First-line surgery in prolactinomas: lessons from a long-term follow-up study in a tertiary referral center.

CONTEXT: Although consensus guidelines recommend dopamine agonists (DAs) as the first-line approach in prolactinomas, some patients may opt instead for upfront surgery, with the goal of minimizing the need for continuation of DAs over the long term. While this approach can be recommended in selected patients with a microprolactinoma, the indication for upfront surgery in macroprolactinomas remains controversial, with limited long-term data in large cohorts. We aimed at elucidating whether first-line surgery is equally safe and effective for patients with micro- or macroprolactinomas not extending beyond the median carotid line (i.e., Knosp grade ≤ 1). METHODOLOGY: Retrospective study of patients with prolactinomas Knosp grade ≤ 1 treated with upfront surgery. The primary endpoint was patients' dependence on DAs at last follow-up. The secondary endpoint was postoperative complications. Independent risk factors for long-term dependence on DAs were analyzed. RESULTS: A microadenoma was noted in 45 patients (52%) and a macroadenoma in 41 (48%), with 17 (20%) harboring a Knosp grade 1 prolactinoma. Median follow-up was 80 months. First-line surgery resulted in long-term remission in 31 patients (72%) with a microprolactinoma and in 18 patients (45%) with a macroprolactinoma (p = 0.02). DA therapy was ultimately required in 11 patients (24%) with microadenomas vs. 20 (49%) with macroadenomas (p = 0.03). As for the latter, DA was required in 13 patients (76%) with Knosp grade 1 macroadenomas vs. 7 patients (29%) with Knosp grade 0 macroadenomas (p = 0.004). There was no mortality, and morbidity was minimal. Knosp grade 1 prolactinomas (OR 7.3, 95% CI 1.4-37.7, p = 0.02) but not adenoma size (i.e., macroprolactinomas) were an independent predictor of long-term dependence on DAs. CONCLUSIONS: First-line surgery in patients with microprolactinomas or macroprolactinomas Knosp grade 0 resulted in a good chance of non-dependency on DA therapy. However, in patients with prolactinomas Knosp grade 1, first-line surgery cannot be recommended, as adjuvant DA therapy after surgery is required in the majority of them over the long term.

Primary Postnatal Dorsal Root Ganglion Culture from Conventionally Slaughtered Calves.

Neurological disorders in ruminants have an important impact on veterinary health, but very few host-specific in vitro models have been established to study diseases affecting the nervous system. Here we describe a primary neuronal dorsal root ganglia (DRG) culture derived from calves after being conventionally slaughtered for food consumption. The study focuses on the in vitro characterization of bovine DRG cell populations by immunofluorescence analysis. The effects of various growth factors on neuron viability, neurite outgrowth and arborisation were evaluated by morphological analysis. Bovine DRG neurons are able to survive for more than 4 weeks in culture. GF supplementation is not required for neuronal survival and neurite outgrowth. However, exogenously added growth factors promote neurite outgrowth. DRG cultures from regularly slaughtered calves represent a promising and sustainable host specific model for the investigation of pain and neurological diseases in bovines.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons.

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease.

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

BK channels affect glucose homeostasis and cell viability of murine pancreatic beta cells.

AIMS/HYPOTHESIS: Evidence is accumulating that Ca(2+)-regulated K(+) (K(Ca)) channels are important for beta cell function. We used BK channel knockout (BK-KO) mice to examine the role of these K(Ca) channels for glucose homeostasis, beta cell function and viability. METHODS: Glucose and insulin tolerance were tested with male wild-type and BK-KO mice. BK channels were detected by single-cell RT-PCR, cytosolic Ca(2+) concentration ([Ca(2+)](c)) by fura-2 fluorescence, and insulin secretion by radioimmunoassay. Electrophysiology was performed with the patch-clamp technique. Apoptosis was detected via caspase 3 or TUNEL assay. RESULTS: BK channels were expressed in murine pancreatic beta cells. BK-KO mice were normoglycaemic but displayed markedly impaired glucose tolerance. Genetic or pharmacological deletion of the BK channel reduced glucose-induced insulin secretion from isolated islets. BK-KO and BK channel inhibition (with iberiotoxin, 100 nmol/l) broadened action potentials and abolished the after-hyperpolarisation in glucose-stimulated beta cells. However, BK-KO did not affect action potential frequency, the plateau potential at which action potentials start or glucose-induced elevation of [Ca(2+)](c). BK-KO had no direct influence on exocytosis. Importantly, in BK-KO islet cells the fraction of apoptotic cells and the rate of cell death induced by oxidative stress (H(2)O(2), 10-100 µmol/l) were significantly increased compared with wild-type controls. Similar effects were obtained with iberiotoxin. Determination of H(2)O(2)-induced K(+) currents revealed that BK channels contribute to the hyperpolarising K(+) current activated under conditions of oxidative stress. CONCLUSIONS/INTERPRETATION: Ablation or inhibition of BK channels impairs glucose homeostasis and insulin secretion by interfering with beta cell stimulus-secretion coupling. In addition, BK channels are part of a defence mechanism against apoptosis and oxidative stress.

Evoked slow muscarinic acetylcholinergic synaptic potentials in rat hippocampal interneurons.

The hippocampus receives an extensive cholinergic input from the medial septal nucleus that ramifies throughout all layers and plays a pivotal modulatory role in cognitive function. Although the pharmacological effects of exogenous application of cholinergic agonists have been extensively studied in hippocampal neurons, much less is known about the effects of synaptically released acetylcholine (ACh). In this respect, most studies have focused on the cholinergic afferent input to pyramidal neurons that produces a characteristically slow depolarizing synaptic response mediated by activation of muscarinic ACh receptors (mAChRs). Here we report that cholinergic afferent stimulation also elicits atropine-sensitive synaptic potentials in hippocampal CA1 interneurons but, in contrast to synaptic responses in pyramidal neurons, these are highly diverse in waveform, although can still be classified into five distinct subtypes. The most common response type (i) 64% of cells) consisted of a slow sustained membrane potential depolarization. The other 36% of responses could be subdivided into responses comprising of (ii) a biphasic membrane potential change in which an initial slow hyperpolarization subsequently transforms into a slow depolarization (20%), (iii) a pure, slow hyperpolarization (13%), and (iv) an oscillatory response persisting for several seconds (2%). Interestingly, there were also interneurons totally insensitive to both synaptic and pharmacological cholinergic challenge. Morphological investigation of recorded cells revealed no obvious correlation between responsiveness to cholinergic afferent stimulation and dendritic and axonal arborization. The current study suggests that synaptic release of ACh results in a complex and differential mAChR-mediated modulation of cellular excitability within the hippocampal interneuron population.

Effects of creatine treatment on survival and differentiation of GABA-ergic neurons in cultured striatal tissue.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by a prominent loss of GABA-ergic medium-sized spiny neurons in the caudate putamen. There is evidence that impaired energy metabolism contributes to neuronal death in HD. Creatine is an endogenous substrate for creatine kinases and thereby supports cellular ATP levels. This study investigated the effects of creatine supplementation (5 mm) on cell survival and neuronal differentiation in striatal cultures. Chronic creatine treatment resulted in significant increased densities of GABA-immunoreactive (-ir) neurons, although total neuronal cell number and general viability were not affected. Similar effects were seen after short-term treatment, suggesting that creatine acted as a differentiation factor. Inhibitors of transcription or translation did not abolish the creatine-mediated effects, nor did omission of extracellular calcium, whereas inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly attenuated the creatine induced increase in GABA-ir cell densities. Creatine exhibited significant neuroprotection against toxicity instigated either by glucose- and serum deprivation or addition of 3-nitropropionic acid. In sum, the neuroprotective properties in combination with promotion of neuronal differentiation suggest that creatine has potential as a therapeutic drug in the treatment of neurodegenerative diseases, like HD.

Effects of creatine treatment on the survival of dopaminergic neurons in cultured fetal ventral mesencephalic tissue.

Parkinson's disease is a disabling neurodegenerative disorder of unknown etiology characterized by a predominant and progressive loss of dopaminergic neurons in the substantia nigra. Recent findings suggest that impaired energy metabolism plays an important role in the pathogenesis of this disorder. The endogenously occurring guanidino compound creatine is a substrate for mitochondrial and cytosolic creatine kinases. Creatine supplementation improves the function of the creatine kinase/phosphocreatine system by increasing cellular creatine and phosphocreatine levels and the rate of ATP resynthesis. In addition, mitochondrial creatine kinase together with high cytoplasmic creatine levels inhibit mitochondrial permeability transition, a major step in early apoptosis. In the present study, we analyzed the effects of externally added creatine on the survival and morphology of dopaminergic neurons and also addressed its neuroprotective properties in primary cultures of E14 rat ventral mesencephalon. Chronic administration of creatine [5 mM] for 7 days significantly increased survival (by 1.32-fold) and soma size (by 1.12-fold) of dopaminergic neurons, while having no effect on other investigated morphological parameters. Most importantly, concurrent creatine exerted significant neuroprotection for dopaminergic neurons against neurotoxic insults induced by serum and glucose deprivation (P < 0.01), 1-methyl-4-phenyl pyridinium ion (MPP+) [15 microM] and 6-hydroxydopamine (6-OHDA) [90 microM] exposure (P < 0.01). In addition, creatine treatment significantly protected dopaminergic cells facing MPP+-induced deterioration of neuronal morphology including overall process length/neuron (by 60%), number of branching points/neuron (by 80%) and area of influence per individual neuron (by 60%). Less pronounced effects on overall process length/neuron and number of branching points/neuron were also found after 6-OHDA exposure (P < 0.05) and serum/glucose deprivation (P < 0.05). In conclusion, our findings identify creatine as a rather potent natural survival- and neuroprotective factor for developing nigral dopaminergic neurons, which is of relevance for therapeutic approaches in Parkinson's disease and for the improvement of cell replacement strategies.

Protein NMR in biomedical research.

Nuclear magnetic resonance (NMR) spectroscopy is a versatile biophysical technique with wide applicability in drug discovery research, particularly for the detection and characterization of molecular interactions. This review highlights in a comprehensive manner the aspects of biomolecular NMR which are most beneficial for pharmaceutical research and presents them as contributions to the different stages of a drug discovery program: target selection, assay development, lead generation and lead optimization. Emphasis is put on the concept of the particular NMR application, rather than on technical details, and on recent examples. Finally, an appendix of frequently asked questions is given.

Transperitoneal or extraperitoneal approach for laparoscopic radical prostatectomy: a false debate over a real challenge.

PURPOSE: With our extensive experience of transperitoneal laparoscopic prostatectomy, we evaluated the advantages and disadvantages of the extraperitoneal approach by comparing 2 consecutive series of each procedure. MATERIALS AND METHODS: We reviewed 200 consecutive procedures performed by 2 surgeons. A total of 100 transperitoneal procedures (designated group 1) were compared to the first 100 extraperitoneal cases (designated group 2). RESULTS: Mean operating time was 173 minutes in group 1 and shorter in group 2 at 163 minutes (p = 0.003). Mean blood loss (360 ml in group 1,375 ml in group 2) and transfusion rates (4% in group 1, 3% in group 2) were equivalent. There were no major complications. Minor complications were 10% in group 1 and 9% in group 2. There was no statistical difference in positive margin rate between groups 1 and 2. CONCLUSIONS: The 2 procedures are equivalent in terms of operative, postoperative and pathological data. Each surgeon has to choose considering personal experience, training and standardization.

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.

Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones.

Glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic neurones against toxic and physical damage. In addition, GDNF promotes differentiation and structural integrity of dopaminergic neurones. Here we show that GDNF can support the function of primary dopaminergic neurones by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, asan inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting that the mode of action for that up-regulation is not directly connected to the regulation of GTPCH I transcription. We conclude that GDNF, in addition to its action in structural differentiation, also promotes differentiation regarding expression and enzymatic activity of a crucial component in the dopaminergic biosynthetic pathway.

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord.

Additive effect of glial cell line-derived neurotrophic factor and neurotrophin-4/5 on rat fetal nigral explant cultures.

Transplantation of embryonic dopaminergic neurons is an experimental therapy for Parkinson's disease, but limited tissue availability and suboptimal survival of grafted dopaminergic neurons impede more widespread clinical application. Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4/5 (NT-4/5) exert neurotrophic effects on dopaminergic neurons via different receptor systems. In this study, we investigated possible additive or synergistic effects of combined GDNF and NT-4/5 treatment on rat embryonic (embryonic day 14) nigral explant cultures grown for 8 days. Contrary to cultures treated with GDNF alone, cultures exposed to NT-4/5 and GDNF+NT-4/5 were significantly larger than controls (1.6- and 2.0-fold, respectively) and contained significantly more protein (1.6-fold). Treatment with GDNF, NT-4/5 and GDNF+NT-4/5 significantly increased dopamine levels in the culture medium by 1.5-, 2.5- and 4.7-fold, respectively, compared to control levels, and the numbers of surviving tyrosine hydroxylase-immunoreactive neurons increased by 1.7-, 2.1-, and 3.4-fold, respectively. Tyrosine hydroxylase enzyme activity was moderately increased in all treatment groups compared to controls. Counts of nigral neurons containing the calcium-binding protein, calbindin-D28k, revealed a marked increase in these cells by combined GDNF and NT-4/5 treatment. Western blots for neuron-specific enolase suggested an enhanced neuronal content in cultures after combination treatment, whereas the expression of glial markers was unaffected. The release of lactate dehydrogenase into the culture medium was significantly reduced for GDNF+NT-4/5-treated cultures only. These results indicate that combined treatment with GDNF and NT4/5 may be beneficial for embryonic nigral donor tissue either prior to, or in conjunction with, intrastriatal transplantation in Parkinson's disease.

Sustained delivery of GDNF: towards a treatment for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive loss of nigral dopaminergic neurons. Although symptomatic therapies to substitute for the missing neurotransmitter dopamine are efficient at the early stages of the disease, the goal is to find alternative therapies which could protect dopaminergic neurons from the degenerative process. We have used two distinct gene therapy approaches to deliver the neuroprotective molecule glial cell line-derived neurotrophic factor (GDNF) in animal models of the disease: (i) an encapsulated genetically engineered cell line releasing GDNF (ex vivo gene therapy); and (ii) a lentiviral vector encoding the GDNF gene (in vivo gene therapy). Both approaches allowed protection of nigral dopaminergic neurons against lesion-induced cell death in rodent as well as monkey models of PD. Behavioral symptoms were also ameliorated in these animals. In addition, co-transplantation of embryonic dopaminergic neuronal grafts and a GDNF-releasing capsule allowed improvement of graft survival and differentiation, thereby accelerating behavioral recovery. These results should lead to clinical application in the near future.

Chemistry and biology of thiamethoxam: a second generation neonicotinoid.

Thiamethoxam is the first commercial neonicotinoid insecticide from the thianicotinyl subclass. It was discovered in the course of our optimisation program on neonicotinoids started in 1985. Novel variations of the nitroimino-heterocycle of imidacloprid led to 4-nitroimino-1,3,5-oxadiazinanes exhibiting high insecticidal activity. Among these, thiamethoxam (CGA 293433) was identified as the best compound and selected for worldwide development. The compound can be synthesised in only a few steps and high yield from easily accessible starting materials. Thiamethoxam acts by binding to nicotinic acetylcholine receptors. It exhibits exceptional systemic characteristics and provides excellent control of a broad range of commercially important pests, such as aphids, jassids, whiteflies, thrips, rice hoppers, Colorado potato beetle, flea beetles and wireworms, as well as some lepidopteran species. In addition, a strong preventative effect on some virus transmissions has been demonstrated. Thiamethoxam is developed both for foliar/soil applications and as a seed treatment for use in most agricultural crops all over the world. Low use rates, flexible application methods, excellent efficacy, long-lasting residual activity and favourable safety profile make this new insecticide well-suited for modern integrated pest management programmes in many cropping systems.

Liposome-mediated gene transfer to fetal human ventral mesencephalic explant cultures.

The feasibility of non-viral gene transfer using liposomes is described for human fetal nigral tissue. Ventral mesencephalic explants from 6 to 12 week old fetuses were grown as free-floating roller tube cultures. For the transfection, a vector coding for beta-galactosidase driven by the Rous Sarcoma Virus promoter was used. The developmental stage of the human tissue, time in vitro and the amount of vector DNA used significantly influenced the transfection efficiency. Optimal transfection results were obtained with tissue from a 10 week old fetus, cultured for 4 days and transfected with mixtures containing 4 microg vector DNA. Histological analysis suggested that a specific population of ventral mesencephalic precursor cells were the target for the gene transfer. This finding might have implications for gene delivery and cell replacement strategies in Parkinson's disease.

NOGO mRNA expression in adult and fetal human and rat nervous tissue and in weight drop injury.

Nogo is a myelin-associated protein known to inhibit growth of neurites. In order to understand possible physiological roles of Nogo, we performed in situ hybridization using rat and human probes complementary to a Nogo-A-specific sequence and a sequence shared by all known Nogo transcripts recognizing nogo-A, -B, and -C. We studied the cellular distribution of nogo-mRNA in fetal and adult human and rat tissues, with a focus on the spinal cord and ganglia. Rat mRNA expression was also studied in a spinal cord weight-drop model and in animals exposed to kainic acid. In human fetal tissue, nogo-A was strongly expressed in the ventral two-thirds of the spinal cord, the dorsal root ganglia, and autonomic ganglia. Similarly, nogo-A mRNA expression was observed in the adult human spinal cord and ganglia. High levels of nogo-A message were observed in neurons, such as motor neurons and sensory ganglia neurons. The distribution of nogo message in rats resembled that seen in human tissues. Thus, nogo mRNA was expressed in neurons and oligodendrocytes, but not astrocytes or Schwann cells. In addition, expression of nogo-A mRNA was observed in human and rat developing muscle tissue. High level of nogo-mRNA were also expressed in the rat trigeminal ganglion and trigeminal pontine nucleus. In fetal rats the adrenal gland and cell clusters in the liver were positive for the nogo-ABC pan-probe, but negative for the nogo-A probe. While neurons in the adult rat brain were generally positive, very prominent nogo-A mRNA and nogo-ABC mRNA signals were obtained from neurons of the hippocampus, piriform cortex, the red nucleus, and the oculomotor nucleus. Nogo-A mRNA expression was markedly reduced in the epicenter of a lesion in the spinal cord of adult rats 6 and 24 h after a weight-drop injury, while no perifocal upregulation of nogo mRNA was seen. No obvious change of nogo expression was detected in kainic acid exposed animals. In conclusion our in situ hybridization study has demonstrated widespread expression of nogo mRNA in the fetal, developing and adult nervous system of rat and man. In addition to oligodendroglial cells, high levels of nogo-A mRNA expression were found in neurons, raising important questions about the function of neuronal nogo mRNA. No obvious regulation of nogo was detected following injury.

Influence of time in culture and BDNF pretreatment on survival and function of grafted embryonic rat ventral mesencephalon in the 6-OHDA rat model of Parkinson's disease.

Embryonic midbrain can be maintained as free-floating roller tube cultures prior to grafting in experimental Parkinson's disease. We examined the influence of pregrafting culture time and pretreatment with brain-derived neurotrophic factor on graft survival and function. Cultures were prepared from solid pieces of embryonic (E14) rat ventral mesencephalon and maintained 4, 8, or 12 days in vitro with or without brain-derived neurotrophic factor (100 ng/ml) and grafted into the striatum of 6-hydroxydopamine-lesioned rats. Graft survival and function were evaluated by amphetamine-induced rotation behavior, number of tyrosine hydroxylase-immunoreactive neurons, striatal reinnervation, and graft volume. Rats receiving untreated tissue cultured for 4 or 8 days displayed no differences in graft quality, while grafts from 12-day-old cultures contained significantly fewer (P < 0.05) tyrosine hydroxylase-immunoreactive neurons (340 +/- 97, 267 +/- 92, and 62 +/- 19) and displayed a lower survival rate (9.6 +/- 2.7, 7.9 +/- 2.7, and 2.6 +/- 0.8% for 4, 8, and 12 days in vitro, respectively). Only rats grafted with 4- and 8-day-old cultures recovered significantly (P < 0.05) from lesion-induced rotations (69.4 +/- 18.6, 70.3 +/- 13.9, and 23.2 +/- 12.1% for 4, 8, and 12 days in vitro, respectively). Striatal reinnervation decreased with increasing culture time (P < 0.05). Pretreatment of the cultures with brain-derived neurotrophic factor affected only graft-induced fiber reinnervation, which was reduced even after short culture times. We therefore suggest that a storage period of 8 days is well suited to maintain embryonic rat ventral mesencephalon with the free-floating roller tube culture technique prior to transplantation. BDNF pretreatment as a new strategy to improve graft survival and function, however, was not effective.