Surgery

The spinal cord is that part of the Central Nervous System (C.N.S.) which elongates into the vertebral canal. In the spinal cord there are (along many other anatomical structures) millions of nerve fibres which transmit to the lower motor neurons the commands coming from the upper motor neuron (brain). When the cord is interrupted the commands from the brain are closed down as is the sensory information coming from the periphery to the brain. The lower motor neurons survive but are disconnected from the brain and released from the control of the tonus, so that the muscles become spastics with clonuses. Also neurological disturbances of the bladder and of the belly occur which cause severe discomfort in everyday activities. Some sexual activity remains, but the absence of sensibility gives a peculiar new sexual life which introduces an unpleasant and intricate mutual adaptation. Luckily pregnancy is possible as well as the ejaculation of fertile sperm which allows young paraplegics to have children. Research has proved that upper motor neurons are able to regenerate their axons, but these cannot progress beyond the lesion. Peripheral nerve fibres, on the contrary, can regenerate and progress up to their targets thanks to the presence of the Schwann cells (which are lacking in the C.N.S.). Spinal cord injuries (S.C.I.s) have been considered incurable for centuries. It was a "dogma" taught to students in medical schools and to paraplegics since the beginning. But various dogma have proven to be false over the course of time. The incurability does not depend on the cord but on our ignorance of its physiopathology. S.C.I.s are known since the ancient Egyptians. The first experiments on monkeys were done in Rome by Galenus during the second century A.D. but 18 centuries had to pass before new research started. The spinal cord after severance does not allow the axons of the upper neurons to descend towards the lower neurons and to stimulate them. The reason for this is not known yet. In 1979-80 Prof. Giorgio Brunelli started a new research : 1 cm of cord was removed at T XI level in rats and the gap was filled in with autologous grafts of peripheral nerves. The grafts were re-inhabited by the axons re-growing from the upper neurons but the progression of the axons stopped as soon as it came in contact again with the C.N.S. environment. To overcome this "non-permissiveness", since 1981 various surgical protocols have been used by the researchers of the Foundation for the Research of Spinal Cord Lesions, aimed at connecting the re-growing axons to the muscular nerves by means of autologous grafts, excluding the lower motor neurons. The upper motor neurons were able to elongate the cytoskeleton of their axons up to the muscles. After the rats research, 4 groups of monkeys (macaca fascicularis) were operated on. Magnetic stimulation of the brain demonstrated good muscular responses. E.M.G. also showed good responses with different latencies according to the different sites of the stimulation. Morphometrical analysis showed good axonal density, good severance area and good myelination of the fibres. While waiting for the permission of the National Ethical committee to transfer to human beings this protocol, the team of the Foundation transferred the ulnar nerve from the upper to the lower limbs (nerve transfers have been in use for decades and did not need special permission). At the begging the patient, mr. Angelo C., must think of moving the arms to get movement of the lower limbs, then, thanks to the brain's plasticity, the patient was able to move the lower limbs by thinking of them. After months of re-education Angelo was able to walk 50-70 steps at a time with the help of a walker. Then having obtained the permission of the Ethical Committee, 3 patients were operated on by C.N.S.-P.N.S. connections according to the latest animal protocol. In 2004 the first patient was able to walk for several minutes with tripode cans and to stand for several hours. In nature all the muscles receive innervation from the lower motor neurons which in turn are stimulated by the upper ones. By means of the C.N.S.-P.N.S. connections they receive innervation from the upper motor neurons while the lower ones are excluded. Good muscular response after stimulation of the cord and of the grafts.only a n allongement of the conduction time is evident according to the different places of stimulation. Furthermore the muscles receive axons coming from different cortical areas of the brain (in fact the C.N.S.- P.N.S. connection is random). Nonetheless the animals and the patients perform selective voluntary movements without co-contractions. Research was continued with the co-operation of the section of Pharmacology, Toxicology and Experimental Therapy of the Medical School of the Brescia University (Prof. PF. Spano, and Prof. M. Pizzi), with the department of Biomedical Sciences and Biotechnologies and the section of Genetics Biology, same University (Prof. S. Barlati and Doc. A. Barbon) and with the department of Neurosciences and Neurophysiopathology of the General Hospital of Brescia (Doc. B. Guarneri). The result of those 3 years of research have been published in June 2005 in the Journal of the National Academy of Science of the U.S.A.: P.N.A.S., 2005, 102, 24, 8752-8757. While in nature all the muscles function thanks to the stimuli coming from the lower motor neurons which use Achetilcolin as a neurotransmitter, after the surgery carried on by the team, muscles functioned due to the excitation coming from the upper motor neuron which uses a different neurotransmitter: the Glutamate. This is possible due to a dedifferentiation of the molecules that form the canals for jons to enter the post synaptic motor end-plates which go back to an embryonic type of synapse. At present, due to the restrictions to the experiments on animals decided by the Italian authorities , no experimental surgery can be performed on living animals in Italy. We now are performing experiment by means of organotopic coltures of the cord also using stem cells and nerve growth factor. This research is done with the collaboration of Department of Clinical and Experimental Science of the Medical School of the University of Brescia, in particular with Marco Cocchi PhD student and Prof. LF Rodella MD MSc, Unit of Anatomy and Physiopathology. But the research is going on with living animals (by means of the original protocol) in 3 foreign research institutes in Spain, Germany and India with which our Foundation has reached an agreement. Their results will validate those we got previously.

Biology

Research in the field of spinal lesions, supported by the use of several experimental models, represents an "indispensable" choice in order to improve the knowledge of the regenerative and reparative processes of the Central Nervous System and to allow the development of innovative therapeutic strategies of the surgical, cellular and pharmacological type. Indeed, as of today, surgical operations allow only to immobilise the vertebral column, but they do not bring any improvement to the palsy since an effective regeneration of the spinal cord is not possible. Why doesn't the spinal cord regenerate? What are the factors that hinder a correct and functional regeneration? Numerous and complex molecular and cellular mechanisms contribute to the failure of the axonal regeneration following the damage: the proteins associated to the myeline (the presence of NOGO above all), the settling of extracellular matrix with inhibitory effect in the lesion sites, the lack or considerable shortage of positive environmental stimuli like the ones provided by growth factors.
On the basis of the considerations above, although the animal model represents a compulsory stage to study the regeneration of the spinal cord, the detailed understanding of its biological aspects and the set of problems related to the use of 'in vivo' models have led scientists to come up with alternative methods.
The marrow's organotypic cultures may be a valid tool to study the axonal regeneration of the spinal damage, proving a useful compromise between the in vitro studies like that of the cellular cultures and the in vivo ones with the animal model. This type of ex vivo culture indeed allows to control the extracellular environment as well as a repeated access, keeping the morphology and the local synaptic connections in vitro practically intact.
The ''Giorgio Brunelli Foundation for the research on Spinal Cord Lesions E.S.C.R.I. ONLUS'', even further to bureaucratic difficulties due to the use of animal experimental models, has decided to take up this course of studies using three elements: platelet preparations, mesenchymal stem cells and nerve growth factors; all this in order to create a microenvironment which favours and allows axonal regeneration capable of contrasting the factors obstructing it.
The platelet concentrates, also called "platelet gels", represent an innovative and interesting methodology used within the tissue regeneration. Such preparations base their regenerative potentiality on the rich content of growth factors that are necessary to induce cellular proliferation, remodelling of the extracellular matrix and the processes of neovascularisation occurring during the various stages of regeneration of different tissues.
The stem cells represent a huge promise to introduce new neurons or glial cells into the damaged nervous system.
Indeed they are a great resource to research and to a potential treatment because of their multi-potentiality, as they can be reproduced in vitro, bound with genetic markers or therapeutic genes implanted in the nervous system.
Finally, the neurotrophic factors like the Nerve Growth Factor (NGF), the Brain Derived Neurotrophic Factor (BDNF) and the Neurotrophin-3 (NT-3) play an essential role.
These three elements combined together are used in cultures and in organotypic co-cultures of spinal cord and encephalic cortex of rats [(a) single slice of spinal cord, (b) co-culture of two slices of spinal cord, (c) co-culture of two slices of cortex and marrow respectively, (d) corticospinal tract in vitro – cortex,bridge,marrow]. Therefore, this is the new path of research of the Foundation.