Stymulacja neuronalnej nadprodukcji BDNF w uszkodzonym rdzeniu kręgowym szczura : ocena zmian molekularnych i morfologicznych w okresie najlepszej poprawy funkcjonalnej : praca doktorska

Głowacka, Anna (1989– )

Stymulacja neuronalnej nadprodukcji BDNF w uszkodzonym rdzeniu kręgowym szczura : ocena zmian molekularnych i morfologicznych w okresie najlepszej poprawy funkcjonalnej : praca doktorska

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Title: Stymulacja neuronalnej nadprodukcji BDNF w uszkodzonym rdzeniu kręgowym szczura : ocena zmian molekularnych i morfologicznych w okresie najlepszej poprawy funkcjonalnej : praca doktorska

Abstract:

Spinal cord injury leads to the disruption and degeneration of neural pathways originating in supraspinal centers. Consequently, patients experience partial or complete loss of motor function, the extent of which depends on the location and severity of spinal damage. Such injuries often result in permanent disability due to limited eﬀectiveness of current treatment methods. One promising strategy involves enriching the neuronal network below the injury site with neurotrophins – low molecular weight proteins that promote neuron survival and synaptic plasticity processes. In this dissertation, adult rats subjected to complete spinal cord transection (SCT) at the lower thoracic segments were administered an intraspinal AAV vector carrying the brain-derived neurotrophic factor gene (AAV-BDNF). Animals overexpressing BDNF demonstrated rapid and signiﬁcant improvement in motor function; by the third week post-injury, they regained the ability for alternating limb movements, weight-bearing, and plantar stepping during treadmill training. These results, obtained in a group of 14 animals, strongly conﬁrmed the therapeutic potential of the AAV-BDNF vector previously observed in a chronic experiment conducted by our Team. In the 6-week experiment, the best functional improvement occurred in the third week post-SCT and AAV-BDNF injection, but side eﬀects in the form of clonic movements emerged in later postoperative periods, likely related to overstimulation of the network. This study aimed to elucidate the molecular basis of BDNF's therapeutic action. To this end, neurochemical and structural changes in the spinal cord and neuromuscular junction were characterized during the period of greatest functional improvement. Given indications of diﬀerent SCT sensitivities in the neuronal circuits of two antagonistic muscles of the ankle joint: the ﬂexor (Tibialis anterior, TA) and extensor (Soleus, Sol), motoneurons and neuromuscular junctions of these two muscles were examined. Combining cellular and subcellular mRNA distribution analysis, assessed via ﬂuorescent in situ hybridization, and protein distribution analysis using immunoﬂuorescence in tissue sections, provided a comprehensive response to questions about the impact of injury and BDNF overexpression on cellular responses in the spinal cord and muscles. Transmission electron microscopy was employed to examine changes in the ultrastructure of the neuromuscular junction. It was demonstrated that, in the third week post-vector administration, BDNF overexpression in the spinal cord concentrated in neurons of the L1-L2 lumbar segments, close to the injection site, while in the area of the hind limb motoneurons, located in the L3-L6 segments, the transgenic protein was observed only in ﬁbers of transduced cells. Based on this characterized distribution of recombinant BDNF, it was concluded that locally introduced vector leads to the transgenic protein's action on neurons located throughout the entire lumbar segment. The therapy was proven to have several positive eﬀects: it maintained reduced SCT post-injury receptivity of motoneurons to BDNF, caused increased transcriptional activity of subsynaptic myocyte nuclei, and contributed to the normalization of neuronal signaling markers in the muscle. Moreover, AAV-BDNF injection preserved the structural integrity of neuromuscular junctions, with the Soleus motor unit responding more favorably to therapy. These results contribute to understanding the mechanism of action of the neurotrophin BDNF and point to a potentially useful research direction: targeted therapy for selected types of motor units.