Impact of P-Glycoprotein-Mediated Active Efflux on Drug Distribution into Lumbar Cerebrospinal Fluid in Nonhuman Primates.

Estimation of unbound drug concentration in the brain (Cu,brain) is an essential part of central nervous system (CNS) drug development. As a surrogate for Cu,brain in humans and nonhuman primates, drug concentration in cerebrospinal fluid (CCSF) collected by lumbar puncture is often used; however, the predictability of Cu,brain by lumbar CCSF is unclear, particularly for substrates of the active efflux transporter P-glycoprotein (P-gp). Here, we measured lumbar CCSF in cynomolgus monkey after single intravenous administration of 10 test compounds with varying P-gp transport activities. The in vivo lumbar cerebrospinal fluid (CSF)-to-plasma unbound drug concentration ratios (Kp,uu,lumbar CSF) of nonsubstrates or weak substrates of P-gp were in the range 0.885-1.34, whereas those of good substrates of P-gp were in the range 0.195-0.458 and were strongly negatively correlated with in vitro P-gp transport activity. Moreover, concomitant treatment with a P-gp inhibitor, zosuquidar, increased the Kp,uu,lumbar CSF values of the good P-gp substrates, indicating that P-gp-mediated active efflux contributed to the low Kp,uu,lumbar CSF values of these compounds. Compared with the drug concentrations in the cisternal CSF and interstitial fluid (ISF) that we previously determined in cynomolgus monkeys, the lumbar CCSF were more than triple for two and all of the good P-gp substrates examined, respectively. Although lumbar CCSF may overestimate cisternal CSF and ISF concentrations of good P-gp substrates, lumbar CCSF allowed discrimination of good P-gp substrates from the weak and nonsubstrates and can be used to estimate the impact of P-gp-mediated active efflux on drug CNS penetration. SIGNIFICANCE STATEMENT: This is the first study to systematically evaluate the penetration of various P-glycoprotein (P-gp) substrates into lumbar cerebrospinal fluid (CSF) in nonhuman primates. Lumbar CSF may contain >3-fold higher concentrations of good P-gp substrates than interstitial fluid (ISF) and cisternal CSF but was able to discriminate the good substrates from the weak or nonsubstrates. Because lumbar CSF is more accessible than ISF and cisternal CSF in nonhuman primates, these findings will help increase our understanding of drug central nervous system penetration at the nonclinical stage.

Direct communication of the spinal subarachnoid space with the rat dorsal root ganglia.

The anatomical position of the subarachnoid space (SAS) in relation to dorsal root ganglia (DRG) and penetration of tracer from the SAS into DRG were investigated. We used intrathecal injection of methylene blue to visualize the anatomical position of the SAS in relation to DRG and immunostaining of dipeptidyl peptidase IV (DPP-IV) for detecting arachnoid limiting the SAS. Intrathecal administration of fluorescent-conjugated dextran (fluoro-emerald; FE) was used to demonstrate direct communication between the SAS and DRG. Intrathecal injection of methylene blue and DPP-IV immunostaining revealed that SAS delimited by the arachnoid was extended up to the capsule of DRG in a fold-like recess that may reach approximately half of the DRG length. The arachnoid was found in direct contact to the neuronal body-rich area in the angle between dorsal root and DRG as well as between spinal nerve roots at DRG. Particles of FE were found in the cells of DRG capsule, satellite glial cells, interstitial space, as well as in small and medium-sized neurons after intrathecal injection. Penetration of FE from the SAS into the DRG induced an immune reaction expressed by colocalization of FE and immunofluorescence indicating antigen-presenting cells (MHC-II+), activated (ED1+) and resident (ED2+) macrophages, and activation of satellite glial cells (GFAP+). Penetration of lumbar-injected FE into the cervical DRG was greater than that into the lumbar DRG after intrathecal injection of FE into the cisterna magna. Our results demonstrate direct communication between DRG and cerebrospinal fluid in the SAS that can create another pathway for possible propagation of inflammatory and signaling molecules from DRG primary affected by peripheral nerve injury into DRG of remote spinal segments.

Delivery of pineal melatonin to the brain and SCN: role of canaliculi, cerebrospinal fluid, tanycytes and Virchow-Robin perivascular spaces.

Historically, the direct release of pineal melatonin into the capillary bed within the gland has been accepted as the primary route of secretion. Herein, we propose that the major route of melatonin delivery to the brain is after its direct release into the cerebrospinal fluid (CSF) of the third ventricle (3V). Melatonin concentrations in the CSF are not only much higher than in the blood, also, there is a rapid nocturnal rise at darkness onset and precipitous decline of melatonin levels at the time of lights on. Because melatonin is a potent free radical scavenger and antioxidant, we surmise that the elevated CSF levels are necessary to combat the massive free radical damage that the brain would normally endure because of its high utilization of oxygen, the parent molecule of many toxic oxygen metabolites, i.e., free radicals. Additionally, the precise rhythm of CSF melatonin provides the master circadian clock, the suprachiasmatic nucleus, with highly accurate chronobiotic information regarding the duration of the dark period. We predict that the discharge of melatonin directly into the 3V is aided by a number of epithalamic structures that have heretofore been overlooked; these include interpinealocyte canaliculi and evaginations of the posterodorsal 3V that directly abut the pineal. Moreover, the presence of tanycytes in the pineal recess and/or a discontinuous ependymal lining in the pineal recess allows melatonin ready access to the CSF. From the ventricles melatonin enters the brain by diffusion and by transport through tanycytes. Melatonin-rich CSF also circulates through the aqueduct and eventually into the subarachnoid space. From the subarachnoid space surrounding the brain, melatonin penetrates into the deepest portions of the neural tissue via the Virchow-Robin perivascular spaces from where it diffuses into the neural parenchyma. Because of the high level of pineal-derived melatonin in the CSF, all portions of the brain are better shielded from oxidative stress resulting from toxic oxygen derivatives.

Primo vascular system in the subarachnoid space of the spinal cord of a pig.

The primo vascular system was recently observed in the central nervous systems of rabbits and rats, but no investigations in large animals have been reported. In the present work we found a putative primo vascular system in the spinal cord of a pig. We obtained spines from four healthy pigs and fixed them with paraformaldehyde. The primo vessels were expected to lie in the subarachnoid space between the pia mater and the arachnoid mater. The composite of three membranes (the pia, the arachnoid, and the dura maters) wrapping the spinal cord was peeled off, isolated from the spine, and put on a slide glass. This composite was stained with 4',6'-Diamidino-2-phenylindole (DAPI) and phalloidin to show the nuclei and the f-actin, respectively, in the cells of the primo vessels. We observed eleven pieces of the putative primo vessels in the subarachnoid space of the spines at the thoracic spinal nerve area. They had the typical rod-shaped nuclei distributed in a broken line, and f-actin signals around nuclei. The lengths of the nuclei were 12-15 µm, and the thicknesses of the primo vessels were 8∼20 µm, which were consistent with other primo vessels that had been observed in the various organs of rabbits, rats, and mice. In addition, we observed branching of the primo vessels, which is again an expected result from previous works. In conclusion, a primo vessel was observed in the subarachnoid space of the spinal cord of a pig. This was the first observation of a primo vessel in a large animal, and the staining method used to observe the primo vessel in a fixed sample was newly developed in this work.

Dynamics of distribution of 3H-inulin between the cerebrospinal fluid compartments.

Since the distribution of substances between various cerebrospinal fluid (CSF) compartments is poorly understood, we studied (3)H-inulin distribution, over time, after its injection into cisterna magna (CM) or lateral ventricle (LV) or cisterna corporis callosi (CCC) in dogs. After the injection into CM (3)H-inulin was well distributed to cisterna basalis (CB), lumbar (LSS) and cortical (CSS) subarachnoid spaces and less distributed to LV. When injected in LV (3)H-inulin was well distributed to all CSF compartments. However, after injection into CCC (3)H-inulin was mostly localized in CCC and adjacent CSS, while its concentrations were much lower in CM and CB and very low in LSS and LV. Concentrations of (3)H-inulin in venous plasma of superior sagittal sinus and arterial plasma were very low and did not differ significantly, while its concentration in urine was very high. In (3)H-inulin distribution it seems that two simultaneous processes are relevant: a) the pulsation of CSF with to-and-fro displacement of CSF and its mixing, carrying (3)H-inulin in all directions, and b) the passage of (3)H-inulin from CSF into nervous parenchyma and its rapid distribution to a huge surface area of capillaries by vessels pulsations. (3)H-inulin then slowly diffuses across capillary walls into the bloodstream to be eliminated in the urine.

Comparison of brain extracellular fluid, brain tissue, cerebrospinal fluid, and serum concentrations of antiepileptic drugs measured intraoperatively in patients with intractable epilepsy.

PURPOSE: The mechanisms of drug resistance in epilepsy are only incompletely understood. According to a current concept, overexpression of drug efflux transporters at the blood-brain barrier may reduce levels of antiepileptic drugs (AEDs) in epileptogenic brain tissue. Increased expression of drug efflux transporters such as P-glycoprotein has been found in brain tissue surgically resected from patients with medically intractable epilepsy, but it is not known whether this leads to decreased extracellular (interstitial) AED concentrations in affected brain regions. This prompted us to measure concentrations of AEDs in the extracellular space of human neocortical tissue by using intraoperative microdialysis (IOMD) in those parts of the brain that had to be removed for therapeutic reasons. For comparison, AED levels were determined in brain tissue, subarachnoid CSF, and serum. METHODS: Concentrations of carbamazepine (CBZ), 10-hydroxy-carbazepine (10-OH-CZ, metabolite of oxcarbazepine), lamotrigine (LTG), levetiracetam (LEV), topiramate, or phenytoin were determined by using one to four catheters during IOMD in the medial temporal gyrus. Furthermore, to calculate the individual recovery of every catheter, an in vitro microdialysis was performed with ultrafiltrate of serum concurrently obtained from the respective patient. In addition, AED levels were determined in the resected brain tissue, CSF, and serum of the same patients. Altogether 22 pharmacoresistant epilepsy patients (nine male, 13 female patients; age 15-54 years) with complex partial seizures or secondarily generalized seizures were involved. In a first series, IOMD samples 40 min after beginning of the microdialysis (flow rate, 1 microl/min), and in a second series, continuous measurements 25, 30, 35, and 40 min from the beginning were evaluated (flow rate, 2 microl/min). With in vitro recovery data of the individual catheters, the concentration in the extracellular space (ECS) was estimated. RESULTS: AED concentrations in the ECS of the cortex measured by catheters located at a distance of 0.6 cm differed markedly in some patients, whereas concentrations in the ultrafiltrate of the serum of the respective patients measured with the same catheters varied only slightly. Furthermore, ECS concentrations related to the ultrafiltrate of serum showed considerable interindividual variations. The high intra- and interindividual variation of ECS concentrations is demonstrated by the low correlation between concentrations in ECS and the ultrafiltrate of serum (CBZ, r= 0.41; 10-OH-CZ, r= 0.42; LTG, r= 0.27) in contrast to the high correlation between brain tissue concentration and the ultrafiltrate of serum (CBZ, r= 0.97; 10-OH-CZ, r= 0.88; LTG, r= 0.98) in the same group of patients. When comparing AED concentrations in the ECS with those in the CSF, ECS concentrations were significantly lower for CBZ, 10-OH-CZ, LTG, and LEV. CONCLUSIONS: The data demonstrate that AED concentrations show a considerable intraindividual and interindividual variation in the ECS of cortical regions. Furthermore, the ECS concentration of several AEDs is significantly lower than their CSF concentration in patients with intractable epilepsy. However, in the absence of data from nonepileptic tissues, it is not possible to judge whether the present findings relate to overexpression of multidrug transporters in the brain. Instead, the present study illustrates the methodologic difficulties involved in performing IOMD studies in patients and may thus be helpful for future approaches aimed at elucidating the role of multidrug transporters in epilepsy.

Dissemination of fat in CSF: a common finding following translabyrinthine acoustic neuroma surgery*.

OBJECTIVES: Following translabyrinthine craniotomy the temporal bone defect is commonly obliterated using a free autologous fat graft. In this series the dura was put back in place but not closed primarily. As the fat graft remains in direct contact with the cerebro spinal fluid (CSF) there is potential for dispersal of fat within the CSF space. This paper aims to determine the frequency of such CSF fat dissemination and its clinical significance. DESIGN: A retrospective review of translabyrinthine acoustic neuroma removal with free fat autograft obliteration of the temporal bone defect between the years 1997 and 2000. SETTING: Tertiary referral oto-neurosurgical centre. Postoperative magnetic resonance (MR) imaging. PARTICIPANTS: All translabyrinthine patients who had postoperative MR imaging were included. Twenty-six cases were identified. Age range was 13-70 years. Fourteen were male patients. MAIN OUTCOME MEASURES: Evidence of CSF fat dissemination on MR and patients' clinical findings. RESULTS: Twenty-two of the 26 scans (85%) demonstrated evidence of fat dissemination into the subarachnoid CSF spaces in the form of microemboli. The cerebellopontine angle was the most common site involved. No evidence of ventricular dilation or any other abnormality was noted. There was no relationship between the presence or extent of fat microembolization and the patients' clinical course. CONCLUSIONS: This study suggests that free fat placed in temporal bone defects commonly migrate into the subarachnoid space and subsequently move around in these spaces. This is not associated with any complications such as hydrocephalus, meningitis or prolonged postoperative headache.

Choroid plexus epithelium (normal and neoplastic) expresses synaptophysin. A potentially useful aid in differentiating carcinoma of the choroid plexus from metastatic papillary carcinomas.

As an incidental finding in paraffin sections of brain tissue used as positive controls for synaptophysin immunostain, the cytoplasm of choroid plexus epithelium present was found to stain strongly positively for this substance. This was subsequently found to be the case in normal choroid plexuses in autopsy material from infancy to old age, as well as in epithelial cells of papillomas and carcinomas of the choroid plexus. The latter findings may prove useful in differentiating choroid plexus carcinomas from metastatic papillary carcinomas of extracerebral origin with the exception of neuroendocrine carcinomas of various sites that are usually positive for synaptophysin.

Identification of cell types producing RANTES, MIP-1 alpha and MIP-1 beta in rat experimental autoimmune encephalomyelitis by in situ hybridization.

The chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta are members of the beta-family of chemokines and potent chemoattractants for lymphocytes and monocytes. To investigate the factors which regulate lymphocyte traffic in experimental autoimmune encephalomyelitis (EAE), we studied, by in situ hybridization analysis, the kinetics of mRNA expression and the potent cellular sources of RANTES, MIP-1 alpha and MIP-1 beta in the central nervous system (CNS) during the course of EAE. RANTES-positive cells appeared in the subarachnoid space and infiltrated the subpial region at around day 10, increased to a peak at days 12-13 and then decreased following the resolution of the acute phase of EAE, though elevated RANTES message expressions still remained on chronic subclinical stage. Most of RANTES positive cells were identified as T-lymphocytes located mainly around blood vessels, by combined studies of in situ hybridization and immunohistochemistry. The remainder of the RANTES-positive cells were astrocytes and macrophages/microglia. MIP-1 alpha and MIP-1 beta mRNA-positive cells appeared around day 10, increased further on days 12-13 and then gradually decreased. Most of the MIP-1 alpha- and MIP-1 beta-positive mononuclear cells were located around blood vessels. The kinetics of RANTES, MIP-1 alpha and MIP-1 beta expression paralleled those of the recruitment of infiltrating inflammatory cells and disease severity. Our observations support the possibility that chemokine production by T-cells, macrophages and astrocytes lead to the infiltration of inflammatory cells into the CNS parenchyma during the acute phase of EAE.

CSF levels of prostaglandins, especially the level of prostaglandin D2, are correlated with increasing propensity towards sleep in rats.

The concentration of PGD2, PGE2, and of PGF2 alpha was measured in the cerebrospinal fluid (CSF) collected from the cisterna magna of conscious rats (n = 29), which, chronically implanted with a catheter for the CSF sampling, underwent deprivation of daytime sleep. Significant elevation of the CSF level of PGD2 was observed following 2.5-h sleep deprivation (SD), and the elevation became more marked following 5- and 10-h SD, apparently reaching the maximum at 5-h SD (703 +/- 140 pg/ml (mean +/- S.E.M.) for baseline vs. 1734 +/- 363 pg/ml for SD, n = 10). The levels of PGE2, and PGF2 alpha also significantly increased following 5- and 10-h SD, but not following 2.5-h SD. It is unlikely that these changes were simply caused by some responses of the animals to stress stimuli, because stress stimuli derived from restraint of the animal at the supine position to a board for 1 h did not produce any acute responses in the CSF levels of prostaglandins (n = 13). In a different group of animals (n = 11) implanted with electrodes for recording electroencephalogram (EEG) and electromyogram (EMG) in addition to the catheter, the levels of the prostaglandins in CSF were determined for slow-wave sleep (SWS) and wakefulness in the day and for SWS and wakefulness in the night. The highest PGD2 value was obtained at daytime SWS, whereas the lowest was at night wakefulness; furthermore, a significant difference was observed between SWS and wakefulness rather than between day and night. The CSF level of PGE2 also showed a similar tendency. In an additional group of animals (n = 6), not only PGD2 but also PGE2 and PGF2 alpha significantly increased the sleeping time of the animal when applied into the subarachnoid space underlying the ventral surface area of the rostral basal forebrain, the previously defined site of action for the sleep-promoting effect of PGD2. The promotion of sleep by PGE2 applied to the subarachnoid space was an effect completely opposite to the well-established awaking effect of the same prostaglandin demonstrated in the hypothalamic region in a series of previous studies. Based on these results, we conclude that increases in CSF levels of prostaglandins, especially that of PGD2, are correlated in rats with heightened propensity towards sleep and further with the depth of sleep under normal as well as SD conditions.

Immunological aspects of kaolin-induced hydrocephalus.

Adult female Sprague Dawley rats were administrated 0.1 ml Kaolin (250 mg/ml) into cisterna magna. One, 4 and 8 weeks later, brains were analyzed using antibodies against MHC class I (OX18), MHC class II (OX6), CD4 (OX38), CD8 (OX8), OX42, ED1, NF, GFAP, AChE and TH. Remarkably high numbers of T lymphocytes, and OX42- and ED1-positive macrophages were found aggregated in subarachnoid spaces, and in the third and fourth ventricles. Marked aggregations of ED1-positive reactive microglial cells were also found in paraventricular structures, medial septum, retrosplenic cortex and commissural structures. However, no such cells were found in hippocampus. ED1-positive areas were also positive for round cells with a rim of MHC I fluorescent cytoplasm as well as for OX42-positive cells and MHC II positive microglial cells. At week 1, in ventro-frontal areas of cortex, CD8-positive cells and MHC I positive astroglial fibers were detected. At week 1, MHC I positive ramified microglial cells were also recognized in almost the entire brain. These positive cells gradually decreased with time and finally remained rounded with a rim of fluorescent cytoplasm. In addition, ED1 positive partly ramified microglial cells could be recognized in corpus callosum, probably representing cells in transition between ramified and reactive microglia. CD8+ cells entered ventral brain structures, and were found in the horizontal diagonal band at week 4, and had disappeared at week 8. Finally in cortex, ED1 positive microglial cells could be identified only in the retrosplenic cortex, and there were also "dark shrunken neurons" in light microscopic stainings. However, there was only a moderate GFAP positive gliosis. In conclusion, kaolin-induced hydrocephalus leads to immune reactions in several defined areas such as cholinergic systems, corpus callosum, circumventricular organs, pontine cerebellar peduncles and the vestibular nucleus.

The neuronal environment after subarachnoid haemorrhage--correlation of amino acid and nucleoside levels with post-operative recovery.

The study explores biochemically the neuronal environment adjacent to a subarachnoid haemorrhage in 11 patients after neurosurgical clipping of an arterial aneurysm. Extracellular fluid (ECF) from the rectus gyrus and subarachnoid fluid (SAF) were sampled with microdialysis probes. The concentrations of amino acids and nucleosides were monitored in 60 min samples collected over 2-4 days. The patients were 33-67 years of age. Surgery was performed 0-5 days after rupture of the aneurysm in 8 patients. One patient was operated on after 15 months. Clipping of aneurysms without prior haemorrhage was performed in two cases. Markedly elevated concentrations of the excitatory amino acid glutamate was observed in the ECF of only one patient who underwent surgery within 8 hours after the haemorrhage. Moderate glutamate elevations were seen in two patients and of aspartate in another patient. Five patients displayed periods of varying length of specifically elevated taurine concentrations in ECF or SAF. Transient periods of high concentrations of glycine and serine were seen in two patients. Even though average concentrations of all amino acids were fairly similar in the ECF and SAF, the pattern of changes vs. time differed markedly in the two compartments. Presently, we conclude that the level of consciousness in the post-operative phase was inversely related to total amino acid concentration in the ECF. Furthermore, while the ECF concentrations of taurine and glycine increased both specifically and transiently in several patients, excitatory amino acid levels were not appreciably elevated subsequent to the neurosurgical intervention.(ABSTRACT TRUNCATED AT 250 WORDS)