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    Originally posted by paolocipolla View Post
    Wise,

    unfortunatly you don't seem to understand my question since you keep mentioning glial scar while I was focusing just on fibroctic scar for a moment.
    Do you even bother to read what Wise posts?

    Oh, yes, there are studies that show axons growing *through* fibrotic scars.

    Comment


      Originally posted by t8burst View Post
      Do you even bother to read what Wise posts?
      Read more carefully...

      Paolo
      In God we trust; all others bring data. - Edwards Deming

      Comment


        Originally posted by paolocipolla View Post
        Read more carefully...

        Paolo
        I read the paper he attached. It seems to be if you have the ability to grow axions across a completely transected spinal cord then you could cut out any fibrotic tissue then bridge the site.

        However I am a simple rocket scientist not a doctor.

        Comment


          Originally posted by Fly_Pelican_Fly View Post
          CrabbyShark - You can't cite text from published papers and then say things like "what if the stem cells do XYZ....what if the lithium does ABC......what if I mix UCBC and orange juice with toothpaste.....maybe that'll work?". Do you realise that just kills your argument in a single heartbeat?
          I'm not really sure what you mean here.
          In the study, MSC promoted axon growth across injury site.
          Lithium enhances proliferation of stem cells and also promotes axon growth.
          Lithium + MSC would probably do an even better job of promoting axon growth across the injury site than MSC by itself.

          Originally posted by Fly_Pelican_Fly View Post
          Why would you suggest that? Although stem cells maybe part of the journey or story - you cannot be as close minded to suggest that every researcher should be working with them. Remember, it's about repairing the spinal cord. And to repair it, you need to understand it. There are researchers working with stem cells - harvested, manufactured and endogenous. There are researchers working on gene expression. There are researchers working with enzymes. There are researchers working with small-chain peptides. There are researchers working on delivery mechanisms like biomaterials, microtubes, viral vectors. There are researchers working immunological targets. All of these and more are aiming to understand the biology of the chronic spinal cord and develop therapeutic targets.
          I agree with you here. Dr. Silver's outcomes generally have been poor. If his focus is:
          Originally posted by jsilver View Post
          I have had a long standing NIH grant entitled "Regeneration beyond the glial scar" for nearly 30 years.
          There's evidence suggesting stem cells are helpful in doing that. His work ignores stem cells. Why? Why not incorporate them into some of what he is doing? Is the goal regeneration past the scar? or is the goal regeneration past the scar using only surgery and chemicals or only surgery and peptides? Stem cell therapies represent a burgeoning field of medical science. He's in an unique position to take advantage of it, if he wanted to.

          Originally posted by Fly_Pelican_Fly View Post
          We get that you're a staunch supporter of UCBC+Lithium. That's fine. The community and field keenly awaits the 12-month data from the trial. However, it's important to acknowledge that there will be critics (part of the territory so get used to it) of any paper that is submitted (and even published) and any line of science that goes to human clinical trial. It's the objective nature of science.
          I'm with you here. I'm a staunch supporter of getting better. I don't care what works. If it's proven, it's safe and it works, I'm down.

          Comment


            Originally posted by t8burst View Post
            I read the paper he attached. It seems to be if you have the ability to grow axions across a completely transected spinal cord then you could cut out any fibrotic tissue then bridge the site.

            However I am a simple rocket scientist not a doctor.
            T8burst,

            The glial scar theory states that proliferation of glial cells form barriers that stop axonal growth. The theory predicts the prevention or removal of glial scar in injured spinal cords would allow regeneration. It also predicts that if the glial scar barrier is not removed, axons cannot grow into the injury site. Based on this theory, many people, including doctors and scientists, believe that the "glial scar" must be removed before any regeneration occurs. The glial scar theory has been used to justify removal of the injury site or even transections of the spinal cord.

            There is no evidence from any animal model indicating that removal of “glial scar” improves functional recovery. In fact, studies from the Sofroniew laboratory [1-2] at UCLA indicate that selective prevention or removal of glial cells at the injury site worsens spinal cord injury and reduces functional recovery rather than enhance regeneration and recovery. In the absence of any data to suggest that removal of “scar” is helpful and the presence of convincing data that it is harmful to the spinal cord, I don’t think removal of glial scar is a good idea.

            At the same time, many studies from the top spinal cord injury laboratories in the world indicate that gliosis or gliosis+fibrosis (glial scar) does not stop axonal growth in a variety of spinal cord injury models, including contusion, hemisection, and transection. In fact, several studies suggest that the gliosis environment paradoxically encourages some axons (such as serotonergic axons, which are important for locomotor recovery) to grow. Let me summarize some of the key evidence again.
            1. In 1997, Beattie, et al. [3] studied many hundreds of contused spinal cords and reported that thousands of axons grow into the contusion site. At first, we did not know where these axons come from. However, in subsequent studies, Hill, et al. [4] from the Beattie laboratory showed that both descending and ascending axons come from the brain, surrounding spinal cord, and dorsal root sensory ganglia.
            2. In 2002, Bradbury, et al. [5] reported the chondroitinase ABC promotes functional recovery after spinal cord injury. Chondroitinase breaks down extracellular matrix proteins called chondroitin-6-sulfate-proteoglycan (CSPG). She found growth of ascending and decending axons across dorsal hemisections and suggested that chondroitinase encourages regeneration of axons to improve functional recovery. Barritt, et al. [6] from the Bradbury laboratory subsequently showed that chondroitinase increases spouting of both injured and intact spinal tracts and these may have contributed to the recovery.
            3. in 2004, Pearce, et al. [7] from the Bunge Laboratory in Miami, showed that if they give drugs that increase cAMP inside cells and transplanted Schwann cells into the contusion site, they can get many serotonergic spinal axons to cross the contusion site.
            4. In 2005, de Castro, et al. [8] from the Stallcup laboratory at Burnham Institute in La Jolla reported that NG2 stimulates rather than inhibit the growth of serotonergic axons. Hawthorne, et al. [9] at the Silver laboratory confirmed this finding. Many scientists have reported the serotonergic axons grow into gliotic areas.
            5. In 2011, Liu, et al. [10] from the He Laboratory at Harvard reported that if they blocked PTEN expression in the cortex of mice, thousands of corticospinal axons grew across hemisection sites.
            6. In 2012, Lu, et al. [11] from the Tuszinsky laboratory at UCSD showed that if they implant neural stem cells with a cocktail of neurotrophins between two stumps of transected rat spinal cords, thousands of axons growing from the transplanted neural stem cells cross the fibrotic glial scar on both sides and travel long distances in the white matter.


            In science, if no evidence supports a theory and much evidence disproves its predictions, the theory is considered wrong. By the way, the above studies also argue against the myelin-based inhibition of axonal growth (i.e. Nogo). The studies support the neurotrophic theory that suggest that spinal axons do not have sufficient growth factors to regenerate and that activation of intracellular messengers (such as cAMP, PTEN/mTOR) with treatments such as rolipram, lithium, Akt stimulators, and neurotrophins stimulate axons to regenerate through gliosis and glial scars.

            Lithum directly stimulates phosphokinase B or Akt (which is the mechanism by which PTEN stimulates mTOR and axon growth). Lithium also inhibits glycogen synthetase kinase 3-beta (GSK-3b), which stimulates neural stem cells to proliferate and cells to produce neurotrophins. Lithium stimulates increased production of neurotrophins in the central nervous system and when combined with umbilical cord blood mononuclear cell transplants. Lithium is a safe way to produce the effects of a PTEN deletion. 30 million people around the world take lithium daily to treat manic depression and the safety profile of the drug is well known and understood.

            Our phase I and II trials have confirmed that lithium can be safely given to people with chronic spinal cord injury. We have safely transplanted cord blood mononuclear cells in 41 patients with spinal cord injury, showing feasibility, safety, and some potential beneficial effects. We are proposing phase III trials where we will transplant HLA-matched umbilical cord blood mononuclear cells (UCBMC) directly into the spinal cord of people with ASIA A/B spinal cord injury. The trials will compare UCBMC transplanted subjects with and without lithium against control subjects that will receive only untethering surgery. In China, we will randomize half of the subjects to intensive 6:6:6 locomotor training and half to 3:3:3 standard rehabilitation.

            Medical tourism clinics around the world have been telling people that they can make them better by injecting UCBMC intrathecally or intravenously (Beike and other groups) and charging them exorbitant prices for such therapy. If our trial shows that the UCBMC transplants have no benefit, we will recommend strongly against their use for chronic spinal cord injury, and go on to other therapies. If our trial shows that UCMBC and lithium are beneficial, it would be a great treatment against which to compare even better treatments. In either case, we have developed a powerful and efficient global platform to assess cell transplant and drug therapies of human spinal cord injury.

            Wise.

            References
            1. Faulkner JR, Herrmann JE, Woo MJ, Tansey KE, Doan NB and Sofroniew MV (2004). Reactive astrocytes protect tissue and preserve function after spinal cord injury. J Neurosci 24: 2143-55. Department of Neurobiology, University of California, Los Angeles, California 90095-1763, USA. Reactive astrocytes are prominent in the cellular response to spinal cord injury (SCI), but their roles are not well understood. We used a transgenic mouse model to study the consequences of selective and conditional ablation of reactive astrocytes after stab or crush SCI. Mice expressing a glial fibrillary acid protein-herpes simplex virus-thymidine kinase transgene were given mild or moderate SCI and treated with the antiviral agent ganciclovir (GCV) to ablate dividing, reactive, transgene-expressing astrocytes in the immediate vicinity of the SCI. Small stab injuries in control mice caused little tissue disruption, little demyelination, no obvious neuronal death, and mild, reversible functional impairments. Equivalent small stab injuries in transgenic mice given GCV to ablate reactive astrocytes caused failure of blood-brain barrier repair, leukocyte infiltration, local tissue disruption, severe demyelination, neuronal and oligodendrocyte death, and pronounced motor deficits. Moderate crush injuries in control mice caused focal tissue disruption and cellular degeneration, with moderate, primarily reversible motor impairments. Equivalent moderate crush injuries combined with ablation of reactive astrocytes caused widespread tissue disruption, pronounced cellular degeneration, and failure of wound contraction, with severe persisting motor deficits. These findings show that reactive astrocytes provide essential activities that protect tissue and preserve function after mild or moderate SCI. In nontransgenic animals, crush or contusion SCIs routinely exhibit regions of degenerated tissue that are devoid of astrocytes. Our findings suggest that identifying ways to preserve reactive astrocytes, to augment their protective functions, or both, may lead to novel approaches to reducing secondary tissue degeneration and improving functional outcome after SCI.
            2. Lepore AC, Dejea C, Carmen J, Rauck B, Kerr DA, Sofroniew MV and Maragakis NJ (2008). Selective ablation of proliferating astrocytes does not affect disease outcome in either acute or chronic models of motor neuron degeneration. Exp Neurol 211: 423-32. Department of Neurology, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Meyer 6-119, Baltimore, MD 21287, USA. Astrocytes play important roles in normal CNS function; however, following traumatic injury or during neurodegeneration, astrocytes undergo changes in morphology, gene expression and cellular function known as reactive astrogliosis, a process that may also include cell proliferation. At present, the role of astrocyte proliferation is not understood in disease etiology of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder that is characterized by a relatively rapid degeneration of upper and lower motor neurons. Therefore, the role of astrocyte proliferation was assessed in both acute and chronic mouse models of motor neuron degeneration, neuroadapted sindbis virus (NSV)-infected mice and SOD1(G93A) mice, respectively. While astrocytes proliferated in the lumbar spinal cord ventral horn of both disease models, they represented only a small percentage of the dividing population in the SOD1(G93A) spinal cord. Furthermore, selective ablation of proliferating GFAP(+) astrocytes in 1) NSV-infected transgenic mice in which herpes simplex virus-thymidine kinase is expressed in GFAP(+) cells (GFAP-TK) and in 2) SOD1(G93A)xGFAP-TK mice did not affect any measures of disease outcome such as animal survival, disease onset, disease duration, hindlimb motor function or motor neuron loss. Ablation of dividing astrocytes also did not alter overall astrogliosis in either model. This was likely due to the finding that proliferation of NG2(+) glial progenitors were unaffected. These findings demonstrate that while normal astrocyte function is an important factor in the etiology of motor neuron diseases such as ALS, astrocyte proliferation itself does not play a significant role.
            3. Beattie MS, Bresnahan JC, Komon J, Tovar CA, Van Meter M, Anderson DK, Faden AI, Hsu CY, Noble LJ, Salzman S and Young W (1997). Endogenous repair after spinal cord contusion injuries in the rat. Exp Neurol 148: 453-63. Department of Cell Biology, Ohio State University College of Medicine, 333 West 10th Avenue, Columbus, Ohio 43210, USA. Contusion injuries of the rat thoracic spinal cord were made using a standardized device developed for the Multicenter Animal Spinal Cord Injury Study (MASCIS). Lesions of different severity were studied for signs of endogenous repair at times up to 6 weeks following injury. Contusion injuries produced a typical picture of secondary damage resulting in the destruction of the cord center and the chronic sparing of a peripheral rim of fibers which varied in amount depending upon the injury magnitude. It was noted that the cavities often developed a dense cellular matrix that became partially filled with nerve fibers and associated Schwann cells. The amount of fiber and Schwann cell ingrowth was inversely related to the severity of injury and amount of peripheral fiber sparing. The source of the ingrowing fibers was not determined, but many of them clearly originated in the dorsal roots. In addition to signs of regeneration, we noted evidence for the proliferation of cells located in the ependymal zone surrounding the central canal at early times following contusion injuries. These cells may contribute to the development of cellular trabeculae that provide a scaffolding within the lesion cavity that provides the substrates for cellular infiltration and regeneration of axons. Together, these observations suggest that the endogenous reparative response to spinal contusion injury is substantial. Understanding the regulation and restrictions on the repair processes might lead to better ways in which to encourage spontaneous recovery after CNS injury.
            4. Hill CE, Beattie MS and Bresnahan JC (2001). Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat. Exp Neurol 171: 153-69. Department of Neuroscience, The Ohio State University, Columbus, Ohio 43210, USA. Contusive spinal cord injury (SCI) results in the formation of a chronic lesion cavity surrounded by a rim of spared fibers. Tissue bridges containing axons extend from the spared rim into the cavity dividing it into chambers. Whether descending axons can grow into these trabeculae or whether fibers within the trabeculae are spared fibers remains unclear. The purposes of the present study were (1) to describe the initial axonal response to contusion injury in an identified axonal population, (2) to determine whether and when sprouts grow in the face of the expanding contusion cavity, and (3) in the long term, to see whether any of these sprouts might contribute to the axonal bundles that have been seen within the chronic contusion lesion cavity. The design of the experiment also allowed us to further characterize the development of the lesion cavity after injury. The corticospinal tract (CST) underwent extensive dieback after contusive SCI, with retraction bulbs present from 1 day to 8 months postinjury. CST sprouting occurred between 3 weeks and 3 months, with penetration of CST axons into the lesion matrix occurring over an even longer time course. Collateralization and penetration of reticulospinal fibers were observed at 3 months and were more extensive at later time points. This suggests that these two descending systems show a delayed regenerative response and do extend axons into the lesion cavity and that the endogenous repair can continue for a very long time after SCI.
            5. Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW and McMahon SB (2002). Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416: 636-40. Sensory Function Group, Centre for Neuroscience Research, Hodgkin Building, Kings College London, Guy's Campus, London Bridge, London SE1 1UL, UK. elizabeth.bradbury@kcl.ac.uk. The inability of axons to regenerate after a spinal cord injury in the adult mammalian central nervous system (CNS) can lead to permanent paralysis. At sites of CNS injury, a glial scar develops, containing extracellular matrix molecules including chondroitin sulphate proteoglycans (CSPGs). CSPGs are inhibitory to axon growth in vitro, and regenerating axons stop at CSPG-rich regions in vivo. Removing CSPG glycosaminoglycan (GAG) chains attenuates CSPG inhibitory activity. To test the functional effects of degrading chondroitin sulphate (CS)-GAG after spinal cord injury, we delivered chondroitinase ABC (ChABC) to the lesioned dorsal columns of adult rats. We show that intrathecal treatment with ChABC degraded CS-GAG at the injury site, upregulated a regeneration-associated protein in injured neurons, and promoted regeneration of both ascending sensory projections and descending corticospinal tract axons. ChABC treatment also restored post-synaptic activity below the lesion after electrical stimulation of corticospinal neurons, and promoted functional recovery of locomotor and proprioceptive behaviours. Our results demonstrate that CSPGs are important inhibitory molecules in vivo and suggest that their manipulation will be useful for treatment of human spinal injuries.
            6. Barritt AW, Davies M, Marchand F, Hartley R, Grist J, Yip P, McMahon SB and Bradbury EJ (2006). Chondroitinase ABC promotes sprouting of intact and injured spinal systems after spinal cord injury. J Neurosci 26: 10856-67. Neurorestoration Group, Wolfson Centre for Age Related Diseases, King's College London, London SE1 1UL, United Kingdom. Chondroitin sulfate proteoglycans (CSPGs) are inhibitory extracellular matrix molecules that are upregulated after CNS injury. Degradation of CSPGs using the enzyme chondroitinase ABC (ChABC) can promote functional recovery after spinal cord injury. However, the mechanisms underlying this recovery are not clear. Here we investigated the effects of ChABC treatment on promoting plasticity within the spinal cord. We found robust sprouting of both injured (corticospinal) and intact (serotonergic) descending projections as well as uninjured primary afferents after a cervical dorsal column injury and ChABC treatment. Sprouting fibers were observed in aberrant locations in degenerating white matter proximal to the injury in regions where CSPGs had been degraded. Corticospinal and serotonergic sprouting fibers were also observed in spinal gray matter at and below the level of the lesion, indicating increased innervation in the terminal regions of descending projections important for locomotion. Spinal-injured animals treated with a vehicle solution showed no significant sprouting. Interestingly, ChABC treatment in uninjured animals did not induce sprouting in any system. Thus, both denervation and CSPG degradation were required to promote sprouting within the spinal cord. We also examined potential detrimental effects of ChABC-induced plasticity. However, although primary afferent sprouting was observed after lumbar dorsal column lesions and ChABC treatment, there was no increased connectivity of nociceptive neurons or development of mechanical allodynia or thermal hyperalgesia. Thus, CSPG digestion promotes robust sprouting of spinal projections in degenerating and denervated areas of the spinal cord; compensatory sprouting of descending systems could be a key mechanism underlying functional recovery.
            7. Pearse DD, Pereira FC, Marcillo AE, Bates ML, Berrocal YA, Filbin MT and Bunge MB (2004). cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat Med 10: 610-6. The Miami Project to Cure Paralysis, University of Miami School of Medicine, 1095 NW 14th Terrace, Miami, Florida 33136, USA. dpearse@miamiproject.med.miami.edu. Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show that cAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.
            8. de Castro R, Jr., Tajrishi R, Claros J and Stallcup WB (2005). Differential responses of spinal axons to transection: influence of the NG2 proteoglycan. Exp Neurol 192: 299-309. Developmental Neurobiology Program, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA. Spinal cord transections were performed in wild type and NG2 proteoglycan null mice in order to study penetration of regenerating axons into the scar that forms in response to this type of injury. Aside from the presence or absence of NG2, the features of the transection scar did not differ between the two genotypes. In both cases, the rostral and caudal spinal cord stumps were separated by collagenous connective tissue that was continuous with the spinal cord meninges. In wild type mice, oligodendrocyte progenitors, macrophages, and microvascular pericytes contributed to up-regulation of NG2 expression in and around the scar. Substantial amounts of non-cell associated NG2 were also observed in the scar. The abilities of two classes of spinal axons to penetrate the transection scar were examined. Serotonergic efferents and calcitonin gene-related peptide-positive sensory afferents both were observed within the lesion, with calcitonin gene-related peptide-positive axons exhibiting a greater capability to penetrate deeply into the scar tissue. These observations demonstrate inherent differences in the abilities of distinct types of neurons to penetrate the scar. Significantly, growth of serotonergic axons into the transection scar was observed twice as frequently in wild type mice as in NG2 knockout mice, suggesting a stimulatory role for the proteoglycan in regeneration of these fibers. These findings run counter to in vitro evidence implicating NG2 as an inhibitor of nerve regeneration. This work therefore emphasizes the importance of including in vivo models in evaluating the responses of specific types of neurons to spinal cord injury.
            9. Hawthorne AL, Hu H, Kundu B, Steinmetz MP, Wylie CJ, Deneris ES and Silver J (2011). The unusual response of serotonergic neurons after CNS Injury: lack of axonal dieback and enhanced sprouting within the inhibitory environment of the glial scar. The Journal of neuroscience : the official journal of the Society for Neuroscience 31: 5605-16. Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106, and Department of Neurosurgery, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or growth-inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer or cortical neurons were cultured on low amounts of laminin with or without relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of growth-associated protein-43 and/or beta1 integrin than cortical neurons. Blocking beta1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.
            10. Liu K, Lu Y, Lee JK, Samara R, Willenberg R, Sears-Kraxberger I, Tedeschi A, Park KK, Jin D, Cai B, Xu B, Connolly L, Steward O, Zheng B and He Z (2010). PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat Neurosci 13: 1075-81. F.M. Kirby Neurobiology Center, Children's Hospital, and Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA. Despite the essential role of the corticospinal tract (CST) in controlling voluntary movements, successful regeneration of large numbers of injured CST axons beyond a spinal cord lesion has never been achieved. We found that PTEN/mTOR are critical for controlling the regenerative capacity of mouse corticospinal neurons. After development, the regrowth potential of CST axons was lost and this was accompanied by a downregulation of mTOR activity in corticospinal neurons. Axonal injury further diminished neuronal mTOR activity in these neurons. Forced upregulation of mTOR activity in corticospinal neurons by conditional deletion of Pten, a negative regulator of mTOR, enhanced compensatory sprouting of uninjured CST axons and enabled successful regeneration of a cohort of injured CST axons past a spinal cord lesion. Furthermore, these regenerating CST axons possessed the ability to reform synapses in spinal segments distal to the injury. Thus, modulating neuronal intrinsic PTEN/mTOR activity represents a potential therapeutic strategy for promoting axon regeneration and functional repair after adult spinal cord injury.
            11. Lu P, Wang Y, Graham L, McHale K, Gao M, Wu D, Brock J, Blesch A, Rosenzweig ES, Havton LA, Zheng B, Conner JM, Marsala M and Tuszynski MH (2012). Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150: 1264-73. Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA. Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.
            Last edited by Wise Young; 25 Jan 2013, 12:51 PM.

            Comment


              Crabby - a few questions you need to ask yourself:

              - Why have the numerous human examples (both legitimate trials as well as pseudo experimental) of intraspinal transplants of various types of stem cells (containing MSCs and/or BMSCs) shown little or no benefit in the chronic setting?

              - Why has Dr Wise Young continued to postulate that he would like to consider adding Chondroitinase and/or Nogo to the UCBC+Lithium combination? For fun? Or is it that he believes (or believed) that the glial scar would still be a hurdle to overcome?

              - Why are the likes of Os Stewart/Zhigang He (working with PTEN/mTor) looking to collaborate with Dr Silver in order to regenerate these "proliferating" CST axons through the glial scar in a chronic setting? Why approach such an average scientist? Why don't they just skip the collaboration and stick some UCBCs into the rodents and witness that bridge in action? Actually, why bother creating a viral vector to knock out PTEN/SOCS3 when they can just grab some lithium off the shelf to activate mTOR? Maybe they are poor scientists too?

              The fact is (whether it matters to you or not), to a scientist, there is an element of speculation. Dont get me wrong, I'm looking forward to seeing the data. If it works, then great for us but don't blame scientists for doing what scientists do - be objective. If you do, we as advocates will have less of them working in our field, and less of them being so patient with us as layman.
              Last edited by Fly_Pelican_Fly; 25 Jan 2013, 10:28 AM.

              Comment


                Thanks for the explanation Wise. So I read this paper you cited below (or tried to), and to me it read as they took rats who had a transected spinal cord (which I think means it has been completely cut), put some stuff made of "fibrin matrices" that had a bunch of drugs and stem cells embedded in it, stuck that between the two halves of the severed spinal cord and nerves not only grew across it but quite a long way past it.

                So assuming I read it correctly (I may not have), intuitively you would want the ends of the spinal cord you are joining to the fibrin matrices to be free of scar tissue because that would make it easier to join to the bridging material (much like dead tissue is debrided from pressure sores to help them heal). If I interpret what you are saying correctly is that is not correct that having the scar tissue doesn't prevent the growth and in fact removing it for whatever reason is actually detrimental.

                Originally posted by Wise Young View Post
                T8burst,
                [*] Lu P, Wang Y, Graham L, McHale K, Gao M, Wu D, Brock J, Blesch A, Rosenzweig ES, Havton LA, Zheng B, Conner JM, Marsala M and Tuszynski MH (2012). Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150: 1264-73. Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA. Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.
                [/list]

                Comment


                  Originally posted by Wise Young
                  T8burst,

                  Does this help?

                  Wise.
                  T5/6, ASIA A, injured 30 Nov 08
                  Future SCI Alumnus.
                  I don't want to dance in the rain, I want to soar above the storm.

                  Comment


                    Originally posted by t8burst View Post
                    Thanks for the explanation Wise. So I read this paper you cited below (or tried to), and to me it read as they took rats who had a transected spinal cord (which I think means it has been completely cut), put some stuff made of "fibrin matrices" that had a bunch of drugs and stem cells embedded in it, stuck that between the two halves of the severed spinal cord and nerves not only grew across it but quite a long way past it.

                    So assuming I read it correctly (I may not have), intuitively you would want the ends of the spinal cord you are joining to the fibrin matrices to be free of scar tissue because that would make it easier to join to the bridging material (much like dead tissue is debrided from pressure sores to help them heal). If I interpret what you are saying correctly is that is not correct that having the scar tissue doesn't prevent the growth and in fact removing it for whatever reason is actually detrimental.
                    T8burst,

                    I am glad that you read the paper. By the way, I apologize to Paolo for my professorial glare when I post papers and he then asks questions that suggest that he has not read the paper. I forget sometimes that he is not my student and has had no training in reading scientific papers.

                    So, let me go through the Lu, et al. paper, as I would go review the paper with my students.

                    1. You should first note the journal. Cell is one of the most highly cited and rigorously reviewed journals in the the field. So, this is a prestigious journal that only publishes what most scientists would consider superb papers. I agree with their choice to publish this study.

                    2. The introduction of the paper starts by mentioning the growth inhibition theories, i.e. the myelin-associated protein and inhibitory extracellular matrix (i.e. the chondroitin-6-sulfate proteoglycans or CSPG) theories. It also talks about the lack of "positive environmental stimuli such as growth factors", which they were too modest to announce as a theory, but what I brazenly call the neurotrophic theory of regeneration (for them). They mention the neuron-intrinsic mechanisms that contribute to axon growth failure (e.g. the cAMP and the PTEN/mTOR theories).

                    3. To assess which of these theories are correct or wrong, they developed this experiment where they transected the spinal cord and then bridged the transection site with neural stem cells placed in a fibrin matrix to hold the cells and to release neurotrophins. The transplanted cells were neural stem cells that were genetically modified to express green fluorescent protein (GFP), so that they and their axons can be readily seen without staining or labelling.

                    4. The transplanted neural stem cells formed neurons that sent axons that grew straight through the glial scar (which forms in the two spinal cord stumps that interface with the fibin matrix), grew long distances in the proximal and distal spinal cords, reconnected with neurons, and improved functional recovery.

                    5. This was not just a few axons. Thousands of GFP axons poured across the glial scar (I have no problems calling this a glial scar because I know that there are both fibroblasts and glial cells that have formed a barrier in the two stumps), disproving the glial scar theory. The axons also had no difficulties growing long distances in white matter (myelinated tissues) and thereby disproved the Nogo and myelin-based axonal inhibitor theory.

                    6. The regeneration was blocked by rapamycin, a drug that blocks mTOR. In fact, mTOR stands for mammalian target of rapamycin. PTEN deletion stimulated Akt and Akt stimulates mTOR. This is the mechanism by which PTEN works to stimulate axonal growth although figure 4 suggests that blocking mTOR only reduces (i.e. does not stop) all the axon growth. So, this experiments suggests strongly that mTOR plays a partial role in the axonal growth. Of course, they placed neurotrophins in the fibrin matrix.

                    7. The treated rats significantly improved their locomotor function, as measured by BBB scores. In figure 5, note that the BBB scores are not particularly impressive, i.e. only went to 6-7 (which are non-walking scores) but nevertheless are significantly different from controls non-treated transected rats (scores of 1-2). They did neurophysiology (i.e. stimulate the spinal cord at one side of the transition and recorded responses in the other side) and showed that the transplanted rats had responses that crossed over to the other side while the controls did not. This strongly argues that axons not only crossed over but made synaptic contacts, accounting for the neurophysiological responses and the BBB scores.

                    8. Finally, they re-transected the injury site and showed that all these neurophysiological and behavioral effects of the transplantation went away, indicating that the regenerated fibers were responsible for the recovery. Figure 6 shows the impressive number of axons that grew from the GFP (green fluorescent protein) expressing cells transplanted into the injury site. They stained the transplantation site with NeuN and found many mature neurons produced by the transplanted neural stem cells. Incidentally, they tried two neural stem cell lines, including one line that was neurally induced form a human embryonic stem cell line.

                    9. Please remember that they are not looking at the host rat's axons but the GFP axons of the transplanted neural stem cells. In fact, this is a real problem for the authors to explain... why are the rats walking? Host axons from the rat must be going into the injury site and making synapses with the transplanted neurons and thereby activate them to produce movement.

                    10. Lu, et al. concluded that properties intrinsic to early stage neurons are sufficient to overcome barriers to growth in the adult CNS. They also pointed out that transplants were effective even when delayed for a week after injury and still restored significant function. Finally, they pointed out that the human embryonic stem cell line that they employed is being tested in a human clinical trial of amyotrophic lateral sclerosis.

                    Does this help?

                    Wise.

                    Comment


                      Originally posted by mcferguson View Post
                      Thanks. Sorry, I accidentally moved my post to T8burst and therefore your post seems as if it occurred before the one that you were referring to. Wise.

                      Comment


                        5. This was not just a few axons. Thousands of GFP axons poured across the glial scar (I have no problems calling this a glial scar because I know that there are both fibroblasts and glial cells that have formed a barrier in the two stumps), disproving the glial scar theory. The axons also had no difficulties growing long distances in white matter (myelinated tissues) and thereby disproved the Nogo and myelin-based axonal inhibitor theory.

                        How does this disprove the glial scar theory? Again, I reiterate, the glial scar is not an impenetrable barrier but a very potent one. You argue that it is an almost non-existant one. In this paper the authors take extreme measures to overpower the still forming scar with the use of immature neurons which have a robust intrinsic growth potential. In addition you fail to mention in your review of the paper that they also use a cocktail of about a dozen neurotrophins and other growth factors to encourage growth of the axons. At the recent Society for Neuroscience meetings Paul Lu showed some new experiments from the Tuszynski lab using the same strategy in clearly chronically lesioned animals with more established scar. Some fibers can still get across however, the numbers of regenerating fibers is now markedly reduced. If one carefully transplants fully adult sensory neurons rostral to a scar the axons grow beautifully until they reach the scar and then stop abruptly. They cannot get through. I'm sure you know that work from our lab quite well. The questions still remain, have you demonstrated in work from your own lab or can you show from the work of others that at least in vitro that LI or UMBC can overcome inhibitory molecules known to be present in the scar that curtail axonal growth? If there is not even evidence in vitro that this combination is sufficiently potent then maybe we should be more tempered in our hope for robust regeneration over long distances in vivo.

                        Comment


                          Dr. Tuszynski discusses the findings reported in the journal "Cell" that showed extensive growth of axons in spinal cord injury using grafts of early stage neural stem cells at the Working 2 Walk 2012 Science and Advocacy Symposium.



                          Video of the Question & Answer Session following presentations by Drs. Hans Keirstead and Mark Tuszynski with Keirstead and Dr. Lu.

                          http://spinalcordresearchandadvocacy.wordpress.com/

                          Comment


                            Originally posted by Wise Young View Post
                            T8burst,


                            So, let me go through the Lu, et al. paper, as I would go review the paper with my students.

                            4. The transplanted neural stem cells formed neurons that sent axons that grew straight through the glial scar (which forms in the two spinal cord stumps that interface with the fibin matrix), grew long distances in the proximal and distal spinal cords, reconnected with neurons, and improved functional recovery.

                            5. This was not just a few axons. Thousands of GFP axons poured across the glial scar (I have no problems calling this a glial scar because I know that there are both fibroblasts and glial cells that have formed a barrier in the two stumps), disproving the glial scar theory. The axons also had no difficulties growing long distances in white matter (myelinated tissues) and thereby disproved the Nogo and myelin-based axonal inhibitor theory.


                            Does this help?

                            Wise.
                            Dr. Young,

                            According to this study (not sure how reputable it is), glial scar is not really formed until around 4 weeks post injury in both rats and dogs. So while the Lu/Tuszynsky paper showing new neuronal relays when given after 2 weeks post injury is getting closer towards a chronic time point, it is not. I would love to see the treatment given 8 weeks after injury to mitigate all doubt. So I'm not so sure that the Paul Lu paper can be used to show that glial scar is not inhibitory to axon growth.

                            The publication in the link was contusion model, while Lu/Tuszynsky paper was on transection, so I'm not sure how that affects anything on timing of scar formation.

                            http://thejns.org/doi/full/10.3171/2010.3.SPINE09190




                            Comment


                              I am deeply disappointed with the progress and the science that has been done in spinal cord regeneration. Even in rats and larger mammals, the debate over mechanisms and strategies are all over the place. There seems to be a ton of work to do that will take longer than I hoped. Yes things grow but protocols for organized regeneration are nowhere near answered. Money is not there and the investment has no immediate upside, so private and public corps steer away. Geron got close but in the end , it was a publicity stunt just to be first to attract money. Money used for other projects that return investments faster. Human Clinical trials with a real treatment success are not even close. All studies show one or two things of a 10,000 piece puzzle. Where are we really? Grafting strategies at Jerry Silvers lab? With zero motor/sensory?
                              I just hope there are more scientist that stand alone with new ideas. Like the one that believed the world was round and everyone else thinking it was flat. Until you prove with complete recovery, you are not asking the right questions or possessing correct answers.
                              Han: "We are all ready to win, just as we are born knowing only life. It is defeat that you must learn to prepare for"

                              Comment


                                Originally posted by Fly_Pelican_Fly View Post
                                - Why have the numerous human examples (both legitimate trials as well as pseudo experimental) of intraspinal transplants of various types of stem cells (containing MSCs and/or BMSCs) shown little or no benefit in the chronic setting?
                                I'm not discounting what you say here. I'd have to see these examples of legitimate trials to better comment on this. How long ago were the trials done? Were the procedures similar to what they are doing now in China?

                                UCBMC alone probably would not very well help chronic SCI walk again. Lithium alone is not going to help chronic SCI walk again. Rehab alone is not going to help chronic SCI walk again. The combination of the three could help chronic SCI walk again. I think this therapy, if shown to work, is in its infancy and is going to evolve and become more effective as scientists figure out what is going on.

                                Originally posted by Fly_Pelican_Fly View Post
                                - Why has Dr Wise Young continued to postulate that he would like to consider adding Chondroitinase and/or Nogo to the UCBC+Lithium combination? For fun? Or is it that he believes (or believed) that the glial scar would still be a hurdle to overcome?
                                Chicken noodle soup and Vitamin C might effectively remedy a cold. Chicken noodle soup, Vitamin C and zinc might better effectively remedy a cold.

                                I don't know what all is inhibiting axonal regeneration across the injury site. I suspect it's a combination of things. Perhaps chondroitinase effectively deals with one of these inhibitors. Perhaps a combination of UCBMC+Li+ch'ase would be more effective than UCBMC+Li alone. This is something I would think would be worth researching.

                                Originally posted by Fly_Pelican_Fly View Post
                                - Why are the likes of Os Stewart/Zhigang He (working with PTEN/mTor) looking to collaborate with Dr Silver in order to regenerate these "proliferating" CST axons through the glial scar in a chronic setting? Why approach such an average scientist? Why don't they just skip the collaboration and stick some UCBCs into the rodents and witness that bridge in action? Actually, why bother creating a viral vector to knock out PTEN/SOCS3 when they can just grab some lithium off the shelf to activate mTOR? Maybe they are poor scientists too?
                                Careful now. I did not say that Jerry Silver is a poor scientist. Part of science is remaining open-minded. I think too often scientists get married to a particular worldview. A five-star chef might not be making the best bread he could be making if he wasn't using the best ingredients available to him. The outcomes of Dr. Silver's current therapeutic interventions involving cutting into the spinal cord have been poor.
                                Originally posted by jsilver View Post
                                It is absolutely clear that at acute stages following spinal cord injury, a variety of different types of stem cells can potently and therapeutically affect the inflammatory cascade that leads to secondary damage.
                                Seems like a pretty damn good ingredient. What else could they potently and therapeutically affect? At what stages? Why not explore this further? Why not try stem cells and peptides? I don't get it. I think Jerry Silver is a great scientist. I would like for his outcomes to be great, too.

                                Originally posted by Fly_Pelican_Fly View Post
                                The fact is (whether it matters to you or not), to a scientist, there is an element of speculation. Dont get me wrong, I'm looking forward to seeing the data. If it works, then great for us but don't blame scientists for doing what scientists do - be objective. If you do, we as advocates will have less of them working in our field, and less of them being so patient with us as layman.
                                I agree with you.
                                Last edited by crabbyshark; 25 Jan 2013, 5:22 PM.

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