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Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70112-1262; the Department of Pathology, Amgen, Inc. (C.L.F.), Thousand Oaks, California 91320-1789; and Geriatric Research Educational and Clinical Center (GRECC), Veterans Administration Medical Center-St. Louis (W.A.B.) and the Division of Geriatrics, Department of Internal Medicine, Saint Louis University School of Medicine (W.A.B.), Saint Louis, Missouri 63106
Address all correspondence and requests for reprints to: Lawrence M. Maness, Ph.D., Veterans Affairs Medical Center, Research Service, 1601 Perdido Street, New Orleans, Louisiana 70112-1262. E-mail: lmaness{at}mailhost.tcs.tulane.edu
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Introduction |
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In experimental procedures, leptin is often delivered directly into the brain of rodents and small mammals by intracerebroventricular (icv) injection, bypassing the BBB transport event. Such an approach quickly delivers large quantities of leptin to the brain and may lower the risk of enzymatic degradation. However, the dynamics of movement within the brain vary significantly from compound to compound (9) and must be taken into account to accurately assess physiological and pharmacological properties of icv administered materials.
Delivery of exogenous leptin to the ventricular system may mimic the hypothesized transport of the blood-borne protein by the CP (7). The subsequent kinetics of distribution after either mechanism of penetration also could be quite comparable. However, the fate of leptin after icv injection has not been thoroughly described. Several possible paths are available subsequent to central injection. The protein may distribute quickly throughout the brain, leading to numerous physiological effects. It is also possible that the protein would be restricted to the ventricular space and be subject to dispersion and removal by the flow of the cerebrospinal fluid (CSF). With CSF removal, leptin would enter the blood, raising the possibility of interactions with its receptor, including those found at the CP. In addition, the degree of degradation of icv administered leptin has not been fully detailed.
In the present study, we investigated the fate of radioiodinated leptin after icv administration in the mouse. The disappearance of leptin from the brain and the appearance of the protein in the blood were measured, and the degrees of intactness in both environments were assessed over time. Furthermore, to determine the degrees of penetration of radioiodinated leptin into the tissues surrounding the ventricular system, we applied film autoradiography to mouse brain sections after icv injection. A similar autoradiographic approach was used to quantify the amount of radioiodinated leptin localized to several regions believed to be involved in the physiological events triggered by leptin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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icv injection
Adult male ICR mice (Charles River Laboratories,
Inc., Wilmington, MA), 20–25 g, were anesthetized with ip
urethane (4.0 g/kg). For each mouse, the skull was exposed, and the
bregma was located. A guarded 26-gauge needle was used to punch a hole
0.2 mm caudal to the bregma and 1.0 mm lateral to the midline at a
depth of 2.25 mm. A 1.0- or 5.0-µl Hamilton syringe (Hamilton, Reno,
NV) was used to inject specified quantities of murine or human I-Lep in
a volume of about 1.0 µl into the right cerebral ventricle.
HPLC
About 5 x 106 cpm I-Lep were injected icv at
time zero. Whole blood from the carotid artery and the whole brain was
removed 2, 5, 10, 20, or 30 min after the icv injection. The blood was
centrifuged at 4 C for 10 min at 5000 x g, and the
serum was collected and lyophilized. The entire brain was homogenized
in 2 ml LR-BSA after removal of the pituitary and pineal. The
homogenate was centrifuged for 10 min at 4 C at 5000 x
g, and the resulting supernatant was lyophilized.
Processing controls were used to determine the amount of degradation that occurred during collection, homogenization, and lyophilization. About 105 cpm was added to the surface of a brain from a mouse that had not been injected with radioactivity and the brain processed as described above. For the serum processing control, about 105 cpm were added to the bottom of a tube before the collection of arterial blood from a mouse that had not received an injection of radioactivity. The blood was then processed as described above.
Brain and serum samples were reconstituted immediately before HPLC analysis with 0.6 ml distilled water with 0.1% trifluoroacetic acid (TFA), vigorously mixed, and centrifuged at 4 C for 15 min at 5200 x g. Of the resulting supernatant, 0.1 ml was injected onto a C4 protein column (Vydac, Hesperia, CA) and eluted with a gradient that linearly progressed from 90% of solution A (0.1% TFA in water) to 90% of solution B (0.1% TFA in acetonitrile) in 50 min, with fractions collected every minute. The levels of radioactivity in the fractions were determined, and the percentage of radioactivity eluting from the column that eluted in the position of I-Lep was calculated. The values for the brain and blood samples were corrected for processing degradation by dividing them by the values of the processing controls for brain (32.5%) and blood (48.1%).
Appearance in blood
Percentage of the injected dose. About 5 x
106 cpm I-Lep were injected icv into 15 mice. Blood from
the carotid artery was collected 2, 5, 10, 20, or 30 min after the icv
injection (n = 3 mice/time point). The blood was centrifuged at 4
C at 5000 x g for 10 min, and the level of
radioactivity in serum was determined. The whole brain was collected,
the pituitary and pineal were removed, and the level of radioactivity
was measured. The levels of radioactivity were corrected by use of the
factors determined above under HPLC. The results were
expressed in two ways. First, the HPLC-corrected level of radioactivity
in 1 ml serum was divided by the amount of radioactivity injected and
multiplied by 100 to yield the percentage of icv injected radioactivity
present in 1 ml serum (percentage per ml). Second, the HPLC values for
radioactivity in brain and serum were used to calculate brain/serum
ratios in units of milliliters per g.
Pharmacokinetics. The brain to blood efflux rate (Q; percentage per min) of I-lep was calculated by the equation: Q = P(Ke)Vd/(1 - e(-)Ket) (Eq I), where P is the percentage per ml at time t, Ke is the inverse of the half-time disappearance rate in minutes of I-Lep from serum after its iv injection multiplied by 0.693, Vd is the volume of distribution in milliliters, and t is time in minutes (10). The half-time of disappearance for I-Lep has been determined to be 5.46 min (to give a Ke of 0.127), and the volume of distribution has been determined to be 2.43 ml (8).
Autoradiography
Two, 10, or 30 min after icv injection of 6 x
105 cpm radioiodinated murine or human leptin (2.65 ng
I-Lep/mouse), mice were decapitated, and the brains were carefully
removed. The intact brains were quickly dipped in -30 C isopentane for
12 sec and moved to a bed of crushed dry ice for 10 min, turning once
for even freezing. The brains were then wrapped in plastic and stored
for 24 h at -70 C. Frozen brains were warmed to -15 C, coronally
sliced at 20 µm, and mounted on gelatin-coated slides.
The mounted sections were desiccated for 24 h at 4 C. Upon warming to room temperature, the slides were put on Hyperfilm 3H (Amersham, Arlington Heights, IL) for 5 days. The film was then developed with D-19 developer (Eastman Kodak Co., Rochester, NY).
Computer-assisted image analysis
Relative optical densities (RODs) of the autoradiographic images
were measured with an MCID image analysis system (Imaging
Research, Inc., St. Catharines, Canada). The total area of
diffusion from the third ventricle was determined from each image by
the autoscan function of the image analyzer. Measurements of maximal
lateral diffusion of I-Lep from the third ventricle were made at two
defined regions: the level of the anterior hypothalamus, 1.0 mm dorsal
to the ventral base of the brain, and the level of the medial arcuate
nucleus, 0.5 mm dorsal to the ventral base of the brain. For each time
point, mean diffusion distances were determined, and the relationship
was analyzed by one-way ANOVA and the Newman-Keuls multiple comparison
test.
D1/2 values, the distance at which the amount of radioactivity equaled half the midline value, also were determined for each time point. A sampling tool, 200 µm in height, was used to measure across the labeled regions at the same coordinates used to determine maximal lateral diffusion from the third ventricle. Transept lines generated from these measurements were then used to determine the distance from the midline/third ventricle needed for the intensity of the labeling to decrease by half. The left and right hemisphere values for each transept line were averaged, and the mean distances of several serial sections were determined. The relationship of the D1/2 values was analyzed by ANOVA and Newman-Keuls multiple comparison test.
Other autoradiograms generated from the 30-min exposure group were analyzed for degrees of labeling at the arcuate nucleus, a region of the brain previously shown to accumulate I-Lep after iv administration (8). ROD values at the arcuate nucleus were compared statistically to other areas labeled by icv I-Lep, including the CP, third ventricle periventricular tissue, and the middle cerebral artery. The specific areas were delineated and measured with templates created from the tissues used to generate the autoradiographic images. Measurements of ROD also were taken at the background of the autoradiographic film and used as correction factors. Multiple measurements were averaged and compared by ANOVA and Newman-Keuls posttests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. For
brain, the percent of radioactivity representing I-Lep ranged from
58–85% over time. As there was no relation between time and percent
intact, the values were combined over time to yield a mean of 72
± 5% (n = 5). This value was used to correct levels of
radioactivity in brain.
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Appearance in blood
Percentage of injected dose. Figure 1A shows the percentage of the icv
injected I-Lep that appeared in 1 ml serum with time. The results were
fitted to a second order polynomial equation: y =
(-)0.00784x2 + 0.367x - 0.669,
where y is percentage per ml, and x is time in
minutes; the regression coefficient was 0.997. Based on this equation,
the maximum value of 3.64%/ml occurred 23.4 min after icv injection.
The brain/serum ratio in units of milliliters per g is shown in Fig. 1B.
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shows
the efflux rate (Q) from brain based on the mean percentage per min
values shown in Fig. 1A
. Between 5 and 30 min, these values averaged
0.943 ± 0.126%/min (n = 4), showing that about 0.94% of
the dose injected icv entered the blood each minute. This calculates to
a half-time disappearance from brain of 73.1 min.
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, A and B, and Fig. 4, A
and B). Subtle degrees of penetration of radiolabeled material from the
ventricular space into the surrounding tissues were observed at 10 min,
but the majority of the labeling appeared to remain ventricular.
However, by 30 min post-icv injection, a different pattern had emerged
(Figs. 3C and 4C). A much greater degree of penetration was now evident
within the periventricular tissues. The overall intensity of the
radioactive labeling also was considerably lower than that at the
shorter exposure times within both the periventricular and ventricular
spaces.
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and 3). At the anterior hypothalamus, the
degree of I-Lep penetration was significantly greater after 30 min than
after 2 or 10 min. Additional significant differences were seen between
2 and 10 min circulation values. At the level of the medial arcuate
nucleus, significant differences also were seen between penetration
distances for all pairings of 2, 10, and 30 min circulation values.
Murine and human I-Lep showed no significant differences in
periventricular penetration at either the hypothalamus or the arcuate
nucleus 30 min after icv injection.
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and 3). At the level of the anterior hypothalamus, the 30 and 2 min
D1/2 values were the only groups to display a statistically
significant difference. At the level of the medial arcuate nucleus, the
30 and 2 min values also displayed a significant difference, as did the
30 and 10 min values. After 30-min circulation periods, ROD values at the arcuate nucleus and all other central regions measured were significantly greater than the mean background RODs (F4,10 = 27.41; P < 0.01 for each). After being corrected for background, ANOVA of the ROD values showed a significant relationship (F3,8 = 8.163; P < 0.01). Newman-Keuls comparisons showed the ROD of the arcuate nucleus (0.178 ± 0.028) to be significantly lower than the RODs of the CP (0.389 ± 0.012; P < 0.01) and the middle cerebral arteries (0.345 ± 0.053; P < 0.05). ROD values corresponding to the tissue adjacent to the third ventricle were not significantly different from the values found at the arcuate nucleus (P > 0.05). The localization of I-Lep within the arcuate nucleus was not as concentrated after icv injection compared with that seen after iv administration of I-Lep (8).
The CP continued to display prominent I-Lep labeling even after the CSF
was essentially cleared of the labeled protein (Fig. 5). Thirty minutes after icv injection
into the ventricle, autoradiographic images showed the CP to be heavily
labeled, although the ventricular space and the surrounding tissues
were not above background levels. The labeling was similar to that seen
at the CP after iv administration of I-Lep (8) and suggested I-Lep
binding on the brain side of the blood-CSF barrier.
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Discussion |
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Levels of I-Lep within the brain after icv injection were always much higher than those previously seen after iv injection (8). However, the results also show that a measurable amount of the leptin administered icv enters the blood. At about 20 min, blood levels after icv injection were similar to those seen after iv injection (8). The rate of brain to blood efflux based on the rate of appearance of I-Lep in serum is consistent with a nonsaturable process such as reabsorption of CSF.
The HPLC results shown in Table 1 give the percentage of radioactivity
in brain and serum representing intact I-Lep after icv injection and
show that it is stable for at least 30 min. This suggests that
iodinated degradation products arising from the catabolism of I-Lep in
brain and blood are cleared at about the same rate as I-Lep. The
relatively high level of radioactivity that represents intact I-Lep
aids in the accurate assessment of the fate of leptin after icv
injection.
I-Lep reaches levels in each milliliter of serum equal to 3.64% of the injected dose 23.4 min after injection. This slow rise is explained by icv injections often having similar pharmacokinetic profiles as those of iv infusions (12). By comparison, I-Lep injected as an iv bolus can be calculated, based on the previously determined (8) volume of distribution of 2.43 ml and the previously determined half-time of disappearance of 5.46 min, to decrease from 41.1%/ml at time zero to 2.11%/ml by 23.4 min. Therefore, at time periods greater than about 20 min, blood levels achieved after an icv injection equal or even exceed those seen after an iv injection.
The brain/serum ratio for I-Lep after icv injection declined in a
logarithmic manner to a value of about 10 ml/g at 30 min. By contrast,
the brain/serum ratio after iv injection is usually measured at about
0.03 ml/g. Therefore, the brain/serum ratio is at least 300 times
higher after icv injection than after iv injection. However, as shown
in Figs. 3C and 4C, most of the radioactivity in the brain 30 min after
icv injection is not evenly distributed throughout the brain but is
concentrated in the periventricular areas.
The efflux rate from brain can be calculated based on the assumption
that the efflux occurs over time and, therefore, is analogous to an iv
infusion. The iv infusion rate (Q) needed to achieve a given level in
the blood can be calculated if the volume of distribution in blood and
the half-time disappearance rate from blood are known (see Eq I).
Figure 2 shows the values for Q computed from the percentage per ml
values of Fig. 1A. The mean value of Q for times between 5 and 30 min
was 0.943%/min. This gives a half-time disappearance from brain of
73.1 min, a rate consistent with entry into the circulation by
reabsorption of CSF. Previous results based on the rate of
disappearance of radioactivity from the CNS after icv injection gave a
value of 53.7 min, a rate that was not statistically different from
that of albumin, which exits the brain by being reabsorbed with the CSF
(8). In that previous study, total levels of radioactivity were used
and not levels corrected to reflect intact I-Lep only; if total
radioactivity appearing in serum had been used here instead of
corrected counts, the efflux rate would have yielded a half-time
disappearance from brain of 53.0 min.
Computer-assisted analysis of the autoradiographic images provided both qualitative and quantitative evidence that after injection into the lateral ventricle of the brain, a measurable degree of I-Lep escapes the ventricular space and enters the periventricular tissue. Since ventricular flow can carry the CSF and CSF-borne I-Lep from the lateral ventricles to the third then to the fourth ventricles, with the fluid then entering the subarachnoid space, periventricular penetration into surrounding tissues may occur along this path. In this study, penetration of I-Lep from the third ventricle into the brain parenchyma was measured at the level of both the anterior hypothalamus and the medial arcuate nucleus. The degree of movement into the tissue was significantly greater with time, indicating time-dependent diffusion in the dispersion of the protein. Detectable amounts of I-Lep were seen over 200 µm from the ventricle-tissue interface after only 2 min and nearly 600 µm from the interface by 30 min. However, turnover of the CSF appeared to be concurrently working against penetration with the simultaneous removal of the protein by bulk flow, since the amount of I-Lep at the ventricular origin was greatly diminished at the later time points.
Lateral diffusion brings I-Lep deeper into periventricular tissue and increases the possibility of interactions of the polypeptide with leptin-sensitive regions within the brain. In this study, D1/2 values, the distances at which leptin-associated radioactivity equaled half the midline value, showed that the amount of protein penetrating the periventricular tissue rapidly decreased with increasing distance from the third ventricle. For example, at the level of the anterior hypothalamus, the concentration of I-Lep had fallen to half of the midline value by about 300 µm, although amounts of radiolabeled protein greater than background could be detected another 300 µm further into the periventricular tissue. Statistical analysis of the D1/2 values showed that only the longest circulation times resulted in D1/2 values significantly greater than those resulting from a short 2-min circulation. The increased time necessary for sufficient amounts of leptin to diffuse from the ventricular space into deeper regions of the brain also increases the possibility of degradation of the protein by endogenous enzymes. Furthermore, only a limited amount of time is available for leptin diffusion to occur due to the removal of the protein with the turnover of the CSF.
Delivery of icv administered leptin to physiologically relevant regions of the brain, therefore, requires that such targets be relatively close to a ventricular space or that relatively large amounts be given. One site that has been implicated in the actions of leptin and is in close proximity to the third ventricle is the arcuate nucleus. Binding studies have shown that the arcuate nucleus possesses a high concentration of leptin receptors, which may be involved in the satiety effects of the protein (6, 13). In a previous investigation, we found that blood-borne leptin is saturably transported into the brain and is selectively localized to the arcuate nuclei, as demonstrated by in vivo autoradiography (8).
The present icv study does not demonstrate the same degree of leptin association with the arcuate nucleus that was seen after iv administration. Although the autoradiographic signal of the arcuate nucleus 30 min after icv injection was significantly greater than background, the degree of labeling at the nucleus was not different from that in the periventricular tissues. This suggests that only minimal amounts of I-Lep were reaching the arcuate from the third ventricle and were not sufficient to selectively label the nucleus, and that the selective labeling of the arcuate nucleus previously seen after iv administration is likely to be due to selective transport by the BBB.
Two other structures did show degrees of labeling significantly different from that in the arcuate nucleus after icv administration. Both the CP and the middle cerebral arteries displayed ROD values significantly greater, not lower, than that in the arcuate nucleus. The substantial labeling of the CP could be anticipated, given the intimate association of the CSF with the fronds of the CP, as well as the previous findings that the CP is another central structure possessing a high concentration of leptin receptors (5, 6, 7). However, the labeling of the CP after icv administration was comparable to the dense labeling of the CP previously seen after iv administration of I-Lep. This suggests that leptin receptors are present on both the CSF and blood sides of the tightly joined choroid epithelial cells that comprise the blood-CSF barrier.
The high ROD values observed at the middle cerebral arteries probably reflect the presence of the labeled protein in the blood after its reabsorption with CSF. CSF flows through the ventricular system and into the subarachnoid space, where the fluid along with CSF-borne materials are removed to the venous circulation at the arachnoid granulations. Thirty minutes after icv injection of I-Lep, a substantial portion of the bolus would have been absorbed out of the ventricular system and into the peripheral circulation. Therefore, some of the radioactivity would be expected to be found in the lumen of the vessels of the cerebral vasculature.
Taken together, these findings show that leptin slowly exits the CNS by being reabsorbed with the CSF to enter the circulation. Due to this efflux, leptin can achieve measurable levels in the serum, where it would be available to stimulate peripheral receptors. Therefore, in designing icv experiments, consideration should be given to dose, time, and potency relative to that of the iv route of administration. Furthermore, the extent of penetration of icv administered leptin into the periventricular tissues also should be taken into account. In smaller animal models, minimal diffusion of leptin from the ventricular space may be sufficient to induce physiological effects. However, in species with the greatest brain mass, comparable degrees of penetration may not be adequate to trigger the anticipated responses.
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Acknowledgments |
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Received January 29, 1998.
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