Local supplementation of FGF-2 and BDNF in the epileptogenic hippocampus. Effects and delivery strategies

Among the epileptic syndromes, temporal lobe epilepsy (TLE) is the most common form in adults. It is the consequence of a brain damage (viral infection, stroke, trauma, cancer ...), capable of triggering a cascade of events culminating in the appearance of spontaneous seizures that are, in many cases, difficult to control with the usual drug therapy. The period that elapses between the initial insult and the development of spontaneous recurrent seizures (SRSs) is defined "epileptogenesis”. The cellular and tissue changes that occur during this phase mainly interest the hippocampal region and include: neurodegeneration, neurogenesis, neuroinflammation and reactive gliosis, angiogenesis, and reorganization of brain circuits. Recently, it was shown that supplementation of neurotrophic factors (NTFs), such as FGF-2 (fibroblast growth factor-2) and BDNF (brain derived neurotrophic factor), has anti-epileptogenic effects by reducing neuronal death, favoring a correct neurogenesis and restoring a proper balance between excitatory and inhibitory circuits. In the main study reported in this thesis, we examined if this treatment can also affect neuroinflammatory processes. We used the pilocarpine model, in which an episode of status epilepticus (SE) is followed by an epileptogenic lesion. After three days, herpes viral vectors expressing FGF-2 and BDNF were injected in the hippocampus. Four, 11 and 25 days after treatment (DAI), animals were sacrificed and their brains removed to analyze the expression of three markers of inflammation: IL-1β, GFAP (a marker of astrocytosis), Ox42 (marker of microglia). The results show a very marked reduction of IL-1β expression, evident as early as 4 days after inoculation of the viral vector, and delayed, but significant, attenuation of the other two markers. The sprouting of mossy fibers is another characteristic of the epileptic hippocampal tissue, in which the axons of granule cells form excitatory synapses with cells not usually innervated, forming a circuit that may favour hyperexcitability. The results show that treatment with neurotrophic factors reduce aberrant sprouting of nerve fibers, in a way that correlates with the attenuation of cellular damage. Parallel behavioral studies have also highlighted the ability of the treatment to reduce the frequency and severity of SRSs that, in this model, begin to occur about 21 days after status epilepticus. Despite the promising results, clinical applicability of neurotrophic factors is limited by the choice of an appropriate route of administration. In the experiments reported in this thesis, herpes viral vectors have been used. These vectors were replication defective and engineered to express the two NTFs. However, their residual toxicity makes them unsuitable for human application. Stem cells modified to express genes of interest, including mesangioblasts (MABs), have demonstrated, in vitro, the ability to promote differentiation, survival and neuronal function. Last but not least, the ability to localize in the damaged site when systemically administered makes these cells viable alternatives to more invasive treatments. Although further investigations are required, the results collected in this thesis are an important contribution to the understanding of the multiple effects of NTFs. In addition, the characterization of an alternative and more applicable route of administration renders gene therapy with neurotrophic factor more applicable for the treatment of several neurodegenerative diseases.