• forelimb
• glioma
• optogenetic stimulation
• plasticity

The aim of this thesis is to investigate plastic changes that occur in peritumoral tissue during glioma growth.
In order to address this issue, we used a classic mouse model of glioma created through the injection of a syngeneic line of murine glioma cells (i.e. GL261 cells) into the motor cortex. With specific motor tests (i.e. Grip Strength and Rotarod) it is possible to longitudinally follow the progression of deficits and identify a pre- and a post-symptomatic stage of the disease. Specifically, the decrease in motor tasks’ performance becomes clear starting from 12 days after tumour induction (Vannini et al., 2017).
My experiments focused on understanding whether a plastic rearrangement of cortical areas might occur in glioma-bearing animals, together with a reduction of motor functionality. To perform this analysis we took advantage of Thy1-ChR2 Transgenic mice (B6.Cg-Tg (Thy1-ChR2/EYFP)18Cfng/J, Jackson Laboratories, USA), that express the gene encoding for the protein ChR2 under the Thymus cell antigen-1 (Thy-1) promoter, i.e. mainly in pyramidal, corticospinal neurons. These mice were optogenetically stimulated at three different time points: baseline (before GL261 cells injection), 14 and 21 days after tumour implantation.
The data that we collected during the optogenetic stimulation were analysed through a custom made algorithm developed in Matlab, which allowed us to measure (i) the intensity of stimulation needed to evoke movement and (ii) the number of body parts activated in every single site of the motor cortex. The cortex was stimulated following a grid with nodes spaced 250 μm. The area analysed (comprising both caudal and rostral forelimb areas) included the cortical surface located from 2 mm posterior to 2.75 mm anterior, and from 0.25 medial to 3 mm lateral – using bregma as the point of reference. Cortical maps at the three different time points were compared. In glioma-bearing mice we found a strong remapping of cortical motor areas that starts 14 days after tumour induction and becomes very remarkable at 21 days. Our data indicate also a significant decrease of the cortical area that evokes forelimb responses (probably due to the tumour growth) and an increase in the threshold required for eliciting a forelimb movement in the peritumoral area.
At the end of the optogenetic experiment, we sacrificed the animals and performed immunohistochemical analysis to evaluate the expression of different markers of plasticity in peritumoral cortical areas. Indeed, we performed staining for perineuronal nets (PNNs), somatostatin and parvalbumin interneurons; we also evaluated the excitation/inhibition ratio with specific markers for glutamatergic and GABAergic terminals. Overall, the data show a downregulation not only of PNNs but also of inhibitory markers in the peritumoral cortex. Conversely, we found no detectable variation in V-Glut1 and vGlut2 expression, indicating a clear shift of the excitation/inhibition ratio in favour of excitation. This event is typically associated with an enhanced potential for plasticity in the adult cortex (Harauzov et al., 2010).
To wrap up, I have obtained data showing that, during glioma growth, the peritumoral tissue rearranges extensively. These data concur with findings in human patients and might help in understanding more thoroughly the cellular mechanisms governing glioma cortical plasticity, which will be useful to develop more effective pharmacological therapies.