- Email: [email protected]
Brain and choroid plexus blood volumes in vertebrates
Comp. Biochem. Physiol., 1968, Vol. 26, pp. 489 to 498. Pergamon Press. Printed in Great Britain
BRAIN
AND
CHOROID PLEXUS BLOOD IN VERTEBRATES*
VOLUMES
S. RICHARD HEISEY t Department of Physiology, Harvard Medical School, Boston, Mass.
A b s t r a c t - - 1 . Brain blood volume per unit brain weight is larger in a cyclostome, teleost, amphibian and reptile than in a bird or mammal. Necturus maculosus and Petromyzon marinus had brain blood volumes approximately 4-5 per cent of brain weight. 2. The hematocrit of blood in the brain was usually less than that of blood drawn from a large peripheral vessel. 3. Choroid plexus blood volume in the rat is 4 per cent of total brain blood volume while in lower vertebrates, choroid plexus blood volume makes up a larger percentage of whole brain blood volume. 4. In Petromyzon marinus, the choroid plexus blood volume is 57 per cent of whole brain blood volume. INTRODUCTION
BOTH studies of brain fluid compartments and of distribution of substances between blood and brain require estimates of brain blood volume. Rosomoff (1961), Davson & Spaziani (1959), and Streicher (1961) using dogs, rabbits and rats, respectively, determined brain blood volumes using either labeled plasma proteins or red blood cells. In a study of organ blood volumes in the dog, Gibson et al. (1946) measured brain blood volume using both a plasma and a red cell label, enabling them to measure, in addition to blood volume, hematocrit in the brain. Such studies indicate that mammalian brain blood volume is approximately 2 per cent of brain wet weight. Neither brain blood volume nor cerebrospinal fluid volume has been measured directly in vertebrates other than mammals. Histological studies, however, suggest that lower vertebrates might have a smaller brain blood volume per gram brain weight than mammals, and that brain blood-cerebrospinal fluid volume ratios might be progressively smaller along the phylogenetic continuum from mammals to fish. Supporting these suggestions, Ariens-Kappers et al. (1960) reported that in Amphioxus the brain parenchyma is avascular. Craigie (1938) noted a paucity of capillary, loops in the brain of a primitive vertebrate, Petrorayzon marinus. Herrick * This investigation was supported by PHS Research Grant No. NB05412 and Research Career Program Award No. 5K03NB07124 from the National Institute of Neurological Diseases and Blindness. t Present Address : Department of Physiology, Michigan State University, East Lansing, Michigan 48823. 489
490
S. RICHARD HEISEY
(1921) was impressed with the large v e n t r i c u l a r cavities a n d choroid plexuses of some of the lower fish a n d a m p h i b i a n s ; he suggested that in some lower vertebrates, the b r a i n m a y be s u p p l i e d with oxygen from blood and in a d d i t i o n m a y receive a collateral s u p p l y of oxygen from the cerebrospinal fluid. I n the p r e s e n t study, b r a i n blood v o l u m e s were m e a s u r e d in representatives of each of the five vertebrate classes. METHODS The white rat (Rattus, sp.), street pigeon (Columba, sp.), fresh water turtle (Pseudemys scripta elegans), bullfrog (Rana catesbeiana), mudpuppy (Necturus maculosus) and goldfish (Carassius auratus) were obtained from commercial sources. The sea lampreys (Petromy~on marinus), obtained from a commercial fisherman who retrieved them from fish traps in the Exeter River near Exeter, New Hampshire, were transported to the laboratory in ice water, and were used on the day of trapping. Rats were anesthetized either with Nembutal (60 mg/kg IP) or with ether; pigeons, Nembutal (6-12 mg IV); turtles, Nembutal (66 mg/kg) or Avertin (250 mg/kg) via an esophageal tube; frogs, immersed in 0.1 ~o tricaine methanesulfonate (MS 222 Sandoz), supplemented with 10 cc of 3°0 urethane injected into the dorsal lymph sac; mudpuppies, goldfish and lampreys were anesthetized with 0'01°o tricaine methanesulfonate in water; lampreys were packed with ice. An isotope dilution method was used for both the red cell and plasma volume measurements. Iodinated human serum albumin labeled with 1131 (RIHSA-Cambridge Nuclear Company) was used to measure plasma volume. The albumin preparations had specific activities of 25-60 t~c/mg with less than 2 per cent of the 113~unbound to protein. Cr sl in the form of Na2Cr~lO4 was used to label red ceils in vitro. Radioactive material was injected at a different site from where peripheral blood samples were drawn. In most animals the radioactive material was injected intravenously and blood samples withdrawn either directly from the heart or from a large artery close to the heart. After injection 5-10 rain were allowed for vascular mixing. A peripheral blood sample was withdrawn and the animal decapitated. The brain was removed, severed from the spinal cord at the level of the obex, blotted and weighed in a counting vial; in some experiments the choroid plexuses were removed and weighed in separate vials. In experiments using either RIHSA or Cr 5~labeled cells, the peripheral blood sample was centrifuged and an aliquot of either plasma or packed red cells was counted ; in some experiments, an aliquot of whole blood was counted, tIematocrits, used for converting either plasma volume (RIHSA) or red cell volume (Cr 51) to blood volume, were determined using an IEC Micro-capillary centrifuge, Model MB. When both R I H S A and Cr 51 labeled red cells were used, the peripheral blood sample was drawn, centrifuged and an aliquot of plasma was counted for RIHSA activity. The cells were washed three times; after the final wash an aliquot of packed red cells was counted for Cr sl activity. All samples of brain and blood were put in 4 ml glass screw cap vials which fit into a crystal scintillation well detector (Baird Atomic Model 810A) and were counted integrally at a discriminator setting of 200 keV. Na2Cr~lO4 was added to heparinized whole blood from a donor animal (20-50 p.c/ml blood) and the mixture allowed to incubate, with occasional mixing, for 1 hr. For rats and pigeons the incubation occurred at room temperature but in the other species at 4°C. Ascorbic acid was added according to the procedure of Read (1954). The blood was centrifuged at 770g for 10 min and the supernatant plasma removed and replaced with 5 ml of cold 0'9~o NaC1 (rats and pigeons) or amphibian Ringer's solution (all other species). The cells were resuspended, recentrifuged and washed twice. Three washes were usually sufficient to bring the tracer activity in the final wash to less than 1 per cent of the activity bound to red blood cells. On the third wash, the tubes were centrifuged at 770g for 10 rain and
BRAIN
AND
CHOROID
PLEXUS
BLOOD
VOLUMES
491
1700g for five min. The packed cells were drawn into a plastic 1-ml syringe for injection into the experimental animal. The hematocrit of the packed red cells was 90-95 per cent.
Calculations a. Single-label experiments. When the plasma-hematocrit method was used, brain blood volume was calculated from the following equations P, IHSA activity/g brain Plasma volume/g brain -- RIHSA activity/ml plasma
(1)
Plasma volume/g brain Blood volume/g b r a i n - - (1-peripheral hematocrit)
(2)
When the red cell-hematocrit method was used, the corresponding equations were Cr sl activity/g brain Red cell volume/g brain -- (~rSt-activity/m1 packed ce~l-ls Blood volume/g brain --
Red cell volume/g brain peripheral hematocrit
(3) (4)
b. Double-label experiments. When RIHSA and Cr ~1 labeled red cells were injected into the same animal, the activity of the brain due to 11~' was separated from that due to Cr 5' by considering that the half life of 1131 is 8 days while that of Cr ~l is 28 days. The amount of activity due to each isotope was determined by solving the following simultaneous equations (Brain counts-bkgd)0 -- Cr051+ I0TM (Brain counts-bkgd) z = klCro 51+ k210TM where kl = exp-[hCr(t~-t0)] ku = exp-[hI(tx-to) ] to -- experimental day t~ -- any day after experimental day hCr = 0'693/27'8 hi = 0'693/8"05 Baker (1963) using a double-label technique to measure blood volume in the dog chose day 0 and day 10 counts to distinguish Cr s* from I TM. In the present study, preliminary experiments showed that brain and blood samples counted on successive days yield slightly different values for I0 TM and Cr0 sl activity and consequently different values for brain plasma and red cell volumes. Therefore, all brain samples were counted several times for up to 25 days with a graphic solution of the simultaneous equations presenting more reliable values of I0131 and Cr051 activity. Such an experiment is shown in Fig. 1. Brain activity was counted seven times, from day 0 to day 15. Brain counts (corrected for background) from days 0, 3, 7, 11, 14 and 15 were substituted in the simultaneous equations and different paired brain counts yielded different values for I0TM and Cr0SL By trial and error one value of Cr03~ was found which, in combination with brain counts and decay constants for all days, yielded a relatively constant value of I0TM (i.e. I0TM counts plotted as a function of days after the experiment scattered about a line with 0 slope). In the experiment of Fig. 1, the estimated value for Cr0 nl was 2"43 x 103 counts/min and this yielded 2"48 × 103 counts/min for I0lsl. Using another set of paired brain counts, and solving the simultaneous equations, Cr0 ~x was 2.31 x 103 counts/min ; this yielded values of I0131which increased with time after the experiment (i.e. I013t counts plotted vs. time scattered about a line with a positive slope). In still another solution of the simultaneous equations, Cr051 was 2"60 x 10s counts/min and yielded I0aSx values which decreased with time after the experiment.
492
S. RICHARD HEISEY TURTLE
X =]'o O=
131
,when
No. C
17
51
ro = 2 3 1 X I O
3
CPM
IO TM ,when CroOI:2.45XIOSCPM
• = ~o
131
51
,when Cro =2.60XIO3CPM
131
]~o , X l O 3CPM
2.80
X
X 2.60
X o O-
-- _O._
O O
o
2.40
O
220
2.00
1.80 DAYS |
I
[
I
J
0
5
I0
15
20
FIG. 1. G r a p h i c solution of two s i m u l t a n e o u s equations w h i c h relate I TM to Cr sz c o u n t s in a turtle brain. W h e n 1 TM c o u n t s on day 0 (10 TM) are plotted as a f u n c t i o n of t i m e after the e x p e r i m e n t a l day, only one value of C r sl c o u n t s o n day 0 (Cr0 s]) yields a c o n s t a n t value for I0 TM counts. In this e x p e r i m e n t 2"43 x 103 c o u n t s / r a i n d u e to C P 1 yielded a line w i t h zero slope (a c o n s t a n t value o f I TM c o u n t s o n day 0). I f the value of C r 51 counts o n day 0 was chosen to b e less t h a n this value, the solution of t h e equations yielded increasing values for 1 TM c o u n t s on day 0; if the value of C r 51 c o u n t s was higher, 10TM c o u n t s decreased w i t h time. All lines were fitted subjectively. O n c e t h e value of C P ] for day 0 was estimated, b r a i n red cell v o l u m e was calculated using e q u a t i o n (3). Similarly calculation of b r a i n plasma v o l u m e u s i n g e q u a t i o n (1) was based u p o n t h e e s t i m a t e d c o u n t s of I TM. Brain blood v o l u m e was calculated as t h e s u m of b r a i n p l a s m a a n d red cell volume.
RESULTS
Brain wet weights as shown in Table I range from 68 mg in the sea lamprey over 2000 mg in the pigeon (Columba sp.). For species comparisons all volumes are expressed per gram of brain wet weight. Brain blood
(Petromyzon marinus) to
BRAIN
AND
CHOROID
PLEXUS
BLOOD
493
VOLUMES
0 ,-4 0 r~ 0 0 e~ Z
Hg'+~
i
+l~_+l~+lg'+l~"
¢'q
¢q
eq
c-~
I'~
¢'q
0o
~
~
eq
~1"
o
,4 ©
"N
<
<
+ l ~,-~ . H ~'~ ~
< M
+1 -,--4
cq ~-.
.t3 ba
t'l
[..., =
~2 z
I ,.4
t~ o~
*Z;
494
S. RIC:HARDHEISEY
volumes determined by the plasma-hematocrit method (RIHSA), red cell-hematocrit method (Cr ~I) and double-label method ( R I I t S A + Cr ~1) show that the phylogenetically lower animals have larger brain blood volumes relative to brain weight than do mammals and birds. The plasma hematocrit method was the only one used in all species and therefore the only one in which a complete comparison is possible. This method shows that the pigeon and rat have a brain blood volume approximately 2.5 per cent of brain weight. The turtle, frog, and goldfish have brain blood volumes approximately 3-3.5 per cent of brain weight. The mudpuppy and the sea lamprey have the largest brain blood volumes (approximately 4"5 per cent of brain weight). Brain plasma volume was also smaller in the mammal and bird than in the lower vertebrates, as was brain red cell volume (with the exception of the goldfish). Sjostrand (1953) states that the cell dilution method results in an underestimation whereas plasma dilution methods lead to an overestimation of total blood volume. In the rat, the plasma-hematocrit method resulted in a larger brain blood volume than the red cell-hematocrit method, supporting Sjostrand's suggestion; however, brain blood volume of Necturus was larger using the red cellhematocrit method (Table 1). With the exception of the double-label experiment in the turtle no attempt was made to prevent blood draining from the large superficial vessels on the brain surface. After the peripheral blood sample was obtained, the turtle neck was tied and severed between double ligatures. When the skull was removed, many large
T A B L E 2 - - H E M A T O C R I T S OF BRAIN AND LARGE SYSTEMIC VESSEL SAMPLES
Animal Rat Pigeon Turtle Frog Necturus
Goldfish Lamprey
Brain Hematocrit (% ± s.e.m.) 31 '0 ± 2.9 (5) 33.3 (2) 17-3 + 3"0* (4) 29.6 +_4.0 (9) -19.8 ± 3.1 (3) --
Peripheral Hematocrit ('~, _ s.e.m.) 43.0 _+_1 '4 (16) 44-6 + 2.3 (8) 24.8 _+_1.2 (27) 25-8 -5 1'1 (30) 20"7 ± I "6 (8) 25.0 ± 2.4 (10) 28.1 _+_2.2 (14)
* Ligatures tied around neck prior to removing, brain. Number of animals is shown in parentheses.
BRAIN
AND
CHOROID
PLEXUS
BI.OOD
495
VOLUMES
F. em
~',
oO
t",l
t"q
:e
o "O ¢#
t,-
0 0 0 Z
<
"~ ~
¢q
~
~,
.,¢. ~
,~-
.,,¢q
~-" tr~ "-," ~ .
~-~
r_; M Ill
,.-i' o
¢,q tt~
t"-
+1 ~" ",O " - " ¢q
o o ,-1 Z
r, Z t",l
-,Oo~ ,~. M 0
eq
¢q
,~. "-~ Ox " - ¢ e,~ ",--" ¢q
+1 "~" t~ v
,d
ea C 0 M
¢0 to3
t~ O
M M t~
o~ o~ o3
0 :11
e-
I M
t~
Z
496
S. RICHARD HEISEY
brain surface vessels were observed to be filled with blood resulting in large calculated brain blood volumes. This was attributable to a greater RIHSA (i.e., plasma) volume. The double-label method provides a means of estimating hematocrits in the brain. A comparison of hematocrits of samples from large peripheral vessels with that of blood in brain vessels is reported in Table 2; except for the frog the latter measurement was always less than the former. These differences are not as great as those reported by Gibson et al. (1946) for the dog. Their methods were designed specifically to measure hematocrit in very small vessels, whereas in the present study, a varying contribution came from larger brain vessels. However, when large brain vessels of the turtle were included (Tables 1 and 2), the brain hematocrit was still less than that of peripheral samples. The value for rat brain hematocrit agrees with that reported by Everett et al. (1956) who used RIHSA and Fe s9 labeled red cells. The wet weight of the choroid plexuses of the rat brain is 0.2 per cent of the brain weight (Tables 1 and 3). In the frog, which has no choroid plexuses in the lateral ventricles, the plexus overlying the fourth ventricle is 3 per cent of the brain weight. The lamprey eel has a huge choroid plexus which lies over the caudal twothirds of the brain and accounts for 8 per cent of the brain weight. Although in the rat only 4 per cent of the brain blood volume is found in the choroid plexuses, this structure of the lamprey eel contains almost 60 per cent of the brain blood volume. Even when the amount of blood contained in the choroid plexuses is subtracted from the total brain blood volume, the lower vertebrate brain blood volume is generally greater than that of the mammal. This correction has the greatest effect on the brain blood volume of the sea lamprey (compare Table 1 and "Fable 3); in this species brain blood volume excluding choroid plexus blood volume is not significantly different from mammalian brain blood volume. DISCUSSION The observation that brain blood volume of lower vertebrates was larger than for mammals was unexpected and conflicts with results of Craigie's anatomical studies (Craigie, 1938). Based upon observed capillary densities in different brain regions, his data imply that brain vascularity increases with a phylogenetic progression from fish to mammals (although all vertebrate classes were not studied). The one exception to this trend in Craigie's study was in Necturus, where the volume of capillaries per unit volume of brain tissue was greater than in any other species studied by him (Craigie, 1940). The present study confirms Craigie's observation on Necturus whose brain blood volume exceeds not only that of Rana catesbeiana but also that of other vertebrate class representatives except Agnatha. A quantitative assessment of vascularity in Petromyzon marinus has not been made previously, but the occurrence of loop-type capillaries (as opposed to a meshtype circulation) has been observed (Craigie, 1938; Wislocki & Campbell, 1937). In the present study, the two animals which have loop-type brain capillaries
B R A I N A N D C H O R O I D PLEXUS B L O O D V O L U M E S
497
(Petromyzon marinus and Necturus maculosus) also have the largest brain blood volumes. Petromyzon marinus, however, appears to have most of its brain blood volume contained within the choroid plexus. The choroid plexuses of the lower vertebrates are large and more conspicuous than those of the mammal or bird. This is reflected in the large percentage of brain blood volume found in the choroid plexuses (Table 3). No choroid plexuses were found in the lateral ventricles of the frog (Rana catesbeiana), however, this structure covering the fourth ventricle contained 35 per cent of the total brain blood volume. The choroid plexus of Petromyzon marinus is large and covers the dorsum of the caudal two thirds of the brain ; its size and vascularity are reflected in the measured blood volume which accounts for approximately 60 per cent of the whole brain blood volume. No sequential change in choroid plexus weight or blood volume was found, just as no predictable changes were found in total brain blood volume through vertebrate classes. In the lower vertebrates the size of the ventricles relative to brain size is bigger than in birds or mammals. Herrick's observation that in lower vertebrates a thin strip of tissue surrounds the lateral ventricles, prompted him to propose that cerebrospinal fluid may have a nutrient function and serve as a source of oxygen, collateral to that of blood (Herrick, 1921). From ventricular size in an amphibian or reptile, one surmises that cerebrospinal fluid volume per gram brain weight is larger than in a mammal. Cerebrospinal fluid volume was not measured in this study, but it has been possible to withdraw 0.8 ml of fluid from the fourth ventricle of an 800 mg turtle brain. '['he cerebrospinal fluid volume contained in the cranium of a goat is approximately 25 per cent of the brain weight (Pappenheimer et al., 1962), while CSF volume is about 9 per cent of the total intracranial contents in the dog (Rosomoff, 1961). From these observations, it would appear that the cerebrospinal fluid volume, as well as the blood volume of lower vertebrate brains is larger than in mammals. The results of the present study indicate (1) that brain blood volume per unit brain weight is the same in birds and mammals, (2) that reptiles, amphibians and fish have a slightly larger brain blood volume per unit brain weight than that of mammals and (3) that in lower vertebrates, the choroid plexus blood volume makes up a larger percentage of total brain blood volume than in mammals. From preliminary observations it appears that the lower vertebrates have a larger cerebrospinal fluid volume per unit brain weight than does the mammal.
Acknowledgements--I wish to thank Mrs. Susan Lautenbacher for valuable technical assistance and Dr. J. R. Pappenheimer for helpful criticisms and suggestions during the course of the experimental work. I am indebted to Drs. Thomas Adams and P. O. Fromm for helpful suggestions with the manuscript. REFERENCES ARIENS-KAPPERSC. U., HUBERG. C. & CROSBYE. C. (1960) The Comparative Anatomy of the Nervous System of Vertebrates Including Man, p. 55. Hafner, New York.
498
S. RICHARD HEISEY
BAKER C. H. (1963) Comparison of blood volume in the dog with Cr ~J, II~l-fibrinogen and T-1824. Am.ft. Physiol. 204, 176-180. CRAGIE E. H. (1938) The comparative anatomy and embryology of the capillary bed of the central nervous system. Assoc. Res. nerv. ment. Dis. 18, 3-28. CRAIGIE E. H. (1940) Vascularity in the brains of tailed amphibians--I I. Necturus maculosus Rafiniesque. Proc. Am. Phil. Soc. 82, 395--410. DAVSON H. 8r SPAZlANI E. (1959) The blood-brain barrier and the extracellular space of brain, ft. Physiol. 149, 135-143. EVERETT N. B. &; SIMMONS B. (1958) Measurement and radioautographic localization of albumin in rat tissues after intravenous administration. Circ. Res. 6, 307-313. EVERETT N. B., SIMMONS B. • LAStlER E. P. (1956) Distribution of blood (Fe sg) and plasma (I TM)volumes of rats determined by liquid nitrogen freezing. Circ. Res. 4, 419-424. GIBSON J. G., SELIGMANA. M., PEACOCK~,~,:. C., AUB J. C., FINE J. & EVANS R. D. (1946) The distribution of red cells and plasma in large and minute vessels of the normal dog, determined by radioactive isotopes of iron and iodine, ft. Clin. Invest. 25, 848-857. HERRICK C. J. (1921) A sketch of the origin of the cerebral hemispheres..)'. Comp. Neurol. 32, 429-454. PAI'PENHEIMERJ. R., HEISEY S. R., JORDAN E. F. & DOWNER J. DEC. (1962) Perfusion of the cerebral ventricular system in unanesthetized goats. Am.ft. Physiol. 203, 763-774. READ R. C. (1954) Red cell volume measurements. New Engl.ff. 31ed. 250, 1021-1027. ROSO.'VIOFFH. L. (1961) Method for simultaneous quantitative estimation of intracranial contents, ft. Appl. Physiol. 16, 395-396. SJOSTRANB T. (1953) Volume and distribution of blood and their significance in regulating the circulation. Physiol. Rev. 33, 202-228. STREICI-tERE. (1961) Thyocyanate space of rat brain. Am.ft. Physiol. 201,334-336. WISLOCKI G. B. & CAMPBELLA. C. P. (1937) The unusual manner of vascularization of the brain of the opossum (Didelphys virginiana). Anat. Rec. 67, 177-191.
BRAIN
AND
CHOROID PLEXUS BLOOD IN VERTEBRATES*
VOLUMES
S. RICHARD HEISEY t Department of Physiology, Harvard Medical School, Boston, Mass.
A b s t r a c t - - 1 . Brain blood volume per unit brain weight is larger in a cyclostome, teleost, amphibian and reptile than in a bird or mammal. Necturus maculosus and Petromyzon marinus had brain blood volumes approximately 4-5 per cent of brain weight. 2. The hematocrit of blood in the brain was usually less than that of blood drawn from a large peripheral vessel. 3. Choroid plexus blood volume in the rat is 4 per cent of total brain blood volume while in lower vertebrates, choroid plexus blood volume makes up a larger percentage of whole brain blood volume. 4. In Petromyzon marinus, the choroid plexus blood volume is 57 per cent of whole brain blood volume. INTRODUCTION
BOTH studies of brain fluid compartments and of distribution of substances between blood and brain require estimates of brain blood volume. Rosomoff (1961), Davson & Spaziani (1959), and Streicher (1961) using dogs, rabbits and rats, respectively, determined brain blood volumes using either labeled plasma proteins or red blood cells. In a study of organ blood volumes in the dog, Gibson et al. (1946) measured brain blood volume using both a plasma and a red cell label, enabling them to measure, in addition to blood volume, hematocrit in the brain. Such studies indicate that mammalian brain blood volume is approximately 2 per cent of brain wet weight. Neither brain blood volume nor cerebrospinal fluid volume has been measured directly in vertebrates other than mammals. Histological studies, however, suggest that lower vertebrates might have a smaller brain blood volume per gram brain weight than mammals, and that brain blood-cerebrospinal fluid volume ratios might be progressively smaller along the phylogenetic continuum from mammals to fish. Supporting these suggestions, Ariens-Kappers et al. (1960) reported that in Amphioxus the brain parenchyma is avascular. Craigie (1938) noted a paucity of capillary, loops in the brain of a primitive vertebrate, Petrorayzon marinus. Herrick * This investigation was supported by PHS Research Grant No. NB05412 and Research Career Program Award No. 5K03NB07124 from the National Institute of Neurological Diseases and Blindness. t Present Address : Department of Physiology, Michigan State University, East Lansing, Michigan 48823. 489
490
S. RICHARD HEISEY
(1921) was impressed with the large v e n t r i c u l a r cavities a n d choroid plexuses of some of the lower fish a n d a m p h i b i a n s ; he suggested that in some lower vertebrates, the b r a i n m a y be s u p p l i e d with oxygen from blood and in a d d i t i o n m a y receive a collateral s u p p l y of oxygen from the cerebrospinal fluid. I n the p r e s e n t study, b r a i n blood v o l u m e s were m e a s u r e d in representatives of each of the five vertebrate classes. METHODS The white rat (Rattus, sp.), street pigeon (Columba, sp.), fresh water turtle (Pseudemys scripta elegans), bullfrog (Rana catesbeiana), mudpuppy (Necturus maculosus) and goldfish (Carassius auratus) were obtained from commercial sources. The sea lampreys (Petromy~on marinus), obtained from a commercial fisherman who retrieved them from fish traps in the Exeter River near Exeter, New Hampshire, were transported to the laboratory in ice water, and were used on the day of trapping. Rats were anesthetized either with Nembutal (60 mg/kg IP) or with ether; pigeons, Nembutal (6-12 mg IV); turtles, Nembutal (66 mg/kg) or Avertin (250 mg/kg) via an esophageal tube; frogs, immersed in 0.1 ~o tricaine methanesulfonate (MS 222 Sandoz), supplemented with 10 cc of 3°0 urethane injected into the dorsal lymph sac; mudpuppies, goldfish and lampreys were anesthetized with 0'01°o tricaine methanesulfonate in water; lampreys were packed with ice. An isotope dilution method was used for both the red cell and plasma volume measurements. Iodinated human serum albumin labeled with 1131 (RIHSA-Cambridge Nuclear Company) was used to measure plasma volume. The albumin preparations had specific activities of 25-60 t~c/mg with less than 2 per cent of the 113~unbound to protein. Cr sl in the form of Na2Cr~lO4 was used to label red ceils in vitro. Radioactive material was injected at a different site from where peripheral blood samples were drawn. In most animals the radioactive material was injected intravenously and blood samples withdrawn either directly from the heart or from a large artery close to the heart. After injection 5-10 rain were allowed for vascular mixing. A peripheral blood sample was withdrawn and the animal decapitated. The brain was removed, severed from the spinal cord at the level of the obex, blotted and weighed in a counting vial; in some experiments the choroid plexuses were removed and weighed in separate vials. In experiments using either RIHSA or Cr 5~labeled cells, the peripheral blood sample was centrifuged and an aliquot of either plasma or packed red cells was counted ; in some experiments, an aliquot of whole blood was counted, tIematocrits, used for converting either plasma volume (RIHSA) or red cell volume (Cr 51) to blood volume, were determined using an IEC Micro-capillary centrifuge, Model MB. When both R I H S A and Cr 51 labeled red cells were used, the peripheral blood sample was drawn, centrifuged and an aliquot of plasma was counted for RIHSA activity. The cells were washed three times; after the final wash an aliquot of packed red cells was counted for Cr sl activity. All samples of brain and blood were put in 4 ml glass screw cap vials which fit into a crystal scintillation well detector (Baird Atomic Model 810A) and were counted integrally at a discriminator setting of 200 keV. Na2Cr~lO4 was added to heparinized whole blood from a donor animal (20-50 p.c/ml blood) and the mixture allowed to incubate, with occasional mixing, for 1 hr. For rats and pigeons the incubation occurred at room temperature but in the other species at 4°C. Ascorbic acid was added according to the procedure of Read (1954). The blood was centrifuged at 770g for 10 min and the supernatant plasma removed and replaced with 5 ml of cold 0'9~o NaC1 (rats and pigeons) or amphibian Ringer's solution (all other species). The cells were resuspended, recentrifuged and washed twice. Three washes were usually sufficient to bring the tracer activity in the final wash to less than 1 per cent of the activity bound to red blood cells. On the third wash, the tubes were centrifuged at 770g for 10 rain and
BRAIN
AND
CHOROID
PLEXUS
BLOOD
VOLUMES
491
1700g for five min. The packed cells were drawn into a plastic 1-ml syringe for injection into the experimental animal. The hematocrit of the packed red cells was 90-95 per cent.
Calculations a. Single-label experiments. When the plasma-hematocrit method was used, brain blood volume was calculated from the following equations P, IHSA activity/g brain Plasma volume/g brain -- RIHSA activity/ml plasma
(1)
Plasma volume/g brain Blood volume/g b r a i n - - (1-peripheral hematocrit)
(2)
When the red cell-hematocrit method was used, the corresponding equations were Cr sl activity/g brain Red cell volume/g brain -- (~rSt-activity/m1 packed ce~l-ls Blood volume/g brain --
Red cell volume/g brain peripheral hematocrit
(3) (4)
b. Double-label experiments. When RIHSA and Cr ~1 labeled red cells were injected into the same animal, the activity of the brain due to 11~' was separated from that due to Cr 5' by considering that the half life of 1131 is 8 days while that of Cr ~l is 28 days. The amount of activity due to each isotope was determined by solving the following simultaneous equations (Brain counts-bkgd)0 -- Cr051+ I0TM (Brain counts-bkgd) z = klCro 51+ k210TM where kl = exp-[hCr(t~-t0)] ku = exp-[hI(tx-to) ] to -- experimental day t~ -- any day after experimental day hCr = 0'693/27'8 hi = 0'693/8"05 Baker (1963) using a double-label technique to measure blood volume in the dog chose day 0 and day 10 counts to distinguish Cr s* from I TM. In the present study, preliminary experiments showed that brain and blood samples counted on successive days yield slightly different values for I0 TM and Cr0 sl activity and consequently different values for brain plasma and red cell volumes. Therefore, all brain samples were counted several times for up to 25 days with a graphic solution of the simultaneous equations presenting more reliable values of I0131 and Cr051 activity. Such an experiment is shown in Fig. 1. Brain activity was counted seven times, from day 0 to day 15. Brain counts (corrected for background) from days 0, 3, 7, 11, 14 and 15 were substituted in the simultaneous equations and different paired brain counts yielded different values for I0TM and Cr0SL By trial and error one value of Cr03~ was found which, in combination with brain counts and decay constants for all days, yielded a relatively constant value of I0TM (i.e. I0TM counts plotted as a function of days after the experiment scattered about a line with 0 slope). In the experiment of Fig. 1, the estimated value for Cr0 nl was 2"43 x 103 counts/min and this yielded 2"48 × 103 counts/min for I0lsl. Using another set of paired brain counts, and solving the simultaneous equations, Cr0 ~x was 2.31 x 103 counts/min ; this yielded values of I0131which increased with time after the experiment (i.e. I013t counts plotted vs. time scattered about a line with a positive slope). In still another solution of the simultaneous equations, Cr051 was 2"60 x 10s counts/min and yielded I0aSx values which decreased with time after the experiment.
492
S. RICHARD HEISEY TURTLE
X =]'o O=
131
,when
No. C
17
51
ro = 2 3 1 X I O
3
CPM
IO TM ,when CroOI:2.45XIOSCPM
• = ~o
131
51
,when Cro =2.60XIO3CPM
131
]~o , X l O 3CPM
2.80
X
X 2.60
X o O-
-- _O._
O O
o
2.40
O
220
2.00
1.80 DAYS |
I
[
I
J
0
5
I0
15
20
FIG. 1. G r a p h i c solution of two s i m u l t a n e o u s equations w h i c h relate I TM to Cr sz c o u n t s in a turtle brain. W h e n 1 TM c o u n t s on day 0 (10 TM) are plotted as a f u n c t i o n of t i m e after the e x p e r i m e n t a l day, only one value of C r sl c o u n t s o n day 0 (Cr0 s]) yields a c o n s t a n t value for I0 TM counts. In this e x p e r i m e n t 2"43 x 103 c o u n t s / r a i n d u e to C P 1 yielded a line w i t h zero slope (a c o n s t a n t value o f I TM c o u n t s o n day 0). I f the value of C r 51 counts o n day 0 was chosen to b e less t h a n this value, the solution of t h e equations yielded increasing values for 1 TM c o u n t s on day 0; if the value of C r 51 c o u n t s was higher, 10TM c o u n t s decreased w i t h time. All lines were fitted subjectively. O n c e t h e value of C P ] for day 0 was estimated, b r a i n red cell v o l u m e was calculated using e q u a t i o n (3). Similarly calculation of b r a i n plasma v o l u m e u s i n g e q u a t i o n (1) was based u p o n t h e e s t i m a t e d c o u n t s of I TM. Brain blood v o l u m e was calculated as t h e s u m of b r a i n p l a s m a a n d red cell volume.
RESULTS
Brain wet weights as shown in Table I range from 68 mg in the sea lamprey over 2000 mg in the pigeon (Columba sp.). For species comparisons all volumes are expressed per gram of brain wet weight. Brain blood
(Petromyzon marinus) to
BRAIN
AND
CHOROID
PLEXUS
BLOOD
493
VOLUMES
0 ,-4 0 r~ 0 0 e~ Z
Hg'+~
i
+l~_+l~+lg'+l~"
¢'q
¢q
eq
c-~
I'~
¢'q
0o
~
~
eq
~1"
o
,4 ©
"N
<
<
+ l ~,-~ . H ~'~ ~
< M
+1 -,--4
cq ~-.
.t3 ba
t'l
[..., =
~2 z
I ,.4
t~ o~
*Z;
494
S. RIC:HARDHEISEY
volumes determined by the plasma-hematocrit method (RIHSA), red cell-hematocrit method (Cr ~I) and double-label method ( R I I t S A + Cr ~1) show that the phylogenetically lower animals have larger brain blood volumes relative to brain weight than do mammals and birds. The plasma hematocrit method was the only one used in all species and therefore the only one in which a complete comparison is possible. This method shows that the pigeon and rat have a brain blood volume approximately 2.5 per cent of brain weight. The turtle, frog, and goldfish have brain blood volumes approximately 3-3.5 per cent of brain weight. The mudpuppy and the sea lamprey have the largest brain blood volumes (approximately 4"5 per cent of brain weight). Brain plasma volume was also smaller in the mammal and bird than in the lower vertebrates, as was brain red cell volume (with the exception of the goldfish). Sjostrand (1953) states that the cell dilution method results in an underestimation whereas plasma dilution methods lead to an overestimation of total blood volume. In the rat, the plasma-hematocrit method resulted in a larger brain blood volume than the red cell-hematocrit method, supporting Sjostrand's suggestion; however, brain blood volume of Necturus was larger using the red cellhematocrit method (Table 1). With the exception of the double-label experiment in the turtle no attempt was made to prevent blood draining from the large superficial vessels on the brain surface. After the peripheral blood sample was obtained, the turtle neck was tied and severed between double ligatures. When the skull was removed, many large
T A B L E 2 - - H E M A T O C R I T S OF BRAIN AND LARGE SYSTEMIC VESSEL SAMPLES
Animal Rat Pigeon Turtle Frog Necturus
Goldfish Lamprey
Brain Hematocrit (% ± s.e.m.) 31 '0 ± 2.9 (5) 33.3 (2) 17-3 + 3"0* (4) 29.6 +_4.0 (9) -19.8 ± 3.1 (3) --
Peripheral Hematocrit ('~, _ s.e.m.) 43.0 _+_1 '4 (16) 44-6 + 2.3 (8) 24.8 _+_1.2 (27) 25-8 -5 1'1 (30) 20"7 ± I "6 (8) 25.0 ± 2.4 (10) 28.1 _+_2.2 (14)
* Ligatures tied around neck prior to removing, brain. Number of animals is shown in parentheses.
BRAIN
AND
CHOROID
PLEXUS
BI.OOD
495
VOLUMES
F. em
~',
oO
t",l
t"q
:e
o "O ¢#
t,-
0 0 0 Z
<
"~ ~
¢q
~
~,
.,¢. ~
,~-
.,,¢q
~-" tr~ "-," ~ .
~-~
r_; M Ill
,.-i' o
¢,q tt~
t"-
+1 ~" ",O " - " ¢q
o o ,-1 Z
r, Z t",l
-,Oo~ ,~. M 0
eq
¢q
,~. "-~ Ox " - ¢ e,~ ",--" ¢q
+1 "~" t~ v
,d
ea C 0 M
¢0 to3
t~ O
M M t~
o~ o~ o3
0 :11
e-
I M
t~
Z
496
S. RICHARD HEISEY
brain surface vessels were observed to be filled with blood resulting in large calculated brain blood volumes. This was attributable to a greater RIHSA (i.e., plasma) volume. The double-label method provides a means of estimating hematocrits in the brain. A comparison of hematocrits of samples from large peripheral vessels with that of blood in brain vessels is reported in Table 2; except for the frog the latter measurement was always less than the former. These differences are not as great as those reported by Gibson et al. (1946) for the dog. Their methods were designed specifically to measure hematocrit in very small vessels, whereas in the present study, a varying contribution came from larger brain vessels. However, when large brain vessels of the turtle were included (Tables 1 and 2), the brain hematocrit was still less than that of peripheral samples. The value for rat brain hematocrit agrees with that reported by Everett et al. (1956) who used RIHSA and Fe s9 labeled red cells. The wet weight of the choroid plexuses of the rat brain is 0.2 per cent of the brain weight (Tables 1 and 3). In the frog, which has no choroid plexuses in the lateral ventricles, the plexus overlying the fourth ventricle is 3 per cent of the brain weight. The lamprey eel has a huge choroid plexus which lies over the caudal twothirds of the brain and accounts for 8 per cent of the brain weight. Although in the rat only 4 per cent of the brain blood volume is found in the choroid plexuses, this structure of the lamprey eel contains almost 60 per cent of the brain blood volume. Even when the amount of blood contained in the choroid plexuses is subtracted from the total brain blood volume, the lower vertebrate brain blood volume is generally greater than that of the mammal. This correction has the greatest effect on the brain blood volume of the sea lamprey (compare Table 1 and "Fable 3); in this species brain blood volume excluding choroid plexus blood volume is not significantly different from mammalian brain blood volume. DISCUSSION The observation that brain blood volume of lower vertebrates was larger than for mammals was unexpected and conflicts with results of Craigie's anatomical studies (Craigie, 1938). Based upon observed capillary densities in different brain regions, his data imply that brain vascularity increases with a phylogenetic progression from fish to mammals (although all vertebrate classes were not studied). The one exception to this trend in Craigie's study was in Necturus, where the volume of capillaries per unit volume of brain tissue was greater than in any other species studied by him (Craigie, 1940). The present study confirms Craigie's observation on Necturus whose brain blood volume exceeds not only that of Rana catesbeiana but also that of other vertebrate class representatives except Agnatha. A quantitative assessment of vascularity in Petromyzon marinus has not been made previously, but the occurrence of loop-type capillaries (as opposed to a meshtype circulation) has been observed (Craigie, 1938; Wislocki & Campbell, 1937). In the present study, the two animals which have loop-type brain capillaries
B R A I N A N D C H O R O I D PLEXUS B L O O D V O L U M E S
497
(Petromyzon marinus and Necturus maculosus) also have the largest brain blood volumes. Petromyzon marinus, however, appears to have most of its brain blood volume contained within the choroid plexus. The choroid plexuses of the lower vertebrates are large and more conspicuous than those of the mammal or bird. This is reflected in the large percentage of brain blood volume found in the choroid plexuses (Table 3). No choroid plexuses were found in the lateral ventricles of the frog (Rana catesbeiana), however, this structure covering the fourth ventricle contained 35 per cent of the total brain blood volume. The choroid plexus of Petromyzon marinus is large and covers the dorsum of the caudal two thirds of the brain ; its size and vascularity are reflected in the measured blood volume which accounts for approximately 60 per cent of the whole brain blood volume. No sequential change in choroid plexus weight or blood volume was found, just as no predictable changes were found in total brain blood volume through vertebrate classes. In the lower vertebrates the size of the ventricles relative to brain size is bigger than in birds or mammals. Herrick's observation that in lower vertebrates a thin strip of tissue surrounds the lateral ventricles, prompted him to propose that cerebrospinal fluid may have a nutrient function and serve as a source of oxygen, collateral to that of blood (Herrick, 1921). From ventricular size in an amphibian or reptile, one surmises that cerebrospinal fluid volume per gram brain weight is larger than in a mammal. Cerebrospinal fluid volume was not measured in this study, but it has been possible to withdraw 0.8 ml of fluid from the fourth ventricle of an 800 mg turtle brain. '['he cerebrospinal fluid volume contained in the cranium of a goat is approximately 25 per cent of the brain weight (Pappenheimer et al., 1962), while CSF volume is about 9 per cent of the total intracranial contents in the dog (Rosomoff, 1961). From these observations, it would appear that the cerebrospinal fluid volume, as well as the blood volume of lower vertebrate brains is larger than in mammals. The results of the present study indicate (1) that brain blood volume per unit brain weight is the same in birds and mammals, (2) that reptiles, amphibians and fish have a slightly larger brain blood volume per unit brain weight than that of mammals and (3) that in lower vertebrates, the choroid plexus blood volume makes up a larger percentage of total brain blood volume than in mammals. From preliminary observations it appears that the lower vertebrates have a larger cerebrospinal fluid volume per unit brain weight than does the mammal.
Acknowledgements--I wish to thank Mrs. Susan Lautenbacher for valuable technical assistance and Dr. J. R. Pappenheimer for helpful criticisms and suggestions during the course of the experimental work. I am indebted to Drs. Thomas Adams and P. O. Fromm for helpful suggestions with the manuscript. REFERENCES ARIENS-KAPPERSC. U., HUBERG. C. & CROSBYE. C. (1960) The Comparative Anatomy of the Nervous System of Vertebrates Including Man, p. 55. Hafner, New York.
498
S. RICHARD HEISEY
BAKER C. H. (1963) Comparison of blood volume in the dog with Cr ~J, II~l-fibrinogen and T-1824. Am.ft. Physiol. 204, 176-180. CRAGIE E. H. (1938) The comparative anatomy and embryology of the capillary bed of the central nervous system. Assoc. Res. nerv. ment. Dis. 18, 3-28. CRAIGIE E. H. (1940) Vascularity in the brains of tailed amphibians--I I. Necturus maculosus Rafiniesque. Proc. Am. Phil. Soc. 82, 395--410. DAVSON H. 8r SPAZlANI E. (1959) The blood-brain barrier and the extracellular space of brain, ft. Physiol. 149, 135-143. EVERETT N. B. &; SIMMONS B. (1958) Measurement and radioautographic localization of albumin in rat tissues after intravenous administration. Circ. Res. 6, 307-313. EVERETT N. B., SIMMONS B. • LAStlER E. P. (1956) Distribution of blood (Fe sg) and plasma (I TM)volumes of rats determined by liquid nitrogen freezing. Circ. Res. 4, 419-424. GIBSON J. G., SELIGMANA. M., PEACOCK~,~,:. C., AUB J. C., FINE J. & EVANS R. D. (1946) The distribution of red cells and plasma in large and minute vessels of the normal dog, determined by radioactive isotopes of iron and iodine, ft. Clin. Invest. 25, 848-857. HERRICK C. J. (1921) A sketch of the origin of the cerebral hemispheres..)'. Comp. Neurol. 32, 429-454. PAI'PENHEIMERJ. R., HEISEY S. R., JORDAN E. F. & DOWNER J. DEC. (1962) Perfusion of the cerebral ventricular system in unanesthetized goats. Am.ft. Physiol. 203, 763-774. READ R. C. (1954) Red cell volume measurements. New Engl.ff. 31ed. 250, 1021-1027. ROSO.'VIOFFH. L. (1961) Method for simultaneous quantitative estimation of intracranial contents, ft. Appl. Physiol. 16, 395-396. SJOSTRANB T. (1953) Volume and distribution of blood and their significance in regulating the circulation. Physiol. Rev. 33, 202-228. STREICI-tERE. (1961) Thyocyanate space of rat brain. Am.ft. Physiol. 201,334-336. WISLOCKI G. B. & CAMPBELLA. C. P. (1937) The unusual manner of vascularization of the brain of the opossum (Didelphys virginiana). Anat. Rec. 67, 177-191.