CELL THERAPIES FOR SPINAL CORD INJURIES IN DOGS: WILL IT WORK?

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1 Vet Times The website for the veterinary profession CELL THERAPIES FOR SPINAL CORD INJURIES IN DOGS: WILL IT WORK? Author : NICOLAS GRANGER, ROBIN FRANKLIN, NICK JEFFERY Categories : Vets Date : March 16, 2009 NICOLAS GRANGER, ROBIN FRANKLIN, NICK JEFFERY explain how their research into spinal cord injuries could have a dramatic influence on both human and veterinary medicine FEW injuries that afflict humans are more devastating than those associated with spinal cord injury (SCI). As vets, we know this also applies to dogs, since we have all had to face the distress caused to dogs and their owners by SCI. These injuries in humans have a high economic and emotional cost: there are around 800 new cases of SCI per year in the UK. Young adults are affected disproportionately, meaning that each patient requires many decades of care the full economic cost is calculated at around 500m per annum for a total of around 30,000 patients living with SCI. The financial and emotional cost of treating dogs with SCI is also a major problem, which often results in euthanasia of the animal. For both dogs and humans, treatment options for SCI are limited to surgery for compressive lesions and supportive care, including rehabilitation and physiotherapy, but these are not sufficiently powerful to restore adequate function to the most severely injured categories of patients. 1 / 19

2 Hopefully, this rather dark picture can now be brightened by developments in SCI research. In the past few years, many interventions have been shown to be realistic treatment options for SCI in humans, and it is probable that cell transplantation will be one of the key components. Current front-runners among cell transplant options are olfactory ensheathing cells (OECs; Figure 1a). Olfactory neurons regenerate throughout mammals lives, and their new axons are supported by the OECs, which are found only in the olfactory system. OECs have many unique properties, and are able to form a bridge between the central nervous system and the peripheral nervous system. Our clinic has received major support from the Medical Research Council to conduct a phase-two clinical trial in dogs with SCI. The aim is to repair the spinal cord with intraspinal injections of OECs, which should give great hope to both human and canine patients with SCI. The trial commenced in October 2008 and will run for three years. Here, we aim to provide a brief background of previous work conducted in our clinic on SCI in dogs, and then outline the current clinical trial, its importance and our expectations. History Despite advances in medical and surgical care for dogs and humans, the current clinical therapies for severe SCI are largely ineffective. Intraspinal transplantation of OECs has been associated with significantly improved functional outcome after a variety of experimental spinal cord lesions in rodent models in many laboratories throughout the world. Our group has previously demonstrated that canine and human OECs can easily be cultured in the laboratory, thus extending the possibility of intraspinal transplantation to those species. We naturally began to translate this work into a clinical context and, in 2005, demonstrated that autologous olfactory glial cell transplantation was reliable and safe in naturally occurring canine SCIs. Nine paraplegic dogs were included in a prospective trial and underwent olfactory bulb biopsy (to obtain OECs), followed by spinal cord transplantation of the cells. However, no control dogs were evaluated, since such a procedure would require the use of experimental dogs, rather than naturally occurring cases. The primary aim was to determine the safety and feasibility of the techniques, rather than to assess the benefits of the procedure. Further work subsequent to this first (phase one) trial has now established that OECs can be harvested and cultured simply and effectively from the olfactory mucosa (nasal cavity or frontal sinus) in dogs, thus avoiding the risks associated with accessing the olfactory bulb (Figure 1b) altogether. We have further developed a new surgical approach (through the temporal bone) that allows a minimally invasive biopsy of the frontal sinus mucosa (Figures 1b and 1c). Lastly, the injection of OECs is made three weeks after the biopsy, which is the time needed to obtain a sufficient number of 2 / 19

3 cells in culture. This injection is made percutaneously under fluoroscopic guidance another technique developed for this project (Figure 1d). New tools for new treatments Over the past few years, we have set up and validated methods to quantify the functional outcome after spinal cord therapies in dogs. They consist of three clinical tests: motion analysis (kinematics), cystometry and recording of evoked potentials. Motion analysis is obtained by recording a dog s gait while it walks on a treadmill (Figure 2a). Infrared cameras detect the movement of reflective markers placed on the dog s skin (Figure 2b). The information generated from this is transferred to a computer to provide a model of the dog walking (Figure 2c) or a graph (Figure 2d). Each point of the graph represents the position of one marker in a given plane (x, y or z) at a certain point in time. Using the set of data derived from each marker, the position of each limb in space can be compared to any other at any time point. In a practical sense, this is very useful, as it allows evaluation of the coordination between the thoracic and pelvic limbs a fundamental prerequisite to assess recovery in dogs with thoracolumbar spinal cord injuries. During cystometry, a urethral catheter is placed to infuse saline into the bladder and measure the pressure during filling. In a normal animal, the pressure slowly increases to the point at which the detrusor will contract, thus initiating micturition (Figure 3a). In paraplegic patients with thoracolumbar injuries, this reflex is exaggerated, because there is no higher centre control a phenomenon called detrusor hyper-reflexia (Figure 3b). Therefore, any recovery of spinal cord autonomic function can be measured with this technique by documenting the disappearance of detrusor hyperreflexia. Finally, evoked potentials are electrical potentials generated by the brain or along the spinal cord. The function of motor pathways is assessed with magnetic motor-evoked potentials (MMEP), in which magnetic stimulation is applied to the motor cortex and the response below the spinal cord lesion is recorded by needles placed in hindlimb muscles (Figures 4a and 4b). Similarly, the function of sensory pathways is assessed by stimulating a peripheral nerve below the spinal cord lesion, and recording somato-sensoryevoked potentials (SSEP) over the brain with subcutaneous needles (Figures 4c and 4d). These three tests have the major advantage that they can be conducted identically in humans and dogs, which makes direct translation of the results from one species to another possible. They are non-invasive, nonpainful and functionally relevant. Patient benefit 3 / 19

4 The expertise in OECs culture, remyelination of the central nervous system, clinical neurology and/or neurosurgery, and outcome measures after SCI therapy have finally been gathered into a unique prospective clinical trial. During the trial, one of two treatments (both known to give functional benefits) will be randomly allocated and injected directly into the spinal cord s damaged region. An MRI scan will be used to determine the epicentre of the lesion before percutaneous injection under fluoroscopic guidance. Outcome measures include motion analysis, cystometry and recording of MMEP and SSEP by a blinded researcher every month for six months, and again at 12 months. Entry into the trial is restricted to dogs that: weigh less than 20kg; have a suitable temperament to comply with the procedures; have a lesion located between T3 and L3, caused by an acute traumatic episode either fracture and/or luxation or intervertebral disc extrusion; have reached a static (for three months) and unacceptable stage of neurological recovery (usually complete loss of motor and sensory function to the pelvic limbs and incontinence); and have owners that are able to return the dog for repeated functional evaluation. This constitutes the first clinical trial carried out in SCI therapy in a large non-human mammal species, thus offering real hope to dogs with unacceptable recovery after SCI and allowing subsequent translation of the results into human medicine. Autologous OEC transplantation in human paraplegia was started in 2005 in Australia using six patients (three transplants and three controls) afflicted with severe spinal cord injury. The results were published in 2008, with the aim of demonstrating the safety of the procedure over time, before largerscale trials. One patient had improved sensory function. Can you help? Power calculations suggest that, using animals that have reached a plateau of unacceptable recovery at three months, a 25 per cent difference in recovery between two groups would be detected with a power of 0.8, using as few as 54 dogs. We must recruit that number of dogs over three years and hope that as many colleagues as possible will join the project with their cases. The treatment is free to the owners, and the project involves a veterinary neurologist and a veterinary nurse both are working full time on the project. 4 / 19

5 Looking forwards This project constitutes a major step forward for veterinary clinical research because it fuses fundamental neuroscience with veterinary medicine to enable the development of novel veterinary therapies. The results of our trial, whether beneficial effects from the transplant are recorded or not, will have a major impact on SCI research. Importantly, it will demonstrate that dogs can be valuable patients for clinical research and that vets can provide biomedical data that is of value to human physicians and basic scientists as well as for our own and our patients benefit. For more published Veterinary Times articles on spinal cord injuries, visit 5 / 19

6 Figure 1a. Olfactory ensheathing cells are specifically stained in red using an antibody against the nerve growth factor receptor they are typically spindle shaped (arrows). Fibroblasts are stained in green using a fibronectin antibody. 6 / 19

7 Figure 1b. A schematic representation of the frontal sinus relative to the olfactory bulb in a dog, demonstrating possible easy access to the frontal sinus mucosa (arrow). FS = frontal sinus, NC = nasal cavity and OB = olfactory bulb. 7 / 19

8 Figure 1c. A new surgical approach (through the temporal bone) has been developed to allow a minimally invasive biopsy (arrow) of the frontal sinus mucosa (blue triangle). 8 / 19

9 Figure 1d. A comparison between a lateral radiograph (upper section) and MRI (lower section) of a dog s thoracolumbar vertebrae and spinal cord. Olfactory ensheathing cells are now injected percutaneously under fluoroscopic guidance (arrows). 9 / 19

10 Figure 2a. To record the gait, the dog is walked on a treadmill. The hindlimbs of paraplegic dogs are supported by a sling, which is placed under the abdomen, thus allowing the vertebral column to be held horizontally. Reflective markers are placed on the skin over selected bones (see arrow). 10 / 19

11 Figure 2b. Infrared cameras detect the movement of the reflective markers. 11 / 19

12 Figure 2c. The position of each marker placed on the dog can be translated on to a computer screen as a coloured dot, thus giving a digital model of the dog walking. 12 / 19

13 Figure 2d. The variation over time of the position of a given marker in a plane (x, y or z) can then be represented as a graph. In this example (a normal dog), the upper trace in blue represents the movement of the right front paw marker over time in the x plane. The bottom trace, in green, represents the corresponding movement of the right hindpaw. Walking movements are cyclical, generating these sinusoidal curves. Curves can be compared mathematically to facilitate an analysis of the coordination between the frontlimbs and hindlimbs. 13 / 19

14 Figure 3a. During cystometry of a normal dog, the bladder pressure slowly increases as it is filled. When the pressure exceeds a certain value (around 20cmH2O in male dogs, for example), micturition will start (arrow). 14 / 19

15 Figure 3b. In paraplegic patients with thoracolumbar lesions, there are premature and unexpected contractions of the detrusor called detrusor hyper-reflexia (see schematisation, including red curves). Those contractions are partially efficient and do not result in complete bladder emptying. 15 / 19

16 Figure 4a (left). Magnetic motor-evoked potentials are generated by the motor cortex when an electromagnet is placed and triggered over the skull (arrow). 16 / 19

17 Figure 4b. Muscle contraction following the stimulation is recorded below the lesion site (hindlimbs, for example) and can be visualised as a large potential. 17 / 19

18 Figure 4c. Somato-sensory-evoked potentials are recorded over the skull by small needles placed under the skin, following stimulation of a peripheral nerve below the spinal cord lesion. 18 / 19

19 Figure 4d. Somato-sensory-evoked potentials recorded over the sensory cortex are usually very large and easily identified potentials. 19 / 19 Powered by TCPDF (