Reappraisal of transcallosal neuron organization in mice and evaluation of their dendritic remodeling and circuit integration following traumatic brain injury

Traumatic Brain Injury (TBI) is an enormous global socio-economic burden since, apart from its high death rate, it is the primary cause of coma worldwide and a prevalent cause of long-term disability. Until today there is no established treatment for dealing with the long-term outcomes of TBI despite many years of research. Although a lot is known about the pathophysiology of TBI in the damaged tissue and the surrounding area in case of focal lesion, only few studies have investigated the structural and functional integrity of the contralateral intact cortex. In order to explore this territory, this study employs a well-established and widely used animal model of focal open skull TBI known as the Controlled Cortical Impact (CCI) model. The first aim of this study was to systematically characterize a specific neuronal population, the transcallosal projection neurons, as they are the ones connecting the intact cortex with the lesioned cortex. The description of the organization of transcallosal neurons and their axonal projections at the contralateral hemisphere was carried out in healthy, non-injured C57Bl6 mice. Retrograde and anterograde tracing methods were implemented to label transcallosal cell bodies and their axonal projections, respectively. In addition, different injection coordinates were used in order to label transcallosal connections at distinct brain regions, including the motor cortex (M1), somatosensory cortex (S1), and barrel cortex, rostral and caudal to Bregma. In agreement with previous research, I observed that transcallosal projections are organized homotopically across the various brain regions, with the axonal terminals spanning the entire cortical column. Interestingly my study describes for the first time a non-negligible fraction of heterotopic transcallosal neurons that, in addition, display a slightly less strict layer distribution pattern compared to the homotopic ones. After the initial characterization of transcallosal neuron organization, I proceeded by investigating how these neurons with projections at the injury site are affected at various timepoints following focal TBI. I used GFPM mice to visualize dendrites and spines of transcallosal and non-transcallosal neurons, in order to examine their structural integrity at different timepoints post-injury. I detected significant differences in dendritic spine density and morphology between controls and injured mice, which were time-dependent. More specifically, the dendritic spine density in transcallosal neurons was strongly decreased as soon as 7days following injury. Interestingly, spine density in non-transcallosal neurons was not changed following TBI. In terms of spine shape, I found a morphological shift only for the apical tuft segments. These results point towards a general sensitivity of transcallosal spines to TBI-induced damage, where loss of spines (preferentially mature) seems to take place at 1-2 weeks post-injury and resolve at 3-6 weeks post-injury, indicative of late plasticity processes. As the anatomically connected neuronal population seems to recover overtime I then decided to further explore whether transcallosal circuit remodeling takes place after TBI. To do so I used the retrograde mono-trans-synaptic tracer SADΔG-GFP (EnvA) Rabies virus. In that way, I was able to distinctively label transcallosal neurons and their presynaptic partners and obtain an overview of the presynaptic population throughout the cortex across brain regions at different post-injury timepoints. This study demonstrates that spine plasticity did not result in adaptive circuit plasticity with the recruitment of other brain regions but rather that initial circuits were re-established. In brief, during this thesis I have demonstrated the adaptive plastic capacities of anatomically connected neurons to the brain injury. I believe that this knowledge may help in unraveling further compensatory plastic mechanisms that could then be therapeutically targeted to improve the outcome following brain injury

Reappraisal of transcallosal neuron organization in mice and evaluation of their dendritic remodeling and circuit integration following traumatic brain injury

Traumatic Brain Injury (TBI) is an enormous global socio-economic burden since, apart from its high death rate, it is the primary cause of coma worldwide and a prevalent cause of long-term disability. Until today there is no established treatment for dealing with the long-term outcomes of TBI despite many years of research. Although a lot is known about the pathophysiology of TBI in the damaged tissue and the surrounding area in case of focal lesion, only few studies have investigated the structural and functional integrity of the contralateral intact cortex. In order to explore this territory, this study employs a well-established and widely used animal model of focal open skull TBI known as the Controlled Cortical Impact (CCI) model. The first aim of this study was to systematically characterize a specific neuronal population, the transcallosal projection neurons, as they are the ones connecting the intact cortex with the lesioned cortex. The description of the organization of transcallosal neurons and their axonal projections at the contralateral hemisphere was carried out in healthy, non-injured C57Bl6 mice. Retrograde and anterograde tracing methods were implemented to label transcallosal cell bodies and their axonal projections, respectively. In addition, different injection coordinates were used in order to label transcallosal connections at distinct brain regions, including the motor cortex (M1), somatosensory cortex (S1), and barrel cortex, rostral and caudal to Bregma. In agreement with previous research, I observed that transcallosal projections are organized homotopically across the various brain regions, with the axonal terminals spanning the entire cortical column. Interestingly my study describes for the first time a non-negligible fraction of heterotopic transcallosal neurons that, in addition, display a slightly less strict layer distribution pattern compared to the homotopic ones. After the initial characterization of transcallosal neuron organization, I proceeded by investigating how these neurons with projections at the injury site are affected at various timepoints following focal TBI. I used GFPM mice to visualize dendrites and spines of transcallosal and non-transcallosal neurons, in order to examine their structural integrity at different timepoints post-injury. I detected significant differences in dendritic spine density and morphology between controls and injured mice, which were time-dependent. More specifically, the dendritic spine density in transcallosal neurons was strongly decreased as soon as 7days following injury. Interestingly, spine density in non-transcallosal neurons was not changed following TBI. In terms of spine shape, I found a morphological shift only for the apical tuft segments. These results point towards a general sensitivity of transcallosal spines to TBI-induced damage, where loss of spines (preferentially mature) seems to take place at 1-2 weeks post-injury and resolve at 3-6 weeks post-injury, indicative of late plasticity processes. As the anatomically connected neuronal population seems to recover overtime I then decided to further explore whether transcallosal circuit remodeling takes place after TBI. To do so I used the retrograde mono-trans-synaptic tracer SADΔG-GFP (EnvA) Rabies virus. In that way, I was able to distinctively label transcallosal neurons and their presynaptic partners and obtain an overview of the presynaptic population throughout the cortex across brain regions at different post-injury timepoints. This study demonstrates that spine plasticity did not result in adaptive circuit plasticity with the recruitment of other brain regions but rather that initial circuits were re-established. In brief, during this thesis I have demonstrated the adaptive plastic capacities of anatomically connected neurons to the brain injury. I believe that this knowledge may help in unraveling further compensatory plastic mechanisms that could then be therapeutically targeted to improve the outcome following brain injury

Heterotopic Transcallosal Projections Are Present throughout the Mouse Cortex

Transcallosal projection neurons are a population of pyramidal excitatory neurons located in layers II/III and to a lesser extent layer V of the cortex. Their axons form the corpus callosum thereby providing an inter-hemispheric connection in the brain. While transcallosal projection neurons have been described in some detail before, it is so far unclear whether they are uniformly organized throughout the cortex or whether different functional regions of the cortex contain distinct adaptations of their transcallosal connectivity. To address this question, we have therefore conducted a systematic analysis of transcallosal projection neurons and their axons across six distinct stereotactic coordinates in the mouse cortex that cover different areas of the motor and somatosensory cortices. Using anterograde and retrograde tracing techniques, we found that in agreement with previous studies, most of the transcallosal projections show a precise homotopic organization. The somata of these neurons are predominantly located in layer II/III and layer V but notably smaller numbers of these cells are also found in layer IV and layer VI. In addition, regional differences in the distribution of their somata and the precision of their projections exist indicating that while transcallosal neurons show a uniform organization throughout the mouse cortex, there is a sizeable fraction of these connections that are heterotopic. Our study thus provides a comprehensive characterization of transcallosal connectivity in different cortical areas that can serve as the basis for further investigations of the establishment of inter-hemispheric projections in development and their alterations in disease

Diffusion kurtosis imaging detects the time-dependent progress of pathological changes in the oral rotenone mouse model of Parkinson's disease

Clinical diagnosis of Parkinson's disease (PD) occurs typically when a substantial proportion of dopaminergic neurons in the substantia nigra (SN) already died, and the first motor symptoms appear. Therefore, tools enabling the early diagnosis of PD are essential to identify early-stage PD patients in which neuroprotective treatments could have a significant impact. Here, we test the utility and sensitivity of the diffusion kurtosis imaging (DKI) in detecting progressive microstructural changes in several brain regions of mice exposed to chronic intragastric administration of rotenone, a mouse model that mimics the spatiotemporal progression of PD-like pathology from the ENS to the SN as described by Braak's staging. Our results show that DKI, especially kurtosis, can detect the progression of pathology-associated changes throughout the CNS. Increases in mean kurtosis were first observed in the dorsal motor nucleus of the vagus (DMV) after 2 months of exposure to rotenone and before the loss of dopaminergic neurons in the SN occurred. Remarkably, we also show that limited exposure to rotenone for 2 months is enough to trigger the progression of the disease in the absence of the environmental toxin, thus suggesting that once the first pathological changes in one region appear, they can self-perpetuate and progress within the CNS. Overall, our results show that DKI can be a useful radiological marker for the early detection and monitoring of PD pathology progression in patients with the potential to improve the clinical diagnosis and the development of neuroprotective treatments. (Figure presented.). © 2021 International Society for Neurochemistr

Diabetes increases mortality in patients with pancreatic and colorectal cancer by promoting cachexia and its associated inflammatory status

Objectives: Cancer is considered an emerging diabetes complication, with higher incidence and worse prognosis in patients with diabetes. Cancer is frequently associated with cachexia, a systemic metabolic disease causing wasting. It is currently unclear how diabetes affects the development and progression of cachexia. Methods: We investigated the interplay between diabetes and cancer cachexia retrospectively in a cohort of 345 patients with colorectal and pancreatic cancer. We recorded body weight, fat mass, muscle mass, clinical serum values, and survival of these patients. Patients were grouped either into diabetic/non-diabetic groups based on previous diagnosis, or into obese/non-obese groups based on body mass index (BMI ≥30 kg/m2 was considered obese). Results: The pre-existence of type 2 diabetes, but not obesity, in patients with cancer led to increased cachexia incidence (80%, compared to 61% without diabetes, p ≤ 0.05), higher weight loss (8.9% vs. 6.0%, p ≤ 0.001), and reduced survival probability (median survival days: 689 vs. 538, Chi square = 4.96, p ≤ 0.05) irrespective of the initial body weight or tumor progression. Patients with diabetes and cancer showed higher serum levels of C-reactive protein (0.919 μg/mL vs. 0.551 μg/mL, p ≤ 0.01) and interleukin 6 (5.98 pg/mL vs. 3.75 pg/mL, p ≤ 0.05) as well as lower serum albumin levels (3.98 g/dL vs. 4.18 g/dL, p ≤ 0.05) than patients with cancer without diabetes. In a sub-analysis of patients with pancreatic cancer, pre-existing diabetes worsened weight loss (9.95% vs. 6.93%, p ≤ 0.01), and increased the duration of hospitalization (24.41 days vs. 15.85 days, p ≤ 0.001). Further, diabetes aggravated clinical manifestations of cachexia, as changes in the aforementioned biomarkers were more pronounced in patients with diabetes and cachexia co-existence, compared to cachectic patients without diabetes (C-reactive protein: 2.300 μg/mL vs. 0.571 μg/mL, p ≤ 0.0001; hemoglobin: 11.24 g/dL vs. 12.52 g/dL, p ≤ 0.05). Conclusions: We show for the first time that pre-existing diabetes aggravates cachexia development in patients with colorectal and pancreatic cancer. This is important when considering cachexia biomarkers and weight management in patients with co-existing diabetes and cancer

PET imaging of the adenosine A2A receptor in the rotenone-based mouse model of Parkinson’s disease with [18F]FESCH synthesized by a simplified two-step one-pot radiolabeling strategy

The adenosine A2A receptor (A2AR) is regarded as a particularly appropriate target for non-dopaminergic treatment of Parkinson’s disease (PD). An increased A2AR availability has been found in the human striatum at early stages of PD and in patients with PD and dyskinesias. The aim of this small animal positron emission tomography/magnetic resonance (PET/MR) imaging study was to investigate whether rotenone-treated mice reflect the aspect of striatal A2AR upregulation in PD. For that purpose, we selected the known A2AR-specific radiotracer [18F]FESCH and developed a simplified two-step one-pot radiosynthesis. PET images showed a high uptake of [18F]FESCH in the mouse striatum. Concomitantly, metabolism studies with [18F]FESCH revealed the presence of a brain-penetrant radiometabolite. In rotenone-treated mice, a slightly higher striatal A2AR binding of [18F]FESCH was found. Nonetheless, the correlation between the increased A2AR levels within the proposed PD animal model remains to be further investigated

A Primeval Mechanism of Tolerance to Desiccation Based on Glycolic Acid Saves Neurons in Mammals from Ischemia by Reducing Intracellular Calcium-Mediated Excitotoxicity

Stroke is the second leading cause of death and disability worldwide. Current treatments, such as pharmacological thrombolysis or mechanical thrombectomy, reopen occluded arteries but do not protect against ischemia-induced damage that occurs before reperfusion or neuronal damage induced by ischemia/reperfusion. It has been shown that disrupting the conversion of glyoxal to glycolic acid (GA) results in a decreased tolerance to anhydrobiosis in Caenorhabditis elegans dauer larva and that GA itself can rescue this phenotype. During the process of desiccation/rehydration, a metabolic stop/start similar to the one observed during ischemia/reperfusion occurs. In this study, the protective effect of GA is tested in different ischemia models, i.e., in commonly used stroke models in mice and swine. The results show that GA, given during reperfusion, strongly protects against ischemic damage and improves functional outcome. Evidence that GA exerts its effect by counteracting the glutamate-dependent increase in intracellular calcium during excitotoxicity is provided. These results suggest that GA treatment has the potential to reduce mortality and disability in stroke patients