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

Direct Isolation, Culture and Transplant of Mouse Skeletal Muscle Derived Endothelial Cells with Angiogenic Potential

Background: Although diseases associated with microvascular endothelial dysfunction are among the most prevalent illnesses to date, currently no method exists to isolate pure endothelial cells (EC) from skeletal muscle for in vivo or in vitro study. Methodology: By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1 +, CD31 +, CD34 dim and CD45 2 cells from skeletal muscles of C57BL6 mice. Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes. When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature. Conclusion: This method, which is highly reproducible, can be used to study the biology and role of EC in diseases such as peripheral vascular disease. In addition this method allows us to isolate large quantities of skeletal muscle derived EC with potential for therapeutic angiogenic applications

LYVE-1 and the response of lymphatic vessel endothelium to inflammation

The lymphatic system provides a conduit for the delivery of antigen-presenting cells from peripheral tissues to draining lymph nodes, both constitutively and during inflammation, when a dramatic increase in the number of trafficking events occurs. This thesis is concerned with the role played by the lymphatic endothelium under pro-inflammatory conditions and the mechanisms regulating entry of leukocytes into the lymphatic capillaries. Monoclonal antibodies against the lymphatic vessel endothelial-specific receptor for hyaluronan, LYVE-1, were raised in rat against the mouse orthologue, for use both as a marker for lymphatics in mouse tissue and to investigate the physiological role of this protein. LYVE-1 was found to be dramatically and specifically down-regulated by TNFa and TNFp, by rapid internalisation and transcriptional down-regulation in primary lymphatic endothelial cells. A significant reduction in LYVE-1 expression was also recorded in intact lymphatic vessels in response to TNFa and in an allergen-induced mouse model of skin inflammation. Furthermore, lymphatic endothelial cells were found to respond to this pro-inflammatory cytokine by instigating a distinct expression programme characterised by up-regulation of the key leukocyte adhesion receptors VCAM-1, ICAM-1 and E-selectin; chemokines and other potential regulators of leukocyte entry. Moreover, the major components of this programme were confirmed in dermal lymphatic vessels in the allergen-induced mouse model of skin inflammation, simultaneous with the down-modulation of LYVE-1 and lymphatic accumulation of migrating leukocytes. The results described in this thesis provide the first evidence that lymphatic endothelial cells play an active role in inflammation. In addition these data indicate that the lymphatic and blood vascular systems share components of the same address code for specific vascular targeting

A structural and developmental study of the posttrabecular aqueous outflow pathway in the mouse eye

Includes abstract.Includes bibliographical references (p. 157-171)

Yap1 promotes sprouting and proliferation of lymphatic progenitors downstream of Vegfc in the zebrafish trunk

Lymphatic vascular development involves specification of lymphatic endothelial progenitors that subsequently undergo sprouting, proliferation and tissue growth to form a complex second vasculature. The Hippo pathway and effectors Yap and Taz control organ growth and regulate morphogenesis and cellular proliferation. Yap and Taz control angiogenesis but a role in lymphangiogenesis remains to be fully elucidated. Here we show that YAP displays dynamic changes in lymphatic progenitors and Yap1 is essential for lymphatic vascular development in zebrafish. Maternal and Zygotic (MZ) yap1 mutants show normal specification of lymphatic progenitors, abnormal cellular sprouting and reduced numbers of lymphatic progenitors emerging from the cardinal vein during lymphangiogenesis. Furthermore, Yap1 is indispensable for Vegfc-induced proliferation in a transgenic model of Vegfc overexpression. Paracrine Vegfc-signalling ultimately increases nuclear YAP in lymphatic progenitors to control lymphatic development. We thus identify a role for Yap in lymphangiogenesis, acting downstream of Vegfc to promote expansion of this vascular lineage.Lin Grimm, Hiroyuki Nakajima, Smrita Chaudhury, Neil I Bower, Kazuhide S Okuda, Andrew G Cox, Natasha L Harvey, Katarzyna Koltowska, Naoki Mochizuki, Benjamin M Hoga

Elucidating how αvβ3-integrin regulates neuropilin-1’s role in tumour angiogenesis in order to improve anti-angiogenic therapy

αvβ3-integrin expression is vastly upregulated in tumour vasculature, and has long been considered a key molecule in promoting tumour angiogenesis. However, its initial promise as a target in anti-angiogenic therapy has wavered since the αvβ3-integrin antagonist cilengitide did not meet its end point in Phase III clinical trials for the treatment of glioblastoma. This failure corresponds with the enhanced tumour growth and angiogenesis observed in β3-integrin-knockout mice, which potentially occurs via a compensatory upregulation of VEGFR2 and enhancement in VEGFR2-neuropilin-1 interactions. Here, I show that tumour growth and angiogenesis are sensitive to neuropilin-1 perturbation even with only a 50% reduction in β3-integrin expression. β3-integrin-heterozygous, but not wild-type, mice show an increased dependence on neuropilin-1 that is not related to changes in neuropilin-1-mediated VEGFR2 function. Rather, the suppression of β3-integrin leads to the activation of a neuropilin-1-dependent endothelial cell migration pathway via a mechanism in which NRP1 is mobilised away from mature focal adhesions following VEGF-stimulation. Concordantly, the simultaneous genetic targeting of both molecules significantly impairs paxillin activation and focal adhesion turnover in endothelial cells, and thus inhibits endothelial cell migration, and tumour growth and angiogenesis, even in established tumours. These findings therefore provide important pre-clinical evidence that pharmacologically targeting both molecules in unison might be an effective anti-angiogenic therapy for patients with advanced cancers

An investigation into the regulatory mechanisms of neutrophil migration into lymphatic vessels in vivo

PhDNeutrophils are recognised to play a pivotal role at the interface between the innate and adaptive immune responses following their rapid recruitment to inflamed tissues and lymphoid organs. Whilst neutrophil trafficking through blood vessels has been extensively studied, the molecular mechanisms regulating their migration into the lymphatic system are still poorly understood. This thesis therefore aimed to investigate the mechanisms involved in neutrophil migration across the lymphatic endothelium during TNF- or Complete Freund’s Adjuvant + antigen (CFA+Ag)-induced inflammation of cremaster muscles in vivo. This work revealed that TNF- or CFA+Ag-stimulation induces a rapid but transient entry of tissue-infiltrated neutrophils into lymphatic vessels, a response associated with the regulation and redistribution of the lymphatic endothelial cell glycocalyx. Interestingly, antigen sensitisation resulted in the production of endogenous TNF within cremaster muscles. Using anti-TNF blocking antibodies and mice deficient in both TNF receptors (p55 and p75), endogenous TNF was demonstrated for the first time to be involved in priming and triggering the migration of neutrophils into tissue-associated lymphatic vessels upon antigen challenge. Additionally, the use of chimeric mice exhibiting neutrophils deficient in both TNFRs demonstrated that TNF directly acts on leukocytes to induce neutrophil migration into lymphatic vessels. Furthermore, the results show that TNF-induced migration of neutrophils into the lymphatic system occurs in a strictly CCR7-dependent manner; blocking CXCR4 or CXCL1 signalling does not affect this response. Finally, both TNF- or CFA+AG-stimulation induced ICAM-1 up-regulation on lymphatic vessels, allowing neutrophils to crawl along the lumen; a response that was demonstrated to be TNF-dependent. These results have provided new insights into the mechanisms that mediate neutrophil migration into lymphatic vessels and their subsequent crawling within these vessels during inflammation. In particular, a new role for TNF as a key regulator of these processes has been demonstrated. Taken together, this work has highlighted potential and effective targets to manipulate the role of neutrophils in adaptive immune responses in vivo.Institute of Bioengineering at Queen Mary University of London (QMUL) and Arthritis Research UK (ARUK