Cerebrospinal fluid outflow: a review of the historical and contemporary evidence for arachnoid villi, perineural routes, and dural lymphatics.

Cerebrospinal fluid (CSF) is produced by the choroid plexuses within the ventricles of the brain and circulates through the subarachnoid space of the skull and spinal column to provide buoyancy to and maintain fluid homeostasis of the brain and spinal cord. The question of how CSF drains from the subarachnoid space has long puzzled scientists and clinicians. For many decades, it was believed that arachnoid villi or granulations, outcroppings of arachnoid tissue that project into the dural venous sinuses, served as the major outflow route. However, this concept has been increasingly challenged in recent years, as physiological and imaging evidence from several species has accumulated showing that tracers injected into the CSF can instead be found within lymphatic vessels draining from the cranium and spine. With the recent high-profile rediscovery of meningeal lymphatic vessels located in the dura mater, another debate has emerged regarding the exact anatomical pathway(s) for CSF to reach the lymphatic system, with one side favoring direct efflux to the dural lymphatic vessels within the skull and spinal column and another side advocating for pathways along exiting cranial and spinal nerves. In this review, a summary of the historical and contemporary evidence for the different outflow pathways will be presented, allowing the reader to gain further perspective on the recent advances in the field. An improved understanding of this fundamental physiological process may lead to novel therapeutic approaches for a wide range of neurological conditions, including hydrocephalus, neurodegeneration and multiple sclerosis

Dynamics of lymphatic regeneration and flow patterns after lymph node dissection

Knowledge about the mechanisms of regeneration of the lymphatic vasculature after surgical trauma is essential for the development of strategies for the prevention and therapy of lymphedema. However, little is known about the alterations of lymphatic flow directly after surgical trauma. We investigated lymphatic function in mice using near-infrared imaging for a period of 4weeks after surgeries that mimic sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND), by removal of the popliteal lymph node (LN) alone or together with the popliteal fat pad, respectively. SLNB-like surgery did not cause changes in lymphatic drainage in the majority of cases. In contrast, lymphatic drainage impairment shown by collecting vessel rupture, dermal backflow and rerouting of lymph flow via collateral vessels were observed after ALND-like surgery. All collateral vessels drained to the inguinal LN. These results indicate that less invasive surgery prevents lymphatic decompensation. They also reveal the development and maturation of collateral lymphatic vessels after extensive surgical trauma, which reroute the flow of lymph towards a different LN. These findings might be helpful for the development of strategies to prevent and/or treat post-surgical lymphedem

Dynamics of lymphatic regeneration and flow patterns after lymph node dissection.

Knowledge about the mechanisms of regeneration of the lymphatic vasculature after surgical trauma is essential for the development of strategies for the prevention and therapy of lymphedema. However, little is known about the alterations of lymphatic flow directly after surgical trauma. We investigated lymphatic function in mice using near-infrared imaging for a period of 4 weeks after surgeries that mimic sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND), by removal of the popliteal lymph node (LN) alone or together with the popliteal fat pad, respectively. SLNB-like surgery did not cause changes in lymphatic drainage in the majority of cases. In contrast, lymphatic drainage impairment shown by collecting vessel rupture, dermal backflow and rerouting of lymph flow via collateral vessels were observed after ALND-like surgery. All collateral vessels drained to the inguinal LN. These results indicate that less invasive surgery prevents lymphatic decompensation. They also reveal the development and maturation of collateral lymphatic vessels after extensive surgical trauma, which reroute the flow of lymph towards a different LN. These findings might be helpful for the development of strategies to prevent and/or treat post-surgical lymphedema

T Cell Migration from Inflamed Skin to Draining Lymph Nodes Requires Intralymphatic Crawling Supported by ICAM-1/LFA-1 Interactions.

T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged T cell migration through afferent LVs in vivo. T cells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic T cell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. In vitro, blockade of endothelial cell-expressed ICAM-1 reduced T cell adhesion, crawling, and transmigration across lymphatic endothelium and decreased T cell advancement from capillaries into lymphatic collectors in skin explants. In vivo, T cell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that T cell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process

Decline of lymphatic vessel density and function in murine skin during aging.

Lymphatic vessels play important roles in the pathogenesis of many conditions that have an increased prevalence in the elderly population. However, the effects of the aging process on the lymphatic system are still relatively unknown. We have applied non-invasive imaging and whole-mount staining techniques to assess the lymphatic vessel function and morphology in three different age groups of mice: 2 months (young), 7 months (middle-aged), and 18 months (aged). We first developed and validated a new method to quantify lymphatic clearance from mouse ear skin, using a lymphatic-specific near-infrared tracer. Using this method, we found that there is a prominent decrease in lymphatic vessel function during aging since the lymphatic clearance was significantly delayed in aged mice. This loss of function correlated with a decreased lymphatic vessel density and a reduced lymphatic network complexity in the skin of aged mice as compared to younger controls. The blood vascular leakage in the skin was slightly increased in the aged mice, indicating that the decreased lymphatic function was not caused by a reduced capillary filtration in aged skin. The decreased function of lymphatic vessels with aging might have implications for the pathogenesis of a number of aging-related diseases

Beyond Trial and Error: A Systematic Development of Liposomes Targeting Primary Macrophages

Monocytes/macrophages are phagocytic innate immune cells playing a pivotal role in tissue homeostasis, inflammation, and antitumor immunity in a microenvironment-dependent manner. By expressing pattern recognition and scavenger receptors on their surface, macrophages selectively take up pathogens, cellular debris, and often—undesirably—drug delivery systems. On the other hand, the propensity of phagocytic cells to internalize particulate drug carriers is used to load them with a cargo of choice, turning the monocytes/macrophages into a diagnostic or therapeutic Trojan horse. Identifying the ideal physicochemical properties of particulate carriers such as liposomes to achieve the most efficient macrophage-mediated drug delivery has been object of extensive research in the past, but the studies reported so far rely solely on trial-and-error approaches. Herein, a design of experiment (DoE) strategy to identify the optimal liposomal formulation is proposed, fully characterized in terms of size, surface charge, and membrane fluidity, to maximize macrophage targeting. The findings are validated using mouse bone marrow-derived macrophages, a primary preparation modeling in vivo monocyte-derived macrophages, thus confirming the robustness and versatility of the systematic and iterative approach and suggesting the promising potential of the DoE approach for the design of cell-targeting delivery systems

Magnetic resonance imaging of cerebrospinal fluid outflow after low-rate lateral ventricle infusion in mice.

The anatomical routes for the clearance of cerebrospinal fluid (CSF) remain incompletely understood. However, recent evidence has given strong support for routes leading to lymphatic vessels. A current debate centers upon the routes through which CSF can access lymphatics, with evidence emerging for either direct routes to meningeal lymphatics or along cranial nerves to reach lymphatics outside the skull. Here, a method was established to infuse contrast agent into the ventricles using indwelling cannulae during imaging of mice at 2 and 12 months of age by magnetic resonance imaging. As expected, a significant decline in overall CSF turnover was found with aging. Quantifications demonstrated that the bulk of the contrast agent flowed from the ventricles to the subarachnoid space in the basal cisterns. Comparatively little contrast agent signal was found at the dorsal aspect of the skull. The imaging dynamics from the two cohorts revealed that the contrast agent cleared from the cranium through the cribriform plate to the nasopharyngeal lymphatics. On decalcified sections, we confirmed that fluorescentlylabeled ovalbumin drains through the cribriform plate and can be found within lymphatics surrounding the nasopharynx. In conclusion, routes leading to nasopharyngeal lymphatics appear to be a major efflux pathway for cranial CSF

Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease model

Increased lymphangiogenesis in joints of mice with inflammatory arthritis

Angiogenesis is involved in the pathogenesis of inflammatory arthritis, but little is known about the role of lymphangiogenesis in this setting. Here, we examined whether tumor necrosis factor (TNF) stimulates osteoclast precursors (OCPs) to produce the lymphatic growth factor, vascular endothelial growth factor-C (VEGF-C), and induce lymphangiogenesis. We used TNF-transgenic (Tg) mice and mice with serum-induced arthritis. OCPs were purified by fluorescence-activated cell sorting of CD11b+/Gr-1-/lo blood or bone marrow cells and subjected to microarray analysis or were generated from spleen or joint cells and treated with TNF. Expression of VEGFs was analyzed and examined by real-time reverse transcription-polymerase chain reaction and Western blotting. Immunostaining and magnetic resonance imaging were used to quantify lymphatic vessels and volumes of synovium and draining lymph nodes. TNF stimulated VEGF-C expression by OCPs and increased nuclear factor-kappa B (NF-κB) binding to an NF-κB sequence in the VEGF-C promoter. OCPs from joints of TNF-Tg mice express high levels of VEGF-C. Lymphatic vessel numbers and size were markedly increased in joint sections of TNF-Tg mice and mice with serum-induced arthritis. The severity of synovitis correlated with draining lymph node size. In summary, TNF induces OCPs to produce VEGF-C through NF-κB, leading to significantly increased lymphangiogenesis in joints of arthritic mice. The lymphatic system may play an important role in the pathogenesis of inflammatory arthritis

Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice.

Cerebrospinal fluid (CSF) has been commonly accepted to drain through arachnoid projections from the subarachnoid space to the dural venous sinuses. However, a lymphatic component to CSF outflow has long been known. Here, we utilize lymphatic-reporter mice and high-resolution stereomicroscopy to characterize the anatomical routes and dynamics of outflow of CSF. After infusion into a lateral ventricle, tracers spread into the paravascular spaces of the pia mater and cortex of the brain. Tracers also rapidly reach lymph nodes using perineural routes through foramina in the skull. Using noninvasive imaging techniques that can quantify the transport of tracers to the blood and lymph nodes, we find that lymphatic vessels are the major outflow pathway for both large and small molecular tracers in mice. A significant decline in CSF lymphatic outflow is found in aged compared to young mice, suggesting that the lymphatic system may represent a target for age-associated neurological conditions