The role of the arachnoid villus in the removal of red blood cells from the subarachnoid space. An electron microscope study in the dog

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1 The role of the arachnoid villus in the removal of red blood cells from the subarachnoid space An electron microscope study in the dog JOHN 17. ALKSNE, M.D., AND ETHEL T. LOVlNGS Division of Neurological Surgery, University of California, San Diego, California, and Division of Neurological Surgery, Medical College of Virginia, Richmond, Virginia ~' q-he electron microscope examination of arachnoid villi from dogs after the subarachnoid injection of blood reveals that erythrocytes accumulate and degenerate in the villi. A continuous layer of endothelium separates the villus from the lumen of the sagittal sinus. No evidence for pores through the endothelium or for the passage of intact erythrocytes from CSF to blood through arachnoid villi has been found. KEY WORDS electron microscopy arachnoid villi subarachnoid hemmorrhage erythrocytes T HE observation by Sprong 9 that the cerebrospinal fluid (CSF) was completely cleared of erythrocytes 5 to 6 days after a head injury stimulated subsequent physiological studies. Courtice and Simmonds 5 injected P~2-tagged red blood cells into the cisterna magna of dogs and found the cerebrospinal flu!d clear of erythrocytes in 48 hours. They detected 7% to 14% of the injected ceils in the circulating blood and found no decrease in the rate of cerebrospinal fluid clearance or in the return of labeled cells to the circulation following ligation of the cervical lymphatics. Coupling these findings with their observation that red blood cells could be seen in the arachnoid villi, Courtice and Simmonds concluded that the villi must be the site of exit. Bradford and Johnson 3 repeated these experiments us- ing Cr ~1- and Fe59-1abeled erythrocytes and found a range of 5% to 65% of the intact cells in circulating blood in 24 hours. Du- Pont, et al., 6 however, found conflicting data; in their experiments less than 0.5% of the labeled erythrocytes entered the bloodstream in 48 hours. Partial explanation for the inconsistency can be found in a report by Adams and Prawirohardjo 1 who counted blood and spinal fluid 24 hours after tagged red cells were injected into the cisterna magna. Their study revealed 20% of the activity in the blood and 4% remaining in the CSF, leaving 76% of the erythrocytes which must be trapped in the arachnoid membranes. Variations in the number of trapped cells secondary to different injection techniques could account for the differing results. No detailed anatomical studies have been 192 I. Neurosurg. / Volume 36 / February, 1972

2 Role of arachnoid villus in removal of red blood cells Fro. 1. Electron micrograph of arachnoid villus fixed by perfusion 4 hours after intracisternal blood injection. The sagittal sinus lumen (SSL) has been cleansed of erythrocytes by the perfusion. An endothelial lining (E) is present separating the cerebrospinal fluid space (CSF) of the araclmoid villus from the sagittal sinus lumen. Numerous red blood ceils (RBC) are present with the villus interspersed among collagen fiber (C) and arachnoidal cells (A). Lead citrate and uranyl acetate, x 13,000. performed to demonstrate a mechanism or site for the proposed erythrocyte passage through the endothelial linings of the arachnoid villus. The experiments by Welch and coworkers 11,12 which suggested the presence of pores in the aracbnoid villus could provide an explanation, but studies with the electron microscope 2 have failed to detect these openings. The present study was undertaken to clarify the role of the arachnoid villi in the removal of red blood cells from the CSF. Materials and Methods Twenty adult mongrel dogs were anesthetized with intravenous nembutal anesthesia, J. Neurosurg. / Volume 36 / February, 1972 ]93

3 John F. Alksne and Ethel Lovings and 8 cc of the animals own venous blood was withdrawn in a heparinized syringe. A cisternal puncture was performed with the dog in the lateral position, and the dog's CSF pressure was measured with a water manometer. Then 6 to 8 cc of cerebrospihal fluid were withdrawn so that the animals' intracranial pressure would not be elevated when the blood was injected. During the period of blood injection, the CSF pressure was continually monitored. If the pressure began to rise above 100 mm of water, the injection was temporarily discontinued to allow the pressure to return to normal. A total of 8 cc of blood was injected in each animal without producing a pressure elevation. The animals were sacrificed by vascular perfusion at 1 hour to 2 weeks after subarachnoid blood injection. The perfusate consisted of 2.5% glutaraldehyde and 2.5% formaldehyde buffered to ph 7.4 with phosphate. Perfusion was performed through a Fro. 2. Electron micrograph of arachnoid villus fixed 24 hours after blood injection. The endothelium (E) with its basement membrane (B) separates the red blood cells in the CSF space from the lumen of the sagittal sinus. A tight junction (J) between two endothelial cells is present. Lead citrate and uranyl acetate, 38,250. ] 94 J. Neurosurg. / Volume 36 / February, 1972

4 Role of arachnoid villus in removal of red blood cells Fi6. 3. Electron micrograph of arachnoid villus fixed 48 hours after intracisternal blood injection. The CSF space is packed with red blood cells but the overlying endothelium remains intact. A junction between two endothelial cells (J) shows no evidence of separation. Lead citrate and uranyl acetate, x 22,160. cannula in the ascending aorta with the descending aorta clamped below the origin of the left subclavian artery. A mechanical pump with a 1500 cc per min output injected 4 liters of perfusate in less than 3 min. After perfusion was completed the sagittal sinus was removed, cut into 1 mm tubular segments, and postfixed in 1% phosphate buffered osmium tetroxide. The tissue was then dehydrated in ascending concentrations of alcohol and embedded in Epon. Arachnoid villi were identified and sectioned by the method previously reported z using an LKB microtome. The sections were stained with lead citrate s and uranyl acetate 1~ and observed with a Hitachi HS 7S electron microscope. Pictures were taken at magnifications of 4000 to 10,000 times and enlarged photographically. Observations Arachnoid villi removed 4 hours after intracisternal blood injection (Fig. 1) demonstrated numerous erythrocytes within the lattice work of the villus. The endothelial covering of the villus provided an intact barrier between the CSF space and the sagittal sinus lumen. The red blood cells within the villus were normal in appearance and configuration. They lay free in the CSF space and showed no evidence of degeneration or phagocytosis. Arachnoid villi fixed 24 hours after intracisternal blood injection appeared similar to the 4-hour villi except that the number of red blood cells was increased. The villus endothelium with its basement membrane remained as an intact barrier between the J. Neurosurg. / Volume 36 / February,

5 John F. Alksne and Ethel Lovings erythrocytes and the lumen of the sagittal sinus (Fig. 2). Tissue removed 48 hours after intracisternal blood injection revealed villi that were densely packed with red blood cells (Fig. 3). The limiting endothelium of the villus was thin, suggesting increased CSF pressure. No defects could be found in the endothelium, however, and no erythrocytes were seen passing from the CSF space to the lumen of the sagittal sinus. Specimens removed 3 to 5 days after blood injection showed a striking change from that described for the earlier villi. A1- though an occasional intact erythrocyte could be identified, the majority of the red blood ceils had degenerated, leaving a fine debris (Fig. 4). Some of the debris could be seen in vacuoles of histiocytes but the majority remained free in the subarachnoid space. The limiting endothelium remained as an intact barrier between the CSF and blood. Arachnoid villi removed 1 and 2 weeks after subarachnoid blood instillation showed definite evidence of phagocytosis of the erythrocyte debris (Figs. 5 and 6). The phagocytic cells appeared to be arachnoid cells rather than migratory cells from the cir- Fro. 4. Electron micrograph of arachnoid villus fixed 4 days after intracisternal blood injection. The CSF space is now filled with a fine debris (D) resulting from red blood cell degeneration. The overlying endothelium has an abnormal granular appearance but remains intact, providing a continuous barrier between CSF and blood. An intact tight junction (J) between two endothelial ceils is seen, indicating that no separations or openings have developed. Lead citrate and uranyl acetate, Neurosurg. / Volume 36 / February, 1972

6 Role of arachnoid villus in removal of red blood cells FIG. 5. Electron micrograph of arachnoid villus fixed 1 week after intracisternal blood injection. Portions of two cells containing dark-stained vacuoles are seen. In the extracellular space adjacent to these cells is residual debris (D) of erythrocyte degeneration. The vacuoles are presumed to contain phagocytized degenerated red blood cells. The fine processes extending from the cells suggest that they are arachnoid cells. Lead citrate and uranyl acetate, 18,900. culation. By the end of 2 weeks most of the material had been removed from the subarachnoid space, but an increase in the arachnoid cell processes was seen (Fig. 7). Discussion The finding that red blood cells injected into the subarachnoid space degenerate within the arachnoid villi supports the observation that no passageway exists through the endothelium of the villus and strengthens the conclusion that the arachnoid villus is not a site where intact erythrocytes can escape from CSF to blood. Two possibilities remain. Either there is no area where such passage can occur and the results obtained with isotope tagging experiments are secondary to an artifact, or some locus other than the arachnoid villus is responsible for the transport. One alternate exit route from the subarachnoid space is the perineural space around cranial nerves. Courtice and Sire- J. Neurosurg. / Volume 36 / February,

7 John F. AIksne and Ethel Lovings monds 5 discounted this as a site for erythrocyte egress, however, because ligation of cervical lymphatics did not alter the clearance. Presumably, any fluid or cells that found exit through the perineural route would have to return to the circulating blood by lymphatic channels. One variable not well controlled in any of the published isotope experiments was injection pressure. Bradford and Johnson 3 noted that a number of their experimental animals succumbed shortly after blood was instilled into the cisterna magna. To prevent a CSF pressure greater than 500 mm H20 with the injection of 8 cc of blood, we found it necessary to withdraw CSF prior to blood injections. Previous authors have not commented adequately on this facet of their procedure. If high pressures were generated at the time of injection, arachnoid villi and other delicate structures could be ruptured allowing abnormally free passage from CSF to blood. FIG. 6. Electron micrograph of arachnoid villus fixed 1 week after intracisternal blood injection. This portion of the villus is made up of normal-appearing arachnoid cell processes which contain three membrane-bound, dark-stained vacuoles. Such vacuoles are not seen in normal villi and presumably are secondary to phagocytosis of degenerated red blood cells. Lead citrate and uranyl acetate, x 27, J. Neurosurg. / Volume 36 / February, 1972

8 Role of arachnoid villus in removal of red blood cells FIG. 7. Electron micrograph of arachnoid villus fixed 2 weeks after intracistemal blood injections. The arachnoid cells (A) have a greater number and density of ceu processes than would normally be expected. Lead citrate and uranyl acetate, 12,300. The observation that blood that has entered the subarachnoid space is trapped in the arachnoid villi, degenerates in this location, and is probably phagocytized by arachnoid cells may explain the decrease in CSF absorption after subarachnoid hemorrhage? Postsubarachnoid hemorrhage hydrocephalus 7 may be an end stage of the same phenomenon if the increased cellularity seen at 2 weeks should proceed to fibrosis. The examination of villi at longer time intervals after blood injection may provide anatomical support for this hypothesis. The demonstration of red blood cells "trapped" in the arachnoid villus is further evidence to support the absence of pores through the endothelial membrane. If openings of the size postulated by Welch and Pollay, 12 (7 to 9 ~) were present, erythrocytes would pass freely into the sagittal sinus. The continued flow of CSF should aid this process so the villi would not become packed with red blood cells. The persistence of erythrocytes within the villus therefore supports the earlier observation that a continuous layer of endothelium is present over the J. Neurosurg. / Volume 36 / February, 1972 ] 99

9 John F. Alksne and Ethel Lovings arachnoid villus even under conditions of increased intracranial pressure. References 1. Adams JE, Prawirohardjo S: Fate of red blood cells injected into cerebrospinal fluid pathways. Neurology (Minneap) 9: , Alksne JF, White LE Jr: Electron-microscope study of the effect of increased intracranial pressure on the arachnoid villus. J Neurosurg 22: , Bradford FK, Johnson PC: Passage of intact iron-labeled erythrocytes from subarachnoid space to systemic circulation in dogs. J Neurosurg 19: , Bradford FK, Sharkey PC: Physiologic effects from the introduction of blood and other substances into the subarachnoid space of dogs. J Neurosurg 19: , Courtice FC, Simmonds WJ: The removal of protein from the subarachnoid space. Aust J Exp Biol Meal Sci 29: , Dupont JR, Van Wart CA, Kraintz L: The clearance of major components of whole blood from cerebrospinal fluid following simulated subarachnoid hemorrhage. J Neuropath Exp Neurol 20: , Foltz EL, Ward AA Jr: Communicating hydrocephalus from subarachnoid bleeding. J Neurosurg 13: , Reynolds ES: The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J Cell Biol 17: , Sprong W: The disappearance of blood from the cerebrospinal fluid in traumatic subarachnoid hemorrhage: the ineffectiveness of repeated lumbar punctures. Surg Gyn Obstet 58: , Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Bioehem Cytol 4: , Welch K, Friedman V: The cerebrospinal fluid valves. Brain 83: , Welch K, Pollay M" Perfusion of particles through arachnoid villi of the monkey. Amer J Physiol 201: , 1961 Received for publication January 25, This investigation was supported by United States Public Health Service Grant Number NB Address reprint requests to: John F. Alksne, M.D., Division of Neurological Surgery, University Hospital, 225 West Dickinson Street, San Diego, California J. Neurosurg. / Volume 36 / February, 1972