[CAVE - A checklist system for preoperative risk evaluation : Guideline-conform cardiopulmonary diagnostics before general and visceral surgical interventions]. / CAVE ­ Ein Checklistensystem zur präoperativen Risikoevaluation : Leitliniengerechte kardiopulmonale Diagnostik vor allgemein- und viszeralchirurgischen Eingriffen.

BACKGROUND: Preoperative evaluation of patient risk is an essential component of patient preparation before surgery. Guidelines provide evidence-based algorithms for preoperative assessment of cardiac risk; however, even experienced physicians correctly apply evidence-based algorithms in only 50% of all cases or less. OBJECTIVE: A checklist system for guideline-based cardiopulmonary risk evaluation in adult patients undergoing abdominal or visceral surgery (CAVE checklists) was created to assist in preoperative cardiopulmonary risk assessment and increase correct application of evidence-based algorithms before elective visceral surgery. MATERIAL UND METHODS: International guidelines were transformed into a checklist system. These checklists were than evaluated in a department of general and visceral surgery. The main goal was to determine whether preoperative examinations, such as electrocardiograph (ECG), chest-x-ray, spirometry and advanced assessment by a cardiologist, are performed according to evidence-based guidelines. The frequency of recommended as well as unnecessary and missed examinations was assessed. RESULTS: In this study 541 patients with a median age of 64.5 years (interquartile range: 52-73 years) were examined using the checklist system. Of the patients 90.4% underwent ECG and 98.5% chest-X-ray as recommended in the guidelines. Spirometry was not recommended in any patient and not performed in any case. Advanced assessment by a cardiologist was performed in 45.5% of cases as recommended in the guidelines. When guidelines did not recommend ECG, x­ray, spirometry or advanced cardiac assessment, 69.4%, 99.6%, 99.3% and 99.8% of patients, respectively, actually did not receive these examinations. Only 2.8% of all patients did not receive an examination that was recommended by the guidelines: 1.5% ECG, 0.2% x­ray and 1.1% advanced cardiological assessment. None of these patients suffered from postoperative cardiopulmonary complications. CONCLUSION: These simple checklists are easy to use and provide a higher degree of evidence-based preoperative cardiopulmonary risk evaluation than previously reported in the literature. Adaptation of the checklists to changing guidelines is easy to perform. Whether the application of these checklists will result in a reduction of morbidity and costs have to be determined in further clinical trials.

Paracentral photoablations to correct higher grades of naturally occurring astigmatism.

PURPOSE: To evaluate the efficacy of paracentral ablations in treating higher degrees of naturally occurring myopic and hyperopic astigmatism. SETTING: Augenklinik, Kreiskrankenhaus Bad Hersfeld, Germany. METHODS: Twenty-five eyes (7 with hyperopia, 18 with myopia) with naturally occurring corneal astigmatism greater than 1.75 diopters (D) were treated by excimer laser. The mean refractive cylinder was -4.05 D +/- 1.46 (SD) (range -1.75 to -7.00 D). The intention was to reduce the astigmatism without consideration of the spherical refractive error. Two paracentral ablations were performed by photorefractive keratectomy with treatment zones of 3.5 mm in the flatter meridian of the cornea. Objective refraction, best corrected visual acuity (BCVA), changes in corneal radius, development of haze, and regression were recorded. RESULTS: The paracentral ablations induced a steepening of the corneal radius in the flatter meridian from 8.12 mm (mean preoperative value) to 7. 84 mm (mean postoperative value) and thus reduced the mean refractive cylinder to -1.12 +/- 0.82 D (range 0.00 to - 3.00 D), corresponding to a mean reduction of 78%. No eye experienced a loss of Snellen lines. The preoperative BCVA (mean 20/25; range 20/50 to 20/20) was unchanged postoperatively CONCLUSIONS: Paracentral ablations resulted in a stable corneal curvature immediately after epithelial healing, with a moderate regression over time. Paracentral ablations with the excimer laser appear to be a safe and effective method to correct higher grades of corneal astigmatism.

[Combined phacoemulsification and goniotrepanation in primary chronic open angle glaucoma and classical pseudoexfoliation glaucoma]. / Kombinierte Phakoemulsifikation und Goniotrepanation bei primärem chronischen Offenwinkelglaukom und klassischem Pseudoexfoliationsglaukom.

BACKGROUND: Combined glaucoma and cataract operation has been demonstrated to be effective in controlling IOP and increasing visual acuity. Because of the differences between patients with primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PXEG), for cataract and glaucoma surgery alone we evaluated the effects and complications for simultanous surgical management. PATIENTS AND METHODS: In a retrospective study 103 patients were examined who underwent a combined phacoemulsification and goniotrephination between January 1993 and January 1997 and had no surgery before (110 eyes with POWG, 22 eyes with PXEG). RESULTS: The average age in the POAG group (75.1 +/- 8.7 years) was significantly less than in the PXEG group (79.3 +/- 5.9 years) (P < 0.05). The mean preoperative IOP in PXEG (31.8 +/- 10.3 mmHG) was significantly higher than in POAG (25.0 +/- 6.4 mmHg) (P < 0.0005). Due to the combined surgery the mean intraocular pressure decreased in both groups < 10 mmHg (days 1 and 7). PXEG had a significantly higher IOP at day 3 than POAG (12.3 +/- 8.4 mmHg versus 8.5 +/- 5.7 mmHg) (P < 0.05) and developed after combined operation IOP peaks > 25 mmHg into a significantly higher level (P < 0.05). Moreover, zonulolysis, rupture of the posterior capsule, vitreous loss and persistence of inflammatory response occurred more often in PXEG, but there was no significant difference compared to POAG. CONCLUSION: PXEG has an higher incidence of typical problems of phacoemulsification, a temporary increase of IOP and prolonged inflammation after combined cataract and glaucoma surgery than POAG, but there is a similar risk compared to a single procedure.

[Cyclocryotherapy in neovascular glaucoma and non-neovascular glaucoma]. / Zyklokryokoagulation bei Neovaskularisierungsglaukomen und Nicht-Neovaskularisierungsglaukomen.

BACKGROUND: There are conflicting reports on the value of cyclocryotherapy and it seems that the success rate is depending on glaucoma conditions, the period of follow-up and the technique. This retrospective study was carried out to assess the efficacy and complication rate of cyclocryosurgery for advanced glaucoma with and without neovascularization. PATIENTS AND METHODS: We induced 76 eyes of 75 patients with inadequately controlled glaucoma, which underwent cyclocryotherapy during the period of 1993 and 1996 (treatment time 60 seconds with -80 degrees C, 6-12 applications (mean 9.8 +/- 2.3), 180-360 degree (median 270 degree), diameter of the probe tip 2.5 mm, 1-2 mm distance from the limbus). Depending on the etiology we distinguished between neovascular (NVG) and non-neovascular glaucoma (nNVG). Pre- and postoperative data from all patients were studied retrospectively, for follow-up after 12-36 months patients were examined. RESULTS: Intraocular pressure (IOP) decreased in all patients from 44.7 +/- 12.6 mm Hg preoperatively to 15.6 +/- 6.5 mm Hg postoperatively after a follow-up of 12-36 months. In 88.2% IOP was lowered to < or = 25 mm Hg. NVG showed a mean IOP reduction from 49.1 +/- 12.5 mm Hg before cyclocryotherapy to 15.6 +/- 5.0 mm Hg at follow-up. In the nNVG group IOP was 40.5 +/- 11.3 mm Hg and 15.7 +/- 7.6 mm Hg after cyclocryotherapy. Pressure was controlled (< or = 25 mm Hg) for 83.8% of NVG and 92.3% of nNVG. A cyclocryotherapy-induced intense inflammation was seen more frequent in NVG (43.2%) than in nNVG (17.9%). 2 patients with NVG and 3 with nNVG developed phthisis postoperatively (total 6.7%). CONCLUSIONS: Cyclocryosurgery is an effective method to reduce IOP in advanced, refractory glaucoma, when other methods have failed. The risk/success rate seems to be acceptable.

[Intraocular lens calculation and ultrasound biometry: immersion and contact procedures]. / IOL-Berechnung und Ultraschallbiometrie: Immersions- und Kontaktverfahren.

BACKGROUND: Ultrasound biometry for axial length measurement may be performed either by directly putting the probe on the cornea or by using a water immersion technique. Our goal was to examine whether there are unsystematic differences between both techniques present besides systematic differences that can be compensated by adjusting calculation formula constants. PATIENTS, MATERIALS AND METHODS: We examined 288 patients in a prospective, randomized trial. There was no ocular pathology present beside cataract. Axial lengths < 21 mm and > 27 mm were excluded. We calculated which IOL power would have given the desired refractive result by using the postoperative refraction and data of the lens implanted. RESULTS: A systematic difference between both techniques is present. With the contact technique, axial length is measured 0.15 mm shorter. This requires adjustment of formula constants. Furthermore, there is an unsystematic difference that leads to 18% greater calculation errors (difference between IOL calculated preoperatively and ideal IOL) with the contact technique. Mean absolute error was 0.43 +/- 0.38 dpt for the immersion group and 0.53 +/- 0.48 dpt for the contact group. CONCLUSIONS: To minimize postoperative refractive errors, ultrasound biometry using immersion technique should be preferred.

[Intraocular pressure values in intubation anesthesia in patients with and without chronic open angle glaucoma]. / Verhalten des intraokularen Drucks in Intubationsnarkose bei Patienten mit und ohne chronischem Offenwinkelglaukom.

BACKGROUND: A number of clinical studies have investigated the influence of hypnotics and relaxants on intraocular pressure. All of them referred to the normal eye. Our goal was to show if there is a difference in intraocular pressure profile during general anesthesia between eyes with and without primary open angle glaucoma. PATIENTS AND METHODS: We prospectively examined two groups of 20 patients with and without primary open angle glaucoma. Intraocular pressure (IOP) readings were taken using a Perkins hand held applanation tonometer during routine surgery on cataract or glaucoma on the fellow eye. Balanced anesthesia was carried out using Etomidate, Vecuronium, Fentanyl, N2O and Enflurane. RESULTS: In the glaucoma group IOP fell from 20.3 +/- 4.1 mm Hg before induction of anesthesia (baseline) to 13.2 +/- 3.8 mm Hg (65% of baseline) after induction and 12.6 +/- 4.2 mm Hg (62%) 5 minutes after induction. In the non-glaucoma group IOP was 16.3 +/- 3.5, 9.7 +/- 3.0 (62%) und 9.6 +/- 3.0 (59%) respectively. Directly after placement of endotracheal tube we read 17.8 +/- 7.6 mm Hg (87%) in the glaucoma and 13.0 +/- 5.9 mm Hg (82%) in the non-glaucoma group. Shortly before removal of endotracheal tube we found IOP to be 21.3 +/- 4.5 mm Hg (107%) in the glaucoma and 17.8 +/- 6.8 mm Hg (106%) in the non-glaucoma group. Back on the ward we measured 19.8 +/- 4.1 mm Hg (94%) and 15.4 +/- 3.5 (93%) respectively. Pressure changes relative to baseline values show no statistically significant differences between the two groups. CONCLUSIONS: General anesthesia lowers IOP to the advantage of intraocular surgery. There is no statistically significant difference in pressure change relative to baseline value in eyes with and without primary open angle glaucoma.

[Significance of optic formula selection for postoperative refraction after cataract operation]. / Bedeutung der Formelauswahl für die postoperative Refraktion nach Katarakt-Operation.

BACKGROUND: Prediction of postoperative patient refraction is of paramount importance in modern cataract surgery. The choice of the IOL calculation formula plays a major role here. The influence of popular calculation formulas is evaluated in a prospective study. MATERIALS AND METHODS: We performed cataract surgery on 539 eyes with PC-IOL implantation in the capsular bag. The IOL power was computed using 10 different calculation formulas with the help of a personal computer. Patient refraction was examined two days and six months after surgery. RESULTS: The mean difference between desired and obtained refraction was -0.11 +/- 0.88 dpt 83% of all patients were in a +/- 1 D interval of desired refraction with a median visual acuity of 20/25. The formula used contributes a major part to the precision of calculation despite all problems due to errors of measurement. The theoretical formulas with variable "pseudophacic anterior chamber depth" are superior to the older ones. Standard deviation of the worst formula tested is 25% higher than that of the best one. With the best formula tested there would have been a good result (error < or = 1 D) in 82% of the cases, with the worst one in only 72%. CONCLUSIONS: The use of older formulas like SRK and Binkhorst should be discontinued and only the newer theoretical formulas (e.g. SRK/T, Holladay, Haigis etc.) be used. This is especially true for short and long eyes. For owners of a personal computer calculation and optimization of those more complex formulas should not be a problem.

Examination of developmental neurotoxicity by the use of tissue culture model systems.

1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity.

Acute and persistent effects of methamphetamine on developing monoaminergic neurons in reaggregate tissue culture.

Three-dimensional, rotation-mediated reaggregate tissue cultures composed of rostral mesencephalic cells and corpus striatal cells were used to examine the short-term and persistent effects of methamphetamine on developing monoamine-containing neurons. Reaggregates were exposed to drug for one week. Reductions in reaggregate endogenous dopamine and serotonin levels occurred following treatment with methamphetamine during days 15-22 of culture over the concentration range 10(-7) to 10(-4) M. The highest methamphetamine concentration reduced dopamine and serotonin levels to 29 and 33%, respectively, of control values. Monoamine levels were reduced from control values after 3 days of exposure to 10(-4) M methamphetamine. No further reduction resulted from 4 additional days of drug treatment. In order to determine whether monoaminergic neurons would recover from the drug-induced deficit, reaggregates were exposed to 10(-4) M methamphetamine for 7 days and then grown in drug-free media for an additional 20 days. During the 20 day recovery period, monoamine levels in the control group increased with time in culture. After an initial rapid increase (recovery days 0-9), the level of monoamines in the recovery group remained at a constant proportion to the level in the control group suggesting that the monoaminergic neurons return to a rate of development similar to that seen in untreated cultures. However, this rate was not sufficient to overcome the reduction in monoamine levels produced by 7 days of methamphetamine treatment. The results indicate that the effects of methamphetamine on developing monoaminergic neurons are marked and persistent.

Survival of developing dopaminergic neurons in reaggregate tissue culture following treatment with methamphetamine.

Developing central dopamine (DA)-containing neurons in rotation-mediated reaggregate tissue culture exposed to 10(-4) M methamphetamine for 1 week (at a time corresponding to the 2nd week of postnatal development) exhibit decreased DA levels. DA neurons were visualized either by a histofluorescent method after 'loading' with exogenous DA, or by tyrosine hydroxylase (TH) immunocytochemistry. An apparent loss of histofluorescent DA cell bodies was observed due to a methamphetamine-induced reduction in exogenous DA accumulation. No decrease in the number of TH-immunoreactive neurons was observed. Thus, developing DA neurons when visualized by TH immunocytochemistry survive despite methamphetamine-induced reductions in DA levels.

Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion.

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenylalanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Correlated light and electron microscopic study of dopaminergic neurons and their synaptic junctions with DARPP-32-containing cells in three-dimensional reaggregate tissue culture.

An antibody to tyrosine hydroxylase has been used in a correlated light and electron microscopic study to characterize dopaminergic neurons and synaptic junctions in three-dimensional reaggregate cell culture. Dissociated fetal mesencephalic cells containing dopamine neurons were coaggregated with dissociated fetal striatal cells in rotatory culture for 21 days. Sections of the coaggregates were stained by the peroxidase anti-peroxidase technique to reveal tyrosine hydroxylase-immunoreactive structures. Clusters of immunoreactive perikarya as well as dendrites and axons were observed. Immunolabeled perikarya were round or oval and approximately 20 microns in diameter. Boutons immunoreactive for tyrosine hydroxylase formed symmetric synapses, primarily with unlabeled dendritic shafts. Symmetric membrane specializations were also observed between tyrosine hydroxylase-positive boutons and unlabeled dendritic spines as well as with the perikaryon of an unlabeled medium-size neuron possessing a slightly indented nucleus. To characterize the neurochemical nature of the neurons postsynaptic to tyrosine hydroxylase-positive boutons in the reaggregates, an antibody against DARPP-32 (a dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein) and an antibody against tyrosine hydroxylase were employed to visualize striatal dopaminoceptive neurons and dopaminergic structures, respectively, in the same section. Examination of reaggregate sections at the light microscopic level demonstrated that DARPP-32-immunoreactive cells were distributed into discrete clusters that were associated with patches of tyrosine hydroxylase-positive axonal varicosities. Ultrastructural analysis of tyrosine hydroxylase-positive boutons in such clusters revealed that dopaminergic axons synaptically contacted DARPP-32-immunoreactive neurons as well as unlabeled neuronal structures.

Selective association of dopamine axons with their striatal target cells in vitro.

The purpose of the present study was to examine the distribution of dopamine (DA) axons after simultaneously confronting DA cells with both target cells of the corpus striatum (CS) and with non-target cells of the tectum (T). Dissociated fetal cells of rostral mesencephalic tegmentum (RMT) containing DA neurons and dissociated non-target tectal cells were exposed to wheat germ agglutinin conjugated to the fluorescent dye, rhodamine, prior to reaggregation in rotatory culture in order to distinguish these cells from non-dyed CS cells in the resulting reaggregates. After 9 days in culture, RMT-T-CS reaggregates were exposed to 10(-5)M DA and processed for DA-fluorescence histochemistry. Single reaggregates were serially sectioned and color photomicrographs prepared from each section. It was found that non-target cells (red rhodamine fluorescence) segregated from the undyed target cells, forming discrete areas containing the two cell types. DA nerve cell bodies and their processes could be distinguished by their green fluorescence. Since it is not possible to distinguish axonal from dendritic processes in a single section, the extent of neuronal dendritic arborization was estimated from reaggregates prepared from cells of the RMT and non-target T cells, in which there is no extensive proliferation of DA axons and fluorescent DA processes (presumably dendrites) are confined to the area near the cell body. Following exclusion of DA dendritic fluorescence, it was found that 85.2% of the presumed axonal fibers were present in the non-dyed areas containing striatal target cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures.

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures.

We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum. In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells. We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system. Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin. Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days. The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase-positive acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations. Representative reaggregate profiles were then re-embedded for electron microscopic examination. In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied. In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration. Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact. In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact. By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates. In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells. In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotrophic effects of hippocampal target cells on developing septal cholinergic neurons in culture.

The influence of hippocampal target cells on the development of cholinergic septal neurons was studied in rotation-mediated reaggregating cell cultures. Brain cells from 15-day-old mouse embryos were obtained from: septum, containing cholinergic cells which project to the hippocampus; hippocampus which contains target cells for the septal cholinergic neurons; and cerebellum, containing cells which are not targets for the septal cholinergic cells. The cells were then cultured for 3 weeks in a rotary incubator in the following combinations: septal cells alone; hippocampal cells alone; cerebellar cells alone; septal-hippocampal cells together; and septal-cerebellar cells together. After harvesting, fixation, and embedding, 50 micron sections were cut and processed for visualization of acetylcholinesterase activity. Sections from reaggregates containing either hippocampal or cerebellar cells alone contained only a few acetylcholinesterase-positive cells, but no positive fibers. Sections from septal-hippocampal coaggregates revealed a pattern of well-defined, fine-caliber acetylcholinesterase-positive fibers with extensive arborizations and varicosities suggesting axonal proliferation. In septal-cerebellar coaggregates, acetylcholinesterase-positive fibers appeared to be degenerating and distinct areas were observed which were essentially devoid of acetylcholinesterase fibers. In some experiments, either cerebellar or hippocampal cells were labeled with wheatgerm agglutinin-rhodamine prior to culture in order to identify these cells in the resulting reaggregates. Analysis of sections from these studies showed that acetylcholinesterase fibers were excluded from regions of coaggregates containing cerebellar cells, but were present in regions of coaggregates containing hippocampal cells. Finally, cell counts of acetylcholinesterase-positive cells in the various combinations revealed that these putative cholinergic neurons were significantly more numerous in septal-hippocampal coaggregates (271 +/- 19 per 10(6) septal cells added) than in septal reaggregates (38 +/- 6 per 10(6) septal cells added) or septal-cerebellar coaggregates (85 +/- 29 per 10(6) septal cells added). These results, taken together, suggest that hippocampal target cells influence the development and survival of cholinergic neurons.

Neurotoxic effects of methamphetamine assessed in three-dimensional reaggregate tissue cultures.

Three-dimensional, rotation-mediated, reaggregate tissue cultures formed from dissociated fetal rostral mesencephalic tegmental (RMT) and corpus striatal (CS) or frontal cortical (FCx) cells were used to study methamphetamine neurotoxicity. Analysis of dopamine (DA), serotonin (5-HT) and gamma-aminobutyric acid (GABA) levels using HPLC techniques revealed decreases in RMT-CS and RMT-FCx reaggregate DA and 5-HT levels after treatment between 14 and 21 days in culture with methamphetamine in concentrations ranging from 10(-6)M to 10(-3)M. Dopamine cell numbers in RMT-CS and RMT-FCx reaggregates were estimated after visualization by histofluorescent techniques. Methamphetamine treatment caused decreases in DA cell numbers which paralleled the decreases in endogenous DA levels. Estimates of the accumulation of exogenous DA by RMT-CS reaggregates treated with methamphetamine showed that the amount of accumulation per cell remained fairly constant despite marked reductions in total DA cell numbers. This suggests that the reductions in endogenous DA levels following methamphetamine were secondary to loss of entire DA neurons rather than of a portion of the terminal axonal fields in the surviving neurons. Reaggregate tissue cultures are a useful tool in the study of potential neurotoxic effects of new or untested psychotherapeutic agents.

Release of dopamine from mesencephalic neurons in aggregate cultures: influence of target and non-target cells.

Spontaneous release of [3H]dopamine (DA) was observed from reaggregates of dissociated cells from fetal rostral mesencephalic tegmentum (RMT) containing DA neurons cocultured with their axonal target cells from striatum (CS) or frontal cortex (FCx), or with non-target cells from occipital cortex (OCx), or tectum. Such release increased in response to 50 mM K+. Tetrodotoxin (TTX) suppressed the spontaneous release from RMT-CS and RMT-FCx reaggregates by 42%; from RMT-tectum reaggregates by 24%, and did not significantly inhibit the release from RMT-OCx cocultures. Since TTX blocks spontaneous neuronal activity, these results suggest that the presence of axonal target cells enhances the activity of the dopamine neurons. DA neurons within RMT-FCx reaggregates released significantly more [3H]DA in response to 50 mM K+ than in RMT-CS cocultures. This result is in accord with the findings in vivo that inhibitory feedback mechanisms on DA release, present in the striatum, are lacking in the frontal cortex.

Enhanced dopamine cell survival in reaggregates containing telencephalic target cells.

Dissociated, 14-day-old embryonic cells of the rostral mesencephalic tegmentum (RMT), including the dopamine neurons of this region, were allowed to reaggregate and develop in rotatory culture for 7 days in the presence of dissociated embryonic cells from the target areas of the dopaminergic neurons, corpus striatum (CS) or frontal cortex (FCx). Alternatively, RMT cells were allowed to reaggregate by themselves or in the presence of dissociated cells from a telencephalic area, occipital cortex (OCx), or mesencephalic area, tectum (T), which are not target areas for the dopamine neurons. Histofluorescence analysis revealed the number of dopamine neurons contained within reaggregates of any given type. Approximately 4 times as many dopamine neurons were found in RMT-CS coaggregates and 1.5 times as many in RMT-FCx coaggregates than in aggregates constituted from cells of the RMT either alone, or in coaggregates from RMT-OCx or RMT-T. Since axonal process formation and maintenance can only be observed in RMT-CS and RMT-FCx coaggregates, the enhanced dopamine neuron survival is probably due to an interaction of dopaminergic axonal processes with target cells within the reaggregates.

Release of dopamine from coaggregate cultures of mesencephalic tegmentum and corpus striatum.

Dissociated dopamine (DA) neurons from 14-day fetal mice were dissected from the rostral mesencephalic tegmentum (RMT) and were allowed to reaggregate in vitro with cells from the corpus striatum (CS). As previously demonstrated under these conditions, DA neurons develop punctate fluorescent varicosities and the capacity to synthesize, accumulate, and retain DA (Kotake, C., P. C. Hoffmann, and A. Heller (1982) J. Neurosci. 2: 1307-1315). After 17 to 22 days in culture, the RMT-CS coaggregates were assessed for their ability to release DA. Coaggregates were incubated in 5.6 x 10(-6) M [3H]DA, washed, and then superfused at 100 microliters/min for 2 hr. Fractions were collected every 2 min. Basal efflux of [3H]DA/2 min was 1% of tissue stores of 3H. K+, 70 mM infused for 8 min induced a peak release of 5.87% of tissue stores of 3H, and 50 mM K+ induced a peak release of 2.13%. The potassium-induced release of [3H]DA was calcium dependent. When d-amphetamine was infused for 12 min, 100 microM solutions induced a peak release of 8.91%, 10 microM induced a peak release of 4.36%, and 1 microM induced a peak release of 1.85% of tissue stores of 3H. Substance P at 100 microM induced a peak release of 2.19% of tissue stores of 3H. Tetrodotoxin (0.5 and 2.5 microM) decreased basal efflux by 40%, blocked substance P-induced release, but did not affect either potassium- or d-amphetamine-induced release of [3H]dopamine.