A quantitative ultrastructural analysis of neurotensin-like immunoreactive terminals in the midbrain periaqueductal gray: analysis of their possible relationship to periaqueductal gray-raphe magnus projection neurons.

The periaqueductal gray of the rat contains significant levels of the putative peptide neurotransmitter neurotensin. The profound anti-nociceptive effects of neurotensin injected into the periaqueductal gray may involve a population of periaqueductal gray neurons having descending projections to the rostral ventral medulla, including nucleus raphe magnus and adjacent reticular nuclei. In this study, electron microscopic immunocytochemistry was used to examine the ultrastructure of periaqueductal gray axon terminals containing neurotensin-like immunoreactive material and to obtain quantitative data regarding the relationship of such terminals to other elements of the neuropil. Of particular interest was the interaction between neurotensin-like immunoreactive terminals and retrogradely labeled neurons that project to nucleus raphe magnus and adjacent reticular nuclei. Within the periaqueductal gray, the sites of retrograde and immuno-labeling were consistent with previous reports. The neurotensin-immunoreactive structures were predominantly axon fibers and terminals. In the ventrocaudal periaqueductal gray, the mean diameter of neurotensin-containing terminals was 0.93 +/- 0.02 micron and they comprised a volume fraction of 0.0010. Most of the neurotensin-positive terminals examined (74.2%) were in contact with or closely apposed to dendrites. The most common anatomical configuration observed was a single neurotensin-immunoreactive terminal juxtaposed to three dendrites. Only 2% of immunoreactive terminals were apposed to perikarya. Neurotensin-immunoreactive terminals were observed to form symmetrical synapses and 96.4% of such terminals were axodendritic. Occasional multiple neurotensin-immunoreactive terminals associated with single dendrites were observed. Although neurotensin-like immunoreactive terminals were quite prominent, only a small percentage made synaptic contact with periaqueductal gray neurons that project to the nucleus raphe magnus and adjacent reticular formation. Among the population of periaqueductal gray neurons retrogradely-labeled from nucleus raphe magnus and adjacent reticular nuclei, the frequency of direct synaptic contact by neurotensin-immunoreactive terminals was 2%. These data suggest that the periaqueductal gray circuitry by which neurotensin ultimately affects descending pathways is complex and may involve a population of local circuit neurons whose transmitters and connections remain to be elucidated.

Ultrastructural morphometric analysis of enkephalin-immunoreactive terminals in the ventrocaudal periaqueductal gray: analysis of their relationship to periaqueductal gray-raphe magnus projection neurons.

The periaqueductal gray plays an important role in the descending modulation of nociception. While the importance of endogenous opioids to periaqueductal gray circuits that modulate nociception is supported by many studies, the ultrastructural relationships between enkephalin-immunoreactive axon terminals and the surrounding periaqueductal gray neuropil have not been quantitated in the rat. Further, the possible interaction between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that project to the rostroventral medulla has not been described. The present study utilized electron microscopic immunocytochemistry to quantitate the normal neuronal associations of enkephalin-immunoreactive terminals in the caudal periaqueductal gray of the rat. A primary focus of this analysis was to ascertain whether any interaction exists between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that were retrogradely-labeled from the nucleus raphe magnus and adjacent medullary reticular nuclei. We examined the ventrolateral periaqueductal gray and the ventral periaqueductal gray immediately subjacent to the aqueduct and found that both the average terminal diameters and the volume fractions of enkephalin-immunoreactive terminals were very similar. In these two regions, most terminals were observed to be in close apposition to either two or three dendrites that were neither retrogradely-labeled nor enkephalin-immunoreactive, although axonal and perikaryal associations were also observed. In the ventrolateral periaqueductal gray, 22% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. In the periaqueductal gray subjacent to the aqueduct, 32% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. Symmetrical synapses with these retrogradely-labeled neurons were formed by 5.5% of enkephalin-immunoreactive terminals in the ventrolateral periaqueductal gray, and by 4.3% of enkephalin-immunoreactive terminals located subjacent to the aqueduct. We also noted that enkephalin-immunoreactive terminals formed symmetrical synapses with non-retrogradely-labeled, enkephalin-immunoreactive dendrites in the periaqueductal gray. Direct opioid input onto putative excitatory periaqueductal gray output neurons that are hypothesized to modulate nociception was an unexpected finding.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of peripheral nerve injury on alpha-2A and alpha-2C adrenergic receptor immunoreactivity in the rat spinal cord.

Neuropathic pain resulting from peripheral nerve injury can often be relieved by administration of alpha-adrenergic receptor antagonists. Tonic activation of alpha-adrenergic receptors may therefore facilitate the hyperalgesia and allodynia associated with neuropathic pain. It is currently unclear whether alpha2A- or alpha2c-adrenergic receptor subtypes are involved in the pro-nociceptive actions of alpha-adrenergic receptors under neuropathic conditions. We therefore investigated the effects of peripheral nerve injury on the expression of these subtypes in rat spinal cord using immunohistochemical techniques. In addition, neuropeptide Y immunoreactivity was examined as an internal control because it has previously been shown to be up-regulated following nerve injury. We observed a decrease in alpha2A-adrenergic receptor immunoreactivity in the spinal cord ipsilateral to three models of neuropathic pain: complete sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. The extent of this down-regulation was significantly correlated with the magnitude of injury-induced changes in mechanical sensitivity. In contrast, alpha2c-adrenergic receptor immunoreactivity was only increased in the spinal nerve ligation model; these increases did not correlate with changes in mechanical sensitivity. Neuropeptide Y immunoreactivity was up-regulated in all models examined. Increased expression of neuropeptide Y correlated with changes in mechanical sensitivity. The decrease in alpha2A-adrenergic receptor immunoreactivity and the lack of consistent changes in alpha2C-adrenergic receptor immunoreactivity suggest that neither of these receptor subtypes is likely to be responsible for the abnormal adrenergic sensitivity observed following nerve injury. On the contrary, the decrease in alpha2A-adrenergic receptor immunoreactivity following nerve injury may result in an attenuation of the influence of descending inhibitory noradrenergic input into the spinal cord resulting in increased excitatory transmitter release following peripheral stimuli.

Production and characterization of a novel monoclonal antibody against neurotensin: immunohistochemical localization in the midbrain and hypothalamus.

We developed a mouse monoclonal antibody against neurotensin (NT), termed NT8, for applications in immunohistochemistry and for ELISA analysis of NT. The antibody's paratope was determined by competitive ELISA using several peptide fragments of NT. That paratope requires intact peptide bonds between NT residues proline-7, arginine-8, and arginine-9. The antibody is of the IgG2B sub-isotype, having an IC50 for intact NT of approximately 3 nM when measured by competitive ELISA. Light microscopic immunohistochemical studies in the periaqueductal gray (PAG) and hypothalamus demonstrated staining patterns that agreed well with previous reports. Neuron perikarya were visualized even in the absence of colchicine pre-treatment, indicating that NT8 antibody is very sensitive in immunohistochemical applications. At the EM level, the antibody stained axon terminals, dendrites, and perikarya in the PAG. In lightly immunoreactive perikarya, rough endoplasmic reticula were visualized, suggesting that biosynthetic precursors to NT might be recognized by NT8.

The effect of acute haloperidol treatment on brain proneurotensin mRNA: in situ hybridization analyses using a novel fluorescence detection procedure.

These studies describe the normal anatomical distribution of neurons containing the mRNA coding for neurotensin (proneurotensin/neuromedin N) in the rat forebrain and midbrain and examine how that distribution is altered by acute administration of the dopamine antagonist haloperidol. A novel fluorescence detection method was developed and employed with biotinylated oligonucleotides to permit the rapid, sensitive visualization of in situ hybridization. The hybridization was temperature-sensitive, eliminated by ribonuclease, and co-localized in neurotensin-immunoreactive perikarya in the midbrain. In the forebrain of control rats, proneurotensin mRNA-containing neurons were found in the dorsomedial and ventrolateral caudate/putamen, in the nucleus accumbens, in the ventral striatum including the olfactory tubercles, and in the septal nuclei. Haloperidol induced significant increases in the frequencies and distributions of hybridization-positive neurons in the striatum and septal nuclei. In the midbrain, the highest frequency of hybridization-positive neurons occurred in the substantia nigra and the superior colliculus. Prominent populations were also present in the dorsal and ventral periaqueductal gray, the oculomotor region, and the medial longitudinal fasciculus. Less prominent were populations of neurons in the dorsomedial deep mesencephalic nuclei and the ventral tegmental area. Haloperidol induced only modest increases in the frequency of pro-neurotensin mRNA-containing neurons in the ventral tegmental area, and had no effects elsewhere in the midbrain. These results show that the fluorescent detection techniques used in this analysis provide a very rapid, reliable method for localizing hybridized mRNA in the rat brain. This study also suggests that a subpopulation of striatal neurons begin to express proneurotensin mRNA in response to haloperidol treatment. This effect of haloperidol on striatal neurons contrasts with results from additional studies of enkephalin mRNA in the striatum, suggesting that the mechanisms of haloperidol stimulation may differ between neurotensin and enkephalin-containing neurons.

Visualization of time-dependent redistribution of delta-opioid receptors in neuronal cells during prolonged agonist exposure.

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.

Evidence that somatostatin inhibits proinsulin synthesis and insulin release by isolated pancreatic islets of the rat.

Conflicting reports on the direction and magnitude of the effect of somatostatin on (pro)insulin synthesis prompted our investigation. Two assays for proinsulin synthesis were designed in which [4,5-3H]-L-leucine incorporation into proinsulin was normalized on the basis of postincubation insulin levels rather than on the number of islets incubated. Somatostatin at a concentration of 10 micrograms/ml inhibited 300 mg/dl glucose-stimulated proinsulin synthesis by 25% from 448 +/- 25 dpm/microunits insulin to 336 +/- 25 dpm/microunits insulin (disintegrations per minute in the proinsulin peak per microunit extractable insulin) (p less than 0.05). Glucagon (10 micrograms/ml) reversed the inhibitory effect of somatostatin on proinsulin synthesis from 336 +/- 25 dpm/microunits insulin to 480 +/- 44 dpm/microunits insulin (p less than 0.02). Somatostatin (10 micrograms/ml) had no significant effect on proinsulin synthesis in the presence of 70 mg/dl or 150 mg/dl glucose. Insulin release in 300 mg/dl glucose was inhibited 38% by 10 micrograms/ml somatostatin from 3.05 +/- 0.40 mU medium/mU tissue to 1.90 +/- 0.10 mU medium/mU tissue (p less than 0.01) over a 45-minute incubation period. These data suggest that somatostatin may act on glucose signal transduction on a level at which both insulin synthesis and secretion are affected. Further, the results are consistent with the hypothesis that cyclic AMP participates in mediating somatostatin effects on B-cell metabolism.

[125I]-insulin metabolism by the rat liver in vivo: evidence that a neutral thiol-protease mediates rapid intracellular insulin degradation.

The subcellular site where insulin is degraded by rat hepatocytes in vivo is controversial. While several potential insulin-degrading enzyme systems, each with its own characteristic cellular location, are known to exist in the liver, questions remain about which of them participates in the degradation of physiologic doses of insulin. These studies examine the proteases that degrade physiologic doses of [125I]-insulin in vivo to determine (1) when and where initial degradation occurs, and (2) which of the potential degradative enzymes is active. Following injection into the mesenteric veins of male rats, intact [125I]-insulin and its labeled degradation products were analysed by reverse-phase high-performance liquid chromatography (RP-HPLC) of biopsy homogenates. [125I]-insulin was rapidly degraded in vivo; the t 1/2 of degradation was approximately 2.7 minutes. To test for extracellular protease activity, an isolated perfused liver system was employed. [125I]-insulin (or [125I]-glucagon) uptake was controlled by changing the temperature of the perfusion medium. Five minutes after [125I]-insulin injection, surface-bound label was recovered in an acidic (pH 3.5) wash. In perfusion at 15 degrees C, both the internalization and degradation of [125I]-insulin were inhibited; 7.2% of unbound hormone was degraded and 5.1% of surface-bound insulin was degraded. Only 11.4% of unbound insulin and 17.4% of surface-bound insulin were degraded at 35 degrees C. In contrast, 95.5% of unbound glucagon and 89.9% of surface-bound glucagon were degraded at 35 degrees C. Thus, although glucagon degradation occurs at the sinusoidal plasmalemma of perfused livers, the same membrane does not mediate the rapid degradation of insulin observed in vivo. Analysis of the RP-HPLC [125I]-insulin elution profiles from liver biopsy homogenates, and comparison of them to profiles produced by purified proteases, suggested that insulin protease is responsible for most hepatic degradation of physiologic doses of insulin. Some cathepsin D-like activity was also observed in vivo, confirming that two pathways exist for insulin metabolism. The time course over which insulin was degraded was more rapid than previous studies in vitro would have predicted. This suggests that more insulin was receptor-bound at the time of its initial degradation, and that the active protease was soluble and was introduced into endocytic peripheral endosomes within seconds after their formation.

The effect of glucose on insulin and proinsulin synthesis in the streptozotocin-nicotinamide--induced rat islet adenoma.

Studies were conducted to examine the insulin and proinsulin synthetic response to glucose in the streptozotocin-nicotinamide--induced rat islet adenoma. Tumors responded to an increase from 3.8 mmol/L to 16.6 mmol/L glucose by increasing incorporation of [3H]-L-leucine into both proinsulin and insulin. Though this response was statistically significant, the stimulation was less than that noted in rat islets, and the variability of incorporation was greater. In addition, the conversion of proinsulin to insulin was generally slow, again with substantial intertumor variation. The recovery of insulin, as well as proinsulin, was significantly higher at the end of four hours of incubation in tumors incubated in 16.6 mmol/L glucose, implicating an insulin-degradative pathway modulated by glucose. Therefore, while the tumor will not replace conventional sources of tissue for insulin biosynthetic experiments, systems utilizing the tumor can serve as an addition to the methodology for studying previously unrecognized or poorly understood intracellular processes within the beta cell.

The synthesis of insulin and proinsulin in a cell-free system derived from the streptozotocin-nicotinamide--induced rat islet adenoma.

Preliminary studies were conducted to develop a cell-free system for insulin biosynthesis using the streptozotocin-nicotinamide--induced rat islet adenoma. Radiolabeled proteins, migrating on steric exclusion chromatography and SDS-gel electrophoresis in the region of insulin and proinsulin, were synthesized in a system prepared from the tumor 800 X g supernatant fraction, rat liver cytosol, and appropriate energy substrates. The proteins were not synthesized by a rat liver cell-free system, and synthesis could be substantially inhibited by the addition of cycloheximide. In addition, it could be shown that the islet proteins were not the products of residual intact cells within the system, nor were they an artifact due to nonspecific binding of [3H]-L-leucine to pre-existing insulin and proinsulin. The radiolabeled material eluting with insulin on steric exclusion chromatography was identified as [3H]-insulin by immunoaffinity column chromatography.

Construction of 1 mm microdialysis probe for amino acids dialysis in rats.

We describe here a microdialysis probe with 1 mm opening for precise and confined dialysis area in the awake, freely moving rat. This probe is designed to allow the local diffusion of the perfusion medium to an area approximately 175 microm high, 266 microm wide (mediolateral direction), and 305 microm in rostrocaudal direction. In addition, the probe allows the local application of drugs to the same precise area of interest. The probe was constructed from a piece of 25 gauge tubing with 1 mm hallowed opening located 0.5 mm from the distal (inserting) end. The dialysis fiber which was inserted into the stainless steel 25 gauge tubing and cemented into place has 200 microm diameter and 5000 molecular weight cut off. We tested the probe diffusion extent by direct infusion of fluorogold through the dialysis cannula. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine a sodium channel activator. All amino acids tested showed a significant 80% times decrease in their recovery concentration when compared to their respective concentrations recovered through 2 mm probe constructed earlier in our laboratory (Renno et al., 1992). Tests in awake rats with probes in the ventrocaudal PAG showed stable amounts of 12 different amino acids during repeated (6-8 times) 12 min samples at 3-5 microl/min collecting rate. Depolarization with 75 microM veratridine resulted in significant elevation in extracellular gamma-aminobutyric acid (GABA), aspartate, glutamate, taurine, glycine and citrulline. This design enables us to apply drugs of interest and measure the concentrations of amino acid neurotransmitters to a more precise, delineated and premeasured areas in the CNS.

Chronic pain increases brainstem proneurotensin/neuromedin-N mRNA expression: a hybridization-histochemical and immunohistochemical study using three different rat models for chronic nociception.

The role of neurotensin in the central nervous system is poorly understood. Exogenous neurotensin has potent antinociceptive effects when injected into the midbrain periaqueductal gray (PAG). Although it is present in terminals, fibers and perikarya within the PAG and other midbrain regions known for their antinociceptive circuits, it is not known whether endogenous neurotensin modulates nociception. We examined the midbrain in three different rat models for nociception to learn whether acute or chronic pain altered neuronal levels of the proneurotensin/neuromedin N mRNA (neurotensin mRNA). The models were: adjuvant-induced polyarthritis, adjuvant-induced unilateral paw inflammation, and unilateral peripheral mononeuropathy caused by ligation of the sciatic nerve. Behavioral observations confirmed that the expected symptoms developed as previously described. Within each of the three experimental models, we performed in situ hybridization histochemistry on coronal sections from three midbrain levels that included the rostral one-third of the PAG, the middle one-third of the PAG, and the caudal one-third of the PAG. At the level of the rostral PAG, we found that neither chronic nor acute nociception altered the frequencies or distributions of neurons containing neurotensin mRNA. In contrast, at the levels of the mid- and caudal one third of the PAG, the early effects of the nociceptive lesions differed from the chronic effects. During the acute phase of each model, increases in either the frequency or field area of neurons that were hybridization-positive for neurotensin mRNA were confined to the ventromedial PAG and the dorsal raphe nucleus. As the nociceptive stimuli became chronic, the early increases in neurotensin mRNA-containing neurons at the level of the middle third of the ventral PAG were diminished but remained above control levels, while increases in neurotensin mRNA began to occur in the midbrain tegmentum lateral to the PAG. The most striking increases in neurotensin mRNA expression were observed 16-17 days after the onset of nociceptive stimuli. At the level of the mid-PAG and caudal PAG, increased hybridization signal intensities and neuron frequencies occurred within the nucleus cuneiformis and the lateral tegmental nuclei, including the pedunculopontine and microcellular tegmental nuclei, as well as the deep mesencephalic nuclei. Hybridization-positive neurons in the tegmental nuclei were not observed at early stages of lesion development, but were a consistent feature of caudal midbrains after nociception became chronic.(ABSTRACT TRUNCATED AT 400 WORDS)

Basal release of Met-enkephalin and neurotensin in the ventrolateral periaqueductal gray matter of the rat: a microdialysis study of antinociceptive circuits.

The periaqueductal gray (PAG) contains neural circuits that participate in descending antinociception. Anatomical and electrophysiological evidence suggests that these circuits might employ opioid peptides and GABA in series to remove a tonic inhibition of descending PAG output neurons. The present studies examined the release of the antinociceptive peptides Met-enkephalin and neurotensin in the ventrolateral PAG, and investigated the interaction between GABA and Met-enkephalin release. In awake and freely moving rats the ventrolateral PAG was dialysed using 25 ga. concentric probes. Basal release of peptide in 12 min or 40 min fractions was determined using radioimmunoassays. To establish how the ventrolateral PAG responds to nociception, dialysis was performed following unilateral hindpaw inflammation using Complete Freund's Adjuvant. Twenty-four hours after inflammation was induced, neurotensin release was increased 133% and Met-enkephalin release was increased 353% compared to control animals. Seven days after inflammation was induced, neurotensin release declined precipitously, while basal Met-enkephalin release remained elevated 313% above controls. Thus, unlike enkephalin, increased basal neurotensin release is not sustained with persistent tonic nociception. In addition, we confirmed in normal animals that the ventrolateral PAG is induced to release Met-enkephalin by systemic morphine. A 43% increase in basal Met-enkephalin release was observed immediately following a 12 mg/kg i.p. morphine injection. Morphine should have the opposite effect (inhibit peptide release) if it acts directly on the enkephalinergic neurons. Thus, we examined the hypothesis that GABAergic interneurons in the PAG mediated morphine-stimulated enkephalin release. When the GABAantagonist bicuculline (0.25 microM to 25 microM) was co-infused with the dialysis medium, Met-enkephalin release increased in a dose-dependent fashion and peaked 68% above pre-infusion levels. These data elucidate the reciprocal inhibitory relationship between GABA and enkephalin in the ventrolateral PAG. We hypothesize that, when nociception induces Met-enkephalin release within this region, the tonic GABAergic inhibition is overcome, resulting in greater sensitivity of PAG enkephalinergic neurons. Ultimately, this enhanced enkephalin release should result in greater excitability of the descending PAG output neurons that are responsible for antinociception.

Nitric oxide synthase is found in some spinothalamic neurons and in neuronal processes that appose spinal neurons that express Fos induced by noxious stimulation.

To determine if nitric oxide (NO) and Fos immunoreactivity induced by noxious stimulation were colocalized in spinothalamic neurons, double-staining immunocytochemical techniques were combined with retrograde neuroanatomical tracing procedures. Initial studies on three rats demonstrated that Fos and nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, did not coexist in spinothalamic tract neurons. However, some spinothalamic neurons were found to contain NOS and some NOS immunoreactive processes were found to appose Fos containing neurons. Thus the remainder of the study: (1) analyzed the relationship of NOS positive neuronal processes with Fos stained neurons using a Fos immunocytochemical technique in combination with either NOS immunofluorescence or NADPH-diaphorase histochemistry; and (2) quantitated the number of NOS containing cells that project to the thalamus using a combined immunofluorescent-retrograde tracing procedure. Both NOS-like immunoreactive (NOS IR) neuronal processes and NADPH-diaphorase positive neuronal processes in the dorsal horn of the lumbar spinal cord were found to appose Fos positive neurons located in laminae I and II of the dorsal horn. Approximately 40% of Fos-labeled cells in these superficial laminae were found to be in apposition to or in close proximity to NOS labeled neuronal processes. Examination of spinal cord sections for NOS-containing spinothalamic tract neurons revealed that lamina X was the only spinal cord region containing such double-labeled neurons. Further quantification revealed that approximately 10% of NOS positive neurons in lamina X were double-labeled with Fluorogold. These findings support the hypothesis that nitric oxide is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus and further indicate that nitric oxide may contribute to the central transmission of spinothalamic information.

Pancreatic islet cell reaggregation systems: efficiency of cell reassociation and endocrine cell topography of rat islet-like aggregates.

Single cells isolated from rat islets of Langerhans were cultured under conditions that support reassociation into islet-like aggregates. Comparisons were made of enzymatic methods of islet dissociation, rotational or static culture conditions, and culture at basal or stimulatory glucose concentrations. Over a period of 4 days the aggregates progressed through three stages of organization: cell coalescence to cellular chains, rearrangement of chains into small spheroids, and growth of spheroids. The numerical yield of aggregates was optimum after islets were dissociated with dispase. Culture under rotation resulted in the production of more aggregates of significantly larger diameter than under static conditions. Medium glucose concentrations of 4 and 11 mM supported cell reassociation under rotator culture, but no aggregation occurred under static culture at the basal (4 mM) glucose level. Aggregates resulting from 4-day rotator culture exhibited endocrine cell distributions similar to intact islets. Islet aggregates released insulin in response to glucose, but nonaggregated cells, maintained in culture, did not. The present comparisons reveal significant variability in the cellular composition, rate of formation, and yield of aggregates, and suggest that the methodology for producing aggregates should be carefully considered in experimental design.

Hospital-based case management: results from a demonstration.

The Flinn Foundation Hospital-based Coordinated Care case management demonstration was designed to help patients discharged from six participating hospitals be linked to community services by a case manager. One unexpected result was that about half of the clients served were referred from the community, not from the hospital. We examine the characteristics of hospital-based case management clients, the predictors of their continuation in case management, and their health status over 1 year, focusing on the differences between hospital- and community-referred clients.

Swing-beds: the Arizona experience.

Swing-beds are acute-care hospital beds temporarily used for long-term care. A demonstration program was developed to evaluate the effectiveness of using swing-beds as catalysts for the expansion of rural hospitals into community health centers to respond better to the needs of older persons in their respective communities. We examined the background and implementation issues of the swing-bed demonstration program in six rural Arizona hospitals.

Geriatric case managers: integration into physician practices.

Integration of case management proved to be a key variable in a national demonstration at nine sites of alternative models designed to enhance primary care of frail elders. Numerous semistructured interviews of participants revealed a number of areas important to achieving successful integration. These areas include making favorable first impressions, building relationships, learning to collaborate, having proximity and contact, communicating, and demonstrating benefits to patients and physicians.

Contract management requires strategic planning.

Contract management is more than just an alternative form of financing. Contract management enables hospitals to avoid costly investments in time, personnel, and capital by using outside sources to provide clinical and support services. However, healthcare organizations that contract for a large number of services run the risk of becoming "hollow hospitals"--institutions where patient care is no longer the hospital's core activity. Hospitals can avoid this situation by carefully reviewing the advantages and risks of contract management in terms of their mission, quality, and organizational objectives.