Changes in Barrier Effect in Pathological States

Factors Influencing Barrier Function

Changes in Barrier Effect in Pathological States LOUIS BAKAY Divisiori of Neitroatrgery, Sture Utiiversitj, of New York a1 BuJyirlo, School of Mediciiie, 462 Gride Street, Bitflulo, N . Y . ( U.S.A.)

Diseases of the central nervous system that are severe enough to alter its structural organization result in localized or generalized increase in the permeability of the blood-brain barrier. From an historical point of view, the principle of the bloodbrain barrier was established by Goldman (1913) who based his theory on two postulates. His “first experiment” revealed that the central nervous system does not stain after the administration of a vital dye into the blood stream. The “second experiment” showed that diffuse coloration of the nervous system occurred when the same dye was injected into the cerebrospinal fluid. Although it was not put in the form of a third postulate, it was accepted from the beginning that lack of staining in the “first experiment” did not include those parts of the brain that were not nervous in structure (choroid plexus, meninges, etc.) or those that were affected by a pathological process. In patho-physiological research, the blood-brain barrier still denotes a hypothetical structure or mechanism with specific vulnerability and almost general rate-limiting importance (Edstrom, 1961). Although a great deal of research, utilizing the latest anatomical and chemical methods, has been carried out over the past years to elucidate the nature of the normal blood-brain barrier, a similar sophistication in aim and methodology was less noticeable in studies of the pathological state. This is perhaps understandable when one considers the enormous impact of any change in permeability of the blood-brain barrier in clinical medicine. The increased permeability or absence of the barrier in various lesions became an accepted fact; the main emphasis was placed on the exploitation of this situation for diagnostic or therapeutic purposes without giving too much consideration to the basic factors that are responsible for its occurrence. From a diagnostic point of view, changes in the blood-brain barrier permeability are used for localization of tumors and other lesions by radioactive brain scanning. The same principle allows for the treatment of cerebral infections by antibiotics and of neoplasms by chemotherapeutic agents ; the substances applied for diagnostic or therapeutic reasons have ready access to the lesion while, on the other hand, they are more or less excluded from the normal brain. A complete review of all pathological conditions affecting the transfer of substances from blood to brain would be prohibitive in size. Consequently, I selected a few specific examples of pathological conditions to illustrate some of the basic problems. Kernicterus and other types of bilirubin pigmentation of the brain were chosen, not R c f i r m w r pp. 336-339

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only because of their clinical importance and intriguing patho-physiology, but also because they were historically the first type of lesion where the blood-brain barrier as the cause of the disorder was suspected. The various types of cerebral edema represent a pathological condition where increased barrier and membrane permeability are determining factors. Brain injuries are characterized by derangement of the barrier; this group is followed by a review of a much morecomplex subject, namely, the state of the barrier in tumors, Our knowledge of the latter, although still incomplete, was increased lately by a great flow of information derived from radioactive brain scanning. B I L I R U B I N A N D T H E B L O O D - B R A I N BARRIER

(Kernicterus)

Perhaps the first application of the blood-brain barrier theory to a clinical problem was its implication in the development of kernicterus. It was recognized early that bilirubin staining of certain portions of the brain, particularly that of the basal ganglia, can frequently be seen in icteric newborn while, on the other hand, adults, even with severe and long-lasting jaundice, do not show any bilirubin deposits in the central nervous system except for that of the choroid plexuses and cerebrospinal fluid. It was originally thought that bile pigments deposit in the newborn brain because of the physiologically undeveloped and more permeable blood-brain barrier which, when fully developed and "leakproof", keeps them out of the mature brain. This hypothesis was further strengthened by the discovery that areas of tissue damage in the adult brain (infarcts, hemorrhages, contusions, tumors, etc.) do stain with bilirubin in hyperbilirubinemia. It seemed logical to assume that kernicterus represents a clinical variant of vital dye experiments, trypan blue being simply substituted by a biological pigment, bilirubin. However, as time passed by, investigators became increasingly aware that kernicterus of the newborn is caused by a combination of circumstances; the relative importance of the individual factors is not yet known. Space does not permit a complete coverage of the vast literature on this subject. A comprehensive review on kernicterus up to 1959 was edited by Sass-Kortsik (1961). The results of most investigations indicate that staining of brain tissue by bilirubin in infants represents more than a simple change in permeability of the blood-brain barrier, although an increase in permeability might be a predisposing factor. According to Hugh-Jones et a / . (1960), kernicterus is most frequently observed in erythroblastosis fetalis; it occurs only rarely in nonerythroblastotic premature infants in association with severe hyperbilirubinemia. Nevertheless, it is known that kernicterus and increased bilirubin in the CSF may occur in conditions other than hemolytic disease of the newborn in rough proportion to the degree of hyperbilirubinemia (Stempfel, 1955). The variability in the occurrence of kernicterus is considerable; well-developed and undamaged infants might not develop it even in the presence of very high serum bilirubin levels (Zuelzer and Brown, 1961). At the same time, Harris et al. (1958) observed kernicterus in conjunction with relatively low levels of bilirubin in premature

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infants. It is generally agreed upon that the presence of indirect bilirubin in erythroblastosis fetalis is important, although its concentration in the plasma by itself is not unequivocally associated with the development of kernicterus. Local tissue injury is assumed to be present in addition to prematurity, increased barrier permeability, and hyperbilirubinemia to account for the staining of the central nervous system. The development of kernicterus might be promoted by the specific metabolic vulnerability of certain areas of the newborn brain, by anoxic damage, or by the direct toxic effect of bilirubin. Bilirubin is a toxic substance (Day, 1956; Waters and Bowen, 1955; Ernster et a / . , 1957), and Ernster et a / . (1957) showed experimentally that it is capable of inhibiting oxidative phosphorylation in neural tissue. The localization of ATP depletion and impaired respiration within the hyperpigmented brain suggested to Schenker et al. (I 966) that impaired phosphorylation may be an important feature of kernicterus. It is important to remember that most of the serum bilirubin is albumin-bound. Unbound and potentially neurotoxic bilirubin, available for transport into the brain, does not usually amount to more than a fraction of total bilirubin. Although the relative proportion of protein-bound bilirubin in plasma is variable, Odell (1959) as well as Blanc and Johnson (1959) believe that unconjugated, and consequently, more toxic bilirubin is the cause of brain damage because it leads to direct necrosis of the nerve cells. Diamond and Schmid (1966) presented conclusive evidence that only unbound bilirubin is able to cross the blood-brain barrier and that the pigment level of the nervous system and bilirubin neurotoxicity are related to the unbound, rather than to the total, pigment concentration in the plasma. These investigators used 14C-labeled bilirubin which lent itself to more accurate determination in the brain tissue than analysis based on color changes. Diamond and Schmid (I 966) could not reach definite conclusions concerning the regional deposition of bilirubin in kernicterus. They thought that this might be related to the binding and retention of the pigment by already damaged cells, but they also considered the possibility that “selective areas of the brain are a priori more vulnerable to bilirubin and once damaged, retain the pigment more avidly.” Some of their data could be considered in favor of anoxia or respiratory acidosis being responsible for bilirubin deposition because acidosis enhanced the accumulation of bilirubin in the brain, but this conclusion remains only tentative. The clinico-pathological syndrome of kernicterus observed in rats with congenital deficiency of glucuronyl transferase (Gunn’s, 1938, strain of rats) is similar to human kernicterus (Blanc and Johnson, 1959). These rats were used for the experimental study of kernicterus without any difinite answer as to its etiology, although it is clear from the experiments of Menken et al. (1966) that hyperbilirubinemia is necessary, but not alone sufficient, for the uptake of [“C]bilirubin by the brain. The brain tissue of kernicteric animals contained significantly more isotope than tissue from healthy jaundiced animals. Other experimental models yielded equivocal results. Nuclear jaundice and pigmentation of the nerve cells closely resembling that of human kernicterus were produced in healthy newborn kittens by repeated intravenous K~J.jrrcvtr05 pp.

336-339

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injections of a bilirubin-albumin solution (Rozdilsky and Olszewski, 1961). However, these investigators were unable to obtain the same effect in newborn puppies and rabbits. In these two species, additional damage to the nervous system was necessary in order to produce kernicterus. Recently, Rozdilsky (1966) came to the tentative conclusion that under these experimental conditions, albumin counteracts the toxic action of bilirubin on the brain. While some of the experiments indicated the toxicity of bilirubin as the primary noxious factor in kernicterus, Polani (1954) concluded from his experiments in rats that the neurological damage may be associated principally with hepatic damage. The combination of anoxic damage and hyperbilirubinemia has also been considered to be the cause of kernicterus. As Dobbing (1961) pointed out in his critical review on the blood-brain barrier, anoxic damage is a frequent condition in premature infants, and the regional distribution of damage in cerebral anoxia and in kernicterus is somewhat similar. Lucey et al. (1 964):observed that the commonly observed “physiological jaundice” in newborn rhesus monkeys does not lead to kernicterus; neither does the administration of indirect-reacting bilirubin. However, kernicterus can be produced in newborn monkeys by rendering them hyperbilirubinemic after a period of temporary asphyxia. Chen et al. (1966) described electron microscopic changes in kernicterus of newborn rabbits associated with asphyxia as well as hyperbilirubinemia. The ultrastructural alterations suggested that the access of bilirubin to the intracellular compartments of the brain is dependent upon the effect of acute anoxia. They assume that anoxia provokes an increase in the permeability of the cerebral capillaries and simultaneously increases the permeability of the cell membranes. The importance of cerebral anoxia in the development of kernicterus has not been accepted without reservations. For instance, Malamud (1963) studied the distribution and degree of CNS damage in newborns in various types of hypoxemia and concluded that the findings in anoxic damage caused by perinatal trauma or convulsions differ from those of kernicterus. “The lesions observed in convulsive disorders could best be defined as a hypoxaemic encephalopathy and those in kernicterus as possibly a bilirubin encephalopathy. Their occasional coexistence does not detract from their specificity” (Malamud, 1963). In contradistinction to the kernicterus of infants, bilirubin in jaundiced adults might simply mark abnormal brain tissue. The relationship between the duration and severity of hyperbilirubinemia and the transfer of bilirubin into the CNS or CSF is not clear. Although bilirubin is frequently found in the CSF in various types of hepatitis, leptospirosis, cirrhosis, and biliary obstruction, Galambos and Rosenberg (1959) could not establish a definite correlation between the duration of jaundice and CSF bilirubin concentration nor between the bilirubin and protein content of the spinal fluid. Table I, compiled from our own patients with known plasma bilirubin values and cerebral lesions studied at autopsy, demonstrates a certain degree of correlation between the presence or absence of staining of the lesion and the plasma bilirubin level, particularly that of indirect-reacting bilirubin. However, the number of these cases is not sufficient to draw definite conclusions, particularly concerning the relative

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TABLE I B I L I R U B I N S T A I N I N G O F CEREBRAL LESIONS

__

Caiise

I.

Total 2 weeks

61.2 mg

2. Ca, liver 3. Cirrhosis 4. Ca, liver

3 weeks 2 months 1 week

23.3 mg 22.0 mg 13.0 mg

5. Ca, liver 6. Hodgkin’s granuloma, liver 7. Colelithiasis

6 weeks

20.4 mg

Hepatitis

12 days Transitory, severe for 8 weeks but resolved 5 weeks before death

9.2 rng 14.0 mg

Indirect

Cerebral Lesion

36.9 mg Infarct (approximately 1 month) 14.1 mg Metastasis 13.2 mg Hemangiorna 4.9 mg Old infarct Recent infarct (few days) 7.5 mg Metastasis ? Contusion 2.0 mg Old infarct Recent infarct (approximately 3 weeks)

Staining

-1-

+

+ -

+

-

importance of protein-bound or diffusible bilirubin. There is no valid theoretical reason against the assumption that protein-bound bile pigment deposits in most of these gross brain lesions without difficulty. Summary

One can state that at the present time, the exact mechanism of bilirubin transfer and deposition in the brain of the hyperbilirubinemic newborn is not known. It seems to be certain that the process involves more than increased barrier permeability. However, the relative importance of pre-existing cellular damage (most likely anoxic) versus secondary tissue damage due to bilirubin toxicity in kernicteruscannot be assessed, yet. CEREBRAL EDEMA

Edema of the brain is produced by the increase of its water content. In addition, there is a variable increase of solutes ranging from electrolytes to large protein molecules. It is, therefore, obvious that this condition is of considerable interest from the point of view of the blood-brain barrier because the excess fluid and particulate matter originates in the blood plasma and could hardly pass into the nervous tissue without a change in membrane permeability. This is particularly true in those types of brain edema where the edema fluid is proteinaceous; the presence of serum albumin in the fluid is prima facie evidence of increased barrier permeability since, under normal circumstances, the exchange of large protein molecules between plasma and brain tissue is minimal. In addition to the blood-brdin barrier aspect, recent electron microscopic investigations of edematous brains led to important discoveries concerning the extracellular Refirmwr pp. 336-339

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space, a compartment that must always be considered when attempting to measure barrier permeability. Under normal conditions, the space between the cells of the central nervous system is narrow, although the narrowness of these clefts does not necessarily disqualify them as important pathways of fluid and small molecules (Nichols and Kuffler, 1964). It seems that in the gray matter, edema is almost exclusively intracellular. In the white matter, excess fluid might accumulate, either between the cells, thereby greatly enlarging the extracellular compartment, or in the cells. Studies on cerebral edema clearly indicate that osmotic changes in plasma and brain fluids, both extra- and intracellular, result in rapid changes in fluid and solute transport between the various tissue compartments. The effect of these fluxes on barrier permeability should not be underestimated. Recent experiments by Klatzo, Wisniewsky, and Smith (1965) indicate that such reversible changes in plasma osmolality as that caused by intravascular infusion of 30 % glucose result in a highly abnormal permeability of the cerebral capillaries for proteins. The “leakiness’ of the blood vessels is of short duration and disappears as soon as the plasma osmolality approaches normal values. Edema of the brain is not a single entity. Some of the various types of brain swellings differ not only in the localization and chemical composition of the excess fluid but also in the permeability of the blood-brain barrier. The chemical and structural characteristics of various brain edemas were recently described by Bakay and Lee (1965); the brief presentation which follows will be limited to the observations on barrier permeability.

Cold-induced edema The edematous white matter shows vital coloration after intravascular trypan blue administration in an experimental model where the swelling of a hemisphere is produced by freezing of the cortical surface. Similar staining was seen after the application of Evan’s blue (Clasen et al., 1962) and fluorescein-labeled serum proteins (Klatzo et al., 1962). The vital staining of the swollen white matter is subject to the time which has elapsed from the injection of the dye (Bakay and Haque, 1964). The dye extravasates at the marginal zone of the injured cortex and infiltrates the edematous white matter gradually, reaching complete distribution only 24 h after its administration. The same spatial and temporal distribution was found by using RlSA as a tracer (Bakay and Haque, 1964), this corroborates previous theories that many vital dyes, including trypan blue, are protein-bound in plasma, and their recovery in the abnormal brain tissue corresponds to that of plasma proteins. A similar, gradual diffusion from the point of entrance (the marginal area of the cortical lesion with its leaky blood vessels) into the area rendered edematous was observed for eleztrolytes, particularly for sodium and chloride. However, since the relative distribution of these cations and anions inside and outside the cell membrane is not well-known, under these circumstances, the conclusions concerning the permeability of the blood-brain barrier are best based on the entrance into the brain of proteins for which the barrier is normally impermeable. In summary, the bloodbrain barrier was found to be grossly altered and increasingly permeable for large

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32 I

particles in cortical freezing at the level of the border zone of the lesion. From there, the extravasated components of plasma penetrate the edematous white matter subsequently; however, the permeability of the blood-brain barrier within the region of the edema, itself, is not perceptibly altered. Edema surrounding cerebral neoplasms and other space-taking lesions has long been known to show coloration with vital dyes and to reveal an increased exchange of other substances including proteins with plasma. The edema of this type as well as the changes in barrier permeability are probably identical with that seen after trauma; the propagation of serum exudate into the surrounding brain tissue is facilitated by the leakiness of the neoplastic vessels.

Other traumatic edemas Vital staining was observed in brains rendered edematous by exposure and manipulation (Prados et a/., 1945), by implantation of dry psyllium seed (Sperl et a/., 1957; Samojarski and Moody, 1957), and by inflating a balloon placed in the extradural space (Ishii et a/., 1959). The dyes applied included trypan blue, acridine dyes, Evan’s blue, and di-iodo-fluorescein. In some of these experiments, tissue proteins and electrolytes were studied. Significant increase of albumin was found in the swollen tissue surrounding implanted psyllium (Hauser et a/., 1963). The greatest concentration was found nearest to the psyllium mass; it decreased progressively as the distance ofthe tissue from the lesion increased. Cutler et al. (1964) studied the movement of 1251labeled serum albumin in brain tissue rendered edematous by balloon compression. They found a close relationship between the penetration of albumin and Evan’s blue, indicating that much of the dye is protein-bound. Just as in cold-induced edema, the passage of albumin from blood into brain tissue occurred at the marginal zone of trauma. From this point of entry, the edematous tissue, which was strictly localized in the white matter, was gradually penetrated by serum albumin, indicating a break in the blood-brain barrier at the level of the lesion but not in the edematous white substance.

Injlammatory edema

In inflammatory edema, which is produced by intracerebral injection of bacterial endotoxins, purified protein derivative of tuberculin, and such additional substances as graphite, the permeability of the blood-brain barrier was increased for Geigy blue (Gonatas e t a / . , 1963). Here, again, the edema is located in the white matter and consists of a protein-rich exudate. There is a considerable freedom in exchange of RISA between plasma and edematous tissue (Katzman et al., 1964). Triethy It in - induced edema Triethyltin-induced edema is of considerable interest regarding the permeability of the blood-brain barrier because it seems to be very different from other types of brain Refiwnccs pp. 336-339

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swelling. Edematous changes are limited to the white matter. The excess fluid, at least in its initial stage, is a non-proteinaceous plasma ultrafiltrate situated in intramyelinic vacuoles which are produced by a split in the myelin sheath (Aleu et al., 1963; Lee and Bakay, 1965). There is no pathological alteration in the ultrastructure of the capillaries, glia cells, or in the size of the extracellular space. It is, therefore, perhaps not surprising that the permeability of the blood-brain barrier for large molecules is not affected. The edematous white matter does not stain with trypan blue and other vital dyes, and large molecules such as albumin do not penetrate the edematous tissue from the blood stream (Magee et al., 1957; Kalsbeck and Cumings, 1962: Katzman et al., 1963; Bakay, 1965). This represents a striking contrast with traumatic and inflammatory edemas. The lack of abnormal morphological changes of the vasculature as well as that of the glia cells is undoubtedly responsible for the normal state of the barrier; unfortunately, this normal state of all structures believed to be instrumental in barrier function does not allow us to draw significant conclusions from triethyltin edema as to the morphological basis of the blood-brain barrier. Edema associated nith cerebral anoxia

Edema associated with cerebral anoxia reflects only one aspect of a complex metabolic disturbance. The exact nature of the changes in tissue structure and metabolism is not adequately known; it is particularly difficult to separate the disturbances pertaining to membrane permeability and to the blood-brain barrier in general from those of anoxic damage to the cells, if such a distinction is, indeed, possible. The complexity of varicus factors involved can be illustrated by the fact that there is very early structural damage in the mitochondria (Bakay and Lee, 1967) that are quite resistant in other types of edema, while on the other hand, the fluid accumulation in anoxia is very moderate. Clearly, edema in anoxic brain damage is only one of many alterations. Studies were directed to investigate the permeability of the capillaries and of the blood-brain barrier, in general, under anoxic conditions because a change in permeability was considered to be essential for the development of edema. The findings obtained with the use of vital dyes were contradictory. Broman’s (1949) experiments on cats showed that complete occlusion of the cerebral circulation caused no disturbance in vascular permeability for trypan blue. Grontoft (1954) came to a similar conclusion, stating that in adults, anoxic injury to the blood-brain barrier cannot be demonstrated with trypan blue, although in infants, barrier damage is closely related to the degree of asphyxia. Becker and Quadbeck (1952) used a more easily permeable vital dye, Astroviolett FF. They were able to demonstrate barrier damage in anoxia produced in a high-altitude chamber and in experimental sub-acute carbon monoxide poisoning at a stage when there was no microscopic evidence of tissue damage. Hodges et al. (1958) used fluorescein to study the relationship between hypoxia and blood-brain barrier permeability with special regard to the relative value of various perfusion techniques in vascular surgery. They found that the appearance of fluorescein in the central nervous system was a sensitive indicator of faulty perfusion technique or inadequate pump-oxygenators with cerebral hypoxia as a result.

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It is, of course, conceivable that permeability changes exist for electrolytes and small molecules, even in the absence of gross changes to vital dyes, because most of the vital dyes used are essentially protein-bound. Factors other than hypoxia, alone, may be responsible for the transfer of substances from blood to brain. Arterial increase in C02 concentration, when severe enough, might be a more important factor. Temporary and easily reversible increase in the permeability of the blood-brain barrier for trypan blue was observed in rabbits by Clemedson, Hartelius, and Holmberg (1957) on inhalation of a gas mixture containing 10 % COZor more. In their opinion, hypercapnia should not be disregarded as a cause of cerebral damage, particularly in asphyxia where the component of anoxia “sometimes seems to have been over-emphasized.” Brierley (I 952) noticed an increase in ~erebral,3~P uptake after inhalation of a C02 and 0 2 mixture, but only when the mixture contained 20 % C02. His tentative conclusion was that the increase in the SZP content of the brain was either due to vasodilatation or to increased capillary permeability, or to both. Goldberg, Barlow, and Roth (1961) showed that exposure to 25 % COZ increased the cerebral concentration of phenobarbital, salicylic acid, acetazolamide, and urea in cats. This increase was not related to changes in blood flow and could not be brought up by inhalation of 5 % C02. Exposure to a high C02 content in the inhaled gas mixture with subsequent severe hypercapnia seems to increase the permeability of the blood-brain barrier, even without additional hypoxia. Goldberg, Barlow, and Roth (1963) studied the effect of 25 % COZ on the cerebral uptake of [35S] sulfate and [14C] urea. They found that although the steady-state sulfate space had not changed, the entry of 35s was greatly enhanced within a few minutes after injection. They also observed regional differences in the cerebral deposition of SSS, including an increased concentration in the central core of the white matter, which suggested irregular changes in vascular permeability. Bakay and Bendixen ( 1 963) separated, experimentally, the various factors involved in asphyxia, namely, anoxia, CO2 retention, and increased venous pressure. They found that real brain swelling occurred only when anoxia was associated with hypercapnia. However, the blood-brain barrier was quite resistant under these circumstances. Although there was an increase in the exchange of electrolytes between plasma and brain tissue, vital staining with trypan blue and significant uptake of albumin by the brain from the blood stream occurred only on extreme hypercapnic hypoxia with arterial 0 2 saturation below 20 % to 25 % and an arterial pH below 6.75. Severe anoxic-ischemic lesions and edema develop following the production of carotid ligation and respiratory hypoxia (Plum et al., 1963). Under such circumstances, there is vital staining and marked swelling of the affected portion of the brain. It is obvious, however, that this experimental model represents infarction and total tissue destruction rather than simple anoxic effect.

Summary Although the amount and distribution of the excess fluid varies in different types of brain edema, they all reveal an increase in tissue water as well as sodium and chloride. Rrjerenrrs pp. 336-339

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However, the exchange of large mole-ules (vital dyes, albumin, etc.) between plasma and edema fluid is limited to those cerebral edemas where structural damage to the small blood vessels can be demonstrated. Furthermore, the point of entry into the nervous system for these large particles is the wall of the injured vessel. B R A I N I N J U R I E S A N D THE B L O O D - B R A I N B A R R I E R

Fresh brain wounds stain immediately with vital dyes injected into the blood stream. They also concentrate various radioactive tracers exclusively or to a much greater extent that the surrounding normal brain tissue. Bakay (1960) found that cerebral lesions exchange sodium with plasma rapidly. This results in an early concentration of z4Na in the injured tissue (Fig. I). However, the ratio of 24Na content between injured

Fig. 1. Transverse section of cat brain (A) and corresponding radioautographs 23 min (B), 70 min (C), and 315 rnin (D)after intravenous injection of NaZ4.(From Bakay, 1960).

and normal brain tissue diminishes during the subsequent few hours because a gradual sodium exchange between normal nervous tissue and blood increases. On the other hand, the 24Na concentration of the injured part remainsconstant and then diminishes due to the fact that the radioactive sodium of the lesion remains in free exchange with that of the plasma, and consequently, decreases in linear proportion to the declining plasma 24Naconcentration. Similar observations were made with 23P (Bakay, 1955). A significantly increased uptake of 32P by the lesion when the tracer was given two hours before death still existed six weeks after trauma. In mild traumas, the increased exchange of substances might be limited to small particles such as the increased uptake of S2Pobserved during temporary concussion by Cassen and Neff (1960). However, injuries associated with visible structural alterations

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of brain tissue usually result in the exchange of serum proteins between plasma and brain. The extent of increased barrier permeability around a lesion, as well as its duration, varies according to the physico-chemical characteristics of the substance used for measurement. Lesions produced by focused ultrasound, as described by Bakay et al. (l956), were used to measure the extent of barrier damage with different indicators. Ultrasonic lesions are very suitable for this purpose because they are spherical and easily reproducible. Furthermore, they are surrounded by normal tissue and are not connected to the surface by needle tract. The results shown in Fig. 2 are based on

32

P

7-7

[I3'I]albumin

trypan blue

Fig. 2. Relative size of increased uptake by spherical ultrasonic lesion in brain tissue of 3zP, trypan blue, and ['3'l]albumin.

measurements of freeze-dried, 25 ,u sections and corresponding contact autoradiograms of cat brains thirty minutes after intravascular administrations of the tracers. Calculations of the volume of tissue with increased permeability are even more revealing. They indicate that the total area of increased uptake of RISA and trypan respectively, of that of 32P. blue is 36 % and 30 The duration of increased permeability also varies according to particle size and other unknown factors. The injured blood vessels seal their walls faster for large molecules than for small particles. As a result, increased uptake of small molecules in the lesion can still be observed at a time when the penetration of vital dyes and protein molecules from the lumen of the vessels is already arrested. Tschirgi (1950) emphasized the importance of protein complexes in the permeability of the blood-brain barrier to dyes. Bakay and Haque (1964) demonstrated the striking similarity between trypan blue and 131I-labeled serum albumin in their exchange between plasma and injured brain tissue. This strongly suggests that the great bulk of trypan blue is protein-bound and that this vital dye can be considered, for all practical purposes, a protein (albumin) tracer. In experiments involving the use of radioactive isotopes, the tracer content of the blood has to be taken into consideration since experimentally-induced cerebral

x,

Rifiwtrces pp. 336-33Y

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lesions often interfere with the blood flow, usually in the direction of stagnation. However, the measurements of Broman et al. (1961) indicated that the false increase in tissue radioactivity caused by pooled blood is less significant than the possible error produced by the rinsing of cerebral vessels. The return to normal of increased blood-brain barrier permeability is gradual, associated with the healing of brain wounds. Normal permeability is established much earlier for large particles than for small molecular substances. Abnormal barrier permeability for serum albumin, an “all-or-none’’ phenomenon , since normally, albumin does not penetrate from the vascular network into the brain substance, was first illustrated by autoradiography in brain injuries by Rozdilsky and Olszewski (1957). Lee and Olszewski (1959) studied the permeability of the blood vessels for RISA at various stages of the healing of brain wounds. The area showing albumin uptake decreased gradually with the passage of time from the injury. The barrier was no more permeable for albumin after three weeks. Abnormal permeability for small molecules, such as electrolytes, exists for a considerably longer time. Similar observations were also made in man in the form of brain scanning of patients with various types of head injuries. It is not surprising that abnormal uptake of various radioactive compounds can be seen in areas of cerebral contusion immediately after the injury (Fig. 3). The process of healing eventually results in a restoration of normal permeability; old scars cannot be visualized by scanning. By using relatively large and metabolically inert compounds, the period of increased permeability can be assessed. Van Vliet et al. (l965), for instance, studied post-surgical brain scans with

Fig. 3. Brain scan in head injury, 4 h after i.v. injection o f 750 pC of [203Hg]~hlormerodrin.Increased concentration of the labeled substance can be seen in the contused frontal lobe.

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polyvinylpyrrolidone (PVP) labeled with l 3 l I to determine the effects of surgical trauma on the vascular permeability of the brain. PVP is an inert substance of high molecular weight which is normally excluded from brain tissue. There was an increased uptake in the crmiotomy site in all patients with cortical incision or resection, or after pressure on the cortex by retraction. The intensity of localized radioactivity diminished as the post-operative period increased. Eventually, the scans became normal ; no positive scan was seen after 79 days following craniotomy. The increasing use of brain scanning in man with different radioactive indicators will contribute, in the future, to our understanding of the spatial and temporal aspects of blood-brain barrier permeability. From a basic point of view, explanations of the blood-brain barrier phenomena can be arranged in two main groups. One theory lays emphasis on the permeability of the capillary wall while the other identifies the selective barrier function with the specific function of the nervous tissue proper. When applied to pathological conditions, this second theory would imply that metabolic, rather than vascular, changes within the altered area of the brain are responsible for the increased concentration of various substances. Although investigative work on the barrier permeability of injured brain tissue involved the use of not only metabolically active but also, inert substances, the relative distribution of the various tracers in the intracellular and extracellular compartments at various time intervals after their transfer into the brain cannot be evaluated from the majority of the published results. The relative importance of the different vascular and cellular membranes remains, therefore, obscure. Hess (1955) thought that a PAS-negative ground substance is responsible for the “blood-brain barrier effect” because this substance, presumably consisting of mucopolysaccharides, disappears in brain wounds to be gradually re-elaborated after a while However, this theory cannot be confirmed by electron microscopy which fails to reveal the presence of an intercellular ground substance. A modified hypothesis (Millen and Hess, 1958) was then put forward that the ground substance immediately surrounding the blood vessels may play an important role in the maintenance of the blood-brain barrier, but this assumption also fails to hold up under electron-microscopic scrutiny. Bakay et a/. (1959) measured the uptake of 32P by normal and injured brain tissue by applying the tracer solution directly to the cerebral tissue by supra-cortical or cisternal application excluding, thereby, vascular channels. During the process of absorption of 32P by the brain tissue, there was no increase in concentration of the isotope within ultrasonically-produced lesions when compared with the surrounding normal brain (Fig. 4). This represented a marked contrast to other experiments that demonstrated high uptake of 32P by the same type of lesions after intravascular administration. The conclusion of Bakay et a/. (1959) was that there is no selective concentration of 32P in the lesion once the isotope has been made equally accessible to injured, as well as normal, tissue. The route by which 32Parrives at the lesion site would be irrelevant if the increased uptake by the lesion werecaused by cellular metabolism alone. Similar observations were made with 24Na; intra- and subcortical lesions did not take up more sodium than normal brain tissue after the direct application of an isotonic 24Na solution over the pia-covered surface. This represents a striking R&rciiczs p p . 336-339

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Fig. 4. Upper: Unstained; dry-frozen section of both upper parietal lobes of cat. Arrow points to vitally stained lesion in the lower layers of the cortex, produced by focused ultrasound 4 h before death. Middle: Radioautograph of same section 200 min after application of isotonic solution containing 32P over thepia-covered cortex. Exposure time: 3 days. Lower: Radioautograph of the same section. Exposure time: 4 days. (From Bakay et a/., 1959).

contrast with the fifteen-to-one ratio between injured and normal brain 24Naconcentration within the first hour after intravascular injection of the isotope (Bakay, 1960). Summary

Structural injury to the brain tissue results in an immediate increase of transfer from blood into traumatized brain of substances that normally are transported slowly or not at all. The abnormal exchange comes to an end at a certain stage of the healing process, earlier for macromolecules than for substances of small particle size. There is some evidence that an increase in vascular permeability characterizes the initial phase of altered barrier permeability; the subsequent migration, retention, and participation of the tracer substances within the nervous tissue is the result of a combination of factors that, at the present time, defy detailed analysis. BRAIN TUMORS

Most brain tumors accumulate various substances, such as vital dyes, various radioactive isotopes, etc., from the blood stream. The exchange of these substances between plasma and tumor tissue is relatively free, or at least much faster than their exchange between plasma and normal brain tissue. The concentration of many radioactive compounds is much greater in the tumor than in the surrounding brain tissue, and thus, a tumor/brain concentration ratio exists which can be measured and exploited for diagnostic purposes. This principle is the basis of radioactive brain scanning for the diagnosis and localization of intracranial neoplasms. In order to determine the possible mechanism responsible for the uptake of sub-

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stances by normal and neoplastic brain tissue, one has to consider differences in vascular permeability, size of extracellular space, and metabolism. Vascular permeability Brain tumors, particularly glioblastomas and metastatic tumors, contain abnormal blood vessels. Some of these vessels take the shape of vascular malformations including tortuous, lacunar, and aneurysmal dilatations, glomeruloids, and arteriovenous shunts. Nystrom (I 960) believes that purely mechanical factors are responsible for the irregularities of the vascular lumen: “Dilatation of the wall in shunts, glomeruloids, and lacunar or aneurysmal vessels was considered to be the result of locallyincreased blood pressure bearing upon a defective, and consequently, weak portion of the vessel wall, the elevated intravascular blood pressure being due to narrowing of lumen distal to the defective portion” (Nystrom, 1960). Such abnormalities could easily be responsible for a change in vascular permeability in terms of increased filtration. In addition, there seems to be a profound metabolic (anaplastic) change in the cellular components of the neoplastic vasculature, at least in malignant brain tumors. This is manifested by endothelial proliferation in the wall ofarterioles, venules, and capillaries. Hagerstrand (I 961) considers the vascular proliferation seen in cerebral metastases as a sign of a specific reaction of the blood vessels of the brain because similar changes cannot be seen either in the primary tumors or in their seedings to other organs but the brain. The possibility of increased permeability across the neoplastic capillary wall by heightened active transport, in contrast with increased diffusion through a structurally deficient vessel, must also be seriously considered. Wright (1963) followed the development of vascularization in artificially-induced ependymoma of mice. Initially, the newly formed arteries were thin-walled and had a poorly defined muscular layer. With the passing of time, the cellular arrangement became more anaplastic; this occurred at the same time as other, profound changes in the vasculature, namely, the development of arterio-venous communications and thin-walled sinusoids that emptied into large, tortuous veins. On electron microscopic examination, the blood vessels of glioblastomas reveal signs of increased metabolic processes in the endothelial cells (accumulation of mitochondria and vesicles) as well as vacuolization and marked variation in breadth of the basement membrane (Nystrom, 1960). Nystrom (1960) also pointed out that the vascular changes were much less pronounced in gliomas of a lesser degree of malignancy. The ultrastructure of the blood vessels of astrocytomas and oligodendrogliomas did not differ essentially from that of normal vessels. This, of course, might be one of the reasons why radioactive brain scan is sometimes “negative” in these gliomas. However, the electron microscopic morphology of the capillaries involved in tumor formation has not been adequately studied, and further investigations are needed. According to Torack (1961), capillary changes vary depending on the involvement of the tissue in the pathological process. In the tumor, itself, the endothelium is hyperplastic, and the perivascular zone is enlarged. In the peripheral zone of the neoplasm, the basement membrane is thickened, R i ~ r r c v i1.5 i pp. 336-339

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fenestrated, and filled with cell processes and dense bodies of lipid material. Torack (1961) related these changes to the increased vascular permeability for electrolytes and colloids, and to the phagocytic activity of the pericapillary cells. Although a parallel between embryonic and neoplastic blood vessels, connoting increased permeability of their walls, is frequently drawn, Tani and Ishii (1963) were unable to arrive at a definite conclusion. As far as their ultrastructure is concerned, embryonic capillaries have a poorly developed basement membrane which might explain their leakiness. However, tumor vessels have many additional features, such as the increased metabolic activity of their endothelial cells and perivascular astrocytes, or the close attachment of tumor cells to the basement membrane, as well as the enlarged extracellular space in the vicinity of the blood vessels; any of these structural peculiarities or a combination of them all could be responsible for the increased exchange of material between plasma and tumor tissue.

Blood content of tumor tissue There is considerable variation in the volume of blood per unit of tumor tissue as well as in the speed of its circulation (Ganshirt and Tonnis, 1956) compared to that of normal brain which averages about 0.5 % in the white matter and 2.5 % in the gray matter. Although the blood content of a mature astrocytoma might not be greater than that of normal brain tissue, most tumors contain more blood vessels per unit of tissue than normal brain. Although we do not have enough data, it is obvious that in a vascular tumor, blood volume is a factor that has to be taken into consideration even without assuming that the vascular permeability is increased. In many tumors, the blood vessels are not only numerous but also distended. Consequently, tracers, including radioactive isotopes, can accumulate in a tumor at a time when their concentration in blood is high. Although the distinction between that part of the tracer which is still contained to the vascular lumen and that which has egressed into the surrounding tissue is probably quite arbitrary after a short interval following its injection into the blood stream, the amount pooled in the blood is important in vascular tumors with a slow transcirculation time, such as some meningiomas. Long et al. (1963) emphasized that those substances which produce a high tumor-to-brain concentration ratio are protein-bound in plasma; this statement implies that a significant portion of these tracers remains in the blood stream. Nevertheless, it seems to me that the importance of radioactive blood contained by the tumor as the main source of some positive brain scans has been over-emphasized in the past. Experimental tumor, such as implanted fibrosarGomas (Matthews and Molinaro, I963), revealed a residual blood volume of only 4.6 % which was less than double that of the normal brain and could hardly be held responsible for the accumulation in the tumor of 1311-labeled albumin, although this large molecular tracer remains in the blood for a considerable length of time. Pinocytosis At this point, it is worth reviewing the possible mechanism of the uptake by tumor of

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substances with large molecular weight. Some of these compounds are very valuable in radioactive brain scanning and in the study of altered barrier permeability. It is also known that they are transferred into the neoplastic tissue from the blood stream by other means than simple diffusion. In contrast, they remain almost entirely within the vascular lumen in normal brain tissue. It is not difficult to explain their presence in necrotic brain or tumor tissue: there, the continuity of small blood vessels is broken, and blood or plasma floods the surrounding tissue, carrying with it almost undiluted amounts of radioactive proteins. However, this mechanism does not apply to neoplasms with reasonably intact vasculature. Few attempts were made to determine the transcapillary exchange of large particles, particularly albumin, in brain tumors ; the conclusions are still somewhat tentative. The exact localization of RISA in brain tumors at the cellular level by light microscopy and microscopic radioautography is all but impossible. Tator et al. ( I 965) were unable to distinguish between vascular and cellular factors in the deposition of radioactive serum albumin in various brain tumors. The incorporation of RISA in cells by pinocytosis is strongly suspected because this mechanism of transfer was observed under other pathological conditions (Klatzo and Miquel, 1960). Incorporation of large molecules into cells of the vascular wall is occasionally seen by electron microscopy in normal brain; the number of pinocytotic vesicles formed by endothelial and glial cells increases greatly in traumatized, and presumably, neoplastic nervous tissue. Raimondi (1964) attempted to localize RISA in human brain tumors at the electron microscopic level. In his opinion, serum albumin is transferred into the capillary endothelium and beyond by pinocytosis. This conclusion is supported by the observation that the number and size of pinocytotic vesicles is increased in tumor tissue, an observation which is also shared by Bakay and Lee (1967). Although pinocytosis is obviously operational in the bulk transport of large molecules, its relative importance in the uptake of these compounds by tumor tissue awaits further clarification. Since pinocytosis is a relatively slow, gradual process, it fails to explain completely the increased barrier permeability that is noticeable immediately after the injection of the tracers.

Extracellular space

It has been suggested by several investigators that the main difference in the uptake of various substances between tumor and normal brain tissue is due to the respective size of their extracellular spaces. This reasoning closely follows the argument that the blood-brain barrier effect in normal brain is not caused by a hindrance to passage through the capillary wall, but rather, at the level of the cell membranes of the central nervous system. The relative impermeability of the normal “barrier” would, then, be based on a functionally inadequate extracellular compartment. An extreme view, no longer accepted, denied the existence of any measurable room between the cells; this theory, however, is contrary to both morphological and physiological evidence. Nevertheless, a comparison of the intercellular space of normal and neoplastic Hifcwnc cs pp. 336-339

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brain tissue should be undertaken to clarify this issue. Unfortunately, there are no sufficient data for this purpose. The exact size of the extracellular compartment is a matter of conjecture even under normal conditions. This subject has been reviewed recently by Bakay and Lee (1965). The present estimate of the extracellular space ranges from 5 % to 20 % of volume; most investigators favor the 15 range. However, there is a difference between the gray and white matter in this respect, and regional differences may exist as well. Although a systematic survey on the size of the extracellular space in brain tumors has never been undertaken, electron microscopic observations by Raimondi el al. (1 962) suggest that an anatomically large extracellular compartment is not likely to be the reason for the increased uptake of different substances by a neoplasm. The intercellular clefts in astrocytomas and glioblastomas are probably not significantly wider than in normal brain tissue, except for those spaces that surround degenerating cells. In meningiomas, which almost invariably concentrate radioactive tracers in high concentration, the cells are positioned closely to one another. Whether the “functional” extracellular space, a compartment that might include some intracellular elements and reflects the distribution of primarily extracellular test substances rather than a visible space, is larger in tumors than in normal brain remains a moot question. Some investigators approached this problem by comparing the uptake by tumor of primarily intracellular and extracellular tracers, respectively. However, such a separation is arbitrary because we are not in a position, at the present time, to determine the relative distribution of these substances in the various compartments of the central nervous system. Analogies taken from their behavior in other organs do not necessarily apply to the brain. Matthews and Molinaro (1963), in their studies on transplanted fibrosarcomas, concluded that “for extracellular substances, tumour concentration depends on extracellular tumour spaces.” However, the weakness of their argument is revealed by the statement that the mean extracellular space of the tumor was 75.5 %; after correction for the Donnan effect and the rdtio of plasma to whole blood concentration, it was 43.6 %. This, although feasible in a few selected tumors, seems to be overly large for members of the glioma group. On the other hand, they allowed only 3.9 for the extracellular space of the normal brain; this estimate is too low because after considering that the residual blood content was 2.4 %, it would indicate a virtual absence of space between the cells. The “extracellular” substances used by these authors included 82Br, BzGa, 95Zr, 1Wb, l311, Ag, 95Nb, and 1311 serum albumin. Although some of these ions probably do not enter the cells, the purely extracellular nature of others, to name 82Br, alone, cannot be accepted. Their conclusion remains, therefore, less categoric: “. . . there also appears to be a ‘barrier’ of some kind to uptake of intracellular substances, and so lack of extracellular space will not explain all the results. Thus, there appear to be two possibilities, either (a) the blood-brain barrier is partly due to lack of extracellular space and partly to some difference in cell permeability or uptake in brain cells compared with cells in other organs, or (b) the blood-brain barrier is due to a physical barrier which is impermeable to extracellular substances but slowly permeable to intracellular substances” (Matthews and Molinaro, 1963).

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Cellular metabolism Although brain tumors do not compare favorably with normal brain tissue in many aspects of their metabolism, such as oxygenation and glucose consumption, metabolism might be responsible for their accumulation of a variety of metabolically active substances. Reasonable as this statement might be, it has to be pointed out that no substance has been found which would achieve a concentration in the brain that would exceed that of the initial blood level or those of such metabolically active tissues as liver or kidney. In fact, as Long et al. (1963) pointed out, the seemingly selective concentration of tracers in brain tumors is due to the low normal background rather than to the abnormally high accumulation in the tumor. There is no conclusive evidence that the concentration in tumors of metabolically active agents is significantly greater than that of inert substances of similar physicochemical properties. Although Selverstone and Moulton (1957) have suggested that the relatively high phospholipid fraction of tumor tissue is instrumental in its 32P uptake, the search for compounds that would selectively concentrate in brain tumors because of their participation in specific metabolic processes has not been successful. The latest investigations of Tator et a/. (1966), for instance, have indicated that the uptake of fatty acids by tumor tissue exceeds that of brain and some other normal organs. The fatty acids are partly oxidized as a metabolic fuel and partly utilized in lipid synthesis by gliomatous tissue. Despite these promising characteristics, no truly significant difference was found between the tumor uptake of [1311]oleic acid and 11:jllIserum albumin except during the initial hour of transfer. Experimental studies furnished important data but failed, so far, to supply us with definite proof concerning the role of metabolism. Mundinger (1965) used in vitro models of tumor cell colonies and investigated the uptake by the cells of various compounds used in radioactive brain scanning before and after the blockage of several metabolic processes by specific inhibitors. His conclusion was that [13lI]albumin was taken up by the cells through pinocytosis; 74As and 1311 were thought to be predominantly extracellular in location, and W u - , 206Bi-, and [2°3Hg]chlormerodrin were mostly intracellular. Some tentative conclusions were reached as to the relative role played by active transport and by diffusion in the uptake of the “extracellular” ions. Interesting as these results are, it should be kept in mind that they were obtained in tissue culture under conditions that could yield only very speculative conclusions when applied to conditions in vivo. Matthews and Molinaro (1963) attempted to correlate the uptake of different radioactive substances by tumor tissue and correlate it with their biological characteristics. The experiments were performed in subcutaneously transplanted fibrosarcomas of rats. Their reasoning in doing this was that any tumor, including brain tumors, has its own vascular structure and metabolism which is comparatively independent from where it is growing. This point, however, can be argued because gliomas, the most frequent type of cerebral neoplasms, possess structural and biological peculiarities which make them, even in their most malignant and anaplastic form, inseparably Refirrirws pp. 336-339

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part of the nervous system. Indeed, one of their characteristic traits is that they do not metastatize into any other organs. Matthews and Molinaro (1963) considered 206Bi-, 43K-, SdRb-, 65Zn-, ”Mnand 2vJHgchlormerodrin] as entirely or predominantly intracellular tracers. However, chlormerodrin is almost completely protein-bound in plasma; we do not know how much of this protein complex is broken down and metabolized in neoplastic brain tissue within the time range involved in brain scanning. Matthews and Molinaro ( I 963) concluded that “for intracellular substances, since tumour concentration is approximately constant, the ratio depends on brain concentration and hence, on blood concentration, and substances which are rapidly cleared from the blood give the best ratio”. However, they point out that no selective concentration of any substance by the tumor relative to other organs was found; the accumulation of the isotopes, even the predominantly intracellular ones, did not depend on some special property of tumor metabolism. Summary

Both structural and metabolic peculiarities could be responsible for the accumulation of substances in brain tumors from the blood stream. The relative importance of the different potential factors involved in this process remains uncertain. The theory that the blood-brain barrier is merely a reflection of cerebral metabolism is rather difficult to reconcile with isotope distribution studies (Long et a]., 1963). The data

Fig. 5. Lateral brain scan, 4 h after i.v. injection of 750 pC of [203Hg]chlormerodrinreveals the presence of a parieto-occipital tumor (glioblastoma).

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Fig. 6. Lateral brain scan, 4 h after i.v. administration of 750 pC of [?03Hg]chlormerodrin.Positive concentration of the isotope in a large area within the right hemisphere; this corresponds to a recent infarct of the brain supplied by the right middle cerebral artery.

obtained, so far, can be readily explained only by the theory that a barrier exists based on selective vascular or cellular membrane permeability, and that this selectivity is reduced or lost in tumor tissue. This assumption is based on the following considerations (Bakay, 1956): 1. Substances which accumulate in brain tumors have different physico-chemical characteristics, such as molecular weight, electric charge, solubility, degree of dissociation, etc., without a common denominator. 2. Inert or metabolically unimportant substances concentrate in the lesion as well, or almost as well, as metabolically active compounds. 3. The concentration of many tracers in tumors is of a similar order of magnitude as in other forms of pathology (brain tissue subjected to trauma, infarction, or infection; Figs. 5, 6). This was also pointed out by Heiser and Quinn (1966) who recentlycompared the brain scan pattern of ischemic infarcts and gliomas by using technetium 99m pertechnetate as a tracer. No significant difference was found between the intensity or homogeneity of the uptake in the two categories. 4. There is no true selectivity in the uptake; none of the agents studied, so far, concentrated in brain tumors to a greater extent than in some other organs of the body. Neither did the tumor concentration ever exceed the initial blood level. 5. Generally speaking, the highest concentration of isotopes is found in nonneurogenic brain tumors; these include a great variety of neoplasms from slow groRcJercnr.e.r f f . 336-339

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wing, benign meningiomas to rapidly expanding, malignant metastases from other organs. Gliomas, the truly characteristic tumors of the central nervous system, do not behave uniformly. Accumulation of tracer substances is most commonly observed in glioblastomas. The uptake rate by astrocytomas, which have a tissue structure that closely resembles that of normal brain, is usually only slightly higher or not higher at all than that of normal brain tissue. The inherent implication of this observation is that really meaningful statements concerning the neoplastic changes of the bloodbrain barrier should be limited to tumors of the glioma group since tumors that are not linked genetically to elements of the nervous tissue could not necessarily share in its organization which includes the blood-brain barrier. ACKNOWLEDGEMENT

The author’s own investigations were supported by research grant NB 03754 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service. REFERENCES R. AND TERRY, R. D. (1963) Fine structure and electrolyte analysis of cerebral ALEU,F. P., KATZMAN, edema induced by alkyl tin intoxication. J. Nerrropathol. Exptl. Neurol., 22, 403-41 3. BAKAY,L. (1955) Studies on blood-brain barrier with radioactive phosphate. V. Effect of cerebral injuries and infarction on the barrier. Arch. Neurol. Psychiat., 73, 2-12. -, (1956) The blood-brain barrier with special regard to the use of radioactive isotopes. Charles C. Thomas, Publisher, Springfield, Illinois. -, (1960) Studies in sodium exchange. Experiments with plasma, cerebrospinal fluid, and normal, injured, and embryonic brain tissue. Neurol., 10, 564-571. -, (1965) Morphological and chemical studies in cerebral edema. Triethyl tin-induced edema. J. Neurol. Sci., 2, 52-67. BAKAY,L., BALLANTINE, JR., H. T. A N D BELL,H. (1959) srP uptake by normal and ultrasonically irradiated brain tissue from cerebrospinal fluid. Arch. Neurol., 1, 59-67. BAKAY,L. AND BENDIXEN, H. H. (1963) Central nervous system vulnerability in hypoxic states: Isotope uptake studies. In: “Selective Vulnerability of the Cerrtral Nervous Systeni in Hypoxaemia”. W. H. McMenemey and J. P. Schade (Eds.). Oxford, Blackwell Scientific Publishers, 63-78. BAKAY, L. AND UL HAQUE,I. (1964) Morphological and chemkal studies in cerebral edema. 1. Cold induced edema. J. Neuropathol. Exprl. Neurol., 23, 393418. BAKAY, L., HUETER, T. F., BALLANTINE, H. T.AND SOSA,D. (1956) Ultrasonically producedchanges in the blood-brain barrier. Arch. Neurol. Psychiar., 76,457461. BAKAY, L. A N D LEE,J. C. (1965) Cerebral Edema. Charles C. Thomas, Publisher, Springfield, Illinois. -, (To be published): Ultrastructural changes in the edematous central net voiis systeni. IV. Hypoxia and hypercapnia. -, (To be published): Ultrastructural changes it1 rlre edenrafous central nervous systenr. V. Peritumoral edema. BECKER, H. ANDQUADBECK, G. (1952)Untersuchungen uber Funktionsstorungen der Blut-Hirnschranke bei Sauerstoffmangel und Kohlenoxidvergiftung mit dem neuen Schrankenindicator Astraviolett FF. Z. Naturforsch. (B), 7, 498-500. BLANC,W. A. AND JOHNSON, L. (1959) Studies on kernicterus. Relationship with sulfonamide intoxication, report on kernicterus in rats with glucuronyl transferase deficiency, and review of pathogenesis. J . Neuropathol. Exptl. Neurol., 18, 165-189. BRIERLEY, J. B. (1952) The penetration of 32Pinto the nervous tissue of the rabbit. J . Pliysiol., 116, 24-25. BROMAN, T. (1949) The Permeability of’Cerebrospinal Vessels in Normal and Pa thological Cotrditions. Copenhagen, Einar Munksgaard. BROMAN, T., EDSTROM, R. AND STEINWALL, 0.(1961) Technical aspects on dyes and radiotracers in the determination of blood-brain barrier damage. Acta Psychiat. Neurol. Scand., 36, 69-75.

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DISCUSSION

D. B. TOWER: I would like to say a word of caution about the presence of ruptured membranes seen in electronmicrographs. When a cell is swollen, one can not necessarily attribute a ruptured membrane to the fact that the cell was swollen beyond its elastic capacity or to other properties of the membrane, because a knife also has had to pass across the section. Certainly, in light-microscopy, knives have been known to create such artifacts. Therefore, I would object to anybody’s being dogmatic about these ruptured membranes. Furthermore, I have a feeling that cells can withstand a tremendous amount of swelling without rupturing the membrane. 1 would also like to suggest that we be cautious about saying that spaces never increase or decrease in size, because I doubt if we understand all the factors influencing these spaces between the time when we sample the tissue and the time when we are able to look at it with the electronmicroscope. I am also disturbed about the apparent constancy which is insisted upon for the size of the spaces between cells. This does not seem quite reasonable from a biological point of view. Finally, I would be cautious concerning ruptured membranes in such conditions as edema, particularly in attempting to interpret where a marker like ferritin might logically be. Dr. Brightman, I remember, called attention to the fact that you can very easily translocate or dislocate ferritin from one place to another if you are not careful with your preparation. I don’t mean this in terms of criticism of Dr. Bakay, or anybody else, but we must be very careful in making statements about these thingswhich are too dogmatic.

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L. BAKAY: I can answer this simply. First of all, I don’t think that the rupture of the cell membrane is of any importance. The point remains that the extracellular space increases in size morphologically in edema in the white matter, whether there is a rupture or not, and it does not increase in the cortex where, incidentally, we never see rupture. As far as the artificial nature of the rupture of the cell is concerned, we never see it in unswollen cells, and you don’t see a ruptured cell membrane even if you use a magnification of 10,000 times with electronmicroscopy. It just simply cannot be seen, ruling out the possibility that this is a random artifact. It might be an artifact associated with swelling, but it is an artifact that is never seen in unswollen cells. As far as the size of the space is concerned, when 1 say that there is no evidence of any increase of the extracellular space in the cortex, even in conditions of edema, and there is one seen in the white matter, 1 mean that purely on the ultrastructural morphological basis. This has no bearing on whatever we mean by a functional extracellular space, and if we assume that some of the extracellular space is intracellular as far as the glia is concerned, that is all very well; but we do not see any evidence of an increase in cortical extracellular space size by electronmicroscopical methods. As far as ferritin is concerned, I know about Dr. Brightman’s work, and I fully agree with much of what he said. But ifit would be artificially translocated in these specimens, wouldn’t you expect that it would be translocated from this extremely narrow extracellular space between these enormously swollen cells? If ferritin would be all over the place, you could say that this has been transferred as an artifact. But if you see two extremely large cells and the narrow cleft inbetween, and the material does not go into the very largely hydrophilic cells on both sides, this is not likely to be an artifact. 0. STEINWALL: I would like to comment on the problem of the neonatal brain barr;er and kernicterus. mentioned by Dr. Bakay. The unconjugated bilirubin is very apt to be bound to protein in the blood and there is reason to believe that, even in the embryo, the blood-brain barrier to protein-bound material is effective. This can be illustrated by a quite recent investigation (together with Klatzo, Olsson and Sourander) on newborn rats and embryos (2 weeks or longer gestation) injected with fluorescein-labeled albumin. As far as could be observed by fluorescence microscopy, there was no cerebral extravasation of the tracer. There might exist conditions, however, in which the proteinbinding of bilirubin is loosened. Being a lipophilic compound the free bilirubin would then pass freely into the brain. I wonder if Dr. Bakay would comment on Grontofts work on human fetuses, where he concluded that the cerebral vessels were impermeable under normal conditions while anoxic fetuses showed signs of blood-brain barrier damage. L. BAKAY:I know about Grontoft’s work, and he emphasizes strongly that it is necessary for the brain to be anoxic to permit a trypan blue infiltration into the brain tissue. The difficulty is that in adult animals you can do the most drastic kind of anoxic and asphyxic experiments, but you always will find tissue necrosis, which there should not be in anoxic as versus ischemic anoxia. I don’t know what kind of damage those infants have been exposed to in addition to anoxia. K. NEAME:I would like to ask Dr. Bakay about the effects of pathological states as seen by brain scanning. Is this a change in the so called blood-brain barrier, or is it a change seen in tissues in general? L. BAKAY: That is a very hard one to answer, because it depends on the tissue. One thing is for certain: that there is no selectivity in the increased uptake in a brain scan due to any condition. The amount of maximal concentration you can get in almost any lesion in the brain corresponds to what you get in normal tissue elsewhere. There is no selective concentration. This answers your question: there is no specific change; in other tissues (muscle, liver or spleen), whether tissue is normal or abnormal, it has about the same concentration, although there could be more of course in the case of a pathological process than there would normally be. What you see as a positive lesion in a brain scan is not a selective concentration but a relatively free exchange of the substance with the blood stream, as occurs in other organs, and it becomes selective only because the suirounding brain does not have completely free exchange. How much of this is an intracellular, how much an extracellular process is a long story which I would like to go into, but do not have the time.

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J. DOBEING: About the spread of edema throughout the white matter of the hemisphere which is being damaged at a focal site in the cortex, I want to ask Dr. Bakay if he thinks that this has any lessons for experimenters who routinely mutilate the brain in the course of the experiments. The question - which is impossible to answer - is, what size of lesion, what extent of trauma is necessary to produce the effect throughout the white matter of the hemisphere? Would ventricular perfusion do it? Would needles, passing through thecerebrum into a ventricle (in most oftheselaboratory animals it is only a potential space anyway) cause such edema? Therefore, the evidence that one is in the ventricle, at least in a small animal, is likely to be at the expense of at least some trauma to the ependyma as well as throughout the tract of the needle? Certainly pharmacological intracerebral injections, which are horrible from the point of view of structures and edema, but will probably suit the purpose of the pharmacologist, would be likely to do this. The question is as to what extent can you inflict damage to the brain before it becomes necessary (particularly when investigating blood-brain barrier effects with drugs and substances concerned with edema) to provide controls rather better than with trqpan blue to discover whether you have this spreading edema from the trauma of your procedure or from the test substances employed.

L. BAKAY: I shall give you an extremely dogmatic answer to this. I don’t know about ventricular perfusion. If it is done without changing tht. pressure conditions and if the solute is not very different in osmolarity or very definitely toxic, I suppose a careful perfusion might not cause any damage, at least on theoretical grounds, in the surrounding brain. Whatever is introduced into the brain causes edema: how much is unpredictable. But you cannot put a lesion in the brain and you cannot put even a simple needle tract in the brain without having a certain amount of edema around it. A. LOWENTHAL: You said that there are two types of edema: one type with an increased protein content and another type with a less increased or with a reduced protein content. Could you say which are those two types?

L. BAKAY:There might be a great number of different types of edema. For example, we don’t know what category anoxia would fit in. The two fairly well defined edemas so far are: ( I ) the traumatic inflammatory peritumoral focal type of edema which seems to be characterized by a fairly free exchange of serum albumin and an obviously greatly disturbed capillary permeability, and (2) the type, which is really only characteristic of traumatic edema and seems to be an intramyelinic accumulation of protein-free plasma ultra-filtrate with no evidence of capillary damage. These are the two extremes, and we don’t really have any other categories as yet.