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.     

Comparison of cerebrospinal fluid transport in fetal and adult sheep

We quantified cerebrospinal fluid (CSF) transport (conductance) and CSF outflow resistance in late-gestation fetal and adult sheep using two methods, a constant pressure infusion method and a bolus injection technique into the lateral ventricles. No significant differences in CSF conductance (fetus 0.013 +/- 0.002, adult 0.014 +/- 0.003 ml x min(-1) x cm H(2)O(-1)) or CSF outflow resistance (fetus 83.7 +/- 9.8, adult 84.7 +/- 19.7 cm H(2)O x ml(-1) x min) were observed. To confirm CSF transport to plasma in fetal animals, (125)I- or (131)I-labeled human serum albumin (HSA) was injected into the lateral ventricles. The tracer entered fetal plasma with an average mass transport rate of 1.91 +/- 0.47% injected/h (n = 9). In two fetuses, we monitored the tracer appearance in plasma and cervical and thoracic duct lymph after injection of radioactive HSA into the ventricular CSF. As was the case in adult animals, fetal tracer concentrations increased in all three compartments over time, with the highest concentrations measured in lymph collected from the cervical lymphatics. These results 1) indicate that global CSF transport parameters in the late-gestation fetus and adult sheep are similar and 2) suggest an important role for extracranial lymphatic vessels in CSF transport before birth.     

The importance of lymphatics in cerebrospinal fluid transport

Despite the fact that the central nervous system parenchyma does not contain lymphatics, extracranial lymphatic vessels play a very important role in volumetric cerebrospinal fluid (CSF) transport. The most important extracranial location at which lymphatics gain access to CSF is in the nasal submucosa after CSF convects through the cribriform plate. At relatively low intracranial pressures (ICPs), the majority of cranial CSF absorption occurs through this pathway. Global CSF transport parameters in the late gestation fetus and adult sheep are very similar, even though significant numbers of arachnoid projections seem to exist only in the adult. Therefore, extracranial lymphatic vessels play an important role in CSF transport before birth and may represent the primary mechanism for CSF absorption in the neonate. Based on these considerations, hydrocephalus may involve reduced CSF transport to, or into extracranial lymphatic absorption sites.     

Does neonatal cerebrospinal fluid absorption occur via arachnoid projections or extracranial lymphatics?

Arachnoid villi and granulations are thought to represent the primary sites where cerebrospinal fluid (CSF) is absorbed. However, these structures do not appear to exist in the fetus but begin to develop around the time of birth and increase in number with age. With the use of a constant pressure-perfusion system in 2- to 6-day-old lambs, we observed that global CSF transport (0.012 +/- 0.003 ml x min(-1) x cmH(2)O(-1)) and CSF outflow resistance (96.5 +/- 17.8 cmH(2)O x ml(-1) x min) were very similar to comparable measures in adult animals despite the relative paucity of arachnoid villi at this stage of development. In the neonate, the recovery patterns of a radioactive protein CSF tracer in various lymph nodes and tissues indicated that CSF transport occurred through multiple lymphatic pathways. An especially important route was transport through the cribriform plate into extracranial lymphatics located in the nasal submucosa. To investigate the importance of the cribriform route in cranial CSF clearance, the cranial CSF compartment was isolated surgically from its spinal counterpart. When the cribriform plate was sealed extracranially under these conditions, CSF transport was impaired significantly. These data demonstrate an essential function for lymphatics in neonatal CSF transport and imply that arachnoid projections may play a limited role earlier in development.     

Blocking cerebrospinal fluid absorption through the cribriform plate increases resting intracranial pressure

Cerebrospinal fluid (CSF) drains through the cribriform plate (CP) in association with the olfactory nerves. From this location, CSF is absorbed into nasal mucosal lymphatics. Recent data suggest that this pathway plays an important role in global CSF transport in sheep. In this report, we tested the hypothesis that blocking CSF transport through this pathway would elevate resting intracranial pressure (ICP). ICP was measured continuously from the cisterna magna of sheep before and after CP obstruction in the same animal. To block CSF transport through the CP, an external ethmoidectomy was performed. The olfactory and adjacent mucosa were removed, and the bone surface was sealed with tissue glue. To restrict our analysis to the cranial CSF system, CSF transport into the spinal subarachnoid compartment was prevented with a ligature tightened around the thecal sac between C1 and C2. Sham surgical procedures had no significant effects, but in the experimental group CP obstruction elevated ICP significantly. Mean postobstruction steady-state pressures (18.0 +/- 3.8 cmH(2)O) were approximately double the preobstruction values (9.2 +/- 0.9 cmH(2)O). These data support the concept that the olfactory pathway represents a major site for CSF drainage.     

High-pressure hydrocephalus: a novel analytical modeling approach

Hydrocephalus is an abnormal accumulation of cerebrospinal fluid (CSF) within ventricles and subarachnoid space (SAS) as a result of disturbances in secretion or absorption procedures. It is believed that arachnoid villi cells, which are microscopic projections of pia-arachnoid mater that extend into venous channels in sagittal sinus, are the main sites for CSF absorption, but it is tempting to speculate that a significant portion of CSF is removed from the SAS by nasal lymphatic vessels around olfactory nerve. Thus, in this paper, we propose an analytical model of CSF-lymphatic-blood circulation, in which these two output pathways for CSF absorption have been considered. Mathematical relations governing the pressures in different interacting compartments of the brain are considered. In addition, for increasing the similarity of our model to the physiological conditions, the bulk flow mechanism, which is supposed to occur during CSF absorption, has been considered in our model. We used our model to simulate hydrocephalus. The results indicate that the lymphatic disorders have more considerable effect in decreasing CSF absorption, compared to the disturbances in arachnoid villi cells. Based on our modeling, we believe that disorders in lymphatic pathway may be a cause of high-pressure hydrocephalus. Surely experimental studies are required to validate our hypothesis.     

Temperature dependence of protein transport across lymphatic endothelium in vitro

The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees, 30 degrees, and 40 degrees C. At 4 degrees C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased. We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperature-dependent and temperature-independent mechanisms. The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory.     

Concentration of aerosolized 99mTc-albumin in the pulmonary lymph of anesthetized sheep

We examined the lymphatic concentration of 99mTc-albumin deposited in the air spaces of anesthetized sheep to determine whether changes in the concentration reflected changes in lung epithelial function. Five control sheep were ventilated with an aerosol of 99mTc-albumin for 6 min, and the lung lymphatic concentration of the tracer was monitored for the next 2 h. During the last 45 min the lymphatic concentration stabilized at a value that was 0.03 +/- 0.01% of the estimated value in the air spaces. Pulmonary vascular hypertension, induced in seven sheep by increasing the left atrial pressure 20 cmH2O for 4 h, increased the lung lymph flow from a base-line value of 3 +/- 2 to 21 +/- 14 ml/h. This caused the concentration of the 99mTc-albumin in the lymph to double to 0.07 +/- 0.03% of the air space concentration (P less than 0.01). Lung injury induced by infusing 0.08-0.10 ml/kg oleic acid intravenously in seven other sheep increased the lymphatic concentration of the 99mTc-albumin 10-fold to 0.31 +/- 0.09% of the air space concentration (P less than 0.01). The increased tracer concentration in the sheep with pulmonary vascular hypertension could be the result of the increased lymph flow causing a diversion of tracer into the lymphatics. However, a mathematical model showed that the 10-fold increase in the lymphatic concentration in the sheep with lung injury was primarily the result of an increase in both permeability and surface area of the epithelium that participated in the transfer of the 99mTc-albumin from the air spaces into the lung tissue drained by the lymphatics.(ABSTRACT TRUNCATED AT 250 WORDS)     

一氧化氮对大鼠胸膜淋巴孔调控及淋巴吸收的影响

实验研究了一氧化氮（nitric oxide,NO）对大鼠胸膜淋巴孔的调控和胸膜腔淋巴吸收的影响。NO供体和NOS（nitric oxide synthase）抑制剂分别经腹腔给药,示踪剂（台盼蓝）胸膜腔内注射后,处死大鼠,测定血清NO和台盼蓝浓度;在扫描电镜下观察各组胸膜淋巴孔,用计算机图像处理,统计学分析。结果显示,NO供体组血清NO浓度为49.34±18.47μmol/L,淋巴孔的面积和密度分别为6.80±1.13 μm2和170.24±66.60/0.1mm2;NOS抑制剂组血清NO浓度为17.72±6.58μmol/L,淋巴孔的面积和密度分别为5.72±1.54μm2和61.71±12.73/0.1mm2。血清NO浓度与淋巴孔开放的面积和密度成正相关（P<0.05）。在胸膜腔给示踪剂后,NO供体组血清台盼蓝的浓度为74.68±33.67mg/L,与对照组比较有显著差异（P<0.05）。提示,NO可以调控胸膜淋巴孔,促进胸膜腔淋巴吸收。     

Intracranial pressure accommodation is impaired by blocking pathways leading to extracranial lymphatics

Tracer studies indicate that cerebrospinal fluid (CSF) transport can occur through the cribriform plate into the nasal submucosa, where it is absorbed by cervical lymphatics. We tested the hypothesis that sealing the cribriform plate extracranially would impair the ability of the CSF pressure-regulating systems to compensate for volume infusions. Sheep were challenged with constant flow or constant pressure infusions of artificial CSF into the CSF compartment before and after the nasal mucosal side of the cribriform plate was sealed. With both infusion protocols, the intracranial pressure (ICP) vs. flow rate relationships were shifted significantly to the left when the cribriform plate was blocked. This indicated that obstruction of the cribriform plate reduced CSF clearance. Sham surgical procedures had no significant effects. Estimates of the proportional flow through cribriform and noncribriform routes suggested that cranial CSF absorption occurred primarily through the cribriform plate at low ICPs. Additional drainage sites (arachnoid villi or other lymphatic pathways) appeared to be recruited only when intracranial pressures were elevated. These data challenge the conventional view that CSF is absorbed principally via arachnoid villi and provide further support for the existence of several anatomically distinct cranial CSF transport pathways.     

The pathogenesis of normal pressure hydrocephalus: A theoretical analysis

Hydrocephalus is an abnormal accumulation of cerebrospinal fluid (CSF) in the cerebral ventricles, usually caused by impaired absorption of the fluid into the bloodstream. Despite obstructed absorption and continued secretion of CSF into the ventricles at a near normal rate, the ventricular CSF pressure (VCSFP) is often normal. We attempt to understand how hydrocephalus can exist with normal VCSFP by exploring the role of the brain parenchyma in absorbing CSF in hydrocephalus. We test three theories: (1) the ventricular wall is impermeable to CSF; (2) ventricular CSF seeps into the parenchyma, from which it is efficiently absorbed; and (3) ventricular CSF seeps into the parenchyma but is absorbed inefficiently. We model the brain as a thick spherical shell consisting of a porous, elastic, solid matrix, containing interstitial fluid and blood. We modify the equations of poroelasticity, which describe flow of fluid through porous solids, to allow for parenchymal absorption. For each of the three theories we calculate the steady state changes in VCSFP and in parenchymal fluid pressure caused by an incremental defect in CSF absorption. We also calculate the steady state changes in fluid content, tissue volume, tissue displacement, and stresses caused by a small increment of VCSFP. We conclude that only the second theory—seepage of CSF with efficient parenchymal absorption—accounts for the clinical features of normal pressure hydrocephalus. These features include sustained ventricular dilatation despite normal VCSFP, increased periventricular fluid content, and localized periventricular white matter damage.     

Length-tension relationships of small arteries, veins, and lymphatics from the rat mesenteric microcirculation

The passive and active length-tension relationships of isolated rat mesenteric lymphatics ( approximately 150 microm ID), and adjacent small arteries ( approximately 240 microm) and veins ( approximately 275 microm) were compared under isometric conditions using a wire myograph. About 60% of the lymphatic vessels developed spontaneous contractions in physiological saline solution at nominal preload. To maximally activate smooth muscle, 145 mM K(+) + 5 x 10(-5) M norepinephrine was used for arteries, and 145 mM K(+) + 1 x 10(-6) M substance P was used for lymphatics and veins. In response, arteries exhibited monotonic force development to a plateau level, whereas lymphatics and veins showed biphasic force development, consisting of a transient force peak followed by partial relaxation to a plateau over approximately 5 min. The passive and the active length-tension curves were similar in shape among all three vessels. However, the maximal active tension of arteries (3.4 +/- 0.42 mN/mm) was significantly greater than peak active tension (0.59 +/- 0.04 mN/mm) or plateau tension (0.20 +/- 0.04 mN/mm) in small veins and greater than peak active tension (0.34 +/- 0.02 mN/mm) or plateau tension (0.21 +/- 0.02 mN/mm) in lymphatics. Maximal active medial wall stress was similar between lymphatics and veins but was approximately fivefold higher in small arteries. For lymphatics, the pressure calculated from the optimal preload was significantly higher than that found previously in isobaric studies of isolated lymphatics, suggesting the capacity to operate at higher than normal pressures for increased responsiveness. Our results represent the first mechanical comparisons of arterial, venous, and lymphatic vessels in the same vasculature.     

Cerebrovascular Permeability Coefficients to Sodium, Potassium, and Chloride

CSF and regional brain concentrations of 42K, 22Na, 36Cl, and [14C]mannitol were determined 3-45 min after intravenous injection of the tracers in pentobarbital-anesthetized rats. Rapid influx of 36Cl and 22Na into ventricular CSF immediately established concentration gradients from CSF to brain extracellular fluid. The CSF contribution to brain uptake of tracers was greatest in periventricular brain regions, where brain 36Cl concentrations were up to ninefold higher than concentrations in regions distant from ventricular CSF. Acetazolamide (20 mg kg-1 i.p.), an inhibitor of CSF formation, decreased 36Cl uptake into CSF and into periventricular brain regions but not into frontal cortex. 36Cl uptake into brain was unidirectional for 10 min after intravenous injection, and, during that period, diffusion from ventricular CSF did not contribute to uptake in the frontal cortex. Therefore, cerebrovascular permeability coefficients could be calculated from tracer concentrations in frontal cortex at 10 min and equaled, in cm s-1, 13.5 X 10(-7) for 42K, 1.4 X 10(-7) for 22Na, 0.9 X 10(-7) for 36Cl, and 1.5 X 10(-7) for [14C]mannitol. The low cerebrovascular permeabilities to K, Na, and Cl, comparable to those of some cell membranes, and the permselectivity (K much greater than Na greater than Cl) suggest that a significant fraction of ion transport across cerebral capillaries is transcellular, i.e., across the endothelial cell membrane.     

Terminal lymphatics: the potential "lethal corner" in the distribution of tissue pO2

BACKGROUND: Terminal lymphatic fluid is the compartment furthest removed from the oxygen supply, and therefore should present the lowest pO(2) in the tissue due to oxygen consumption by the tissue and the lymphatic vessel wall. METHODS AND RESULTS: The distribution of pO(2) was determined in the tissue, the lymphatic microvessels, and arterioles and venules of the hamster chamber window model, which is studied without anesthesia with the tissue isolated from the environment. Lymphatic fluid pO(2) was measured with the phosphorescence oxygen quenching method. Small terminal lymphatic fluid pO(2) was 18.4 +/- 2.6 mmHg, and 18.0 +/- 2.4 mmHg in collecting lymphatics. Tissue pO(2) averaged 24.6 +/- 2.7 mmHg. The significant difference between tissue and intralymphatic pO(2) was due in part to the presence of an oxygen gradient across the lymphatic wall, which ranged from 3.7 +/- 1.3 mmHg for terminal lymphatics, to 6.0 +/- 1.2 mmHg for collecting lymphatics. This gradient is assumed to be due to the oxygen consumption by the cellular component of the lymphatic wall. CONCLUSION: The increased vessels wall gradient found in collecting lymphatics was reconciled by the findings that these microlymphatic vessels tend to be contiguous to the arterioles, whereas the terminal lymphatics are dispersed in the tissue. These findings indicate that terminal lymphatic present the lowest oxygen tension in the tissue, and therefore are the locations at risk to develop anoxia when the microvascular oxygen supply becomes limited.     

Lymphatic drainage of cerebrospinal fluid in mammals – are arachnoid granulations the main route of cerebrospinal fluid outflow?

The outflow of the cerebrospinal fluid (CSF) in animals was over the years the subject of detailed analysis. For a long time it was stated that arachnoid granulations of the venous sinuses play a key role in CSF circulation. However, recent studies on this subject have shown that a considerable part of the CSF is drained to the lymphatic vessels. Moreover, disorders in the CSF passage may result in severe central nervous system diseases such as e.g. hydrocephalus. In this paper, we summarize the current knowledge concerning the lymphatic drainage of the CSF in mammals. We present in detail comparative anatomy of different species taking into account cranial and spinal compartment. In addition, we clarified role of the lymphatic vessels in the CSF outflow and the relationship between impairment in this transport and central nervous system diseases. In the author’s opinion knowledge on CSF circulation is still poorly examined and therefore required comment.     

Positive-pressure ventilation alters blood-to-brain and blood-to-CSF transport in neonatal pigs

These experiments examine the transfer of sucrose, urea, sodium, and albumin from blood to brain in new-born pigs exposed to an increase in ventilation pressure. We also studied the movement of urea and sodium from blood to cerebrospinal fluid (CSF). By use of a standard time-cycled pressure-limited infant respirator, mean airway pressure (Paw) was increased from approximately 3 to 17 cmH2O. Urea and albumin transfer into the brain were unchanged with increased Paw. Sodium transport decreased significantly in all brain regions, while sucrose transfer was increased in the cerebrum [transfer constant (Kin) = 3.5 +/- 0.04 vs. 9.9 +/- 1.0 cm3.g-1.s-1.10(6)] at the increased Paw. Transport of urea nd sodium from blood to CSF decreased to half of control values with increased Paw. Thus, in newborn pigs, increasing Paw selectively alters blood-to-brain transport. In addition, movement of tracers from blood to CSF was severely restricted, possibly by a decrease in CSF production. It appears likely that the increased cerebral venous pressure causes the observed changes in tracer transport. Such altered blood-to-brain transport could adversely affect neuronal function.     

Brain-blood permeability: TNF-alpha promotes escape of protein tracer from CSF to blood

The objective of this study was to determine the effect of tumor necrosis factor (TNF)-alpha on the efflux of protein from the central nervous system to blood based on assessing the clearance of radiolabeled albumin from the cerebrospinal fluid (CSF) to blood in rats. (125)I-labeled human serum albumin ((125)I-HSA) was injected into a lateral ventricle, and venous blood was sampled hourly to determine the basal CSF protein clearance into the blood. After this, rats were intraventricularly infused with 10 microliter TNF-alpha and 10 microliter (131)I-HSA (n = 6) or 10 microliter saline and 10 microliter (131)I-HSA (n = 6). Venous blood was sampled hourly for 3 h. (131)I-HSA tracer recovery increased threefold in the venous blood and was significantly higher in the spleen, muscles, and skin in animals treated with TNF-alpha. No significant changes were observed in control animals treated with saline. The data suggest that TNF-alpha promotes the clearance of protein macromolecules from the CSF to the venous blood.     

Transmural pressure in rat initial subpleural lymphatics during spontaneous or mechanical ventilation

The role played by the mechanical tissue stress in supporting lymph formation and propulsion in thoracic tissues was studied in deeply anesthetized rats (n = 13) during spontaneous breathing or mechanical ventilation. After arterial and venous catheterization and insertion of an intratracheal cannula, fluorescent dextrans were injected intrapleurally to serve as lymphatic markers. After 2 h, the fluorescent intercostal lymphatics were identified, and the hydraulic pressure in lymphatic vessels (P lymph) and adjacent interstitial space (P int) was measured using micropuncture. During spontaneous breathing, end-expiratory P lymph and corresponding P int were -2.5 +/- 1.1 (SE) and 3.1 +/- 0.7 mmHg (P < 0.01), which dropped to -21.1 +/- 1.3 and -12.2 +/- 1.3 mmHg, respectively, at end inspiration. During mechanical ventilation with air at zero end-expiratory alveolar pressure, P lymph and P int were essentially unchanged at end expiration, but, at variance with spontaneous breathing, they increased at end inspiration to 28.1 +/- 7.9 and 28.2 +/- 6.3 mmHg, respectively. The hydraulic transmural pressure gradient (DeltaP tm = P lymph - P int) was in favor of lymph formation throughout the whole respiratory cycle (DeltaP tm = -6.8 +/- 1.2 mmHg) during spontaneous breathing but not during mechanical ventilation (DeltaP tm = -1.1 +/- 1.8 mmHg). Therefore, data suggest that local tissue stress associated with the active contraction of respiratory muscles is required to support an efficient lymphatic drainage from the thoracic tissues.     

Role of natriuretic factor in central nervous system (CNS)-induced natriuresis

The presence of a natriuretic factor in the plasma of rats in which a 350 mM Na (high Na) artificial cerebrospinal fluid (CSF) was infused into the lateral ventricle was tested. Blood was obtained from control rats and rats which received an infusion of high Na CSF intraventricular (IVT) for 15 min. The plasma was incubated for 30 min at room temperature, acidified, placed in a boiling-water bath, and then centrifuged. The plasma supernate was assayed for natriuretic activity in pentobarbital anesthetized bioassay rats. Sodium excretion increased 6.5 +/- 1.1 mueq/kg X min in rats which received an infusion of a control saline solution, 13.3 +/- 3.2 mueq/kg X min in rats which received infusion of control plasma supernates, and 32.1 +/- 8.3 mueq/kg X min in those rats which received plasma supernates from rats infused with high Na CSF IVT. Blood pressure was unchanged in all groups. The increment in sodium excretion elicited by plasma supernate from the high Na IVT group was significantly greater than that elicited by either control saline solution or control plasma extracts. Therefore, it is concluded that a heat-stable and nonpressor natriuretic factor is present in the plasma of rats infused IVT with high Na CSF.