Cerebrospinal fluid absorption (CSF) at the cribriform plate is mediated by direct extracranial connections to the lymphatic system. Given the accessibility of these pharmacologically responsive vessels we hypothesized that the rate of CSF outflow can be modulated via the intranasal delivery of drugs known to affect lymphatic contractile activity.

INTRODUCTIONLymph nodes are often the target of radiotherapy procedures. Unfortunately, the impact of nodal irradiation on lymphatic function is uncertain. In this study, our aim was to quantify the impact of lymph node irradiation on lymph flow.METHODS AND RESULTSThe popliteal node or the nodal excision site of rabbits was treated with four daily 8 Gy doses of radiation. A FITC-dextran tracer was infused into a prenodal popliteal lymphatic. The area under the tracer blood recovery curve (AUC) indicated lymphatic functionality and the inflow pressure versus flow rate relationship inferred resistance through the system. Fluoroscopic and histological examination provided supporting data. Radiation of intact nodes decreased lymph transport significantly at 1 week, 1 month, and 6 months post-treatment (AUCs of 207.9 ± 79.87, 191.6 ± 62.95, and 250.44 ± 46.45) in comparison to controls (667.32 ± 104.18). Surprisingly, this functional decline was similar to that detected with a combination of node removal and irradiation of the excision site. The pressure-flow relationships in all treatment groups were significantly different from controls. This may be due in part to fibrosis and the thickening of the nodal capsules and trabeculae observed at 1 and 6 months. Fluoroscopy and Evans blue dye studies revealed vigorous new lymphatic vessel growth and occasionally, vessels anastomosed with local veins.CONCLUSIONSIrradiation of the popliteal lymph node impaired lymph transport and increased the pressure required to maintain flow through the system. New vessel formation and the growth of lymph-venous anastomoses indicated the development of alternative drainage pathways as a compensatory response.

The objective of this study was to examine host-shunt interactions in sheep with kaolin-induced hydrocephalus.

Perhaps the greatest paradox in the hydrocephalus field is the failure of researchers to consistently measure transmantle pressure gradients (ventricle to subarachnoid space) in either human or animal models of the communicating form of the disorder. Without such a gradient, conceptualization of how ventricular distention occurs is difficult. Based on evidence from both a mathematical model [35] and experiments in skin [51], we observed that the intraventricular injection of anti-β1 integrin antibodies in rat brains results in a reduction of periventricular pressures to values below those monitored in the ventricles. In addition, many of these animals developed hydrocephalus [30]. We conclude that the dissociation of β1 integrins from the surrounding matrix fibers generates pressure gradients favouring ventricular expansion suggesting a novel mechanism for hydrocephalus development. Several issues, however, need further clarification. If hydrostatic pressure declines in the periventricular tissues then fluid absorption must occur. Aquaporin-4 (AQP4) is a likely candidate for this absorption as it is the predominant water channel in the brain. Indeed, when capillary function is negated, periventricular interstitial fluid pressures increase after anti-β1 integrin antibody administration. This suggests that capillary absorption of parenchymal water may play a pivotal role in the generation of pressure gradients in our hydrocephalus model. Focusing on these issues, we present two poroelastic models to investigate the role of intramantle pressure gradients in ventriculomegaly and to determine if integrin-matrix disassociation represents a complete causative mechanism for hydrocephalus development.

Lymphedema is a frequent consequence of lymph node excision during breast cancer surgery. Current treatment options are limited mainly to external compression therapies to limit edema development. We investigated previously, postsurgical lymphedema in a sheep model following the removal of a single lymph node and determined that autologous lymph node transplantation has the potential to reduce or prevent edema development. In this report, we examine the potential of lymphangiogenic therapy to restore lymphatic function and reduce postsurgical lymphedema.

We recently reported a lymphatic cerebrospinal fluid (CSF) absorption deficit in a kaolin model of communicating hydrocephalus in rats with ventricular expansion correlating negatively with the magnitude of the impediment to lymphatic function. However, it is possible that CSF drainage was not significantly altered if absorption at other sites compensated for the lymphatic defect. The purpose of this study was to investigate the impact of the lymphatic absorption deficit on global CSF absorption (CSF outflow resistance).

Intraocular pressure (IOP) is the most important risk factor for glaucoma development and progression. Most anti-glaucoma treatments aim to lower IOP by enhancing aqueous humor drainage from the eye. Aqueous humor drainage occurs via well-characterized trabecular meshwork (TM) and uveoscleral (UVS) pathways, and recently described ciliary body lymphatics. The relative contribution of the lymphatic pathway to aqueous drainage is not known. We developed a sheep model to quantitatively assess lymphatic drainage along with TM and UVS outflows. This study describes that model and presents our initial findings.Following intracameral injection of I-125-bovine serum albumin (BSA), lymph was continuously collected via cannulated cervical lymphatic vessels and the thoracic lymphatic duct over either a 3-h or 5-h time period. In the same animals, blood samples were collected from the right jugular vein every 15 min. Lymphatic and TM drainage were quantitatively assessed by measuring I-125-BSA in lymph and plasma, respectively. Radioactive tracer levels were also measured in UVS and "other" ocular tissue, as well as periocular tissue harvested 3 and 5 h post-injection. Tracer recovered from UVS tissue was used to estimate UVS drainage. The amount of I-125-BSA recovered from different fluid and tissue compartments was expressed as a percentage of total recovered tracer. Three hours after tracer injection, percentage of tracer recovered in lymph and plasma was 1.64% +/- 0.89% and 68.86% +/- 9.27%, respectively (n = 8). The percentage of tracer in UVS, other ocular and periocular tissues was 19.87% +/- 5.59%, 4.30% +/- 3.31% and 5.32% +/- 2.46%, respectively. At 5 h (n = 2), lymphatic drainage was increased (6.40% and 4.96% vs. 1.64%). On the other hand, the percentage of tracer recovered from UVS and other ocular tissue had decreased, and the percentage from periocular tissue showed no change. Lymphatic drainage increased steadily over the 3 h post-injection period, while TM drainage increased rapidly reaching a plateau at 30 min.This quantitative sheep model enables assessment of relative contributions of lymphatic drainage, TM and UVS outflows, and may help to better understand the effects of glaucoma agents on outflow pathways. (C) 2011 Elsevier Ltd. All rights reserved.

Background: Our objective was to develop an animal model of postsurgical lymphedema that would permit quantitation of edema and lymphatic function after the removal of a single popliteal lymph node in sheep. Methods: As a measure of lymph transport, 125I-human serum albumin was injected into prenodal vessels at 8, 12 and 16 weeks after nodal excision, and plasma levels of the protein tracer were used to calculate the transport rate of the tracer to blood (percent injected per hour). Edema was quantified from the circumferential measurement of the hind limbs. Results and Conclusions: Following nodal excision, the limbs became progressively more edematous up to 3 days after nodectomy. After this, the swelling decreased but had not resolved even at 16 weeks after surgery. Compared with control limbs (17.2 ± 0.6; n = 7), lymphatic function was depressed at 8 weeks after surgery (10.6 ± 1.5; n = 7). At 12 (14.4 ± 1.0; n = 7) and 16 weeks (13.9 ± 1.0; n = 6), regeneration of lymphatic vessels at the excision site helped to restore about 80% of lymphatic capacity. These techniques may be helpful in understanding the pathophysiology associated with cancer-related postsurgical lymphedema and may facilitate the development of new strategies to treat or prevent this condition.

Background: The authors' objective was to test whether the transplantation of an autologous lymph node into a nodal excision site in sheep would restore lymphatic transport function and reduce the magnitude of postsurgical lymphedema. Methods: As a measure of lymph transport, iodine-125 human serum albumin was injected into prenodal vessels at 8 and 12 weeks after surgery, and plasma levels of the protein were used to calculate the transport rate of the tracer to blood (percent injected per hour). Edema was quantified from the circumferential measurement of the hind limbs. Results: The transplantation of avascular lymph nodes at 8 (n = 6) and 12 weeks (n = 6) produced lymphatic function levels of 12.3 ± 0.5 and 12.6 ± 0.8, respectively. These values were significantly less (p < 0.001) than those measured at similar times in the animals receiving sham surgical procedures (16.6 ± 0.7, n = 6; and 16.1 ± 0.7, n = 6, respectively). When vascularized transplants were performed, lymphatic function was similar to the sham controls and significantly greater (p < 0.001) than that of the avascular group (8 weeks, 15.8 ± 0.9, n = 8; 12 weeks, 15.7 ± 1.0, n = 10). Lymph transport correlated significantly with the health of the transplanted nodes (scaled with histologic analysis) (p < 0.0001). The vascularized node transplants (n = 18) were associated with the greatest clinical improvement, with the magnitude of edema in these limbs exhibiting significantly lower levels of edema (p = 0.039) than nontreated limbs (n = 18). Conclusions: The successful reimplantation of a lymph node into a nodal excision site has the potential to restore lymphatic function and facilitate edema resolution. This result has important conceptual implications in the treatment of postsurgical lymphedema.

Impaired aqueous humor flow from the eye may lead to elevated intraocular pressure and glaucoma. Drainage of aqueous fluid from the eye occurs through established routes that include conventional outflow via the trabecular meshwork, and an unconventional or uveoscleral outflow pathway involving the ciliary body. Based on the assumption that the eye lacks a lymphatic circulation, the possible role of lymphatics in the less well defined uveoscleral pathway has been largely ignored. Advances in lymphatic research have identified specific lymphatic markers such as podoplanin, a transmembrane mucin-type glycoprotein, and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Lymphatic channels were identified in the human ciliary body using immunofluorescence with D2-40 antibody for podoplanin, and LYVE-1 antibody. In keeping with the criteria for lymphatic vessels in conjunctiva used as positive control, D2-40 and LYVE-1-positive lymphatic channels in the ciliary body had a distinct lumen, were negative for blood vessel endothelial cell marker CD34, and were surrounded by either discontinuous or no collagen IV-positive basement membrane. Cryo-immunogold electron microscopy confirmed the presence D2-40-immunoreactivity in lymphatic endothelium in the human ciliary body. Fluorescent nanospheres injected into the anterior chamber of the sheep eye were detected in LYVE-1-positive channels of the ciliary body 15, 30, and 45 min following injection. Four hours following intracameral injection, Iodine-125 radio-labeled human serum albumin injected into the sheep eye (n = 5) was drained preferentially into cervical, retropharyngeal, submandibular and preauricular lymph nodes in the head and neck region compared to reference popliteal lymph nodes (P < 0.05). These findings collectively indicate the presence of distinct lymphatic channels in the human ciliary body, and that fluid and solutes flow at least partially through this system. The discovery of a uveolymphatic pathway in the eye is novel and highly relevant to studies of glaucoma and other eye diseases.

In some tissues, the injection of antibodies to the beta(1)-integrins leads to a reduction in interstitial fluid pressure, indicating an active role for the extracellular matrix in tissue pressure regulation. If perturbations of the matrix occur in the periventricular area of the brain, a comparable lowering of interstitial pressures may induce transparenchymal pressure gradients favoring ventricular expansion. To examine this concept, we measured periventricular (parenchymal) and ventricular pressures with a servo-null micropipette system (2-microm tip) in adult Wistar rats before and after anti-integrin antibodies or IgG/IgM isotype controls were injected into a lateral ventricle. In a second group, the animals were kept for 2 wk after similar injections and after euthanization, the brains were removed and assessed for hydrocephalus. In experiments in which antibodies to beta(1)-integrins (n = 10) but not isotype control IgG/IgM (n = 7) were injected, we observed a decline in periventricular pressures relative to the preinjection values. Under similar circumstances, ventricular pressures were elevated (n = 10) and were significantly greater than those in the periventricular interstitium. We estimated ventricular to periventricular pressure gradients of up to 4.3 cmH(2)O. In the chronic preparations, we observed enlarged ventricles in many of the animals that received injections of anti-integrin antibodies (21 of 29 animals; 72%) but not in any animal receiving the isotype controls. We conclude that modulation/disruption of beta(1)-integrin-matrix interactions in the brain generates pressure gradients favoring ventricular expansion, suggesting a novel mechanism for hydrocephalus development.

Communicating hydrocephalus (CH) occurs frequently, but clinically-relevant animal models amenable to diagnostic imaging and cerebrospinal fluid shunting are not available. In order to develop and characterize models of subarachnoid space (SAS) obstruction at the basal cisterns (BC) or cerebral convexities (CX), 25% kaolin was injected in adult female Sprague-Dawley rats following halothane anesthesia; intact- or saline-injected animals served as controls. For BC animals (n=28 hydrocephalics, n=20 controls), an anterior approach to the C1-clivus interval was employed and 30 microl of kaolin or saline was injected. For CX injections (n=13 hydrocephalics, n=3 controls), 50-60 microl of kaolin was injected bilaterally after separating the partitions in the SAS. In BC-injected rats, kaolin was observed grossly in the basal cisterns but not in the cisterna magna or at the foramina of Luschka, indicating that communicating (or extra-ventricular)--not obstructive--hydrocephalus had been induced. Following ketamine/xylazine anesthesia, magnetic resonance imaging (MRI) of gadolinium injected into the lateral ventricle also demonstrated CSF flow from the foramina of Luschka. MRI also revealed that ventriculomegaly progressed steadily in BC animals and by 2 weeks post-kaolin the mean Evan's ratio (frontal horn) increased significantly (mean 0.45 compared to 0.31 in intact- and 0.34 in saline-injected controls; p<0.001 for each). CX animals exhibited kaolin deposits covering approximately 80% of the cerebral hemispheres and developed noticeable ventriculomegaly (mean Evan's ratio 0.40), which was significant relative to intact animals (p=0.011) but not saline-injected controls. Surprisingly, ventriculomegaly following CX injections was less severe and much more protracted, requiring 3-4 months to develop compared to ventriculomegaly produced by BC obstruction. No hydrocephalic animals demonstrated obvious neurological deficits, but BC-injected animals that subsequently developed more severe ventriculomegaly exhibited nasal discharges and "coughing" for several days following kaolin injection. The new BC model is relevant because the clinical presentation of CH in children is often associated with obstruction at this site, while the CX model may be more representative of late adult onset normal pressure hydrocephalus.

Several parameters associated with the cerebrospinal fluid (CSF) system show a change in the later stages of life, including elevated CSF outflow resistance. The latter implies a CSF absorption deficit. As a significant portion of CSF absorption occurs into extracranial lymphatic vessels located in the olfactory turbinates, the purpose of this study was to determine whether any age‐related impediments to CSF absorption existed at this location. In previous studies, we observed rapid movement of the CSF tracer into the olfactory turbinates in young rats (peaking 30 min after injection), with the concentration of the tracer being much higher in the turbinates than in any other tissue measured. In the study reported here, 125I‐human serum albumin was injected into the lateral ventricles of 3‐, 6‐, 12‐ and 19‐month‐old Fisher 344 rats. The animals were sacrificed at various times after injection of the radioactive tracer, and appropriate tissue samples were extracted. At 30 min post injection, the average tracer values expressed as per cent injected/g tissue were 6.68 ± 0.42 (n = 9, 3 months), 4.78 ± 0.67 (n = 9, 6 months), 2.49 ± 0.31 (n = 9, 12 months) and 2.42 ± 0.72 (n = 9, 19 months). We conclude that lymphatic CSF transport declines significantly with age. In concert with the known drop in CSF formation, the reduction in lymphatic CSF absorption may contribute to a decrease in CSF turnover in the elderly.

We examined the impact of lymph flow obstruction in large post-nodal lymphatic vessels in sheep. A silk ligature was placed 2 cm downstream from the prescapular or popliteal lymph node and tightened to interrupt flow. At 6, 12 and 16 weeks after lymph flow blockage, a network of small interconnecting lymphatics (approximately 10-40 microm in diameter) could be observed in the vicinity of the ligature. These were identified using antibodies to the lymphatic endothelial markers LYVE-1 or VEGFR-3 or unequivocally, with the upstream intraluminal injection of the non-specific cell dye CFDA-SE. The observed lymphangiogenesis coincided with increased levels of Prox1, Tie2 (Y992) phosphorylation, MAPK activation, and decreased Akt activition. In the popliteal preparations, saline was infused into the prenodal ducts upstream of the regeneration site. The slopes of the inflow pressure versus flow relationships were 17.3+/-3.6, immediately after vessel obstruction, 36.2+/-9.6 at 6 weeks and 15.0+/-5.3 at 12-16 weeks. For comparison, the average slope in a completely intact popliteal system was 3.1+/-0.3 (from a previous publication). The resistance to flow remained high up to 12-16 weeks after flow obstruction suggesting that normal flow parameters had not been achieved over this time. The lymph node appeared to have some role in limiting the impact of post-nodal lymph obstruction, a function that appeared to be compromised by lymph stasis.

It has been assumed that the pathogenesis of hydrocephalus includes a cerebrospinal fluid (CSF) absorption deficit. Because a significant portion of CSF absorption occurs into extracranial lymphatics located in the olfactory turbinates, the purpose of this study was to determine whether CSF transport was compromised at this location in a kaolin-induced communicating (extraventricular) hydrocephalus model in rats. Under 1–3% halothane anesthesia, kaolin ( n = 10) or saline ( n = 9) was introduced into the basal cisterns of Sprague-Dawley rats, and the development of hydrocephalus was assessed 1 wk later using MRI. After injection of human serum albumin (125I-HSA) into a lateral ventricle, the tracer enrichment in the olfactory turbinates 30 min postinjection provided an estimate of CSF transport through the cribriform plate into nasal lymphatics. Lateral ventricular volumes in the kaolin group (0.073 ± 0.014 ml) were significantly greater than those in the saline-injected animals (0.016 ± 0.001 ml; P = 0.0014). The CSF tracer enrichment in the olfactory turbinates (expressed as percent injected/g tissue) in the kaolin rats averaged 0.99 ± 0.39 and was significantly lower than that measured in the saline controls (5.86 ± 0.32; P < 0.00001). The largest degree of ventriculomegaly was associated with the lowest levels of lymphatic CSF uptake with lateral ventricular expansion occurring only when almost all of the lymphatic CSF transport capacity had been compromised. We conclude that lymphatic CSF absorption is impaired in a kaolin-communicating hydrocephalus model and that the degree of this impediment may contribute to the severity of the induced disease.

The purpose of this investigation was to enhance our understanding of cerebrospinal fluid (CSF) absorption pathways. To achieve this, Microfil (a coloured silastic material) was infused into the subarachnoid space (cisterna magna) of sheep post mortem, and the relevant tissues examined macroscopically and microscopically. The Microfil was taken up by an extensive network of extracranial lymphatic vessels in the olfactory turbinates. In addition however, Microfil also passed consistently through the dura at the base of the brain. Microfil was noted in the spaces surrounding the venous network that comprises the cavernous sinus, in the adventitia of the internal carotid arteries and adjacent to the pituitary gland. Additionally, Microfil was observed within the endoneurial spaces of the trigeminal nerve and in lymphatic vessels emerging from the epineurium of the nerve. These results suggest several unconventional pathways by which CSF may be removed from the subarachnoid space. The movement of CSF to locations external to the cranium via these routes may lead to its absorption into veins and lymphatics outside of the skull. The physiological importance of these pathways requires further investigation.

Previous studies suggested that a major portion of cerebrospinal fluid (CSF) is absorbed by extracranial lymphatics located in the olfactory turbinates. The objective of this study was to determine the impact of elevated intracranial pressure (ICP) on downstream cervical lymphatic pressures in the rat. Pressures were measured in the deep cervical lymph nodes using a servo-null micropressure system. A catheter was placed in a lateral ventricle and fluid was infused from a reservoir at defined ICPs. When Ringer’s solution was infused, elevations of ICP from 10 to 50 cm H2O resulted on average in a reduction of diastolic cervical node pressures. In contrast, when a diluted plasma solution (80% plasma in Ringer’s) was infused, downstream diastolic lymphatic pressures increased as ICP was elevated to 50 cm H2O. These data are consistent with the view that much of the CSF-derived water that convects into the lymphatics is absorbed into the ethmoidal or nodal blood vessels. This study supports the concept of fluid continuity between the subarachnoid space and extracranial lymphatics and suggests that this loss of CSF-derived water may act as a safety mechanism to reduce the volume load to the downstream lymphatic vessels.

The textbook view that cerebrospinal fluid (CSF) absorption occurs mainly through the arachnoid granulations and villi is being challenged by quantitative and qualitative studies that support a major role for the lymphatic circulation in CSF transport. There are many potential sites at which lymphatics may gain access to CSF but the primary pathway involves the movement of CSF through the cribriform plate foramina in association with the olfactory nerves. Lymphatics encircle the nerve trunks on the extracranial surface of the cribriform plate and absorb CSF. However, the time during development in which the CSF compartment and extracranial lymphatic vessels connect anatomically is unclear. In this report, CSF–lymphatic connections were investigated using the silastic material Microfil and a soluble Evan’s blue-protein complex in two species; one in which significant CSF synthesis by the choroid plexus begins before birth (pigs) and one in which CSF secretion is markedly up regulated within the first weeks after birth (rats). We examined a total of 46 pig fetuses at embryonic (E) day E80–81, E92, E101, E110 (birth at 114 days). In rats, we investigated a total of 115 animals at E21 (birth at 21 days), postnatal (P) day P1–P9, P12, P13, P15, P22, and adults. In pigs, CSF–lymphatic connections were observed in the prenatal period as early as E92. Before this time (E80–81 fetuses) CSF–lymphatic connections did not appear to exist. In rats, these associations were not obvious until about a week after birth. These data suggest that the ability of extracranial lymphatic vessels to absorb CSF develops around the time that significant volumes of CSF are being produced by the choroid plexus and further support an important role for lymphatic vessels in CSF transport.

Bifida Meeting abstracts - A single PDF containing all abstracts in this supplement is available here .