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Introduction: Human induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process. Methods: In this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter. Results: The retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types. Discussion: Our results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.
RESUMEN
OBJECTIVES: To compare post-concussion symptoms in patients who sustained a sports-related mild traumatic brain injury (TBI) to those with non-sports-related mild TBI at 7 and 90 days post-injury. METHODS: This prospective multicentre cohort study took place in seven Canadian Emergency Departments (ED). Non-hospitalized patients aged ≥ 14 years with a documented mild TBI that occurred ≤ 24 h of ED visit and a Glasgow Coma Scale score ≥ 13 were included. Main outcome measure was the presence of ≥ 3 symptoms on the Rivermead Post-concussion Questionnaire at 90 days post-injury. Secondary outcomes were the prevalence of (1) individual Rivermead Post-concussion Questionnaire symptom, (2) total Rivermead Post-concussion Questionnaire score ≥ 21 and (3) return to normal daily activities. Adjusted risk ratios (RR) were calculated. RESULTS: 1727 patients were included, 363 (21.0%) sustained a sports-related mild TBI. Similar proportions of patients with ≥ 3 symptoms, a Rivermead Post-concussion Questionnaire score ≥ 21 and those who returned to their normal daily activities were observed at 7 and 90 days post-injury. Sports-related mild TBI patients were at higher risk of poor concentration [RR: 1.3 (95% CI 1.05-1.54)] and non-return to sports activities [RR: 2.2 (95% CI 1.69-2.94)] at 7 days post-injury. At 90 days, sports-relate -mild TBI patients reported less fatigue [RR: 0.7 (95% CI 0.51-0.98)] and feeling of dizziness [RR: 0.6 (95% CI 0.35-0.99)]. CONCLUSION: Patients who sustained sports-related mild TBI could be at lower risk of experiencing symptoms such as fatigue and dizziness 90 days post-injury. Clinicians should be mindful that non-sports-related mild TBI patients may experience more post-concussion symptoms and that the level of physical activity may influence the patient's rehabilitation.
RéSUMé: OBJECTIFS: Comparer les symptômes post-commotionnels chez les patients ayant subi un traumatisme cranio-cérébral léger (TCCL) lié au sport à ceux qui ont un TCCL non lié au sport à 7 et 90 jours post-traumatisme. MéTHODES: Cette étude prospective de cohorte multicentrique s'est déroulée dans sept Départements d'urgence (DU) du Canada. Les patients non hospitalisés âgés de ≥ 14 ans ayant subi un TCCL ≤ 24 heures avant leur visite au DU et présentant un score de Glasgow de ≥ 13 ont été inclus. Le critère principal de jugement était la présence d'au moins 3 symptômes au Rivermead Post-concussion Questionnaire 90 jours après le traumatisme. Les critères de jugement secondaires étaient la prévalence de 1) chacun des symptômes du Rivermead Post-concussion Questionnaire 2) score total du Rivermead Post-concussion Questionnaire ≥ 21 et 3) retour aux activités habituelles. Des risques relatifs ajustés (RR) ont été calculés. RéSULTATS: Un total de 1727 patients a été inclu, dont 363 (21,0 %) ont subi un TCCL lié au sport. Des proportions similaires de patients présentant ≥ 3 symptômes, un score ≥ 21 au Rivermead Post-concussion Questionnaire et de participants ayant repris leurs activités habituelles ont été observés à 7 et 90 jours après la blessure. Les patients avec TCCL lié au sport présentaient un risque plus élevé de manque de concentration (RR: 1,3 [IC à 95 % = 1,05-1,54]) et de non-retour aux activités sportives (RR: 2,2 [IC à 95 % = 1,69-2,94]) à 7 jours après la blessure. À 90 jours, les patients avec TCCL lié au sport ont signalé moins de fatigue (RR: 0,7 [IC à 95 % = 0,51-0,98]) et de sensation de vertige (RR: 0,6 [IC à 95 % = 0,35-0,99]). CONCLUSION: Les patients qui ont subi un TCCL lié au sport seraient moins à risque de présenter des symptômes tels que de la fatigue et des étourdissements 90 jours après la blessure. Les cliniciens doivent être conscients que les patients avec TCCL non lié au sport peuvent présenter davantage de symptômes post-commotionnels et que le niveau d'activité physique peut influencer la réadaptation du patient.