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Autoradiographic study of nucleic acid precursor incorporation in nerve cells of the mouse brain
.Yt~roi~ncc I977Vol 2.pp.10771084PergamonPress. Prmed
in GreatBritain.
AUTORADIOGRAPHIC STUDY OF NUCLEIC ACID PRECURSOR INCORPORATION IN NERVE CELLS OF THE MOUSE BRAIN H. PAKKENBERG and R. FOG Laboratory of Neurology, Hvidovre Hospital, 2650 Hvidovre, Denmark Abstract-Following intravenous administration of five tritiated nucleic acid bases and five nucleosides in mice the intensity of labelling of the cerebral cortex cells and the liver cells show the following pattern. Cytidine and uridine cauSe intense labelling; uracil, adenine, adenosine, guanosine, guanine, thymine and cytosine only a weak labelling. The choroid plexus is labelled intensely by cytidine and uridine, moderately by guanosine and adenosine. weakly by other substances. In vitro experiments and intracerebral injection with uridine, uracil, adenine and adenosine give the same labelling pattern as intravenous injection. Inhalation of CO, does not alter the labelling of nerve cells apart from that due to the uridine group. where a weaker labelling of the nerve cells is observed. It is concluded that, in the case of the nucleic acid precursors used here, metabolic specificities are of greater significance for the labelling pattern of the nerve cells than the transport specificities of the blood-brain barrier.
[5-3H]uridine (spec. act. 25 Ci/mmol), [I-‘Hlguanine sulphate (spec. act. 500 mCi/mmol), [8-“Hlguanosine (spec. well as into liver cells (PAKKESBERG& FOG, 1972, act. 16 Ci/mmol), [6-3H]thymine (spec. act. 26 Ci/mmol). 1973) following intravenous and intraperitoneal injec- [6-3H)thymidine (spec. act. 23 Ci/mmol), [5-3H]cytidine (spec. act. 29 Ci/mmol. [3H]cystosine was not commertion. A number of factors are still not clear regarding cially available, but 5-iodocytosine was tritiated by catathe passage of nucleic acid precursors through the lytic hydrogenation and purified by The Radiochemical blood-brain barrier and their utilization in the nuc- Centre, Amersham. A total of sixteen animals were used leic acid metabolism of the nerve cells (BASKIN, for each substance. Under chloroform anaesthesia they MA~IARZ& AGRANOFF, 1972; F~URGET & TREMBLAY, were killed after 1, 6 and 24 h or 1I days, four animals 1972; GEIGER & YAMASAKI,1956; HOGANS,GUROFF in each group. They were immediately perfused through & UDENFRIEND,1971). C&NFORD & OLDENDORF the heart with Lillie’s fluid (formalin 4%) after the right (1975) found by the Oldendorf technique that out of atrium had been opened. The histological and autoradiographic procedures used seventeen compounds examined, measurable, saturwere the same as explained in an earlier paper (PAKKENable uptakes were established for adenine, adenosine, BERG & FOG, 1973). The dipping method with Ilford guanosine and uridine. One transport system for Nuclear Research Emulsion K 5 was used. The exposure adenine and one for adenosine, guanosine, inosine time (at 4°C) had to be varied, as otherwise the number of and uridine were demonstrated. grains would be too small in some of the preparations. The present study examines the incorporation of In the case of uridine and cytidine, 5 days’ exposure was a series of nucleic acid precursors following intraused for choroid plexus cells and liver sections, IO days’ venous injection, in vitro uptake, intracerebral injec- exposure was used for nerve cells. In the case of the other tion and CO, inhalation. This might indicate if the substances, 14 days’ exposure was used for plexus cells barrier function or other factors (precursor pool size, and liver sections, 42 days for nerve cells. However. all synthesis rate. rate of metabolism of precursor to grain counts were corrected for these differences before other compounds) are of the greatest significance for drawing the curves. The grain count over the nuclei and cytoplasm of the various cell types was also corrected to the incorporation pattern. an area of 1OOfirn’(corresponding approximately to the area of the nuclei of the plexus cells). The counting was MATERIALS AND METHODS performed over hepatocytes, great nerve cells in the fifth layer of the cerebral cortex and over the epithelial cells Intravenous injection of precursors of the choroid plexus. A total of 160 white male mice were used, weight 25-27 g. An amount of 0.25 mCi of the radioactive substance (determined from the specification supplied by the In~racerehral injection In a total of forty white mice. weight 25-27g, a guide producers) was dissolved in 0.25 ml distilled water and injected into the tail vein. The following substances were cannula was inserted in the skull. so that a thinner needle could be passed through this guide cannula and the needle used (The Radiochemical Centre, Amersham, England): tip placed in the right corpus striatum. Twenty-four hours [8-3H]adenine (spec. act. 21 Ci/mmol), [2-3H]adenosine later one microliter (Hamilton syringe) of one of the fol(spec. act. 26 Ci/mmol, [6-3H]uracil (spec. act. 24 Ci/mmol, WF. HAVE previously poration of uridine
studied the kinetics of the incorinto nerve and plexus cells, as
1077
1078
H. PAKKENBERG and R. FOG
lowing substances was injected: [5-‘Hluridine (spec. act. 29 Ci/mmol), [6-3H]uracil (spec. act. 21 Ci/mmol), [8-3H]adenine (spec. act. 26 Ci/mmol), [2-‘Hladenosine (spec. act. 21 Ci/mmol) (The Radiochemical Centre, Amersham. England). In sixteen animals 1PCi of uridine was injected, and in sixteen animals the same quantity of uracil, without anaesthesia. After intervals of 5 min, 30 min. 60 min or 4 h, four animals in each lot were killed with chloroform and perfused immediately with Lillie’s fluid through the left ventricle, as described above. Adenine or adenosine was injected in the same way into a total of eight mice, four with each substance. The animals were killed after 30min and perfused as described. Autoradiograms were made as described. The blocks were cut serially, the intention being to hit the tip of the injection track. The autoradiograms were exposed for 48 h. Grains were counted over ten nerve cell nuclei lying nearest to the tip of the injection track which showed the highest grain count. In vitro uptake Another technique used to by-pass the blood-brain barrier was to place slices of mouse brain from a decapitated mouse in modified Ringer’s solution (NaCl 120m~. NaHCO, 15 mM, KC1 5 mM, CaCI, (2H,O) 5 mM, MgSO, (7 H,O) 1 mM, glucose 5.106 mM). To this, 1OOpCi [S-“Hluridine or [6-3H]uracil or [8-3H]adenine or [Z-‘Hladenosine (spec. act. as given above) was added in 5 ml solution. The solution was maintained at 38°C in a water-bath, a gas mixture of O2 9S%, CO, 5%, being bubbled through. After 15 min the slices were removed and placed in 4”,‘, formalin for 3 days. Sections 4 pm in thickness were then cut and autoradiograms prepared as described above. The exposure time was 2 days.
substance, and four control animals were injected with the same nucleic acid precursor, but without CO* inhalation. Autoradiograms were prepared as described. The period of exposure in the case of uridine was 14 days, but three times longer (42 days) in the case of the other substances. Grains were counted over twenty-five nerve cells in the cortex and twenty-five nerve cells in the thalamus. In the same manner
the background
were determined.
RESULTS Intravenous
injection
of precursors
As the curves for the incorporation cytosine
are almost
of thymine and with the uracil curves,
identical
only those for the latter are shown (Fig. I). The guanine incorporation curves are like the adenine inURACIL
2*SE PN PC H
G ti 100 10-I
0
0
0
1
6
2L
URACIL G 100
26L h 2xSE NNy NCNP *
fl 10
NN-r
0
0
b
i
2L
6
COZ inhalation Preliminary trials were made with Evans’ blue. These will be described briefly. White mice were placed in a plastic box, capacity about 2l., and a mixture of 25% CO, and 75% 0, passed into the box at the rate of 3 I./min. 0.25 ml of the following mixture was injected i.v.: Evans’ blue ICQmg, albumin 5OOmg, physiological saline 10 ml. After about 30 min half the animals developed convulsions and died. The experiment was repeated with a gas mixture of 15% CO2 and 85% 0,. which resulted in the survival of the.animals. After injection of the Evans’ blue the animals were kept in the box under the above conditions for 1h, the gas mixture being supplied at the rate of 3 I./min. The animals were then killed as already described, and frozen sections prepared, 10pm in thickness. These were embedded in a glycerol-water mixture and examined under a fluorescence microscope fitted with an Osram HBO 200 lamp and ilters BG 38, BG 12, KP 790 and K 530. For the actual CO, experiment, white male mice were employed as above. The animals were placed in a plastic box of about 21. in volume. A gas mixture of 85% 0, and 15% CO, was passed into this after bubbling through water, at a rate of 3 I./min. After 15min, 0.25 mCi of the tritiated compounds mentioned earlier (uridine, uracil, adenine and adenosine) were injected i.v. The animals were then returned to the plastic box immediately and the 0,C02 mixture continued for 45 min. The animals were killed with chloroform and perfused as described earlier. Four animals were used for each
counts
26Lh
FIG. 1. ADENINE GE
2xSE
261
h
2XSE NN u NC +, NP Y B *
0
0
i
24
i FIG. 2.
2&h
1079
Nucleic acid precursor uptake
ADENOS I NE 2xSE PN PC w LN * LC 3 0* PN -
ADENOSINE G
‘v%
LOO
M
ZXSE NN 3 NC * NP * B”
NN -L.
0
6
I
264
21
h
0
FIG.
6
I
2L
26L h
3.
2xSE
PN--,
2500
.
PN PC LN LC I3
50
3 i *
2xSE NN NC )--i NP +t3+
IJRIDINE G 1600
vc
LO 1600
NN -
1
NC -
::::_::::
\ 900
30 9w
LN--,
LOO
20 -
LOO
PC-_, LC_
100
IO-
100
NP .
6-e.
.
B-e* 0
0
0
0
I
6
24
264
h
i
;
6
ZL
FIG. 4. FIGS 1-I. Number of grains covering 25 choroid plexus cells, liver cells and cortical nerve cells, 1 h to 264 h after i.v. injection of four different nucleic acid precursors. All values are corrected for differences in area, using the area of the choroid plexus cell nuclei as unit (IOO~m’). Four animals in each group. PN = plexus cell nuclei. PC = plexus ceil cytoplasm. LN = liver cell nuclei. LC = liver cell cytoplasm. NN = nerve cell nuclei. NP = neuropil. NC = nerve cell cytoplasm. B = background. SE = standard error of the mean. G = total grain count. fi is used for statistical reasons (PAKKEW BERG & FOG, 1973).
2&
h
H. PAKKEYNBERG and R. Foe
1080
TABLE I. INCORPORATION OF NUCLEIC ACIDPRECURSORS INTOCELL NCCLEI FOLLOWING INTRACER~W.AL INJL~W
n 5min
3
30min 60min 4h
I I
Uridine mean
3
13 32 24 26
range
,i
622 19-44 1435 1441
2 3 4 4
Uracil mean 2 3 3 4
range
n
1 ~3 l-9
2
Adenine mean 3
range
P!
I 7
4
Adenosinc mean
.!
range
I x
l-13 l-12
Following intracerebral injection of one pC of uridine, uracil, adenine or adenosine in mice the animals were killed with chloroform after 5, 30, 60 min or 4 h (uridine, uracil), the adenine- and adenosine-injected animals after 30 min. In autoradiograms (exposure time 2 days) the number of grains were counted in ten cell nuclei close to the end of the iniection track. The table shows the averaee number (mean) and range of grain number per nucleus. II. number of animals.
corporation curves
(Fig. 2) and the guanosine incorporation curves like those for adenosine incorporation (Fig. 3). Finally the cytidine incorporation curves are of the same shape as the uridine incorporation curve (Fig. 4). The incorporation curves for the five bases have certain features in common. The labelling of the nerve cells and liver cells is at a low level. In the case of the choroid plexus, it is moderate for adenine and guanine, lower for the other substances. The four nucleosides (as expected, no grains were found over the cells in the case of thymidine, apart from cells in division) can be grouped into two types: guanosine and adenosine show moderate incorporation into the choroid plexus, but the incorporation is low in liver cells and nerve cells. Uridine and cytidine are avidly taken up by the choroid plexus, and moderately by liver cells and nerve cells. lntracerehi-al injection of precursors The results are shown in Table 1. The labelling following uridine injection is significant; labelling by the other three substances is only about 10% of that of uridine. This corresponds to the findings upon intravenous injection. The nerve cells at the tip of the injection track are not morphologically changed, while shrunken cells are found along the track. In a few cases, some of the injected fluid has apparently penetrated into the ventricle, without this having affected the incorporation into the cells. Labelling of cells some few hundred pm from the tip of the injection track is minimal, and does not increase in those groups of animals living longest after the injection. Thus, it is unlikely that there is any significant diffusion or other form of transport of the injected substances. It may also be mentioned that the labelling of the cells 5 min after the injection is only about half the labelling after a period between 30 min and 4 h. This agrees with the observation that after intravenous injection, the maximum count is not found until a lapse of 30 min (PAKKENBERG& FOC, 1972). In vitro uptake of precursors
As shown in Figs 5-9, there is a marked uptake of uridine. while the other three substances are incor-
porated only to a very limited extent. A number of the cells in the preparations have been altered morphologically, in particular showing shrinkage, and these cells have a low uptake of uridine. Choroid plexus tissue occurs in some slices, and here the cells are moderately labelled, both with uridine and adenine. This is in agreement with the labelling found in the intravenous experiment. It might be mentioned that the labelling of cells in the margins of the slice is somewhat capricious, many of the cells being labelled in some regions, while few or none are labelled in other regions. No cells are labelled which are deeper than 200~ into the slice, presumably because of the presence of hypoxia here. EfSect of CO,-inhalation The injection of Evans’ blue + albumin produced pronounced fluorescence in the liver. kidneys and lungs, but in the brain fluorescence was seen only in the walls of a few vessels, not in the nerve cells. This was the case in both control animals and CO,-treated animals. The results of the experiments with nucleic acid bases and nucleosides are shown in Table 2. In the uridine-injected animals, the grain counts are seen to be significantly lower in the cortex and thalamus following CO, inhalation than when CO2 was not administered. In the case of the uracil, adenine and adenosine-injected animals, however, the grain counts showed no significant differences between CO2 administration and no CO2 administration.
DISCUSSION There was a considerable difference in the labelling of the three cell types studied, following intravenous administration of the radio-labelled nucleic acid bases and nucleosides. The method does not permit an evaluation of the manner in which the labelled substances are metabolized in the cells, but it is accepted that uridine participates in RNA synthesis, cytidine in RNA and DNA synthesis. Although the other sub stances give rise to only a slight labelling of nerve cells in the cortex, it is nevertheless obvious that this occurs with all the substances examined with the exception of thymidine.
FIGS 5-9. Autoradiograms from in uifro experiments. Slices of mouse brain in 5 ml modified Ringe solution with 100 PCi tritiated uridine (Fig. 5), adenine (Fig. 6). adenine, choroid plexus (Fig. 7), ader sine (Fig. 8) or uracil (Fig. 9) for 15 min. Exposure time 2 days.
1083
Nucleic acid precursor uptake TABLE2. EFFECTOF INHALATION OF CARBON DIOXIDE ONINCORPORA~ON OF NUCLEIC ACIDPRECURSORS FOLIoWINGINTRAVENOUS INJECTiON Uridine -co, + coz Cortex Nerve cell nuclei Nerve cell cytoplasm Neuropil Thalamus Nerve cell nuclei Nerve cell cytoplasm
Uracil -co, + co2
+co,
Adenine -co,
Adenosine -co, +co1
141
220*
51
63
12
18
40
30
10
13
14
12
8
12
11
10
29
31
64
87
24
42
38
46
104
2527
75
70
13
13
24
18
15
16
19
17
I
15
11
8
Mice were placed in a mixture of 85% O2 and 15% CO, for 15 min. 0.25 mC of uridine, uracil, adenine or adenosine was injected iv. and the mice placed in the O,-CO2 mixture for 45 min. In autoradiograms (exposure time 14 days (uridine), or 42 days (uracil, adenine, adenosine)) the total number of grains covering twenty-five nerve cells was counted in cortex and thalamus. The number is an average of counts from four animals. -CO, indicate control animals. * = P <%.005. t = P < o.ooo5. Compared to the nerve cells, the labelling of the choroid plexus cells is far greater for most of the substances examined, except for cytosine, uracil and thymine, for which the labelling is low for both cell types. The same is found on comparing choroid plexus cells and liver cells, also with the exception of cytosine, uracil and thymine. A comparison of nerve cells and liver cells shows a relatively low degree of incorporation for seven of nine of the substances examined. In the case of uridine, the incorporation into nerve cells is high and twice as great as for liver cells, while the reverse is the case for cytidine. We have noted that the endothelial cells of vessel walls are strongly labelled in animals injected with adenine (especially in the liver) and with adenosine (both in liver and brain). This labelling is maintained for at least 11 days, especially in the animals receiving adenosine. ALTMAN& CHOROVER(1963) have similarly found strong labelling of vascular cells after administration of adenine. On comparing adenine and uracil, they found a somewhat different distribution of grain density over nerve cells and neuropil than was found in the present investigation, but they used cats as well as injection into the third ventricle, which may explain the differences. It is striking that liver cells are labelled strongly only by those same two nucleosides, uridine and cytidine, which also label nerve cells strongly. This might suggest that the blood-brain barrier is not of restrio tive significance for the passage of these substances into the brain, and that incorporation of precursors (the salvage pathway) is used to a certain extent by these cell types. The fact that intravenous injection, by ensuring a high concentration in the blood, makes labelling over the nerve and liver cells possible with a further seven of the ten precursors employed, even though only to a modest degree, may be due to ‘unphysiological’ conditions initially. However, our
results with intraperitoneal injection provide evidence against this conclusion (PAKKENBERG& FOG, 1973). The results of the experiments where precursors were given by intracerebral injection confirm that the blood-brain barrier can hardly be of great significance for the transport of the four precursors used since the pattern of incorporation is the same as in intravenous injection. It is likewise the same in the in uitro experiments. As far as the CO,-inhalation experiments are concerned, it is striking that no signs are found of Evans’ blue-albumin passing the blood-brain barrier following CO2 inhalation, since these were the findings in experiments using this complex or tryptan blue in cats, rabbits and guinea-pigs (CLEMEJXON,HARTELIUS & HOLMBERG, 1958) and in dogs (JOHANSSON8~ LINDER, 1974). Nor are there any signs of increased passage of tritiated nucleic acid precursors following the injection of these substances. On the contrary, labelling following C02-inhalation was lower in the cortical and thalamic cells in the case of uridine. This agrees with the tinding by Restarts, KENNEDY, SHINOHARA& &iKOLO~ (1976) of reduced glucose uptake in the rat brain during CO,-inhalation. STEPHENS (1951) found non-specific histopathologic changes in the nerve cells of rats after inhalation of 2043% CO*. ln conclusion, the CO1 experiment was performed to see if ‘opening’of the barrier would increase the labelling of the nerve cells. The opposite was found and we suppose that this means that other factors than the barrier function are of importance for the incorporation of uridine. As the pattern of labelling following intracerebral injection and the in oitro pattern is the same as the distribution of labelling following intravenous injection, we conclude that the blood-brain barrier is not an important restrictive factor in the incorporation pattern. The different incorporation patterns of the
1084
H. PAKKENBERC; and R. Foe
precursors might be explained by differences in pool size, synthesis rate, transport systems, etc. (CQRNFORD & OLDENDORF, 1975).
Acknowledyemenl-We have received financial from the Witco foundation.
support
REFERENCES ALTMAN.I. & CHOROVER S. (1963) Autoradiographic investigation of the distribution and utilization of mtraventricularly injected adenine-C3H], uracil-[3H] and thymidine-C3H] in the brain of cats. J. Physiol.. Lond. 169, 770-779. BASKINF., MASIARZ F. & ACRANOFFB. (1972) Effect of various stresses on the incorporation of [3H]orotic acid into goldfish brain RNA. Brain Res. 39, 151-162. BOURGETP. & TREMBLAY G. (1972) Pyrimidine biosynthesis in rat brain. J. Neurochem. 19, 1617-1624. CLEMED~ON C.-J., HARTELIUSH. & HOLMBERGG. (1958) The influence of carbon dioxide inhalation on the cerebral vascular permeability to trypan blue (‘the blood-brain barrier’). Acta path. microbial. stand. 42, 137.. 149. CORNFORDE. & OLDENDORF W. (1975) Independent blood-brain barrier transport systems for nucleic acid precursors. Biochem. biophys. Acta 394, 21 l-219.
GEIGERA. & YAMASAKI S. (1956) Cytidine and uridine requirement of the brain. J. Neurochem. 1, 93.-100. HOGANSA.. GUROFF G. & UDENFRIENDS. (1971) Studies on the origin of pyrimidines for biosynthesis of neuron RNA in the rat. J. Neurochem. 18, 1699-1710. JOHANSWNB. & LINDERL.-E. (1974) Blood-brain barrier dysfunction in acute arterial hypertension induced by clamping of the thoracic aorta. Acta neural. stand. 50, 360-365. PAKKENBERG H. & Foci R. (1972) Kinetics of C3H-Sluridine incorporation in brain cells of the mouse. Expl Nrurol. 36, 40-410.
PAKKENBERG H. & Fee R. (1973) Kinetics of the incorporation of some tritiated nucleic acid precursors and C3H]lysine into mouse brain cells following intraperitoneal injection. J. Neurochem. 21, 841-848. ROWERSM., KENNEDYC., SHINOHARA M. & SOKOLOFFL. (1976) Effects of CO2 on local cerebral glucose utilization in the conscious rat. Neurology 26. 346.
(Accepted 28 June 1977)
in GreatBritain.
AUTORADIOGRAPHIC STUDY OF NUCLEIC ACID PRECURSOR INCORPORATION IN NERVE CELLS OF THE MOUSE BRAIN H. PAKKENBERG and R. FOG Laboratory of Neurology, Hvidovre Hospital, 2650 Hvidovre, Denmark Abstract-Following intravenous administration of five tritiated nucleic acid bases and five nucleosides in mice the intensity of labelling of the cerebral cortex cells and the liver cells show the following pattern. Cytidine and uridine cauSe intense labelling; uracil, adenine, adenosine, guanosine, guanine, thymine and cytosine only a weak labelling. The choroid plexus is labelled intensely by cytidine and uridine, moderately by guanosine and adenosine. weakly by other substances. In vitro experiments and intracerebral injection with uridine, uracil, adenine and adenosine give the same labelling pattern as intravenous injection. Inhalation of CO, does not alter the labelling of nerve cells apart from that due to the uridine group. where a weaker labelling of the nerve cells is observed. It is concluded that, in the case of the nucleic acid precursors used here, metabolic specificities are of greater significance for the labelling pattern of the nerve cells than the transport specificities of the blood-brain barrier.
[5-3H]uridine (spec. act. 25 Ci/mmol), [I-‘Hlguanine sulphate (spec. act. 500 mCi/mmol), [8-“Hlguanosine (spec. well as into liver cells (PAKKESBERG& FOG, 1972, act. 16 Ci/mmol), [6-3H]thymine (spec. act. 26 Ci/mmol). 1973) following intravenous and intraperitoneal injec- [6-3H)thymidine (spec. act. 23 Ci/mmol), [5-3H]cytidine (spec. act. 29 Ci/mmol. [3H]cystosine was not commertion. A number of factors are still not clear regarding cially available, but 5-iodocytosine was tritiated by catathe passage of nucleic acid precursors through the lytic hydrogenation and purified by The Radiochemical blood-brain barrier and their utilization in the nuc- Centre, Amersham. A total of sixteen animals were used leic acid metabolism of the nerve cells (BASKIN, for each substance. Under chloroform anaesthesia they MA~IARZ& AGRANOFF, 1972; F~URGET & TREMBLAY, were killed after 1, 6 and 24 h or 1I days, four animals 1972; GEIGER & YAMASAKI,1956; HOGANS,GUROFF in each group. They were immediately perfused through & UDENFRIEND,1971). C&NFORD & OLDENDORF the heart with Lillie’s fluid (formalin 4%) after the right (1975) found by the Oldendorf technique that out of atrium had been opened. The histological and autoradiographic procedures used seventeen compounds examined, measurable, saturwere the same as explained in an earlier paper (PAKKENable uptakes were established for adenine, adenosine, BERG & FOG, 1973). The dipping method with Ilford guanosine and uridine. One transport system for Nuclear Research Emulsion K 5 was used. The exposure adenine and one for adenosine, guanosine, inosine time (at 4°C) had to be varied, as otherwise the number of and uridine were demonstrated. grains would be too small in some of the preparations. The present study examines the incorporation of In the case of uridine and cytidine, 5 days’ exposure was a series of nucleic acid precursors following intraused for choroid plexus cells and liver sections, IO days’ venous injection, in vitro uptake, intracerebral injec- exposure was used for nerve cells. In the case of the other tion and CO, inhalation. This might indicate if the substances, 14 days’ exposure was used for plexus cells barrier function or other factors (precursor pool size, and liver sections, 42 days for nerve cells. However. all synthesis rate. rate of metabolism of precursor to grain counts were corrected for these differences before other compounds) are of the greatest significance for drawing the curves. The grain count over the nuclei and cytoplasm of the various cell types was also corrected to the incorporation pattern. an area of 1OOfirn’(corresponding approximately to the area of the nuclei of the plexus cells). The counting was MATERIALS AND METHODS performed over hepatocytes, great nerve cells in the fifth layer of the cerebral cortex and over the epithelial cells Intravenous injection of precursors of the choroid plexus. A total of 160 white male mice were used, weight 25-27 g. An amount of 0.25 mCi of the radioactive substance (determined from the specification supplied by the In~racerehral injection In a total of forty white mice. weight 25-27g, a guide producers) was dissolved in 0.25 ml distilled water and injected into the tail vein. The following substances were cannula was inserted in the skull. so that a thinner needle could be passed through this guide cannula and the needle used (The Radiochemical Centre, Amersham, England): tip placed in the right corpus striatum. Twenty-four hours [8-3H]adenine (spec. act. 21 Ci/mmol), [2-3H]adenosine later one microliter (Hamilton syringe) of one of the fol(spec. act. 26 Ci/mmol, [6-3H]uracil (spec. act. 24 Ci/mmol, WF. HAVE previously poration of uridine
studied the kinetics of the incorinto nerve and plexus cells, as
1077
1078
H. PAKKENBERG and R. FOG
lowing substances was injected: [5-‘Hluridine (spec. act. 29 Ci/mmol), [6-3H]uracil (spec. act. 21 Ci/mmol), [8-3H]adenine (spec. act. 26 Ci/mmol), [2-‘Hladenosine (spec. act. 21 Ci/mmol) (The Radiochemical Centre, Amersham. England). In sixteen animals 1PCi of uridine was injected, and in sixteen animals the same quantity of uracil, without anaesthesia. After intervals of 5 min, 30 min. 60 min or 4 h, four animals in each lot were killed with chloroform and perfused immediately with Lillie’s fluid through the left ventricle, as described above. Adenine or adenosine was injected in the same way into a total of eight mice, four with each substance. The animals were killed after 30min and perfused as described. Autoradiograms were made as described. The blocks were cut serially, the intention being to hit the tip of the injection track. The autoradiograms were exposed for 48 h. Grains were counted over ten nerve cell nuclei lying nearest to the tip of the injection track which showed the highest grain count. In vitro uptake Another technique used to by-pass the blood-brain barrier was to place slices of mouse brain from a decapitated mouse in modified Ringer’s solution (NaCl 120m~. NaHCO, 15 mM, KC1 5 mM, CaCI, (2H,O) 5 mM, MgSO, (7 H,O) 1 mM, glucose 5.106 mM). To this, 1OOpCi [S-“Hluridine or [6-3H]uracil or [8-3H]adenine or [Z-‘Hladenosine (spec. act. as given above) was added in 5 ml solution. The solution was maintained at 38°C in a water-bath, a gas mixture of O2 9S%, CO, 5%, being bubbled through. After 15 min the slices were removed and placed in 4”,‘, formalin for 3 days. Sections 4 pm in thickness were then cut and autoradiograms prepared as described above. The exposure time was 2 days.
substance, and four control animals were injected with the same nucleic acid precursor, but without CO* inhalation. Autoradiograms were prepared as described. The period of exposure in the case of uridine was 14 days, but three times longer (42 days) in the case of the other substances. Grains were counted over twenty-five nerve cells in the cortex and twenty-five nerve cells in the thalamus. In the same manner
the background
were determined.
RESULTS Intravenous
injection
of precursors
As the curves for the incorporation cytosine
are almost
of thymine and with the uracil curves,
identical
only those for the latter are shown (Fig. I). The guanine incorporation curves are like the adenine inURACIL
2*SE PN PC H
G ti 100 10-I
0
0
0
1
6
2L
URACIL G 100
26L h 2xSE NNy NCNP *
fl 10
NN-r
0
0
b
i
2L
6
COZ inhalation Preliminary trials were made with Evans’ blue. These will be described briefly. White mice were placed in a plastic box, capacity about 2l., and a mixture of 25% CO, and 75% 0, passed into the box at the rate of 3 I./min. 0.25 ml of the following mixture was injected i.v.: Evans’ blue ICQmg, albumin 5OOmg, physiological saline 10 ml. After about 30 min half the animals developed convulsions and died. The experiment was repeated with a gas mixture of 15% CO2 and 85% 0,. which resulted in the survival of the.animals. After injection of the Evans’ blue the animals were kept in the box under the above conditions for 1h, the gas mixture being supplied at the rate of 3 I./min. The animals were then killed as already described, and frozen sections prepared, 10pm in thickness. These were embedded in a glycerol-water mixture and examined under a fluorescence microscope fitted with an Osram HBO 200 lamp and ilters BG 38, BG 12, KP 790 and K 530. For the actual CO, experiment, white male mice were employed as above. The animals were placed in a plastic box of about 21. in volume. A gas mixture of 85% 0, and 15% CO, was passed into this after bubbling through water, at a rate of 3 I./min. After 15min, 0.25 mCi of the tritiated compounds mentioned earlier (uridine, uracil, adenine and adenosine) were injected i.v. The animals were then returned to the plastic box immediately and the 0,C02 mixture continued for 45 min. The animals were killed with chloroform and perfused as described earlier. Four animals were used for each
counts
26Lh
FIG. 1. ADENINE GE
2xSE
261
h
2XSE NN u NC +, NP Y B *
0
0
i
24
i FIG. 2.
2&h
1079
Nucleic acid precursor uptake
ADENOS I NE 2xSE PN PC w LN * LC 3 0* PN -
ADENOSINE G
‘v%
LOO
M
ZXSE NN 3 NC * NP * B”
NN -L.
0
6
I
264
21
h
0
FIG.
6
I
2L
26L h
3.
2xSE
PN--,
2500
.
PN PC LN LC I3
50
3 i *
2xSE NN NC )--i NP +t3+
IJRIDINE G 1600
vc
LO 1600
NN -
1
NC -
::::_::::
\ 900
30 9w
LN--,
LOO
20 -
LOO
PC-_, LC_
100
IO-
100
NP .
6-e.
.
B-e* 0
0
0
0
I
6
24
264
h
i
;
6
ZL
FIG. 4. FIGS 1-I. Number of grains covering 25 choroid plexus cells, liver cells and cortical nerve cells, 1 h to 264 h after i.v. injection of four different nucleic acid precursors. All values are corrected for differences in area, using the area of the choroid plexus cell nuclei as unit (IOO~m’). Four animals in each group. PN = plexus cell nuclei. PC = plexus ceil cytoplasm. LN = liver cell nuclei. LC = liver cell cytoplasm. NN = nerve cell nuclei. NP = neuropil. NC = nerve cell cytoplasm. B = background. SE = standard error of the mean. G = total grain count. fi is used for statistical reasons (PAKKEW BERG & FOG, 1973).
2&
h
H. PAKKEYNBERG and R. Foe
1080
TABLE I. INCORPORATION OF NUCLEIC ACIDPRECURSORS INTOCELL NCCLEI FOLLOWING INTRACER~W.AL INJL~W
n 5min
3
30min 60min 4h
I I
Uridine mean
3
13 32 24 26
range
,i
622 19-44 1435 1441
2 3 4 4
Uracil mean 2 3 3 4
range
n
1 ~3 l-9
2
Adenine mean 3
range
P!
I 7
4
Adenosinc mean
.!
range
I x
l-13 l-12
Following intracerebral injection of one pC of uridine, uracil, adenine or adenosine in mice the animals were killed with chloroform after 5, 30, 60 min or 4 h (uridine, uracil), the adenine- and adenosine-injected animals after 30 min. In autoradiograms (exposure time 2 days) the number of grains were counted in ten cell nuclei close to the end of the iniection track. The table shows the averaee number (mean) and range of grain number per nucleus. II. number of animals.
corporation curves
(Fig. 2) and the guanosine incorporation curves like those for adenosine incorporation (Fig. 3). Finally the cytidine incorporation curves are of the same shape as the uridine incorporation curve (Fig. 4). The incorporation curves for the five bases have certain features in common. The labelling of the nerve cells and liver cells is at a low level. In the case of the choroid plexus, it is moderate for adenine and guanine, lower for the other substances. The four nucleosides (as expected, no grains were found over the cells in the case of thymidine, apart from cells in division) can be grouped into two types: guanosine and adenosine show moderate incorporation into the choroid plexus, but the incorporation is low in liver cells and nerve cells. Uridine and cytidine are avidly taken up by the choroid plexus, and moderately by liver cells and nerve cells. lntracerehi-al injection of precursors The results are shown in Table 1. The labelling following uridine injection is significant; labelling by the other three substances is only about 10% of that of uridine. This corresponds to the findings upon intravenous injection. The nerve cells at the tip of the injection track are not morphologically changed, while shrunken cells are found along the track. In a few cases, some of the injected fluid has apparently penetrated into the ventricle, without this having affected the incorporation into the cells. Labelling of cells some few hundred pm from the tip of the injection track is minimal, and does not increase in those groups of animals living longest after the injection. Thus, it is unlikely that there is any significant diffusion or other form of transport of the injected substances. It may also be mentioned that the labelling of the cells 5 min after the injection is only about half the labelling after a period between 30 min and 4 h. This agrees with the observation that after intravenous injection, the maximum count is not found until a lapse of 30 min (PAKKENBERG& FOC, 1972). In vitro uptake of precursors
As shown in Figs 5-9, there is a marked uptake of uridine. while the other three substances are incor-
porated only to a very limited extent. A number of the cells in the preparations have been altered morphologically, in particular showing shrinkage, and these cells have a low uptake of uridine. Choroid plexus tissue occurs in some slices, and here the cells are moderately labelled, both with uridine and adenine. This is in agreement with the labelling found in the intravenous experiment. It might be mentioned that the labelling of cells in the margins of the slice is somewhat capricious, many of the cells being labelled in some regions, while few or none are labelled in other regions. No cells are labelled which are deeper than 200~ into the slice, presumably because of the presence of hypoxia here. EfSect of CO,-inhalation The injection of Evans’ blue + albumin produced pronounced fluorescence in the liver. kidneys and lungs, but in the brain fluorescence was seen only in the walls of a few vessels, not in the nerve cells. This was the case in both control animals and CO,-treated animals. The results of the experiments with nucleic acid bases and nucleosides are shown in Table 2. In the uridine-injected animals, the grain counts are seen to be significantly lower in the cortex and thalamus following CO, inhalation than when CO2 was not administered. In the case of the uracil, adenine and adenosine-injected animals, however, the grain counts showed no significant differences between CO2 administration and no CO2 administration.
DISCUSSION There was a considerable difference in the labelling of the three cell types studied, following intravenous administration of the radio-labelled nucleic acid bases and nucleosides. The method does not permit an evaluation of the manner in which the labelled substances are metabolized in the cells, but it is accepted that uridine participates in RNA synthesis, cytidine in RNA and DNA synthesis. Although the other sub stances give rise to only a slight labelling of nerve cells in the cortex, it is nevertheless obvious that this occurs with all the substances examined with the exception of thymidine.
FIGS 5-9. Autoradiograms from in uifro experiments. Slices of mouse brain in 5 ml modified Ringe solution with 100 PCi tritiated uridine (Fig. 5), adenine (Fig. 6). adenine, choroid plexus (Fig. 7), ader sine (Fig. 8) or uracil (Fig. 9) for 15 min. Exposure time 2 days.
1083
Nucleic acid precursor uptake TABLE2. EFFECTOF INHALATION OF CARBON DIOXIDE ONINCORPORA~ON OF NUCLEIC ACIDPRECURSORS FOLIoWINGINTRAVENOUS INJECTiON Uridine -co, + coz Cortex Nerve cell nuclei Nerve cell cytoplasm Neuropil Thalamus Nerve cell nuclei Nerve cell cytoplasm
Uracil -co, + co2
+co,
Adenine -co,
Adenosine -co, +co1
141
220*
51
63
12
18
40
30
10
13
14
12
8
12
11
10
29
31
64
87
24
42
38
46
104
2527
75
70
13
13
24
18
15
16
19
17
I
15
11
8
Mice were placed in a mixture of 85% O2 and 15% CO, for 15 min. 0.25 mC of uridine, uracil, adenine or adenosine was injected iv. and the mice placed in the O,-CO2 mixture for 45 min. In autoradiograms (exposure time 14 days (uridine), or 42 days (uracil, adenine, adenosine)) the total number of grains covering twenty-five nerve cells was counted in cortex and thalamus. The number is an average of counts from four animals. -CO, indicate control animals. * = P <%.005. t = P < o.ooo5. Compared to the nerve cells, the labelling of the choroid plexus cells is far greater for most of the substances examined, except for cytosine, uracil and thymine, for which the labelling is low for both cell types. The same is found on comparing choroid plexus cells and liver cells, also with the exception of cytosine, uracil and thymine. A comparison of nerve cells and liver cells shows a relatively low degree of incorporation for seven of nine of the substances examined. In the case of uridine, the incorporation into nerve cells is high and twice as great as for liver cells, while the reverse is the case for cytidine. We have noted that the endothelial cells of vessel walls are strongly labelled in animals injected with adenine (especially in the liver) and with adenosine (both in liver and brain). This labelling is maintained for at least 11 days, especially in the animals receiving adenosine. ALTMAN& CHOROVER(1963) have similarly found strong labelling of vascular cells after administration of adenine. On comparing adenine and uracil, they found a somewhat different distribution of grain density over nerve cells and neuropil than was found in the present investigation, but they used cats as well as injection into the third ventricle, which may explain the differences. It is striking that liver cells are labelled strongly only by those same two nucleosides, uridine and cytidine, which also label nerve cells strongly. This might suggest that the blood-brain barrier is not of restrio tive significance for the passage of these substances into the brain, and that incorporation of precursors (the salvage pathway) is used to a certain extent by these cell types. The fact that intravenous injection, by ensuring a high concentration in the blood, makes labelling over the nerve and liver cells possible with a further seven of the ten precursors employed, even though only to a modest degree, may be due to ‘unphysiological’ conditions initially. However, our
results with intraperitoneal injection provide evidence against this conclusion (PAKKENBERG& FOG, 1973). The results of the experiments where precursors were given by intracerebral injection confirm that the blood-brain barrier can hardly be of great significance for the transport of the four precursors used since the pattern of incorporation is the same as in intravenous injection. It is likewise the same in the in uitro experiments. As far as the CO,-inhalation experiments are concerned, it is striking that no signs are found of Evans’ blue-albumin passing the blood-brain barrier following CO2 inhalation, since these were the findings in experiments using this complex or tryptan blue in cats, rabbits and guinea-pigs (CLEMEJXON,HARTELIUS & HOLMBERG, 1958) and in dogs (JOHANSSON8~ LINDER, 1974). Nor are there any signs of increased passage of tritiated nucleic acid precursors following the injection of these substances. On the contrary, labelling following C02-inhalation was lower in the cortical and thalamic cells in the case of uridine. This agrees with the tinding by Restarts, KENNEDY, SHINOHARA& &iKOLO~ (1976) of reduced glucose uptake in the rat brain during CO,-inhalation. STEPHENS (1951) found non-specific histopathologic changes in the nerve cells of rats after inhalation of 2043% CO*. ln conclusion, the CO1 experiment was performed to see if ‘opening’of the barrier would increase the labelling of the nerve cells. The opposite was found and we suppose that this means that other factors than the barrier function are of importance for the incorporation of uridine. As the pattern of labelling following intracerebral injection and the in oitro pattern is the same as the distribution of labelling following intravenous injection, we conclude that the blood-brain barrier is not an important restrictive factor in the incorporation pattern. The different incorporation patterns of the
1084
H. PAKKENBERC; and R. Foe
precursors might be explained by differences in pool size, synthesis rate, transport systems, etc. (CQRNFORD & OLDENDORF, 1975).
Acknowledyemenl-We have received financial from the Witco foundation.
support
REFERENCES ALTMAN.I. & CHOROVER S. (1963) Autoradiographic investigation of the distribution and utilization of mtraventricularly injected adenine-C3H], uracil-[3H] and thymidine-C3H] in the brain of cats. J. Physiol.. Lond. 169, 770-779. BASKINF., MASIARZ F. & ACRANOFFB. (1972) Effect of various stresses on the incorporation of [3H]orotic acid into goldfish brain RNA. Brain Res. 39, 151-162. BOURGETP. & TREMBLAY G. (1972) Pyrimidine biosynthesis in rat brain. J. Neurochem. 19, 1617-1624. CLEMED~ON C.-J., HARTELIUSH. & HOLMBERGG. (1958) The influence of carbon dioxide inhalation on the cerebral vascular permeability to trypan blue (‘the blood-brain barrier’). Acta path. microbial. stand. 42, 137.. 149. CORNFORDE. & OLDENDORF W. (1975) Independent blood-brain barrier transport systems for nucleic acid precursors. Biochem. biophys. Acta 394, 21 l-219.
GEIGERA. & YAMASAKI S. (1956) Cytidine and uridine requirement of the brain. J. Neurochem. 1, 93.-100. HOGANSA.. GUROFF G. & UDENFRIENDS. (1971) Studies on the origin of pyrimidines for biosynthesis of neuron RNA in the rat. J. Neurochem. 18, 1699-1710. JOHANSWNB. & LINDERL.-E. (1974) Blood-brain barrier dysfunction in acute arterial hypertension induced by clamping of the thoracic aorta. Acta neural. stand. 50, 360-365. PAKKENBERG H. & Foci R. (1972) Kinetics of C3H-Sluridine incorporation in brain cells of the mouse. Expl Nrurol. 36, 40-410.
PAKKENBERG H. & Fee R. (1973) Kinetics of the incorporation of some tritiated nucleic acid precursors and C3H]lysine into mouse brain cells following intraperitoneal injection. J. Neurochem. 21, 841-848. ROWERSM., KENNEDYC., SHINOHARA M. & SOKOLOFFL. (1976) Effects of CO2 on local cerebral glucose utilization in the conscious rat. Neurology 26. 346.
(Accepted 28 June 1977)