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Department of Endocrinology and Metabolism (S.F., C.S., H.L.), Magdeburg University Medical School, 39120 Magdeburg, Germany; and Warwick Medical School (H.L.), Coventry University Hospital, Coventry CV2 2DX, United Kingdom
Address all correspondence and requests for reprints to: Dr. Carla Schulz, Department of Endocrinology and Metabolism, Otto-von-Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany. E-mail: carla.schulz{at}medizin.uni-magdeburg.de.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In animal experiments, chronically increased peripheral leptin concentrations after ip or sc injection led to reduced food intake in lean and, to a lesser extent, in diet-induced obese mice. Over time, however, both groups developed a peripheral leptin resistance, which could be overcome by intracerebroventricular injection of a 4000-fold lower leptin dose than applied peripherally (7, 8).
Previously published data from our group (9) and Shimizu et al. (10) indicate that the poor delivery of peripherally administered leptin to its site of action in the brain can be improved by intranasal (i.n.) application. To reach the brain, i.n. delivered leptin has to be absorbed by the epithelium lining the nasal cavity. For other peptides (e.g. nerve growth factor, albumin, and horseradish peroxidase), leakage through extracellular clefts as well as transcytosis processes have successfully been shown (11, 12, 13). Transcytosis in particular appears to be relevant in supporting cells, probably as well as in respiratory and olfactory epithelia (14). After entry into the subepithelium, leptin can leak into perineural spaces as well as lymphoid and blood vessels.
The brain parenchyma may then be reached by diffusion within the CSF after transport to the subarachnoid space via the perineural spaces (15), which are located between olfactory or trigeminal axons and ensheathing Schwann cells or along a not yet specified connection between lymphoid vessels and CSF (16). In experiments employing i.n. delivery of radioiodinated IGF-I, a 7.65-kDa peptide, Thorne et al. (16) found radioactivity within the brain parenchyma but not within the CSF, suggesting that there is an additional, not yet clarified, pathway from the nasal cavity to brain parenchyma.
The aim of this study was to investigate the access of leptin from the nasal cavity to the central nervous system (CNS) and to assess whether this takes place under circumvention of the BBB. Furthermore, we addressed the question of whether unlabeled iv injected leptin competitively hinders the uptake of i.n. applied leptin into the CNS. In this context, the hypothalamus, as the main area involved in appetite regulation, was of particular interest.
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Materials and Methods |
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The experimental protocols for animals and their care were in accordance with German law and were approved by the committee on animal care. All experiments met the highest standards of humane animal care.
Solutions
Three different leptin solutions (0.2, 0.1, and 0.03 mg/kg) were prepared by dissolution of lyophilized murine recombinant leptin (Sigma-Aldrich, Taufkirchen, Germany) in physiological saline (0.9% NaCl), containing 1% sodium taurodihydrofusidiate (STDHF) as permeation enhancer to yield a final dilution containing the dose required for one animal in 24 µl.
Radiochemicals
Lyophilized 125I-labeled mouse leptin was labeled in four batches by Anawa Trading SA, Wangen Zurich, Switzerland, and purchased at Biotrend Chemicals, Cologne, Germany. The specific activity in the different batches was between 61.2 TBq/mmol and 67.6 TBq/mmol at the reference date. The radioiodinated leptin was custom labeled upon our order with an enzymatic method (lactoperoxidase); it was dissolved (see below) and used immediately after delivery. All experiments performed with one batch of [125I]leptin were carried out within 8 d. Between experiments, the solution was stored at –80 C, according to the manufacturer’s recommendation. Upon completion of the experiments, trichloroacetic acid (TCA) precipitation was performed with the remaining aliquot, confirming greater than 90% of integrity of [125I]leptin.
Lyophilized [125I]leptin was dissolved in each of the unlabeled leptin solutions (3.7 kBq/µl). The radiolabeled leptin in the solutions was not included in concentration calculations, because the [125I]leptin changed the total leptin concentrations by a negligible amount. It represented 0.4% weight per volume of total leptin at the most.
Anesthesia
Anesthesia was initiated by 90 mg/kg ketamine ip, followed by im injection of xylazine (13 mg/kg) and atropine sulfate (45 µg/kg) as soon as the animals were sufficiently sedated. The body temperature of the animals was maintained at 36.5 C during the whole experiment by a regulated heating pad.
Experimental procedure
Distribution of i.n. applied leptin in nonperfused animals.
All animals (n = 8 per group) were equipped with a femoral vein catheter for collection of blood. Subsequently, the trachea was cannulated, the nasopalatine ducts blocked with Histoacryl-blue, and the pharynx obstructed with sc tissue fixed with Histoacryl-blue to avoid leakage of leptin solution from the nasal cavity into the digestive tract. Furthermore, we equipped the animals with a self-manufactured mini-catheter to draw CSF samples from the cisterna magna for the determination of radioactive leptin. Results are not shown here, because the recovery of radiolabeled leptin was low and samples could not be quantified consistently and evaluated statistically. Total duration of surgery was approximately 120 min.
Before i.n. application, animals were allowed to recover for 30 min. Then 24 µl of one of the three leptin solutions, containing [125I]leptin, was applied alternately into the nares, while the animals were positioned on their backs. Five, 10, 15, 20, and 30 min after the application, blood samples (200 µl each) were taken. Subsequent to the last sampling, the animals were killed. The brain was immediately removed and rinsed in ice-cold saline and freed from obvious blood vessels. It was then dissected into nine regions (Table 1
), tracing defined anatomical structures as dissection markers, in a number of consecutive steps: dorsally—1) dissection of the olfactory bulb by coronal section frontal to the cortex (olfactory bulb); 2) V-shaped section after the delineation of the cortex, rostral of the corpora quadrigemina; and 3) transection of pedunculi cerebelli and excerption of cerebellum (cerebellum), mesencephalon, and rhombencephalon as one portion (mes- and rhombencephalon); ventrally—4) dissection of frontal cortex and a portion of the striatum by vertical section directly rostral to the optic chiasm (frontal cortex); 5) dissection of somatosensory cortex directly caudal to the optic chiasm, including septum, portion of the striatum, and areas ventral of these (somatosensory cortex); 6) dissection of the hypothalamus by rectangular incision, dorsally confined by the delineation of the third ventricle, laterally confined by the fissure between di- and telencephalon (hypothalamus); 7) separation of the remaining brain portion by a sagittal incision; 8) excoriation of cortices from the remaining diencephalic structure with excision of hippocampi (hippocampus); and 9) dissection of occipital and entorhinal cortices including amygdala (occipital and entorhinal cortices, including amygdala) from thalami (thalamus).
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). The samples were weighed on a Mettler AE200-S analytical scale and analyzed in a multicrystal 
-counter (LB 2111; EG&G Berthold, Bad Wildbad, Germany). The measured counts per minute per gram were used to calculate the total leptin concentration (nanograms per gram) in each sample (in blood samples, data are presented per milliliter instead of per gram).
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Brain average values for each animal were achieved by division of the sum of the counts per minute of each brain’s samples by the sum of the weight in grams of these samples and processed as described above, to yield the total i.n.-derived leptin concentration and the percent applied activity per gram.
To take into account the decay of radiolabeled leptin added to the i.n. leptin solution, a calibration curve was prepared daily and included in the calculations.
Distribution of intranasally applied leptin in transcardially perfused animals and effects of excess unlabeled leptin in the circulation.
Animals were surgically equipped as stated under Distribution of i.n. applied leptin in nonperfused animals, with the exception of the cisterna magna mini-catheter. In addition, a second femoral vein catheter was placed at the other hind limb for the application of 0.2 mg/kg unlabeled leptin, dissolved in 500 µl saline or saline as control, respectively. Total duration of surgery was approximately 60 min.
Simultaneously with the i.n. application, unlabeled leptin or saline was injected iv. At the end of the experiment, animals with unlabeled leptin iv were transcardially perfused for 3 min with 25 ml/min saline (n = 7) to flush blood vessels and remove [125I]leptin. Animals with saline iv were either transcardially perfused (n = 7) or decapitated without previous transcardial perfusion (n = 7).
All other treatments, handling of samples, evaluation, etc. were identical to those described above.
TCA precipitation
The method used was adapted from Banks et al. (17), who have shown that TCA precipitation is equally effective as HPLC to examine the degradation of radiolabeled leptin in samples with more than 50% integrity. In these samples, only intact leptin, [125I]tyrosine, and free 125I were detected after separation by HPLC. No labeled large peptide fragments, which would precipitate by the addition of TCA and thus may have been misinterpreted as leptin, were found.
In preparation for TCA precipitation, peripheral tissues were chopped into small pieces. The dissected parts of the brain were combined into two equivalents of a hemisphere, each handled separately in the following manner. Samples were weighed and measured for -radiation. After subsequent homogenization with a Sonopuls HD 60 ultrasonic homogenizer, tissues were processed following Banks’ protocol for brain tissue. Plasma samples were treated similarly to serum samples in Banks’ experiment (17). Briefly, intact iodinated leptin in tissue extracts and plasma was precipitated with the help of 30% TCA in lactated Ringer solution and separated by centrifugation. After withdrawal of the supernatant, radioactivity of the precipitate was determined in a -counter, and the percentage of intact leptin was calculated.
Statistical analysis
The effects of the different doses of leptin applied were studied either as a function of time for blood or as a function of brain area or type of peripheral tissue. Therefore, statistical analysis of the data was by ANOVA for repeated measures. If there was a significant difference between treatment groups over time, all areas, or all types of tissue, a post hoc Bonferroni test would be performed. P 
0.05 was accepted as the level of statistical significance. All data are expressed as mean ± SEM.
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Results |
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-radiation. The amount of exogenous i.n. leptin, indicated by [125I]leptin, differed between the regions. It was significantly higher in hypothalamus and olfactory bulb than in the brain average. Compared with brain average, occipital and entorhinal cortex/amygdala as well as cerebellum contained significantly lower quantities of iodinated leptin, whereas hippocampus content was not different from brain average (Fig. 1A). Moreover, the application of the three solutions led to a significantly different total leptin concentration (i.e. i.n.-derived labeled plus unlabeled leptin, calculated from the amount of [125I]leptin) over all brain areas (P = 0.025; F = 4.586). A post hoc Bonferroni test revealed that the difference was between the groups receiving 0.03 and 0.2 mg/kg i.n. (P = 0.023).
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).
Details of these data and P and F values are listed in Table 1.
Blood.
The variation in leptin concentration (indicated by [125I]leptin) in blood within the observation period was investigated for 0.03 mg/kg (n = 7) and 0.1 mg/kg (n = 5) i.n. administered leptin. The percent applied activity per milliliter as well as leptin content (indicated by [125I]leptin) increased with time in both groups. Comparison between groups showed that the percent applied activity per milliliter in blood was significantly lower in the 0.1 mg/kg i.n. group (P = 0.006; F = 12.339) in all samples (Fig. 2B). Total leptin concentrations (i.e. i.n.-derived labeled plus unlabeled leptin, calculated from the amount of [125I]leptin) were significantly different between groups as well (P = 0.005; F = 12.980) (Fig. 2A), indicating that a greater amount of i.n.-derived leptin gained access to the circulation in the 0.1 mg/kg i.n. group.
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). Considering all tissue samples, there was no significant difference in leptin content (indicated by [125I]leptin) between groups (Fig. 3A). The percent applied activity per gram in the 0.1 mg/kg i.n. group and the 0.2 mg/kg i.n. group were significantly lower than in the 0.03 mg/kg i.n. group (P1/2 = 0.034; P1/3 < 0.001) (Fig. 3B).
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A comparison of transcardially perfused animals with excess unlabeled leptin iv and their respective controls did not reveal any significant differences between brain areas over all groups. Leptin content in selected brain areas was as follows: olfactory bulb (43.5 ± 17.0 and 34.0 ± 13.0 ng/g), hypothalamus (50.7 ± 32.7 and 42.0 ± 22.6 ng/g), and brain average (18.4 ± 5.9 and 19.3 ± 11.1 ng/g) in saline iv and leptin iv, respectively. A third group of animals that had received saline iv but was not transcardially perfused at the end of the experiment was statistically compared with animals with saline iv and transcardial perfusion; a comparison between brain areas over all groups did not reveal statistical differences. Leptin content in nonperfused animals was 72.6 ± 55.5 ng/g in the olfactory bulb, 41.1 ± 28.3 ng/g in the hypothalamus, and 11.9 ± 3.2 ng/g in brain average.
Blood.
For statistical evaluations, blood samples from all time points were included; absolute values are shown only for time points 5 and 30 min.
In transcardially perfused animals, i.n.-derived leptin content in blood (indicated by [125I]leptin) did not differ significantly between groups over the time course between animals receiving unlabeled leptin vs. saline iv, respectively. At time points 5 and 30 min, leptin content in blood was as follows for unlabeled leptin iv and saline iv animals, respectively: 5 min (115.7 ± 25.3 and 143.7 ± 32.8 ng/ml) and 30 min (111.8 ± 29.4 and 101.2 ± 18.1 ng/ml). Blood leptin content in animals that received saline iv but were not subject to subsequent transcardial perfusion were 93.55 ± 29.8 ng/ml at 5 min and 90.6 ± 20.7 ng/ml at 30 min.
Peripheral tissues.
In statistical evaluations, peripheral tissues as mentioned in Table 2 were included; absolute values are shown only for selected organs.
A statistical evaluation of transcardially perfused animals with iv unlabeled leptin compared with iv saline animals did not reveal any significant differences in i.n.-derived leptin content (indicated by [125I]leptin) between tissues over all groups. The i.n.-derived leptin content in selected tissues was as follows in unlabeled leptin iv and saline iv animals, respectively: heart (19.6 ± 5.4 and 14.9 ± 1.9 ng/g), lung (53.9 ± 16.8 and 50.6 ± 15.9 ng/g), liver (93.0 ± 59.1 and 35.7 ± 5.9 ng/g), and kidney (246.1 ± 75.7 and 345 ± 77.1 ng/g). Animals that received saline iv but were not transcardially perfused at the end of the experiment were statistically compared with animals with saline iv and transcardial perfusion. There were no statistical differences between tissues over all groups. The i.n.-derived leptin content (indicated by [125I]leptin) in nonperfused animals was 41.1 ± 10.4 ng/g in heart, 50.8 ± 4.9 ng/g in lung, 70.1 ± 25.4 ng/g in liver, and 233.6 ± 76.9 ng/g in kidney.
Degradation of radioiodinated leptin
The highest quantity of intact radioiodinated leptin was found in brain tissue with 81.5 ± 4.8%. In peripheral tissues, the least degradation was found in the heart with 72.5 ± 12.1% intact [125I]leptin; the lowest quantity of intact leptin in the peripheral tissues was found in lung with 49.7 ± 11.6% (Fig. 4). In blood, the integrity of radioiodinated leptin decreased to 39.1 ± 4.8% within 30 min after application (Fig. 5).
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Discussion |
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), whereas the percent applied activity per gram, which reflects the amount of [125I]leptin within the samples, did not differ in a statistically significant manner between dosage groups for all brain areas (Fig. 1B). Because the amount of iodinated leptin in the three solutions applied (0.03, 0.1, and 0.2 mg/kg) was identical, these results show that the access of the iodinated portion was not affected by the amount of unlabeled leptin, indicating an unhindered transport of leptin from the nasal cavity into the brain within the concentration range tested.
Although there is no statistical difference between groups over all areas in the percent applied activity per gram, there are notable exceptions in distinct areas, in particular in the hypothalamus, where application of the lowest leptin concentration leads to the highest percent applied activity per gram (Fig. 1B).
Even the lowest amount of i.n. applied leptin found in the hypothalamus (5.90 ± 4.17 ng/g), as calculated from [125I]leptin, is supraphysiological. The physiological leptin concentration in the hypothalamus of lean rats is estimated to be in the range of 40 pg/g (18), which is about 150 times lower than the concentration determined in our experiments. Yet it has to be taken into account that in our first set of experiments, in addition to radioiodinated leptin that entered the brain parenchyma, also circulating [125I]leptin within brain blood vessels was detected. However, the results of our transcardial perfusion experiments suggest that there is only a minor contribution of blood [125I]leptin to total brain [125I]leptin, because we did not detect significant differences between transcardially perfused and nonperfused animals over all brain areas.
Taking into account that about 81% of the measured activity in brain tissue originated from intact iodinated leptin, i.n. delivery leads to a leptin concentration (as indicated by [125I]leptin) more than 120 times the physiological concentration, corroborating the hypothesis of direct delivery of leptin from the nasal cavity into the brain.
The applied solution in the 0.1 mg/kg i.n. group was more than three times as concentrated as in the 0.03 mg/kg i.n. group containing the same amount of radioiodinated leptin. However, the resulting blood concentration of i.n.-derived leptin (indicated by [125I]leptin) in the 0.1 mg/kg i.n. group exceeded the concentration of the 0.03 mg/kg i.n. group only 1.4 to 2-fold (Fig. 2A), whereas the amount of radioiodinated leptin was lower in the group that received the higher amount of unlabeled leptin i.n. (the 0.1 mg/kg i.n. group) as indicated by the percent applied activity per milliliter (Fig. 2B). These results suggest that i.n. delivery of increasing leptin doses does not lead to an adequate increase in blood leptin (indicated by [125I]leptin). The concentrations of i.n. leptin (indicated by [125I]leptin) found in peripheral tissues did not differ between groups (Fig. 3A), and the percent applied activity per gram decreased with increasing amounts of unlabeled leptin (Fig. 3B). In addition, these figures show that i.n. applied leptin enters the tissues to different extents. This may be due in part to differences in blood supply, but the amount of exogenous i.n. leptin (indicated by [125I]leptin) in kidneys, liver, and spleen is different despite a similar blood supply of these organs. We assume that the very high amount of [125I]leptin found in kidneys reflects their role as the main site for leptin catabolism. The leptin receptor Ob-Ra is expressed in high quantity in the kidney (19), suggesting a role in the degradation of its ligand. In transfection experiments, employing Chinese hamster ovary cells, it was shown that Ob-Ra ligand complexes are internalized for intracellular degradation (20). We assume that this process takes part in the kidney as well and thus accounts for the accumulation of iodinated leptin in this organ.
The degradation of [125I]leptin was higher in blood than in peripheral tissues, although leptin reaches the peripheral organs via the circulation. This finding may be explained by lesser contact with catabolic enzymes in peripheral tissues through uptake into cells or receptor binding. The percentage of degradation in brain was lower than in plasma samples. This is further supporting a proposed direct access of leptin from the nasal cavity into the brain (17).
A finding of particular interest is that supraphysiological levels (21) of unlabeled leptin in peripheral blood do not interfere with CNS uptake after i.n. application. In this experimental setting, i.n. leptin (indicated by [125I]leptin) that has reached the circulation competes at the BBB with the excess of unlabeled leptin administered i.v. Nevertheless, brain uptake of [125I]leptin is not diminished. This further supports the notion that access of leptin from the nasal cavity into brain is primarily independent of the circulation. The finding that high peripheral leptin levels do not impede CNS uptake of i.n. leptin is of great interest for possible future therapeutic approaches, because it suggests that this mode of application will also be effective in hyperleptinemia, which is associated with obesity. Additional experiments will be necessary to investigate CNS access of i.n. leptin in animals with diet-induced obesity, for this animal model, which best resembles human obesity, is characterized by leptin resistance at the BBB (7).
An interesting and completely unexpected finding was the marked difference in leptin uptake into blood and peripheral tissues, and to a minor extent also into CNS, in the two sets of experiments we have performed. In the first study, employing three different doses of leptin (each containing an identical amount of radiolabeled leptin) in animals killed without previous transcardial perfusion, leptin content derived from i.n. leptin (as indicated by [125I]leptin), for example in kidney was 11.6 ± 1.7 ng/g in animals with 0.2 mg/kg leptin i.n. However, in the second study, designed to investigate the effects of iv unlabeled leptin and of transcardial perfusion on CNS and tissue content of leptin, leptin concentration derived from i.n. application (indicated by [125I]leptin) in kidney was 233.6 ± 76.9 ng/g in the matching group, i.e. the 0.2 mg/kg i.n. leptin group with saline iv and without transcardial perfusion. Similar differences were found in other peripheral tissues and blood. As noted above, differences in i.n.-derived leptin content between the two sets of experiments were smaller in brain than in blood and peripheral tissues but were nevertheless notable; e.g. in the first study, i.n.-derived leptin content in hypothalamus was 13.8 ± 5.7 ng/g as opposed to 41.1 ± 28.3 ng/g in the matching group of the second set of experiments.
Animals from both sets of experiments were treated identically during the experiments, except for a second iv catheter and the absent mini-catheter at the cisterna magna in the second group. These differences in instrumentation associated with the different groups of animals resulted in different durations of surgery: 120 min in the first study as opposed to 60 min in the second study. From the present data, one can only speculate about reasons accounting for the marked differences in uptake in either tissue, such as for example the effects of anesthesia on circulation and thus uptake into the periphery. However, this does not explain the similarly observed, although somewhat smaller, differences in CNS uptake.
As mentioned in the Introduction, there are at least three potential routes of transport for peptides from the nasal cavity into brain that are applicable in the given timeframe of our observation: leakage from CSF to brain parenchyma after entry into perineural spaces between olfactory or trigeminal nerves and ensheathing Schwann cells and/or delivery from lymphatics along a still unknown connection to the subarachnoidal space as well as a not yet clarified direct access to brain parenchyma.
In our experimental design, the first two routes appear less likely than a direct access into brain tissue, because there was no decrease of i.n.-derived leptin (indicated by [125I]leptin) with increasing distance from brain surface, which would be characteristic for entrance into parenchyma by diffusion from the CSF. Cerebellum and frontal cortex/striatum as well as occipital and entorhinal cortex/amygdala share a vast contact area with the CSF, but the amount of exogenous i.n. leptin (as indicated by [125I]leptin) is significantly lower than the one calculated for brain average. In contrast, the hippocampus, which is enclosed by brain tissue, contained a percent applied activity per gram similar to that calculated for brain average.
As mentioned in Materials and Methods (under Distribution of i.n. applied leptin in nonperfused animals), we were unable to determine [125I]leptin in the CSF; nevertheless, a participation of CSF in the transport process cannot be ruled out. In recent experiments by Shimizu et al. (10), it was shown that the i.n. application of leptin led to a 3-fold increase in CSF leptin within 60 min, although the dose used was more than 10-fold the maximum dose used in our experiments. Furthermore, in Shimizu’s experiment, leptin was dissolved in ethanol, possibly provoking a severe damage of the nose epithelium, which certainly enhanced the uptake of the peptide to the subepithelium, from where further transport to the brain took place. To minimize epithelial damage, we used saline as a solvent, together with 1% STDHF. The amphiphilic STDHF has been shown to enhance the uptake of peptides into the nasal submucosa, probably through formation of micelles, the latter facilitating contact between membrane lipids and protein, leading to increased endocytosis. Human volunteers reported tolerable initial disturbances after i.n. application of 1% STDHF solution, which faded within 10 min (22). The surface irritation of the rat nasal mucosa after application of STDHF was low compared with other permeation enhancers (23).
Our results in several ways indicate that the access of i.n. leptin into the brain circumvents the BBB. In experiments employing animals without transcardial perfusion before being killed, the amount of i.n.-derived leptin (indicated by [125I]leptin) found in the hypothalamus exceeded the physiological level by 120 times, although transport across the BBB has been shown to be already saturated at the given blood concentration (18). Even more so, an excess of peripheral leptin, induced by iv application of 0.2 mg/kg unlabeled leptin, as in the second set of experiments, does not significantly reduce CNS uptake of i.n. leptin (indicated by [125I]leptin), suggesting that the access is not via blood. The uptake of leptin (as indicated by [125I]leptin) from the nasal cavity into brain tissue did not appear to be attenuated by an increase in i.n. applied dosage, whereas the uptake into the blood appeared to be hindered by increasing doses. However, additional experiments are necessary to investigate this in more detail. Furthermore, our experiments for the first time show that i.n. delivery of leptin leads to a rapid, dose-dependent increase in leptin in brain. In particular, in the prime center of appetite regulation, the hypothalamus, the resultant leptin content markedly exceeds the physiological concentration. This is in agreement with earlier results of our work, reporting decreased food intake and changes in neuropeptide expression in response to long-term treatment of Wistar rats with i.n. applied leptin (9).
The i.n. delivery of leptin appears to be a promising therapeutic perspective in the treatment of obesity, particularly in light of our finding that i.n. leptin reaches the CNS also in the state of peripheral hyperleptinemia. It will be crucial to further investigate this mode of application and its physiological effects in an animal model of acquired leptin resistance, i.e. diet-induced obesity.
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Acknowledgments |
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STDHF was generously provided by Leo Pharmaceuticals, Ballerup, Denmark.
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Footnotes |
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First Published Online February 9, 2006
1 S.F. and C.S. contributed equally to the manuscript. 
Abbreviations: BBB, Blood-brain barrier; CNS, central nervous system; CSF, cerebrospinal fluid; STDHF, sodium taurodihydrofusidiate; TCA, trichloroacetic acid.
Received August 9, 2005.
Accepted for publication January 27, 2006.
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, 0.03 mg/kg i.n.; 
, 0.1 mg/kg i.n. Data are given as mean ± SEM. P < 0.05.
