Bacterial enzyme aids recovery

I have posted before on the Glial scar and an inhibitory component of this, chondroitin sulphate proteoglycans, or CSPGs. The Glial scar is a particular consequence of CNS injury but CSPGs are also found everywhere in the extra cellular matrix (ECM) of the central nervous system. CSPGs have many known and unknown functions. For example, they form lattice-like nets around synapses between cells to stabilise these signalling junctions. They are likely to influence how cells move around the CNS, thus playing an important part in repair by resident stem and precursor cells which need to migrate to the area in need of repair, and they may harbour important molecules that sustain cells. Recently, two putative receptors have been identified giving insight into how CSPGs might act at the molecular level.
The inhibitory aspects of CSPGs are most problematic. The bacterial enzyme chondroitinase ABC (ChABC) degrades CSPGs, and experimentally, it has been shown to have a number of reparative effects making it a very promising treatment option for SCI. Nevertheless, there are concerns relating to how we should deliver this bacterial protein in the clinic. For one, ChABC activity is short-lived and therefore repeated injections or delivery by intrathecal catheter is needed which is unattractive.

Gene therapy approaches may provide the answer. Modified viruses have long been used as a tool to carry inserted genes into cells, “infecting” them with a copy of the gene which in turn causes the cell to produce (express) the protein. Obviously, concerns surround gene therapy as well, but the prospect of only needing a single administration of a therapeutic viral vector which gives rise to long-term expression of the therapeutic gene is compelling.

A number of groups are looking at a gene therapy approach for the delivery of chondroitinase. K Bartus (#892.21) presented a poster in the last session of the SfN meeting which got much attention. The poster presented data on sustained and widespread degradation of CSPGs following a single early injection of a lentiviral vector carrying a gene of ChABC optimised for expression in mammalian cells. The treatment led to a dramatic reduction of lesion pathology: sparing of neuronal tissue and dramatic reduction in cavity volume. The treatment also significantly improved signal conduction across the injury site coupled with improved performance on a horizontal ladder test.

With such sustained and active remodelling of the CSPG content of the cord there was a worry that there might be adverse effects such as development of pain syndromes. The authors tested for this and found no evidence of heightened sensitivity in the treated group of animals. The injury site was heavily vascularised with many tissue bridges associated with numerous axons.

Perhaps the most striking aspect of this study was that it was done in a contusion injury model, the most clinically relevant model of injury.

Recovery of bladder function worth the wait

One of the major clinical problems after complete SCI is the so-called neurogenic bladder. In this state, there is a lack of coordinated activity between the bladder and the external urethral sphincter which results in decreased voiding efficiency and increased pressure within the bladder. There is still relatively limited research into regenerative approaches to this important problem.

Yu-Shang Lee (#562.10) wanted to examine whether using a peripheral nerve graft to circumvent a transection injury (at the T8 level), in combination with chondroitinase and acidic fibroblast growth factor (aFGF) treatment would restore functional reflexes of the bladder. As before, the group implanted the ends of the nerve graft either side of the injury having first injected these sites with chondroitinase. The chondroitinase helps regenerating axons to both enter the graft and leave again at the other end. The fibroblast growth factor transformed astrocyte cells into a more elongated form – helping to support directional organised regeneration of axons. The group performed a number of treatment and control experiments and monitored bladder function over 6 months. All groups displayed bladder dysfunction initially. However, over the course of 6 months the full treatment group (nerve graft + chondroitinase + growth factor) began to show markedly improved function. At 6 months this treatment group had lower residual volume in the bladder after voiding, normalised bladder pressure and voiding intervals. Lower volumes and pressures would indicate less incontinence and distension - the latter being the most common trigger for autonomic dysreflexia.

The timeframe of recovery (many months) would suggest regeneration was involved. To test this hypothesis they recut the nerve graft and found the improved bladder function was abolished. Furthermore, tracing studies established that the regenerating axons had come from regions of brain normally associated with bladder control.

Recovery of function using nerve bridges

I wrote earlier on biomaterials. This fits nicely with a poster I saw today from the Silver lab (Case Western University, Ohio) who have been using peripheral nerve (PN) bridges to circumvent a complete hemisection injury of the cervical cord. Such an injury interrupts signals from respiratory control system in the brain to motor neurons in the spinal cord that drive breathing. The result in this injury is paralysis of the diaphragm on one side. It is an excellent system to investigate both regeneration (through the grafted nerve bridge) and plasticity, which is to say, the reorganisation of the connections in the respiratory system after injury. The group published earlier in the year on their findings demonstrating successful long-distance regeneration through the bridge and out again into the cord tissue below the injury where they connect with respiratory circuits. This new circuitry could be “trained” to restore breathing.

Obviously the loss of the peripheral nerve in patients with high cervical injuries with poor respiratory function is an insignificant concern when compared with the possibility of restored breathing but the clinical potential may be tempered if peripheral nerve bridges are to be used for those with lower injuries.

Unfortunately, sourcing from donor tissue would be associated with immune response issues. Recently, a group led by Schmidt (an author on the poster) have developed a technique that can take peripheral nerves and strip them of all the cells leaving only the structural proteins and extracellular matrix (ECM) as a lattice-like scaffold. The ECM is important as it acts as a permissive surface over which cells and axons can grow. The value of this technique is that donor tissue can be used as it will not illicit an immune response now that the cells have been removed. In their poster (#441.01), the group compared the acellular bridging grafts with standard nerve grafts for their ability to support axonal growth after a cervical C3/C4 hemisection injury. This type of injury allowed them to assess forelimb function which they did in a variety of ways. Immediately after injury they found animals in all groups lost the ability to use the affected limb during vertical exploration. Recovery in both the acellular and conventional nerve graft groups was seen and similar. In one test This is an important new finding as it suggests there is clinical potential in this donor-sourced material in SCI repair strategies.

Old concepts revisited and new models

The Tuszynski lab went a little old school in a couple of papers presented during today's morning session. Verena Haringer (#158.10) from the group presented on the properties of cells they had isolated from rodent embryonic spinal cord tissue. Tissue from the cord was dissociated and progenitor cells isolated and grown up. They found these cells would spontaneously become neuronal cell lineages. They could manipulate these to form an excitatory class of neuronal cell which they hoped might form connected neuronal circuit relays after transplantation to re-establish signal transmission lost after trauma to the spinal cord. Despite the tendency of these embryonic cord progenitor cells to turn into neutrons in culture, unfortunately when transplanted into the injured cord they formed non-neuronal cell types (predominately of a glial lineage). One key experiment that needs to be done is to graft these cells into the uninjured cord to see if it is biological signals arising from the damage that ensures this fate or something more general to the in vivo setting.

Switching tack just slightly, Paul Lu (#158.11) from the same group took tissue from E14 rats (embryonic cord tissue) and grew up neuronal cells which they grafted (via injection) into a complete transection (T3) injury. One of the key questions they were asking was, do neurons have an intrinsic capacity to overcome the inhiitory injury environment of the adult CNS? After injection, cells survived and matured, but did not migrate. These grafted cells sent out large numbers of axonal projections over remarkably long distances, both up and down the cord from the injection site. They studied possible mechanisms and established that mTOR signalling was at least partially involved in this rebust ability to grow long axons. Importantly, they found evidence for integration and synapse formation between host and graft cells as well as electrophysiological evidence that these synapses functioned appropriately. As a result the animal recovered some hindlimb function. One of the advantages of this work was te availability of transgenic strains that allowed the graft cells to come pre-labelled with a flourescent marker making identification of the graft cells more positive.

There were also a few posters describing the use of pigs as a model for SCI. Much has been learned from our study of the consequences of injury in the rodent but some in the field are now using pigs to help establish and refine surgical procedures and confirm safety etc.. The pig is significantly larger than the rodent and therefore closer in anatomical scale to humans. It also has a greater volume of cerebral spinal fluid (CSF) and amenable to training so that functional outcomes can be assessed. Brain Kwon (J. Lee #160.04) and others are developing standard outcome scores so that severity of injury and recovery can be better assessed. The use of this larger species may prove to be a useful translational tool.

Washington plays host to SfN 2011

Official SfN 2011 Header

It has been a whole year and once again the Society for Neuroscience annual meeting decends on another major US city - this time Washington , DC.

Each year I try to give a flavour of the meeting and write about some of the interesting things I see at the meeting. Washington at this time of year is looking pretty good; cold but sunny. Now the meeting has started in earnest, though, there will be little time to appreciate this as most of the day will be spent underground in the large Hall C where the exhibitors and scientists do there stuff.

Today we had two poster sessions devoted to spinal cord injury. Homework done, I headed off to take a look at a number of papers looking at the potential of various biomatrials in SCI. SCI causes many problems including neuronal cell death, damage to the axons that convey information up and down the cord from the brain to the body and back again, Glial scar formation as well as up-regulation of scar-like material in areas surrounding the injury site. This makes repair and regeneration of the spinal cord very difficult. Another important clinical consequence of SCI is often the formation of large (cystic) cavities. It is proposed that biocompatible materials may help to overcoming this and other problems associated with SCI. With biomaterials, for instance, it may be possible to create a scaffold structure through which regenerating nerves may grow. They may be able to bridge the cystic cavity which are otherwise no-go areas for regrowing axons.

They can have other properties which we might be able to utilise. There is no one biomaterial. Indeed, the term "biomaterials" really is just a generic description of a class of materials that are biocompatible. They are often polymers - chains of single units or groups of units that are strung together. The type of units that these polymers are made of dictates their properties. They may be naturally occuring or man-made. On top of that they may be stable and permanent or biodegradable. The science and use of biomaterials in many medical fields is advancing and there were certainly quite a few posters describing the effects of biomaterials in SCI today.

J. Hyun (presentation number #64.12) presented on a 3D glass fibre scaffold implanted into the gap created by the complete transection of the spinal cord. The glass fibre was created in such a way to incorporate collagen to act as a suitable base material over which regenerating axons could grow. They found improvements in locomotor function (as determined by the BBB scale).

The biomaterial described above was pre-formed before implantation but a number of investigators in this session have been examining materials that can be injected as liquids before turning into more solid structures within the tissue. T. Novosat (#64.13) described a “reverse thermal gel” which, simply put, is a material that undergoes a change in structure as the temperature changes. It is described as “reverse” because it is liquid at low temperatures but becomes gel-like at higher temperatures such as body temp. This property is particularly useful as it can be injected at a low temperature as a liquid, spread like a liquid and as its temperature increases within the tissue it becomes gel-like. One can imagine therefore it integrating into the tissue much better and offering a substrate on which axons and other cells can now regrow. The purpose of the study was to check the biocompatibility of the material which they reported as good, eliciting no obvious adverse effects.

L. Conova (#64.14) used a very similar concept but pre-mixed their liquid polymer with the neurotrophin BDNF (growth supporting molecule) and or fibroblast cells to look at the effect the biomaterial had on graft cell survival rates. As above, they did not report any adverse effects from the injected material and provided evidence that cells survived better when delivered in the biomaterial. The material also appeared permissive for axon growth when delivery included the neurotrophin BDNF.

Y. Liu (#64.15) from the Fehlings lab, used a novel small peptide that on injection self-assembles to form fibres “that resemble the native” cord microstructure. Injection of this material lead to dramatically less neuronal cell death, a reduction of Glial scarring and inflammation and a significant promotion of axonal preservation and regeneration. They also reported improvements in the quality of the electrical signal carried along axons and better functional outcomes.