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.

Lhermitte-Duclos disease with atypical vascularization--case report and review of the literature.

OBJECTIVE: A case of Lhermitte-Duclos disease (LDD, dysplastic gangliocytoma) with atypical vascularization is reported. LDD is a rare cerebellar mass lesion which may be associated with Cowden's syndrome and the PTEN germline mutation. CASE MATERIAL: A 61-year-old male presented 15 years before with a transient episode of unspecific gait disturbance. Initial magnetic resonance (MR) imaging revealed a right-sided, diffuse, nonenhancing cerebellar mass lesion. No definitive diagnosis was made at that time, and the symptoms resolved spontaneously. 15 years later, the patient presented with acute onset of vomiting associated with headache and ataxic gait. MR imaging showed a progression of the lesion with occlusive hydrocephalus. The lesion depicted a striated pattern characteristic for LDD with T1-hypointense and T2-hyperintense bands, nonenhancing with contrast. After resection of the mass lesion, the cerebellar and hydrocephalic symptoms improved rapidly. The pathological examination confirmed the diagnosis of dysplastic gangliocytoma (WHO Grade I) with enlarged granular and molecular cell layers, reactive gliosis and dysplastic blood vessels. No other clinical features associated with Cowden's syndrome were present. CONCLUSIONS: This case illustrates that LDD with atypical vascularization is a slow-growing posterior fossa mass lesion which may remain asymptomatic for many years. Timing of surgical treatment and extent of resection in patients with LDD is controversial. The typical features on standard T1-/T2-weighted MR imaging allow a diagnosis without surgery in most cases. The authors believe that the decision to treat in these cases should be based on clinical deterioration.

Contribution of the apparent diffusion coefficient in perilesional edema for the assessment of brain tumors.

OBJECTIVES: Diffusion-weighted MRI is sensitive to molecular motion and has been applied to the diagnosis of stroke. Our intention was to investigate its usefulness in patients with brain tumor and, in particular, in the perilesional edema. METHODS: We performed MRI of the brain, including diffusion-weighted imaging and mapping of the apparent diffusion coefficient (ADC), in 16 patients with brain tumors (glioblastomas, low-grade gliomas and metastases). ADC values were determined by the use of regions of interest positioned in areas of high signal intensities as seen on T2-weighted images and ADC maps. Measurements were taken in the tumor itself, in the area of perilesional edema and in the healthy contralateral brain. RESULTS: ADC mapping showed higher values of peritumoral edema in patients with glioblastoma (1.75 x 10(-3)mm(2)/s) and metastatic lesions (1.61 x 10(-3)mm(2)/s) compared with those who had low-grade glioma (1.40 x10(-3)mm(2)/s). The higher ADC values in the peritumoral zone were associated with lower ADC values in the tumor itself. CONCLUSIONS: The higher ADC values in the more malignant tumors probably reflect vasogenic edema, thereby allowing their differentiation from other lesions.

Identification and validation of P311 as a glioblastoma invasion gene using laser capture microdissection.

The mRNA expression profiles from glioblastoma cells residing at the tumor core and invasive rim of a human tumor resection were compared. From a single tumor specimen, 20,000 single cells from each region were collected by laser capture microdissection. Differential expression of 50-60 cDNA bands was detected. One of the sequences overexpressed by the invasive cells showed 99% homology to the P311 gene, the protein product of which is reported to localize at focal adhesions. Relative overexpression of P311 by invading glioblastoma cells compared with tumor core was confirmed by quantitative reverse transcription-PCR of six glioblastoma specimens after laser capture microdissection collection of rim and core cells. In vitro studies using antisense oligodeoxynucleotides and integrin activation confirmed the role of P311 in supporting migration of malignant glioma cells. Immunochemistry studies confirmed the presence of the P311 protein in tumor cells, particularly at the invasive edge of human glioblastoma specimens.

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.

Death-associated protein 3 (Dap-3) is overexpressed in invasive glioblastoma cells in vivo and in glioma cell lines with induced motility phenotype in vitro.

PURPOSE: To discover the genetic determinants of glioma invasion in vivo, we compared the mRNA expression profiles of glioblastoma cells residing at the tumor core versus those at the invasive rim of a human tumor resection. EXPERIMENTAL DESIGN: From a single glioblastoma specimen, 20,000 individual cells from each region (core and invasive rim) were collected by laser capture microdissection and analyzed by mRNA differential display. Differential expression of gene candidates was confirmed by laser capture microdissection and quantitative reverse transcription-PCR in additional glioblastoma multiforme specimens, and the role in migration was further evaluated in glioma cell lines in vitro. RESULTS: Reproducible overexpression the death-associated Protein 3 (Dap-3) mRNA (NM 004632, GenBank; also reported as human ionizing resistance conferring protein mRNA, HSU18321, GenBank) by invasive cells was identified. Although the full-length Dap-3 protein has been described as proapoptotic, the NH(2)-terminal fragment can act in a dominant negative way resulting in protection from programmed cell death. In glioma cell lines T98G and G112 with an induced motility phenotype, Dap-3 was up-regulated at the mRNA and protein level as assessed by quantitative reverse transcription-PCR, cDNA microarray, and Western blot analysis. These cells showed an increased resistance to undergo camptothecin-induced apoptosis, which was overcome by effective Dap-3-antisense treatment. Antisense treatment also decreased the migration ability of T98G cells. CONCLUSIONS: Dap-3 is up-regulated in invasive glioblastoma multiforme cells in vivo and in glioma cells with an induced motility phenotype in vitro. When migration is activated, Dap-3 is up-regulated and cells become resistant to apoptosis. These findings suggest that Dap-3 confers apoptosis-resistance when migration behavior is engaged.

Cerebral revascularization model in a swine.

The purpose of this study was to analyze the suitability of the cerebral vasculature of the pig regarding a revascularization procedure. In two 60 kg pigs the femoral artery was exposed and canulated for selective angiography and interventional procedures. After the angiography, the pigs were brought to the animal OR for craniotomy and analysis of the intracranial cerebral arteries and the surgical exposure of the carotid arteries under the microscope. Angiography demonstrated the presence of a true internal-, external carotid artery and vertebral arteries. Both the vertebral and internal carotid arteries are feeding a rete mirabilis both at the cranial base and the cranio-cervical junction. At these sites further advancement of the angiography catheter was not possible. Out of these rete mirabilis, an intracranial carotid artery and an intracranial vertebral artery were formed, respectively. The intracranial cerebral vessels were of the dimension of 1 mm and less. The extracranial portion of the internal carotid artery was 2.5 mm of diameter. From these findings, we conclude that a direct cerebral revascularization procedure of the intracranial vessels is not possible in the swine. However, a global revascularization procedure on the extracranial portion of the internal carotid artery is thus feasible, both using a low- and high-flow anastamosis technique.

Dependency of blood flow velocity in the middle cerebral artery on end-tidal carbon dioxide partial pressure--a transcranial ultrasound Doppler study.

The end-tidal carbon dioxide partial pressure (PCO2) response curves for the flow velocity in the middle cerebral artery were studied in 31 normal subjects with transcranial Doppler techniques. An exponential curve with an exponent of 0.034 mm Hg-1 was found to be a good fit to the recorded data. By means of this relationship, recordings of flow velocity in cerebral arteries can be normalized to a standard value of PCO2. Physiological aspects of cerebrovascular reactivity to PCO2 and the clinical implications of the PCO2 response curve are discussed. The normal material provides a reference for assessing pathological responses.

NGF increases neuritic complexity of cholinergic interneurons in organotypic cultures of neonatal rat striatum.

The influence of NGF on cholinergic interneurons in organotypic roller tube cultures of 4 day postnatal rat striatum was examined after 13 to 16 days in vitro. Cultures were divided into four groups. The medium of the NGF treated group was supplemented with 5 ng/ml NGF, whereas control groups were cultured either without NGF, by adding 20 ng/ml neutralising anti-NGF antibody, or by adding both NGF and anti-NGF antibody to the medium. Two different cell populations were identified by an image analysis system which measured acetylcholinesterase staining intensity. It was demonstrated that NGF promotes survival of the large, intensely stained population. Eighty computer-assisted reconstructions of intensely stained cells, 20 for each treatment group, were performed in a random order by means of a neuron tracing system. Axons and dendrites were analysed separately. NGF enhanced complexity of neuritic, predominantly axonal trees by increasing the number of axonal segments by 91% to 100% (P < 0.01), the number of dendritic segments by 33% to 63% (P = 0.09 to P < 0.01), maximal axonal branch order by 37% to 50% (P < 0.05), and maximal dendritic branch order by 22% to 37% (P < 0.05). Further evidence of more complex neuritic trees was given by Sholl concentric sphere analysis. Anti-NGF antibody could block all these effects. General rules of branching architecture were not affected by NGF treatment as shown by analysing mean segment length in relation to the branch order, branch point exit angles, total tortuosity, Rall's ratio, and tapering of neuritic trees.

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.

Somatostatin receptor scintigraphy in central nervous system tumors: role of blood-brain barrier permeability.

UNLABELLED: Somatostatin receptors are expressed in meningiomas and low-grade gliomas, raising the hope that scintigraphy with 111In-DTPA-D-Phe1-octreotide might be helpful in the in vivo localization, differential diagnosis and postoperative/postradiotherapy brain tumor follow-up. METHODS: Indium-111-DTPA-D-Phe1-octreotide scintigraphy and brain scintigraphy using 99mTc-DTPA as a nonspecific tracer for blood-brain barrier integrity were simultaneously performed in 60 patients with CNS tumors using dual-isotope acquisition mode SPECT. For 23 patients, the scintigraphic findings were also compared with in vitro somatostatin receptor autoradiography of surgical biopsy specimens. RESULTS: In meningiomas (located outside the blood-brain barrier), the somatostatin receptor scan showed all tumors and scintigraphic signal intensity correlating with in vitro SSR density positive in all meningiomas. Less contrast was seen on 99mTc-DTPA scans. In all tumors inside the blood-brain barrier, the 111In-DTPA-D-Phe1-octreotide scan visualized the tumors with a disrupted blood-brain barrier, as seen by 99mTc-DTPA scintigraphy. Discrepancies, however, were observed between somatostatin receptor scintigraphy and in vitro receptor autoradiography. CONCLUSION: Combined somatostatin receptor and 99mTc-DTPA scintigraphy may be helpful for noninvasive differentiation between meningiomas and other CNS tumors. False-negative scans were observed as a result of shielding by the intact blood-brain barrier. Interpretation of negative and positive somatostatin receptor scans in CNS tumors must therefore be done with caution.

Effects of combined BDNF and GDNF treatment on cultured dopaminergic midbrain neurons.

Neural transplantation is an experimental therapy for Parkinson's disease. Pretreatment of fetal donor tissue with neurotrophic factors may improve survival of grafted dopaminergic neurons. Free-floating roller tube cultures of fetal rat ventral mesencephalon were treated with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), or a combination of both. Dopamine content of the culture medium, the number of tyrosine hydroxylase-immunoreactive neurons, and culture volumes were moderately increased in the BDNF- and GDNF-treated cultures but significantly increased by 6.8-, 3.2- and 2.4-fold, respectively after treatment with the combination of both factors. We conclude that pretreatment of dopaminergic tissue in culture with a combination of BDNF and GDNF may be an effective means to improve the quality of tissue prior to grafting.

A mathematical model for the estimation of human embryonic and fetal age.

Precise determination of donor age in human embryonic and fetal tissue is crucial for cell transplantation due to the existence of distinct time windows within which successful grafting is possible. This study demonstrates that between 4-12 wk postconception embryonic and fetal age can be estimated based on various morphometric parameters measured on a routine basis in suction abortion material. The greatest length, the neck-rump length, the foot length, and the proximal and distal arm and leg length were correlated with the anamnestic and ultrasonographically estimated age. Multivariate regression analyses showed a linear correlation between age and the logarithmic value of the various morphometric parameters. The best correlation was found for a mathematical model combining the limb parameters (r = 0.904; p < 0.001; n = 37). A prospective follow-up study (n = 40) was carried out to test the validity of the mathematical model. A high correlation was found between the calculated age and the estimated age based on anamnestic data (r = 0.749, p < 0.001). Outliers due to errors in the anamnestic data were readily identified by comparing anamnestic with calculated age. This method allows determination of embryonic and fetal age within and beyond the age group of the Carnegie classification and may, therefore, be useful for the needs of experimental and clinical cell transplantation.

GDNF increases the density of cells containing calbindin but not of cells containing calretinin in cultured rat and human fetal nigral tissue.

Among the dopaminergic neurons in substantia nigra pars compacta and in the ventral tegmental area, subpopulations express the calcium-binding proteins calbindin (CB) and calretinin (CR), and the CB-containing neurons are supposed to be less prone to degeneration in Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic neurons. Using free-floating roller-tube (FFRT) cultures derived from fetal rat (E14) ventral mesencephalon we found that GDNF (10 ng/ml) significantly increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons. The possible effects of GDNF treatment on CB-immunoreactive (CB-ir) and CR-ir neurons in such cultures were examined in the present study. The neuronal cell densities were measured by quantifying the numbers of CB-ir and CR-ir neurons in areas of sections through the most extensive parts of the spherical cultures. In 4-day-old and 8-day-old cultures GDNF treatment increased the density of CB-ir neurons by 50% and 59%, respectively. Partial co-existence of TH and CB was shown using the method of double immunolabeling. The density of CR-containing neurons was unaffected by GDNF treatment as confirmed by Western blotting for CR. Parallel effects of GDNF treatment were obtained for cultures of human fetal ventral mesencephalon (8 weeks postconception). In conclusion, our findings identify GDNF as a potent factor for fetal rat and human nigral CB-ir neurons able to promote their survival in culture. Referring to a suggested neuroprotective role of CB, the results may be of relevance in the context of neuronal transplantation of patients suffering from severe Parkinson's disease.

Long-term survival of dopaminergic neurones in free-floating roller tube cultures of human fetal ventral mesencephalon.

Transplantation of human fetal ventral mesencephalon (VM) to Parkinsonian patients has shown beneficial effects in several clinical trials. However, further improvements in the transplantation technique are needed. Delayed surgery, i.e., the in vitro maintenance of the tissue prior to transplantation would present several advantages. The roller tube technique as initially described by Gähwiler (1981) was modified in several aspects for the long-term maintenance of dopaminergic neurones of human fetal VM. Tissue cultures were maintained free-floating in the medium for up to 42 days. The human fetal material was obtained from legal induced suction abortions. The embryonic age ranged from 5 to 12 weeks post-conception. Identification of VM was possible in 43% of the cases. Neurones in cultures were demonstrated by means of immunohistochemistry for tyrosine hydroxylase (TH) and gamma-amino butyric acid (GABA), by electron microscopy and by hybridisation histochemistry using a TH-mRNA-sensitive probe. A high variability in the number of TH-positive cells in individual cultures derived from the same embryo was observed. In 20 microns frozen sections of such tissue cultures the mean +/- SEM of TH-positive cells was 6.5 +/- 1.2/0.1 mm2 (n = 79; range: 0-73). The technique described insures the growth of long-term cultures of human fetal VM.

Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function.

Free-floating roller tube (FFRT) cultures of fetal rat and human nigral tissue are a means for tissue storage prior to grafting in experimental Parkinson's disease. In the present study, FFRT cultures prepared from embryonic-day-14 rat ventral mesencephalon were maintained for 4, 8, 12, or 16 days in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4-5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase immunoreactive neurons was significantly higher for BDNF treated cultures (2729+/-300) at DIV 8, as compared to controls (1679+/-217). At DIV 12, the culture volume was significantly increased by BDNF (1.05+/-0.12 vs. 0.71+/-0.04 mm3). Similar results were obtained for total protein. Western blot analysis demonstrated increasing signals for GFAP with increasing time in culture, but levels for control and BDNF treated cultures did not differ at any time-point investigated. In conclusion, it is suggested that the time window for effective storage of dopaminergic tissue prior to grafting can be extended by using the FFRT culture technique and that the in vitro storage may be further prolonged by treatment with BDNF.

Dilemmas in the management of patients with arteriovenous malformations.

An arteriovenous malformation (AVM) consists of one or more arteries that drain through one or several small openings directly into one or more veins. The capillary system between arteries and veins is missing. The natural history of an unruptured AVM demonstrates a 1-2% bleeding rate and once ruptured a 2-4% annual risk of rebleeding. There is a risk of dying of AVM up to 1% per year, a 1% annual risk of developing de novo epilepsy, and a 1% chance of disability per year. Small AVMs are more likely to rupture than large AVMs. The goal of treatment is complete obliteration of the malformation. The risk of surgical treatment depends mainly on its size, location and drainage pattern. Size and angioarchitecture determine the risks of embolotherapy and radiotherapy. AVMs in Spetzler-Martin grades I-III should be treated by microneurosurgery or a combination of embolotherapy and microneurosurgery. They can be excised with low surgical mortality and morbidity. For AVMs in Spetzler-Martin grades IV and V staged treatment approaches such as embolotherapy followed by surgery or radiotherapy should be considered. Rarely is embolotherapy or radiotherapy alone indicated. There are AVMs in Spetzler-Martin grades IV and V that may be inaccessible for surgical or any other treatment, and that should be left alone. Prospective randomised trials on the optimal management of AVMs are lacking. All our knowledge on AVMs stems from open series and indirect comparisons.

Severe symptomatic vasospasm after rupture of an arteriovenous malformation.

A 31-year-old woman had intracerebral and intraventricular hemorrhage from an arteriovenous malformation. Vasospasm of the internal carotid arteries developed and was treated with angioplasty. On initial CT scans, only traces of blood were seen in the basal cisterns; thus, the development of symptomatic vasospasm was an unexpected complication.

Noninvasive dopamine determination by reversed phase HPLC in the medium of free-floating roller tube cultures of rat fetal ventral mesencephalon: a tool to assess dopaminergic tissue prior to grafting.

The low availability of dopamine containing neurons for grafting in Parkinson's disease is a general problem. Free-floating roller tube (FFRT) cultures allow storage of fetal mesencephalic tissue prior to transplantation. Preoperative functional testing permits to select an optimized set of individual cultures for transplantation. Rat fetal ventral mesencephali (E13) were dissected out and divided into four equally sized pieces each and individually prepared as FFRT cultures. After 4, 8, 12, and 16 days in vitro (DIV) the medium of each culture was collected during routine medium change and immediately stabilized. Dopamine was extracted and probes were determined with reversed phase HPLC using electro-chemical detection. After 16 DIV cultures were fixed and cell counts performed in tyrosine hydroxylase (TH)-immunostained serial sections. The mean dopamine content +/- SEM In culture conditioned media was at 4 DIV: 21 +/- 2 pg, n = 38; at 8 DIV: 37 +/- 4 pg, n = 40; at 12 DIV: 52 +/- 7 pg, n = 38; and at 16 DIV: 39 +/- 5 pg, n = 38. In all cultures devoid of dopamine after 4 and 8 DIV (12.5%) levels remained below detectability at 12 and 16 DIV. Cultures derived from the rostral mesencephalon showed significantly higher dopamine values than those from the caudal mesencephalon at 12 DIV. The mean number of TH-immunoreactive (-ir) cells/culture +/- SEM after 16 DIV was 556 +/- 51, n = 40. The correlation between TH-ir cell number (CN) and dopamine content of rostrally derived cultures at 16 DIV was: CN = 7.4 (dopamine [pg]) + 248; R = 0.75; n = 19; p < 0.001. No dopamine was present in cultures without TH-ir cells. These results demonstrate that sequential noninvasive screening of dopamine in single cultures is feasible and that the dopamine content is correlated to the number of surviving TH-ir cells. This permits to select cultures rich in dopaminergic neurons for transplantation.

Effect of nimodipine on cerebrovascular response to CO2 in asymptomatic individuals and patients with subarachnoid hemorrhage: a transcranial Doppler ultrasound study.

The cerebrovascular response to CO2 was evaluated by measuring relative changes in blood flow velocity within the middle cerebral artery by transcranial Doppler ultrasonography during normo-, hypo-, and hypercapnia. In seven patients without subarachnoid hemorrhage (five with unruptured arteriovenous malformations and two with aneurysms), the CO2 vasoreactivity was tested on the side of the middle cerebral artery with normal flow velocities opposite the lesion. A baseline CO2 reactivity test was obtained in each patient and then repeated under constant intravenous infusion of nimodipine, 2 mg/hr. Nine patients with ruptured aneurysms who were rated at Hunt and Hess Grades 1 or 2 were operated on within 1 to 3 days after the hemorrhage and treated with nimodipine, 2 mg/hr, given intravenously. In these patients, CO2 vasoreactivity was tested during the second week after the hemorrhage, when the middle cerebral artery velocity was increased by at least 50% of the initial value or more. Nimodipine was then discontinued and, 48 hours later, when the middle cerebral artery velocity was still in the same range, CO2 vasoreactivity was tested again. Two months later, after full recovery from the subarachnoid hemorrhage and normalization of the velocities, a third measurement of CO2 reactivity was obtained as a baseline control. No significant effect of nimodipine on CO2 vasoreactivity could be demonstrated in any of the test periods. In the second week after a subarachnoid hemorrhage, a significant reduction of the cerebrovascular response to CO2 was found (P less than 0.005).