Gene transfer to the spinal cord neural scar with lentiviral vectors: predominant transgene expression in astrocytes but not in meningeal cells.

Viral vector-mediated overexpression of neurotrophins in cells constituting the neural scar may represent a powerful approach to rendering scar tissue of a central nervous system (CNS) lesion permissive for neuronal regrowth. In this study a lentiviral vector encoding green fluorescent protein (LV-GFP) was injected in and around the neural scar 2 weeks after a dorsal column lesion in the rat spinal cord in order to analyze transduction characteristics of the neural scar after 4, 7, and 14 days. GFP expression was found at all points after injection and increased from 4 to 7 days, with no apparent difference observed between 7 and 14 days. The core of the lesion was virtually devoid of GFP signal despite direct vector injections in this area. The colocalization of GFP with specific cell markers (GFAP, vimentin, Raldh2, NeuN, OX-42, ED-1, and NG-2) indicated that the predominant cells transduced in the rim of the lesion were astrocytes, with neurons, microglia, oligodendrocyte precursors, and macrophages transduced to a lesser extent. None of the Raldh2-positive meningeal cells, present in the core of the scar, expressed GFP. In vitro meningeal cells were readily transduced, indicating that in vivo the formation of an extracellular matrix might prevent LV particles from transducing cells in the core of the scar. Because astrocytes are important cellular constituents of the glial scar after CNS injury, transduction of astrocytes with LV vectors encoding neurotrophic factors like BDNF or NT-3 may be used to enhance regeneration of severed axonal tracts through or along boundaries of a CNS lesion.

A new HBsAg screening assay designed for sensitive detection of HBsAg subtypes and variants.

The design of a new HBsAg screening assay, the Hepanostika HBsAg Ultra is based on the use of monoclonal antibodies raised against native wild-type HBsAg and reactive with HBsAg in which the common 'a'-determinant is modified by site-directed mutagenesis of four of the cysteine moieties. The design was checked using the same cysteine variants and samples from patients known to be infected with HBsAg variants. The results found were compared with other state-of-the-art commercial screening assays. The design of the Hepanostika HBsAg Ultra enabled detection of all variant HBsAg-positive samples in contrast to the other commercial assays. An additional 980 samples were tested to assess the specificity and sensitivity of the Hepanostika HBsAg Ultra. Screening of presumed negative serum and plasma samples resulted in a specificity of 100%. This makes the Hepanostika HBsAg Ultra the first screening assay with a design able to detect HBsAg variants with high sensitivity and specificity.

[Comments on the Dutch Institute for Health Care Improvement's standard code of practice entitled, "Donation of fetal tissue"]. / Opmerkingen bij het CBO-modelreglement 'Terbeschikkingstelling van foetaal weefsel'.

Under the auspices of the Kwaliteitsinstituut voor de Gezondheidszorg CBO [Dutch Institute for Healthcare Improvement] a standard code of practice was developed as a template for local institutional codes to implement the Law on Foetal Tissue. It is a useful model code, but arguments should have been outlined more explicitly, notably in instances where the code adopts a somewhat stricter position than the Law. The following remarks pertain to the model code: 1. It may be argued that the inclusion or exclusion of 12-15-year-old pregnant girls should be relative to the privacy-related sensitivity of the use of foetal tissue. 2. Transplantation requires additional tests for the safety of the recipient, including testing for HIV/AIDS. A pregnant woman's permission for such testing should not be taken for granted. 3. The abortion technique may be modified in view of the subsequent use of foetal tissue if the woman consents to the modification, with the prerequisites that the modification does not harm the woman and that any potential sensation of pain by the foetus must be minimised.

Adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function.

To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding for green fluorescent protein or saline. AAV-BDNF- and AAV-NT-3-transduced 293 human kidney cells produced and secreted BDNF or NT-3, respectively, in vitro. The secreted neurotrophins were biologically active; they both promoted outgrowth of sensory neurites in vitro. In vivo, transgene expression was observed predominantly in neurons for at least 16 weeks after injection. Compared with controls, a modest though significant improvement in hind-limb function was found in rats that received AAV-BDNF and AAV-NT-3. Retrograde tracing demonstrated that twice as many neurons with processes extending toward the Schwann cell graft were present in the second lumbar cord segment of AAV-BDNF- and AAV-NT-3-injected animals compared with controls. We found no evidence, however, for growth of regenerated axons from the Schwann cell implant into the caudal cord. Our results suggest that AAV vector-mediated overexpression of BDNF and NT-3 in the cord caudal to a Schwann cell bridge modified the local lumbar axonal circuitry, which was beneficial for locomotor function.

Vincristine induced apoptosis in acute lymphoblastic leukaemia cells: a mitochondrial controlled pathway regulated by reactive oxygen species?

Vincristine (VCR), a microtubule interfering anti-cancer agent, plays a key role in the treatment of childhood acute lymphoblastic leukaemia (ALL). The route of VCR induced apoptosis in ALL cells is not well defined. In this study we demonstrated caspase-9 and -3 activation in vivo in bone marrow leukaemic cells of a child with newly diagnosed ALL, after treatment with a single dose of VCR. We hypothesized that VCR induced apoptosis in ALL cells proceeds by a mitochondrial controlled pathway. We further studied the route of VCR induced apoptosis in Jurkat acute lymphoblastic leukaemia cells. First we showed that VCR induces activation of caspase-9 and -3 in Jurkat cells. With the caspase-9 inhibitor Z-LEHD-FMK we proved that caspase-9 was activated prior to caspase-3. Loss of mitochondrial transmembrane potential was independent of caspase-9 activation. To confirm the mitochondrial role in VCR induced apoptosis, the effect of blocking the mitochondrial route upstream of caspase-9 activation was investigated at two different levels: reactive oxygen species (ROS) scavenging and Bcl-2 overexpression. Generation of ROS was detected early in Jurkat cells during VCR exposure. Ascorbic acid, a ROS scavenger, inhibited ROS generation as well as caspase-9 and -3 activation and cell death induced by VCR. Furthermore, in Bcl-2 overexpressing Jurkat cells mitochondrial membrane potential changes, caspase-9 and -3 activation and cell death upon VCR exposure were decreased, in comparison to parental Jurkat cells. However, generation of ROS was not decreased in Jurkat cells with Bcl-2 overexpression. We concluded that ROS play a regulatory role in the initial phase of a mitochondrial controlled pathway of VCR induced apoptosis in Jurkat cells.

Intravitreal injection of adeno-associated viral vectors results in the transduction of different types of retinal neurons in neonatal and adult rats: a comparison with lentiviral vectors.

Replication-deficient viral vectors encoding the marker gene green fluorescent protein (GFP) were injected into the vitreous of newborn, juvenile (P14), and adult rats. We tested two different types of modified virus: adeno-associated viral-2-GFP (AAV-GFP) and lentiviral-GFP vectors (LV-GFP). The extent of retinal cell transduction in different-aged animals was compared 7, 21, and 70 days after eye injections. At all postinjection times, LV-GFP transduction was mostly limited to pigment epithelium and cells in sclera and choroid. In contrast, transduction of large numbers of neural retinal cells was seen 21 and 70 days after AAV-GFP injections. AAV-GFP predominantly transduced neurons, although GFP-positive Müller cells were seen. All neuronal classes were labeled, but the extent of transduction for a given class varied depending on injection age. After P0 injections about 50% of transduced cells were photoreceptors and 30-40% were amacrine or bipolar cells. After adult injections 60-70% of transduced cells were retinal ganglion cells. In adults many GFP-positive retinal axons were traced through the optic nerve/tract and terminal arbors were visualized in central targets.

[A European discussion about stem cells for therapeutic use]. / Een Europese discussie over stamcellen voor therapie.

Stem cells as a source material for growing cellular transplants to repair dysfunctional organs appear to be a new challenge for medical science. Though stem cells are also present in foetal and adult organs, embryonic stem cells from the pre-implantation embryo in particular have the potency to proliferate easily in vitro and the capacity to differentiate into all the body's organ-specific cells. Therefore, these are the ideal cells for developing new cell transplantation therapies for diseases such as Parkinson's disease, diabetes mellitus and heart failure. The use of spare in vitro fertilization (IVF) embryos or pre-implantation embryos specially created to harvest human embryonic stem cells is, however, controversial and an ethical problem. In a European discussion platform organised by the European Commission Research Directorate-General, the status quo of the progress was presented and subsequently commented upon and discussed in terms of medical-ethical, social, industrial and patient interests. The expectations of this new medical technology were high, but clinical trials seem only acceptable once the in vitro differentiation of stem cells can be adequately controlled and once it is known how in vitro prepared stem cells behave after implantation. The ethical justification of the use of in vitro pre-implantation embryos remains controversial. The prevailing view is that the interests of severely ill patients for whom no adequate therapy exists, surmounts the interest of protection of a human in vitro pre-implantation embryo, regardless of whether it was the result of IVF or of transplantation of a somatic cell nucleus of the patient in an enucleated donor egg cell (therapeutic cloning).

Ethical guidance on human embryonic and fetal tissue transplantation: a European overview.

This article presents an overview of regulations, guidelines and societal debates in eight member states of the EC about a) embryonic and fetal tissue transplantation (EFTT), and b) the use of human embryonic stem cells (hES cells) for research into cell therapy, including 'therapeutic' cloning. There appears to be a broad acceptance of EFTT in these countries. In most countries guidance has been developed. There is a 'strong' consensus about some of the central conditions for 'good clinical practice' regarding EFTT. International differences concern, amongst others, some of the informed consent issues involved, and the questions whether an intermediary organisation is necessary, whether the methods of abortion may be influenced by the possible use of EFT, and whether EFTT should only be used for the experimental treatment of rare disorders. The potential use of hES cells for research into cell therapy has given a new impetus to the debate about (human) embryo research. The therapeutic prospects with regard to the retrieval and research use of hES cells appear to function as a catalyst for the introduction of less restrictive regulations concerning research with spare embryos, at least in some European countries. It remains to be seen whether the prospect of treating patients suffering from serious disorders with transplants produced by therapeutic cloning will decrease the societal and moral resistance to allowing the generation of embryos for 'instrumental' use.

Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord.

Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We report, to our knowledge for the first time, that purified cultures of primary OEG are capable of expressing a foreign gene following adenoviral (AdV) and lentiviral (LV) vector-mediated gene transfer. OEG implants subjected to AdV vector-mediated gene transfer expressed high levels of transgenic protein in both intact and lesioned spinal cord at 7 days after implantation. However, the levels of transgene expression gradually declined between 7 and 30 days after implantation in lesioned spinal cord. Infection with LV vectors resulted in stable transduction of primary OEG cultures and transgene expression persisted for at least 4 months after implantation. Genetic engineering of OEG opens the possibility of expressing additional neurotrophic genes and create optimal 'bridging' substrates to support spinal axon regeneration. Furthermore, stable transduction of OEG allows us to reliably study the behaviour of implanted cells and to obtain better understanding of their regeneration supporting properties.

Adenoviral vector-mediated expression of neurotrophin-3 increases neuronal survival in suprachiasmatic nucleus grafts.

To improve transplantation results of fetal suprachiasmatic nucleus (SCN) in SCN-lesioned (SCNX) rats, grafts were ex vivo transduced with an adenoviral vector encoding for neurotrophin-3 (AdNT-3) before implantation. Mock- and AdLacZ-transduced grafts were used as controls. First, transplants were evaluated microscopically and by image analysis for the presence of vasopressinergic (VPergic) and vasoactive intestinal polypeptidergic (VIPergic) SCN neurons at 10 weeks or later postgrafting. Ex vivo AdNT-3-transduced transplants displayed increased volume areas of VPergic and VIPergic SCN cells in comparison with those in mock- and AdLacZ-transduced transplants, but significantly improved graft-to-host VPergic and VIPergic SCN fiber growth was not reached (though AdNT-3-transduced transplants tended to grow more VPergic fibers into the brain of VP-deficient SCNX Brattleboro rat recipients, which were chosen as recipients to circumvent the presence of non-SCN VP fiber staining). Second, a small group of arrhythmic Wistar rats received AdNT-3- or control-treated SCN grafts while continuously on-line for the monitoring of overt circadian activities in the pre- and postgrafting periods. The results indicated that ex vivo transduced SCN grafts can still restore arrhythmia, but that the NT-3-mediated anatomical improvements of the grafting results were not sufficient to enhance efficacy of reinstatement of circadian rhythm in SCN-lesioned rats. However, in this group VIP staining volume area, not VP staining volume area, correlated significantly with reinstatement of circadian rhythm.

The suprachiasmatic nucleus and the circadian time-keeping system revisited.

Many physiological and behavioral processes show circadian rhythms which are generated by an internal time-keeping system, the biological clock. In rodents, evidence from a variety of studies has shown the suprachiasmatic nucleus (SCN) to be the site of the master pacemaker controlling circadian rhythms. The clock of the SCN oscillates with a near 24-h period but is entrained to solar day/night rhythm by light. Much progress has been made recently in understanding the mechanisms of the circadian system of the SCN, its inputs for entrainment and its outputs for transfer of the rhythm to the rest of the brain. The present review summarizes these new developments concerning the properties of the SCN and the mechanisms of circadian time-keeping. First, we will summarize data concerning the anatomical and physiological organization of the SCN, including the roles of SCN neuropeptide/neurotransmitter systems, and our current knowledge of SCN input and output pathways. Second, we will discuss SCN transplantation studies and how they have contributed to knowledge of the intrinsic properties of the SCN, communication between the SCN and its targets, and age-related changes in the circadian system. Third, recent findings concerning the genes and molecules involved in the intrinsic pacemaker mechanisms of insect and mammalian clocks will be reviewed. Finally, we will discuss exciting new possibilities concerning the use of viral vector-mediated gene transfer as an approach to investigate mechanisms of circadian time-keeping.

Intercostal nerve implants transduced with an adenoviral vector encoding neurotrophin-3 promote regrowth of injured rat corticospinal tract fibers and improve hindlimb function.

Following injury to central nervous tissues, damaged neurons are unable to regenerate their axons spontaneously. Implantation of peripheral nerves into the CNS, however, does result in axonal regeneration into these transplants and is one of the most powerful strategies to promote CNS regeneration. In the present study implantation of peripheral nerve bridges following dorsal hemisection is combined with ex vivo gene transfer with adenoviral vectors encoding neurotrophin-3 (Ad-NT-3) to examine whether this would stimulate regeneration of one of the long descending tracts of the spinal cord, the corticospinal tract (CST), into and beyond the peripheral nerve implant. We chose to use an adenoviral vector encoding NT-3 because CST axons are sensitive to this neurotrophin and Schwann cells in peripheral nerve implants do not express this neurotrophin. At 16 weeks postimplantation of Ad-NT-3-transduced intercostal nerves, approximately three- to fourfold more of the anterogradely traced corticospinal tract fibers had regrown their axons through gray matter below the lesion site when compared to control animals. Regrowth of CST fibers occurred over more than 8 mm distal to the lesion site. No regenerating CST fibers were, however, observed into the transduced peripheral implant. Animals with a peripheral nerve transduced with Ad-NT-3 also exhibited improved function of the hindlimbs when compared to control animals treated with an adenoviral vector encoding LacZ. Thus, transient overexpression of NT-3 in peripheral nerve tissue bridges is apparently sufficient to stimulate regrowth of CST fibers and to promote recovery of hindlimb function, but does not result in regeneration of CST fibers into such transplants. Taken together, combining an established neurotransplantation approach with viral vector-gene transfer promotes the regrowth of injured CST fibers through gray matter and improves the recovery of hindlimb function.

The use of adenoviral vectors and ex vivo transduced neurotransplants: towards promotion of neuroregeneration.

Regeneration of injured axons following injury depends on a delicate balance between growth-promoting and growth-inhibiting factors. Overexpression of neurotrophin genes seems a promising strategy to promote regeneration. Trophic genes can be overexpressed at the site of injury at the axonal stumps, or at the perikaryal level of the injured neuron. Transduction of the neural cells can be achieved by applying adenoviral vectors, either directly in vivo or-in the case of neurotransplantation as an ex vivo approach. In both cases it would create a more permissive environment for axonal growth and therefore in functional regeneration. In this article, the feasibility of the use of adenoviral vectors in several neuroregeneration models--in particularly in spinal cord lesion models and the biological clock transplantation model--is illustrated. The results show that the adenoviral vectors can be a powerful tool to study the effects of overexpression of genes in an in vivo paradigm of nerve regeneration or nerve outgrowth. The potential use of adenoviral vectors and ex vivo transduced neurotransplants is discussed.

Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition.

Enoyl acyl carrier protein reductase (ENR) is involved in fatty acid biosynthesis. In Escherichia coli this enzyme is the target for the experimental family of antibacterial agents, the diazaborines, and for triclosan, a broad spectrum antimicrobial agent. Biochemical studies have suggested that the mechanism of diazaborine inhibition is dependent on NAD(+) and not NADH, and resistance of Brassica napus ENR to diazaborines is thought to be due to the replacement of a glycine in the active site of the E. coli enzyme by an alanine at position 138 in the plant homologue. We present here an x-ray analysis of crystals of B. napus ENR A138G grown in the presence of either NAD(+) or NADH and the structures of the corresponding ternary complexes with thienodiazaborine obtained either by soaking the drug into the crystals or by co-crystallization of the mutant with NAD(+) and diazaborine. Analysis of the ENR A138G complex with diazaborine and NAD(+) shows that the site of diazaborine binding is remarkably close to that reported for E. coli ENR. However, the structure of the ternary ENR A138G-NAD(+)-diazaborine complex obtained using co-crystallization reveals a previously unobserved conformational change affecting 11 residues that flank the active site and move closer to the nicotinamide moiety making extensive van der Waals contacts with diazaborine. Considerations of the mode of substrate binding suggest that this conformational change may reflect a structure of ENR that is important in catalysis.

Vincristine pharmacokinetics after repetitive dosing in children.

PURPOSE: We studied vincristine disposition after 169 weekly i.v. bolus injections in 32 children with acute lymphoblastic leukemia, non-Hodgkin lymphoma, or Wilms' tumor. The aim of the study was to determine intrapatient and interpatient variability in vincristine disposition and demographic, clinical, and biochemical characteristics influencing this variability. METHODS: Vincristine plasma concentrations were measured by a high-performance liquid chromatography assay with electrochemical detection. A limited sampling strategy was used based on a bayesian parameter estimation algorithm that is part of the ADAPT II software package. A two-compartment, first-order model was fitted to the data, and pharmacokinetic parameters were calculated from the model using the ADAPT II software. For statistical analysis, analysis of variance (ANOVA), t test, simple and multiple regression analysis, and non-parametric or robust equivalents were used. RESULTS: Results showed a large intrapatient and interpatient variability in distribution half-life, elimination half-life, total body clearance, apparent volume of distribution at steady state, and area under the concentration-time curve. Intrapatient variability was significantly smaller than interpatient variability for all these parameters except distribution half-life. The diagnosis or treatment protocol turned out to be the most predictive characteristic; leukemia and non-Hodgkin lymphoma patients had a significantly higher total body clearance than Wilms' tumor patients. CONCLUSIONS: We conclude that both intrapatient and interpatient variability in vincristine pharmacokinetics is large in pediatric cancer patients and that variability, although significantly influenced by diagnosis, largely remains unpredictable.

Ontogeny of neurotrophin receptor trkC expression in the rat forebrain and anterior hypothalamus with emphasis on the suprachiasmatic nucleus.

There is little information about neurotrophic regulation in the developing rat hypothalamus. In the present study, we therefore examined the expression of neurotrophin receptor TrkC in the developing forebrain and hypothalamus. In situ hybridization of coronal sections revealed that on the 15th day of gestation, trkC messenger RNA expression is homogeneously distributed over the neocortex, septum, thalamus, hypothalamus, hippocampus, rhinencephalon and the amygdala. Exceptions were the anteroventral nucleus of the hypothalamus and the striatum, which showed higher levels of trkC messenger RNA expression, and the germinal zones which were devoid of trkC messenger RNA. After birth, the homogeneous staining pattern changes into a heterogeneous staining pattern like that found in adulthood. TrkC expression is observed in the area of the suprachiasmatic nucleus as early as E17 and continues until adulthood. The presence of the TrkC receptor in the E17 suprachiasmatic nucleus suggests that neurotrophin-3 plays a role in development of this structure and that application of neurotrophin-3 could stimulate neuronal survival and neuritic outgrowth in a suprachiasmatic nucleus transplantation model.

Sequences surrounding the transcription initiation site of the Arabidopsis enoyl-acyl carrier protein reductase gene control seed expression in transgenic tobacco.

The NADH-specific enoyl-acyl carrier protein (ACP) reductase, which catalyses the last reducing step during the fatty acid biosynthesis cycle, is encoded in Arabidopsis thaliana encoded by a single housekeeping gene (ENR-A) which is differentially expressed during plant development. To identify elements involved in its tissue-specific transcriptional control, a fragment comprising the 1470 bp region directly upstream of the ATG start codon of the ENR-A gene was fused to the uidA (GUS) reporter gene and analysed in transgenic Nicotiana tabacum plants. GUS activity found during development of the transgenic plants was similar to endogenous ENR protein levels found in both tobacco and Arabidopsis plants, except for developing flowers. In floral tissue the promoter fragment showed very little activity in contrast to the relatively high level of endogenous ENR expression. Successive deletions from the 5' and 3' regions of the promoter fragment revealed the presence of at least three elements which control GUS expression in different stages of development in the transgenic tobacco plants. First, expression in young developing leaves required both the presence of sequences between -329 to -201 relative to the transcription start and part of the untranslated leader comprising the first intron. Second, root-specific GUS expression was still observed after deletion of the 5'-upstream sequences up to 19 bp of the transcription initiation site. Further, the additional removal of the intron from the untranslated leader increased root-specific expression by ca. 4- to 5-fold. Third, high expression in seeds was still observed with the minimal upstream promoter segment of 19 bp. This seed expression level was found to be independent of the presence or absence of the intron in the untranslated leader. Finally, 3' deletion of the leader sequence up to 17 bp of the transcription start greatly impaired GUS activity during all stages of plant development, suggesting that the deleted sequence of the leader either functions as an enhancer for transcription initiation or stabilizes the mRNA.

Vasopressin-deficient suprachiasmatic nucleus grafts re-instate circadian rhythmicity in suprachiasmatic nucleus-lesioned arrhythmic rats.

It was investigated whether grafts of the suprachiasmatic nucleus could re-instate circadian rhythmicity in the absence of its endogenous vasopressin production and whether the restored rhythm would have the long period length of the donor. Grafts of 17-days-old vasopressin-deficient homozygous Brattleboro rat fetuses, homotopically placed in arrhythmic suprachiasmatic nucleus-lesioned Wistar rats, re-instated circadian drinking rhythm within 20-50 days similar as seen for grafts of heterozygous control fetuses. Period length of the recovered rhythm revealed a similar difference (average 24.3 vs. 23.8 h) as reported for the rhythm between the adult Brattleboro genotypes. In all transplants, also those of the two-third non-recovery rats, a surviving suprachiasmatic nucleus was visible as a vasoactive intestinal polypeptide-positive neuronal cell cluster, whereas heterozygous transplants also revealed the complementary vasopressinergic cell part. Explanation of the absence of recovery failed since no undisputable correlation emerged between recovery of rhythm and vasoactive intestinal polypeptide, vasopressin and/or somatostatin immunocytochemical characteristics of the suprachiasmatic nucleus of the transplant. Special focus on the somatostatinergic neurons revealed their presence only occasionally near or in between the vasoactive intestinal polypeptidergic and (in the case of heterozygous grafts) vasopressinergic cell cluster. However their aberrant cytoarchitectural position appeared not to have affected the possibility to restore drinking rhythm of the suprachiasmatic nucleus-lesioned arrhythmic rat. It was concluded that grafted Brattleboro fetal suprachiasmatic nucleus develop their intrinsic rhythm conform their genotype and that vasopressin is not a crucial component in the maintenance nor in the transfer of circadian activity of the biological clock for drinking activity. Vasopressin of the suprachiasmatic nucleus may instead serve modulation within the circadian system.

Circadian rhythmicity of vasopressin levels in the cerebrospinal fluid of suprachiasmatic nucleus-lesioned and -grafted rats.

Transplantation of the fetal suprachiasmatic nucleus (SCN) in arrhythmic SCN-lesioned rats can reinstate circadian drinking rhythms in 40% to 50% of the cases. In the current article, it was investigated whether the failure in the other rats could be due to the absence of a circadian rhythm in the grafted SCN, using a circadian vasopressin (VP) rhythm in the cerebrospinal fluid (CSF) as the indicator for a rhythmic SCN. CSF was sampled in continuous darkness from-intact control rats and SCN-lesioned and -grafted rats. VP could be detected in all samples, with concentrations of 15 to 30 pg/ml in the control rats and 5 to 15 pg/ml in the grafted rats. A circadian VP rhythm with a two- to threefold difference between peak and nadir values was found in all 7 control rats but in only 4 of 13 experimental rats, despite the presence of a VP-positive SCN in all grafts. A circadian VP rhythm was present in 2 drinking rhythm-recovered rats (6 of 13) and in 2 nonrecovery rats. Apparently, in these latter rats, the failure of the grafted SCN to restore a circadian drinking rhythm cannot be attributed to a lack of rhythmicity in the SCN itself. Thus, the presence of a rhythmic grafted SCN, as is deduced from a circadian CSF VP rhythm, appears not to be sufficient for restoration of a circadian drinking rhythm in SCN-lesioned arrhythmic rats.