Pre- and post-shunting observations in adult sheep with kaolin-induced hydrocephalus.

BACKGROUND: The objective of this study was to examine host-shunt interactions in sheep with kaolin-induced hydrocephalus. METHODS: Forty-two sheep (29-40 kg) were utilized for this study. In 20 animals, various kaolin doses were injected into the cisterna magna including 10 and 50 mg/kg as well as 2-4 ml of a 25% kaolin suspension. Based on animal health and hydrocephalus development, 3 ml of a 25% kaolin suspension was chosen. In 16 animals, kaolin was administered and 6-8 days later, the animals received a custom made ventriculo-peritoneal shunt. In 8 animals ventricular CSF pressures were measured with a water manometer before kaolin administration and 7-8 days later. The sheep were allowed to survive for up to 9-12 weeks post-kaolin or until clinical status required euthanasia. Brains were assessed for morphological and histological changes. Ventricle/cerebrum cross sectional area ratios (V/C) were calculated from photographs of the sliced coronal planes immediately anterior to the interventricular foramina. RESULTS: Intraventricular pressures increased from 12.4±1.1 cm H2O to 41.3±3.5 cm H2O following kaolin injection (p < 0.0001, n = 8). In all animals, we observed kaolin on the basal surface of the brain and mild (V/C 0.03-0.10) to moderate (V/C >0.10) ventricular expansion. The animals lost weight between kaolin administration and shunting (33.7±1.2 kg versus 31.0±1.7 kg) with weights after shunting remaining stable up to sacrifice (31.6±2.2 kg). Of 16 shunted animals, 5 did well and were sacrificed 9-12 weeks post-kaolin. In the remainder, the study was terminated at various times due to deteriorating health. Hydrocephalus was associated with thinning of the corpus callosum, but no obvious loss of myelin staining, along with reactive astroglial (glial fibrillary acidic immunoreactive) and microglial (Iba1 immunoreactive) changes in the white matter. Ventricular shunts revealed choroid plexus ingrowth in 5/16, brain tissue ingrowth in 1/16, problems with shunt insertion in 3/16, occlusion by hemorrhagic-inflammatory material in 5/16, or no obstruction in 2/16. Free flowing CSF indicated that the peritoneal catheter was patent. CONCLUSIONS: Cerebrospinal fluid shunts in hydrocephalic sheep fail in ways that are reminiscent of human neurosurgical experience suggesting that this model may be helpful in the development of more effective shunt technology.

Identification of lymphatics in the ciliary body of the human eye: a novel "uveolymphatic" outflow pathway.

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.

Possible role of the cavernous sinus veins in cerebrospinal fluid absorption.

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.

Lymphangiogenesis following obstruction of large postnodal lymphatics in sheep.

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.

Quantification of cerebrospinal fluid transport across the cribriform plate into lymphatics in rats.

A major pathway by which cerebrospinal fluid (CSF) is removed from the cranium is transport through the cribriform plate in association with the olfactory nerves. CSF is then absorbed into lymphatics located in the submucosa of the olfactory epithelium (olfactory turbinates). In an attempt to provide a quantitative measure of this transport, 125I-human serum albumin (HSA) was injected into the lateral ventricles of adult Fisher 344 rats. The animals were killed at 10, 20, 30, 40, and 60 min after injection, and tissue samples, including blood (from heart puncture), skeletal muscle, spleen, liver, kidney, and tail were excised for radioactive assessment. The remains were frozen. To sample the olfactory turbinates, angled coronal tissue sections anterior to the cribriform plate were prepared from the frozen heads. The average concentration of 125I-HSA was higher in the middle olfactory turbinates than in any other tissue with peak concentrations achieved 30 min after injection. At this point, the recoveries of injected tracer (percent injected dose/g tissue) were 9.4% middle turbinates, 1.6% blood, 0.04% skeletal muscle, 0.2% spleen, 0.3% liver, 0.3% kidney, and 0.09% tail. The current belief that arachnoid projections are responsible for CSF drainage fails to explain some important issues related to the pathogenesis of CSF disorders. The rapid movement of the CSF tracer into the olfactory turbinates further supports a role for lymphatics in CSF absorption and provides the basis of a method to investigate the novel concept that diseases associated with the CSF system may involve impaired lymphatic CSF transport.

Development of cerebrospinal fluid absorption sites in the pig and rat: connections between the subarachnoid space and lymphatic vessels in the olfactory turbinates.

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.

Evidence of connections between cerebrospinal fluid and nasal lymphatic vessels in humans, non-human primates and other mammalian species.

BACKGROUND: The parenchyma of the brain does not contain lymphatics. Consequently, it has been assumed that arachnoid projections into the cranial venous system are responsible for cerebrospinal fluid (CSF) absorption. However, recent quantitative and qualitative evidence in sheep suggest that nasal lymphatics have the major role in CSF transport. Nonetheless, the applicability of this concept to other species, especially to humans has never been clarified. The purpose of this study was to compare the CSF and nasal lymph associations in human and non-human primates with those observed in other mammalian species. METHODS: Studies were performed in sheep, pigs, rabbits, rats, mice, monkeys and humans. Immediately after sacrifice (or up to 7 hours after death in humans), yellow Microfil was injected into the CSF compartment. The heads were cut in a sagittal plane. RESULTS: In the seven species examined, Microfil was observed primarily in the subarachnoid space around the olfactory bulbs and cribriform plate. The contrast agent followed the olfactory nerves and entered extensive lymphatic networks in the submucosa associated with the olfactory and respiratory epithelium. This is the first direct evidence of the association between the CSF and nasal lymph compartments in humans. CONCLUSIONS: The fact that the pattern of Microfil distribution was similar in all species tested, suggested that CSF absorption into nasal lymphatics is a characteristic feature of all mammals including humans. It is tempting to speculate that some disorders of the CSF system (hydrocephalus and idiopathic intracranial hypertension for example) may relate either directly or indirectly to a lymphatic CSF absorption deficit.