Methods to study glymphatic system in the rodent brain during physiological and pathological processes

Over the past decade, our understanding of brain-waste clearance underwent a revolution after the discovery of the so-called glymphatic system. The glia-lymphatic (abbreviated, glymphatic) system was first described in 2012 as a paravascular macrosystem involved in the distribution of nutrients and clearance of metabolic waste from the brain parenchyma. The main actor of the system is the cerebrospinal fluid (CSF), which from the subarachnoid space (SAS) flow into the perivascular space (PVS) of brain penetrating arteries, and from there into the brain parenchyma, facilitated by aquaporin-4 (AQP4) water channels on astrocitic endfeet surrounding blood vessels.Intriguingly, once in the parenchyma, the CSF mixes with the interstitial fluid (ISF), releasing nutrients and collecting metabolic wastes and toxic proteins, including Aβ and Tau, two proteins involved in the pathogenesis of Alzheimer´s disease (AD).Importantly, recent studies have shown that the glymphatic system, and therefore its function in “cleaning” the brain from toxic wastes and proteins, is impaired during aging. Age is the main risk factor for developing neurodegenerative disease caused by accumulation of toxic proteins, e.g. AD or Parkinson´s diseases (PD). Therefore, the understanding of how glymphatic system functions is of utmost importance.Because of the novelty of this field, it is needed to find consensus on the techniques and methodology to study glymphatic system, as well as interpretation of the results.The aim of this thesis was then to describe methodologies useful for glymphatic studies in rodents, and to apply these methods to study CSF movement in the brain both in physiological and pathological settings (i.e., hypothermia and PD).Specifically, in Paper I we described the CM injection method to study glymphatic function in the rodents´ brain. In Paper II, we quantitatively compare the efficiency of different lectins and staining methods to label vasculature in rodents, as an important step for AQP4 polarisation studies. In Paper III, we investigate how AQP4 is affected byantisense oligonucleotides (ASO) targeting Aqp4 mRNA. In Paper IV, we investigated glymphatic system function in two different mouse models of PD, and test whether glymphatic system is involved in the clearance of α-syn (a protein involved in PD pathogenesis) from the brain parenchyma. Finally, in Paper V we explored glymphaticfunction and AQP4 in hypothermia, a condition often related to anaesthesia.Overall, this thesis helped the field of brain fluid dynamics investigation by providing a description of the techniques that can be used in pre-clinical research to investigate glymphatic function on a macroscopic level and dissect the microscopic players of the system. The methods described in this thesis can be adapted to the investigation of glymphatic function in different physiological and pathological settings, as well as different preclinical models. Advancing pre-clinical research with reproducible and standardised methods is fundamental for following translational applications

Not all lectins are equally suitable for labeling rodent vasculature

The vascular system is vital for all tissues and the interest in its visualization spans many fields. A number of different plant-derived lectins are used for detection of vasculature; however, studies performing direct comparison of the labeling efficacy of different lectins and techniques are lacking. In this study, we compared the labeling efficacy of three lectins: Griffonia simplicifolia isolectin B4 (IB4); wheat germ agglutinin (WGA), and Lycopersicon esculentum agglutinin (LEA). The LEA lectin was identified as being far superior to the IB4 and WGA lectins in histological labeling of blood vessels in brain sections. A similar signal-to-noise ratio was achieved with high concentrations of the WGA lectin injected during intracardial perfusion. Lectins were also suitable for labeling vasculature in other tissues, including spinal cord, dura mater, heart, skeletal muscle, kidney, and liver tissues. In uninjured tissues, the LEA lectin was as accurate as the Tie2–eGFP reporter mice and GLUT-1 immunohistochemistry for labeling the cerebral vasculature, validating its specificity and sensitivity. However, in pathological situations, e.g., in stroke, the sensitivity of the LEA lectin decreases dramatically, limiting its applicability in such studies. This work can be used for selecting the type of lectin and labeling method for various tissues

Adolescents with Mental Disorders: The Efficacy of a Multiprofessional Approach

In the book "Mental Illnesses - Evaluation, Treatments and Implications" attention is focused on background factors underlying mental illness. It is crucial that mental illness be evaluated thoroughly if we want to understand its nature, predict its long-term outcome, and treat it with specific rather than generic treatment, such as pharmacotherapy for instance. Additionally, community-wide and cognitive-behavioral approaches need to be combined to decrease the severity of symptoms of mental illness. Unfortunately, those who should profit the most by combination of treatments, often times refuse treatment or show poor adherence to treatment maintenance. Most importantly, what are the implications of the above for the mental health community? Mental illness cannot be treated with one single form of treatment. Combined individual, community, and socially-oriented treatments, including recent distance-writing technologies will hopefully allow a more integrated approach to decrease mental illness world-wide

Traumatic brain injury: rehabilitation

Este estudo revisou artigos nas bases de dados do MEDLINE (PubMed) e demais fontes de pesquisa, sem limite de tempo. Para tan-to, adotou-se a estratégia de busca baseada em perguntas estruturadas na forma (P.I.C.O.) das iniciais: "Paciente"; "Intervençao"; "Controle" e "Outcome". Com esses descritores efetivaram-se cruzamentos de acordo com o tema proposto em cada tópico das perguntas (P.I.C.O.). Analisado esse material, foram selecionados os artigos relativos às perguntas e, por meio do estudo dos mesmos, estabeleceram-se as evidências que fundamentaram às diretrizes do presente documento.This study revised articles from the MEDLINE (PubMed) databases and other research sources, with no time limit. To do so, the search strategy adopted was based on (P.I.C.O.) structured questions (from the initials "Patient"; "Intervention"; "Control" and "Outcome". With the above keywords crossings were performed according to the proposed theme in each topic of the (P.I.C.O.) questions. After analyzing this material, articles regarding the questions were selected and, by studying those, the evidences that fundamented the directives of this document were established

Not All Lectins Are Equally Suitable for Labeling Rodent Vasculature

The vascular system is vital for all tissues and the interest in its visualization spans many fields. A number of different plant-derived lectins are used for detection of vasculature; however, studies performing direct comparison of the labeling efficacy of different lectins and techniques are lacking. In this study, we compared the labeling efficacy of three lectins: Griffonia simplicifolia isolectin B4 (IB4); wheat germ agglutinin (WGA), and Lycopersicon esculentum agglutinin (LEA). The LEA lectin was identified as being far superior to the IB4 and WGA lectins in histological labeling of blood vessels in brain sections. A similar signal-to-noise ratio was achieved with high concentrations of the WGA lectin injected during intracardial perfusion. Lectins were also suitable for labeling vasculature in other tissues, including spinal cord, dura mater, heart, skeletal muscle, kidney, and liver tissues. In uninjured tissues, the LEA lectin was as accurate as the Tie2–eGFP reporter mice and GLUT-1 immunohistochemistry for labeling the cerebral vasculature, validating its specificity and sensitivity. However, in pathological situations, e.g., in stroke, the sensitivity of the LEA lectin decreases dramatically, limiting its applicability in such studies. This work can be used for selecting the type of lectin and labeling method for various tissues

The spiraled knowledge: the story of Mestre Alcides de Lima Tserewaptu and the purpose for a shared knowledge production

Esta pesquisa de mestrado se destina a trazer uma reflexão, a partir da proposta de Produção Partilhada do Conhecimento, acerca do encontro entre a diversidade de saberes orais - presente na cultura oral e popular do Brasil -, e as atuais propostas de transformação das pesquisas nas Ciências Humanas. Por meio da História Oral, etnografia e pesquisa participante contextualizamos academicamente como pode acontecer esta parceria para a produção de conhecimento, em diálogo com a história de vida de Mestre Alcides de Lima Tserewaptu e relatos de vivências seus e de parceiras(os) do Centro de Estudos e Aplicação da Capoeira (CEACA), na cidade de São Paulo. A narrativa de sua jornada pela tradição oral e cultura popular em ambientes institucionais de ensino, tais como a Universidade de São Paulo USP e a Escola Municipal de Ensino Fundamental Desembargador Amorim Lima, contando com o congado e a capoeira, manifestações de resistência e luta pela valorização cultura afro-brasileira, proporcionou a análise de possibilidades de caminhos para se pensar a interlocução destes saberes orais com as propostas teóricas nas áreas de história, cultura, antropologia e educação. A antropologia visual foi um dos meios de partilha e registro dos processos da pesquisa (entrevistas, rituais, batizados, reuniões). A prática - já desenvolvida pelo CEACA na produção de material audiovisual -, junto ao reconhecimento de que as tradições das culturas orais não podem ser transpostas para o escrito, garantiram uma colaboração mais coletiva e a legitimação desse método de pesquisa. Um dos resultados do trabalho é um registro audiovisual no qual o Mestre Alcides e algumas pessoas do CEACA colaboraram com a narrativa, inserção de materiais que representam a essência do grupo, seus valores, motivações e práticas em diálogo com as discussões teóricas.This masters research intends to bring a reflection - from the proposal of Shared Knowledge Production - to the encounter between the diversity of oral knowledge - present in the oral and popular culture of Brazil -, and the current proposals for the transformation of researches in the Human Sciences. Through Oral History, ethnography and participant research, we seek to contextualize academically how this partnership for the production of knowledge, in dialogue with the life history of Mestre Alcides de Lima Tserewaptu, griô, and among the narratives of his experiences and from his partners at Center for the Study and Application of Capoeira (CEACA), in the city of São Paulo. The narrative of his journey through oral tradition and popular culture in educational institutions such as University of São Paulo and School Amorim Lima, counting on the congado and capoeira, manifestations of resistance and struggle for the valorization of Afro- Brazilian culture, provided the analysis of possibilities of ways to think the interlocution of these oral knowledge with theoretical proposals in the areas of history, culture, anthropology and education. Visual anthropology was one of the means of sharing and recording research processes (interviews, rituals, baptisms, meetings). The practice already developed by CEACA in the production of audiovisual material, allied with the recognition that oral traditions of oral cultures can not be transposed into writing, ensured a more collective collaboration and the legitimation of this method of research. One of the results of the work is an audiovisual record in which Mestre Alcides and some people of CEACA collaborated with the narrative, insertion of materials that represent the essence of the group, their values, motivations and practices in dialogue with the theoretical discussions

Activity-Induced Amyloid-β Oligomers Drive Compensatory Synaptic Rearrangements in Brain Circuits Controlling Memory of Presymptomatic Alzheimer's Disease Mice

Background: A consistent proportion of individuals at risk for Alzheimer's disease show intact cognition regardless of the extensive accumulation of amyloid-β (Aβ) peptide in their brain. Several pieces of evidence indicate that overactivation of brain regions negative for Aβ can compensate for the underactivation of Aβ-positive ones to preserve cognition, but the underlying synaptic changes are still unexplored. Methods: Using Golgi staining, we investigate how dendritic spines rearrange following contextual fear conditioning (CFC) in the hippocampus and amygdala of presymptomatic Tg2576 mice, a genetic model for Aβ accumulation. A molecular biology approach combined with intrahippocampal injection of a γ-secretase inhibitor evaluates the impact of Aβ fluctuations on spine rearrangements. Results: Encoding of CFC increases Aβ oligomerization in the hippocampus but not in the amygdala of Tg2576 mice. The presence of Aβ oligomers predicts vulnerability to network dysfunctions, as low c-Fos activation and spine maturation are detected in the hippocampus of Tg2576 mice upon recall of CFC memory. Rather, enhanced c-Fos activation and new spines are evident in the amygdala of Tg2576 mice compared with wild-type control mice. Preventing Aβ increase in the hippocampus of Tg2576 mice restores CFC-associated spine changes to wild-type levels in both the hippocampus and amygdala. Conclusions: Our study provides the first evidence of neural compensation consisting of enhanced synaptic activity in brain regions spared by Aβ load. Furthermore, it unravels an activity-mediated feedback loop through which neuronal activation during CFC encoding favors Aβ oligomerization in the hippocampus and prevents synaptic rearrangements in this region

Blood transfusions and steatohepatitis are independent risk factors for complications following liver resection for colorectal cancer liver metastases

none9The aim of the present study was to evaluate the risk factors for postoperative complications following liver resection for colorectal cancer liver metastases. Patients who underwent hepatic resection for colorectal cancer liver metastases were stratified according to chemotherapy administration and body mass index (BMI) to eliminate potential confounding factors. A univariate analysis was conducted to identify potential risk factors for postoperative complications following liver resection. Variables that exhibited a potential association were evaluated by multivariable logistic regression analysis to identify those independently associated with postoperative morbidity. Between January 2012 and March 2012, 100 patients underwent hepatic resection for liver metastases from colorectal carcinoma at the Treviso Regional Hospital (Treviso, Italy) and at the Regina Elena National Cancer Institute (Rome, Italy). Of the 100 patients, 61 received preoperative oxaliplatin- or irinotecan-based chemotherapy. A total of 25 the patients had a BMI of ≥28 kg/m(2). On univariate analysis, BMI ≥28 kg/m(2) was found to be positively correlated with the presence of steatosis (P<0.01) and steatohepatitis (P<0.01). The administration of preoperative chemotherapy was correlated with the development of steatosis (P<0.01), steatohepatitis (P=0.02) and postoperative complications (P=0.03). Even following stratification for the use of preoperative chemotherapy, BMI ≥28 kg/m(2) maintained its positive association with steatohepatitis. On multivariate analysis, steatohepatitis (P=0.005, HR=0.118, 95% CI: 0.027-0.518) and blood transfusions (P=0.001, HR=0.131, 95% CI: 0.038-0.452) were independently associated with postoperative complications. BMI ≥28 kg/m(2) (P=0.004, HR=8.30, 95% CI: 2.39-28.7) and irinotecan treatment (P=0.016, HR=0.16, 95% CI: 0.037-0.711) were independent risk factors for steatohepatitis. In conclusion, steatohepatitis and perioperative blood transfusions were found to be the main determinant of postoperative complications following liver resection for colorectal liver metastases. Overweight patients may be more prone to the cytotoxic effects of irinotecan, harboring a higher risk of developing steatohepatitis.noneMassani, Marco; Capovilla, Giovanni; Ruffolo, Cesare; Bonariol, Roberta; Maccatrozzo, Paola; Tuci, Francesco; Battistella, Giuseppe; Grazi, Gian Luca; Bassi, NicolòMassani, Marco; Capovilla, Giovanni; Ruffolo, Cesare; Bonariol, Roberta; Maccatrozzo, Paola; Tuci, Francesco; Battistella, Giuseppe; Grazi, Gian Luca; Bassi, Nicolo

Glecaprevir/Pibrentasvir is safe and effective in Italian patients with chronic hepatitis C aged 75 years or older: A multicentre study

Background: Glecaprevir and Pibrentasvir (G/P) determine high rates of sustained virological response (SVR) with optimal safety profile in patients with chronic hepatitis C virus (HCV) infection. The efficacy and safety of G/P in Caucasian patients aged 75 years and older have not been widely analysed. Methods: This is a retrospective multicentre real-world study enrolling all consecutive patients 75 years and older who received G/P between October 2017 and January 2022 at five referral centres in Italy. SVR was analysed by intention-to-treat (ITT) and per-protocol analyses (PP). Results: A total of 570 patients met the inclusion criteria and were analysed: mean age was 80 (75-97) years, 356 (62%) were females, 52% (298/570) had HCV-1, 44% (252/570) had HCV-2 and 137 (24%) patients had liver cirrhosis. Four hundred and sixty-three (81%) patients were taking at least one concomitant drug, with 144 (25%) taking ≥5 concomitant drugs. G/P was given for 8 weeks in 488 patients (86%). During treatment, 48 patients (8%) reported side effects, with 10 (2%) patients discontinuing treatment prematurely. Two patients developed treatment-unrelated serious adverse events. Overall, the SVR rate was 97.9% (558/570) by ITT analysis and 99.6% (558/560) by PP analysis. SVR rates remained consistently high among subgroup analysis stratified by genotype, treatment duration, fibrosis stage and concomitant medications. Conclusions: Treatment with G/P achieved 97.9% SVR rates in HCV patients older than 75 years of age. Safety was optimal with only 2% of patients discontinuing early