Miles G. Johnston

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

BackgroundThe 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.MethodsStudies 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.ResultsIn 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.ConclusionsThe 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.

Integration of the subarachnoid space and lymphatics: Is it time to embrace a new concept of cerebrospinal fluid absorption?

In most tissues and organs, the lymphatic circulation is responsible for the removal of interstitial protein and fluid but the parenchyma of the brain and spinal cord is devoid of lymphatic vessels. On the other hand, the literature is filled with qualitative and quantitative evidence supporting a lymphatic function in cerebrospinal fluid (CSF) absorption. The experimental data seems to warrant a re-examination of CSF dynamics and consideration of a new conceptual foundation on which to base our understanding of disorders of the CSF system. The objective of this paper is to review the key studies pertaining to the role of the lymphatic system in CSF absorption.

Drainage of CSF through lymphatic pathways and arachnoid villi in sheep: measurement of 125I-albumin clearance.

We investigated lymphatic drainage pathways of the central nervous system in conscious sheep and quantified the clearance of a cerebrospinal fluid (CSF) tracer into lymph and blood. In the first group of studies, 125I‐HSA was injected into the lateral ventricles of the brain or into lumbar CSF and after 6 h, various lymph nodes and tissues were excised and counted for radioactivity. Multiple lymphatic drainage pathways of cranial CSF existed in the head and neck region defined by elevated 125I‐HSA in the retropharyngeal/cervical, thymic, pre‐auricular and submandibular nodes. Implicated in spinal CSF drainage were mainly the lumbar and intercostal nodes. In a second group of experiments, multiple cervical vessels and the thoracic duct were cannulated and lymph diverted from the animals. Transport of tracer through arachnoid villi was taken from recoveries in venous blood. Following intraventricular administration, the 6 h recoveries of 125I‐HSA in the lymph (sum of cervical and thoracic duct) and blood were 8.2%± 3.0 and 12.5%± 4.5 respectively and at 22 h, 25.1%± 6.9 and 20.8%± 4.1 respectively. When 125I‐HSA was injected into lumbar CSF, the 6 h recoveries of tracer in thoracic duct and blood were 11.6%± 2.7 and 16.3%± 3.7 respectively. Total lymph and blood recoveries were not significantly different in any experiment. We conclude that the clearance of 125I‐HSA from the CSF is almost equally distributed between lymphatic and arachnoid villi pathways.

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.

Determination of volumetric cerebrospinal fluid absorption into extracranial lymphatics in sheep

We estimated the volumetric clearance of cerebrospinal fluid (CSF) through arachnoid villi and extracranial lymphatics in conscious sheep. Catheters were inserted into both lateral ventricles, the cisterna magna, multiple cervical lymphatics, thoracic duct, and jugular vein. Uncannulated cervical vessels were ligated.125I-labeled human serum albumin (HSA) was administered into both lateral ventricles.131I-HSA was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. From mass balance equations, total volumetric absorption of CSF averaged 3.37 ± 0.38 ml/h, with 2.03 ± 0.29 ml/h (∼60%) removed by arachnoid villi and 1.35 ± 0.46 ml/h (∼40%) cleared by lymphatics. With projected estimates for noncannulated ducts, total CSF absorption increased to 3.89 ± 0.33 ml/h, with 1.86 ± 0.49 ml/h (48%) absorbed by lymphatics. Additionally, we calculated total CSF drainage to be 3.48 ± 0.52 ml/h, with 54 and 46% removed by arachnoid villi and lymphatics, respectively, using previously published mass transport data from our group. We employed estimates of CSF tracer concentrations that were extrapolated from relationships observed in the study reported here. We conclude that 40-48% of the total volume of CSF absorbed from the cranial compartment is removed by extracranial lymphatic vessels.We estimated the volumetric clearance of cerebrospinal fluid (CSF) through arachnoid villi and extracranial lymphatics in conscious sheep. Catheters were inserted into both lateral ventricles, the cisterna magna, multiple cervical lymphatics, thoracic duct, and jugular vein. Uncannulated cervical vessels were ligated. 125I-labeled human serum albumin (HSA) was administered into both lateral ventricles. 131I-HSA was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. From mass balance equations, total volumetric absorption of CSF averaged 3.37 +/- 0.38 ml/h, with 2.03 +/- 0.29 ml/h (approximately 60%) removed by arachnoid villi and 1.35 +/- 0.46 ml/h (approximately 40%) cleared by lymphatics. With projected estimates for noncannulated ducts, total CSF absorption increased to 3.89 +/- 0.33 ml/h, with 1.86 +/- 0.49 ml/h (48%) absorbed by lymphatics. Additionally, we calculated total CSF drainage to be 3.48 +/- 0.52 ml/h, with 54 and 46% removed by arachnoid villi and lymphatics, respectively, using previously published mass transport data from our group. We employed estimates of CSF tracer concentrations that were extrapolated from relationships observed in the study reported here. We conclude that 40-48% of the total volume of CSF absorbed from the cranial compartment is removed by extracranial lymphatic vessels.

Subarachnoid injection of Microfil reveals connections between cerebrospinal fluid and nasal lymphatics in the non-human primate

Based on quantitative and qualitative studies in a variety of mammalian species, it would appear that a significant portion of cerebrospinal fluid (CSF) drainage is associated with transport along cranial and spinal nerves with absorption taking place into lymphatic vessels external to the central nervous system. CSF appears to convect primarily through the cribriform plate into lymphatics associated with the submucosa of the olfactory and respiratory epithelium. However, the significance of this pathway for CSF absorption in primates has never been established unequivocally. In past studies, we infused Microfil into the subarachnoid compartment of numerous species to visualize CSF transport pathways. The success of this method encouraged us to use a similar approach in the non‐human primate. Yellow Microfil® was injected post mortem into the cisterna magna of 6 years old Barbados green monkeys (Cercopithecus aethiops sabeus, n = 6). Macroscopic and microscopic examination revealed that Microfil was (1) distributed throughout the subarachnoid compartment, (2) located in the perineurial spaces associated with the fila olfactoria, (3) present within the olfactory submucosa, and (4) situated within an extensive network of lymphatic vessels in the nasal submucosa, nasal septum and turbinate tissues. We conclude that the Microfil distribution patterns in the monkey were very similar to those observed in many other species suggesting that significant nasal lymphatic uptake of CSF occurs in the non‐human primate.

Lymphatic cerebrospinal fluid absorption pathways in neonatal sheep revealed by subarachnoid injection of Microfil

There is mounting evidence that a significant portion of cerebrospinal fluid drainage is associated with transport along cranial and spinal nerves with absorption taking place into lymphatic vessels external to the central nervous system. To characterize these pathways further, yellow Microfil® was infused into the cisterna magna of 2–7‐day‐old lambs post mortem to perfuse either the cranial or spinal subarachnoid compartments. In some animals, blue Microfil was perfused into the carotid arteries simultaneously. Microfil was observed in lymphatic networks in the nasal mucosa, covering the hard and soft palate, conchae, nasal septum, the ethmoid labyrinth and the lateral walls of the nasal cavity. Many of these lymphatics drained into vessels located on the lateroposterior wall of the nasopharynx and from this location drained to the retropharyngeal lymph nodes. Additionally, lymphatics containing Microfil penetrated the lateral wall of the nasal cavity and joined with superficial lymphatic ducts travelling towards the submandibular and preauricular lymph nodes. In two cases, lymphatic vessels were observed anastomosing with deep veins in the retropharyngeal area. Microfil was also distributed within the nerve trunks of cranial and spinal nerves. The contrast agent was located in longitudinal channels within the endoneurial space and lymphatics containing Microfil were observed emerging from the mesoneurium. In summary, Microfil distribution patterns in neonatal lambs illustrated the important role that cranial and spinal nerves play in linking the subarachnoid compartment with extracranial lymphatics.

The modulation of enhanced vascular permeability by prostaglandins through alterations in blood flow (hyperemia)

The enhanced vascular permeability induced by histamine or bradykinin in the skin of the guinea-pig and rabbit was significantly augmented by small amounts of prostaglandins of the E type. When injected alone these prostaglandins had little effect on vascular permeability. Furthermore, E type prostaglandins were found to be more potent at inducing hyperemia than either histamine or bradykinin. Prostaglandin F2α did not enhance the vascular permeability induced by histamine or bradykinin nor did it produce hyperemia in the skin. In the rat, prostaglandins alone enhanced vascular permeability but they also increased the effect of histamine, serotonin and bradykinin. Using85Sr-microspheres to measure blood flow a correlation was found between the degree of hyperemia produced by prostaglandins and the degree to which they augmented enhanced vascular permeability due to histamine, serotonin or bradykinin. Prostaglandins therefore can directly mimic the hyperemia of the inflammatory process and can also modulate the changes in vascular permeability caused by other mediators of inflammation.

Effects of arachidonic acid and its cyclo-oxygenase and lipoxygenase products on lymphatic vessel contracility in vitro

Lymphatic vessels exhibit rhythmical contractility in vivo and in vitro and this activity appears to regulate lymph flow. A technique for measuring the circular muscle contractions of isolated bovine mesenteric lymphatic vessel segments has been devised and utilized to study the pharmacological properties of these vessels. Non-contracting lymphatic vessels can be induced to contract rhythmically with a variety of mediators, the most potent being a stable PGH2 analogue (compound U46619), and the leukotrienes B4, C4 and D4 (threshold concentrations in the nanomolar range). Prostaglandin F2 alpha, noradrenaline, serotonin and histamine also elicited rhythmical activity but much higher concentrations were required. PGE2 and PGE1 were potent inhibitors of spontaneous contractions or those induced with U46619. In keeping with the diverse pharmacological effects of the metabolites of arachidonic acid, the addition of arachidonate to an isolated lymphatic vessel generated both stimulatory and inhibitory activities. It is concluded that arachidonic acid products (produced in the lymphatic vessel or entering the vessel in lymph draining the tissues) regulate lymph flow through their effects on lymphatic smooth muscle.

Raised intracranial pressure increases CSF drainage through arachnoid villi and extracranial lymphatics

We demonstrated previously that about one-half of cerebrospinal fluid (CSF) removed from the cranial vault was cleared by extracranial lymphatic vessels. In this report we test the hypothesis that lymphatic drainage of CSF increases as intracranial pressure (ICP) is elevated in anesthetized sheep. Catheters were inserted into both lateral ventricles, cisterna magna, cervical lymphatics, and jugular vein. A ventriculocisternal perfusion system was employed to regulate CSF pressures and to deliver a protein tracer (125I-labeled human serum albumin) into the CSF compartment. 131I-labeled human serum albumin was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. ICP was controlled by adjusting the height of the inflow reservoir and the cisterna magna outflow catheter appropriately. The experimental design consisted of a 3-h period of lower pressure followed by a 3-h period of higher pressure in the same animal (10-20 or 20-30 cmH2O). We determined that incremental changes in ICP were associated with higher CSF transport through lymphatic and arachnoid villi routes in all eight animals tested (P = 0.004).We demonstrated previously that about one-half of cerebrospinal fluid (CSF) removed from the cranial vault was cleared by extracranial lymphatic vessels. In this report we test the hypothesis that lymphatic drainage of CSF increases as intracranial pressure (ICP) is elevated in anesthetized sheep. Catheters were inserted into both lateral ventricles, cisterna magna, cervical lymphatics, and jugular vein. A ventriculocisternal perfusion system was employed to regulate CSF pressures and to deliver a protein tracer (125I-labeled human serum albumin) into the CSF compartment.131I-labeled human serum albumin was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. ICP was controlled by adjusting the height of the inflow reservoir and the cisterna magna outflow catheter appropriately. The experimental design consisted of a 3-h period of lower pressure followed by a 3-h period of higher pressure in the same animal (10-20 or 20-30 cmH2O). We determined that incremental changes in ICP were associated with higher CSF transport through lymphatic and arachnoid villi routes in all eight animals tested ( P = 0.004).