have a hard time accepting these structures as regenerating axonal bundles. Even if they might be axonal then they will be considered to have been spared but somehow their visualization with DTI has changed over time. As I mentioned earlier if you can show that such rectilinear bundles continue to elongate at their distal tips over extended periods of time then, I think, you will be in business. None-the-less, without showing convincing data using modern anatomical techniques that such unprecedented regeneration can occur in a chronic animal model using your strategy, there will always be skeptics that you have proven that long distance axonal regeneration can occur at such lengthy chronic stages in a human. As far as correlation between behavior and the presence of these structures, I thought I heard you present at Bedford that there wasn't any.
                          Last edited by jsilver; 31 Jan 2013, 1:10 PM.

                          Comment


                            All we need now is for the Neurological scores to correlate with the findings. Patience is a virtue. Will find out more next five months.
                            In any case, the dialogue between the two professors is extremely informative.
                            Please Dr. Young, Keep sharing with us all the good news.

                            Comment


                              Originally posted by jsilver View Post
                              Thanks for citing this one from the Fehlings lab because it demonstrates nicely the role of scar associated inhibitory molecules in curtailing stem cell migration especially at chronic stages after injury. You and Wise should actually read it. Just look at the title. The authors demonstrate yet again the need for a combinatorial strategy that breaks down CSPGs (via chondroitinase) and adds a cocktail of neurotrophins to keep stem cells alive in order to allow them to escape the lesion, migrate remarkable distances and remyelinate axons. Without chondroitinase at chronic stages the stem cells remained trapped within the scar. This is not a paper about axonal regeneration to restore function but likely remyelination of axons or possibly sprouting effects.
                              From attached:
                              In this report, we studied the role of glycogen synthase kinase-3 (GSK-3) inactivation on neurite and axon growth from adult neurons via combined in vitro and in vivo approaches. We found that the major CNS inhibiting substrates including chondroitin sulfate proteoglycans could inactivate protein kinase B (Akt) and activate GSK-3 signals in neurons.
                              Systemic application of the GSK-3 inhibitor lithium to spinal cord-lesioned rats suppresses the activity of this kinase around lesion.
                              Application of GSK-3 inhibitors stimulates axon formation and elongation of mature neurons whether in presence or absence of inhibitory substrates.
                              Originally posted by jsilver View Post
                              None-the-less, it is a very nice paper giving hope that a proper combinatorial strategy employing stem cells can restore some useful function at chronic time points following SCI.
                              Excellent!
                              Treatments with GSK-3 inhibitors including a clinical dose of lithium to rats with thoracic spinal cord transection or contusion injuries induce significant descending corticospinal and serotonergic axon sprouting in caudal spinal cord and promote locomotor functional recovery.
                              Our studies suggest that GSK-3 signal is an important therapeutic target for promoting functional recovery of adult CNS injuries and that administration of GSK-3 inhibitors may facilitate the development of an effective treatment to white matter injuries including spinal cord trauma given the wide use of lithium in humans.

                              Comment


                                My favorite part of the paper:

                                "We recently reported that pain reliever ibuprofen improved axon
                                growth and functional recovery in SCI rodents when applied at a
                                dose of 60 mg/kg/d, which is a higher dose than that used clinically
                                as a cyclooxygenase inhibitor (Fu et al., 2007)."
                                http://spinalcordresearchandadvocacy.wordpress.com/

                                Comment

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