REGENERATING A DAMAGED SPINAL CORD

Unlike most cells in the body, cells from the spinal cord are unable to spontaneously regenerate, meaning they lack a mechanism allowing them to replace dead or damaged cells independently.

An injury to the spinal cord is usually caused by a contusion injury (a region of injured tissue or skin in which blood capillaries have been ruptured; a bruise) from a blunt force trauma at the cervical/neck level, for example as a result of a car crash, resulting permanent sensory, motor and autonomic impairment alongside possible paralysis, loss of coordination, hand function and sensation, sexual dysfunction and secondary complications involving bladder and lung infections and spasticity.

Animal models have been crucial to understanding the spinal cord, allowing us to mimic a human spinal cord’s functional and behavioural traits in peer reviewed articles, in particular in mice, rats, cats, primates and dogs, all allowing the study of treatments, physiology and injury pathology of a spinal injury.

The injury pathology of a damaged spinal cord in a mouse represented in the image above represents a dense scar tissue forming at the site of impact through progressive degradation due to the inability to regenerate the adult central nervous system is due to 3 main mechanisms; 1) Nogo – a molecule found within the myelin sheathe of nerve fibres in the CNS leading to inflammation and blocking axonal growth, 2) Formation of glial scars resulting in cyst formation and 3) Chondroitin sulfate proteoglycans which limit regeneration and neuroplasticity (continuous processing allowing short-term, medium-term, and long-term remodelling of the neurosynaptic organization).

NOGO-A in the adult CNS acts as a negative regulator or neuronal growth exerted on myelin in the mammalian brain and spinal cord and therefore, stabilizing of the CNS at the expense of neuroplasticity, the formation of new neural connections, post-injury. The neutralization or blockage of NOGO-A enhances regeneration and neuroplasticity. This is achieved through monoclonal antibody production, which can then be injected and bind to NOGO-A receptor sites.

Recently we have seen optimism regarding promoting regeneration from Kings College research using chondroitinase, a bacterial enzyme which works to break down proteoglycan sugars making the extracellular matrix more permissive (illustrated to the right) enabling more growth and neuroplasticity, resulting in reduced tissue damage from cell death, enhanced growth and functional recovery, hence improvements in coordination and limb function. However, the enzyme degrades quickly, meaning for an effective treatment the patient will need to receive a prolonged stable delivery to a widespread area for the whole spinal cord.

chABC (chondroitinase ABC enzyme) digests Chondroitin sulfate proteoglycan chains making the extra-cellular matrix more permissive for neuroplasticity and can therefore overcome CSPG-mediated inhibition however is denatured at 37 °C therefore necessitating repeat injections or local infusions over a long period of time which is generally invasive, infection prone and clinically problematic.

To overcome this problem gene therapy is applicable in order to insert the bacterial enzyme into the human genome, this requires a lentiviral vector from the human immunodeficiency virus (HIV-1). This is used as it has the ability to mediate potent transduction (where foreign DNA is introduced to a cell via a viral vector) and stable expression into dividing and non-dividing cells in vivo (within a living organism) and in vitro (outside a living organism). The virus is usually produced in HEK 293T cells and the essential lentiviral gene must be expressed in the cell to allow the general of lentiviral particles, usually expressed by several plasmids, 3 examples all w different functions.

After culturing a sample can undergo cell fractionation where the cells are homogenised and then placed in an ultracentrifuge to take the supernatant to transduce the target cells, this viral RNA is reverse transcribed and then imported into the nucleus, allowing it to be stably integrated into the host genome.

However in clinical practice we are unable to regulate control over gene expression, therefore the application of a dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulation, effectively an on/off switch, allowing removal of treatment after removal of doxycycline, therefore allowing short and long term gene therapy options in treatment exemplified in a rat model, however not read for human trials as a small part of the gene remained active even when switched off, therefore requiring further development and trials in larger species before it can be introduced as a viable treatment. This helped mitigate the issue of constant release of active chondroitinase, meaning excessive growth and neuroplasticity leading to additional complications, worsening the patients’ condition.

The pathology of the immune response for regenerating the spinal cord has been modelled in a mouse, presenting that axons could now conduct ‘really well’ after repair after 8 weeks of chondroitinase treatment represented by increased coordination and motor neuron skills, regaining the ability to pick up sugar treats placed in front of the mouth through a small window in which the mouse would have to reach and grab, hence representing the long term gene expression had resulted in massively increased neuroplasticity and advanced coordinated motor neuron ability. This also represents the importance of animal models allowing scientists to mimic human behavioural and functional traits in order to study effects of treatments and other stimuli effectively.

References:

https://www.medicinenet.com/script/main/art.asp?articlekey=40362
https://www.spinalcord.com/blog/what-you-need-to-know-about-spinal-cord-regeneration
https://www.sciencedaily.com/releases/2015/03/150312173806.htm
http://www.jneurosci.org/content/34/14/4822.short
https://www.nature.com/articles/nrn2936
https://www.pnas.org/content/107/8/3340
https://www.nature.com/articles/35000219
https://academic.oup.com/brain/article/141/8/2362/5036378
https://www.sciencedaily.com/releases/2018/06/180614213734.htm
https://www.ncbi.nlm.nih.gov/pubmed/18418947
https://www.ncbi.nlm.nih.gov/pubmed/8602510
Naldini et al., 1996; Hendriks et al., 2007
https://www.invivogen.com/sites/default/files/invivogen/old/docs/Insight_201004.pdf?PHPSESSID=a81552686acd6275994a013320f5602c

Photo Credits due to: https://now.tufts.edu/articles/hope-spinal-cord-injuries

Arjun Patel