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1. We have examined the effects of tolmetin and meclofenamate on isolated human PMN functions under FMLP stimulating conditions. 2. In a dose dependent manner, tolmetin and meclofenamate inhibited all PMN functions, except that tolmetin stimulated PMN chemotaxis. 3. Meclofenamate was much more potent than tolmetin as an inhibitory agent. 4. We also conducted competitive receptor binding assays for tolmetin, meclofenamate and ibuprofen on the FMLP receptor. 5. All three NSAID inhibited FMLP binding in a dose dependent manner with the potency order being meclofenamate greater than ibuprofen greater than tolmetin.

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In animal models of Fetal Alcohol Syndrome, ethanol causes a number of changes in brain development, with many of these changes being very transient. This is especially true for the process of synaptogenesis in different brain areas. Our quantitative electron microscopic study of synaptogenesis in the molecular layer of the rat dentate gyrus supports the above statement, by demonstrating that ethanol has no effect on the appearance of synapses in the dentate gyrus during early postnatal life (10-30 days old). However, prenatal ethanol exposure does appear to affect the process of synapse turnover, which is indicated by the significantly delayed appearance of complex (curved) synapses and multiple synaptic contacts on single axonal terminals. Efficient synapse turnover is thought to be required for the normal maintenance of neuronal plasticity, which in turn ensures an animal's ability to respond to novel environments, tasks and injuries. It would seem that the prenatal neurotoxicology of ethanol may manifest itself by more subtle mechanisms at sites of structural and functional importance.

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The process of reactive synaptogenesis has been demonstrated in several areas of the central nervous system, including the hippocampal dentate gyrus. After a complete unilateral entorhinal lesion, approximately 85% of the input to the outer two-thirds of the ipsilateral dentate molecular layer is lost. Bilateral fluctuations in synaptic density within non-denervated zones of the dentate molecular layer predict further alterations in neural circuitry at sites located transneuronally to the denervated dentate granule cells. Using quantitative electron microscopy, our study demonstrates a complete cycle of synapse loss and reacquisition within the ipsilateral but not contralateral CA4/hilus region of the hippocampal formation. This area is one of the terminal fields for the dentate granule cell mossy fiber axons. In addition the granule cell mossy fiber axons sprout during the postlesion time course and form a significantly increased number of new mossy fiber terminals within the ipsilateral and contralateral CA4/hilus area. Our results indicate that responses to brain injury may no longer be confined to a local denervated site, but probably include polyneuronal circuitry loops, which may encompass one or more areas of the central nervous system. Previous difficulties in providing a close behavioral or functional correlation to localized structural events may be explained by a more global brain response to an injury.

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Quantitative electron microscopy was used to examine the effect of circulating glucocorticoids on the removal of degenerating synapses and the replacement of lost synaptic contacts in young adult rats that follow partial denervation of the hippocampal dentate gyrus. Subjects were adrenalectomized prior to subcutaneous implantation of pellets containing a specified concentration of corticosterone and subsequent unilateral ablation of the entorhinal cortex. Animals maintained at high circulating concentrations of glucocorticoids were significantly retarded in the early phase of degenerating synapse removal and in the rate of synaptic replacement. Subjects maintained at extremely low concentrations of glucocorticoids were also significantly retarded in the early stages of synapse removal but showed an early replacement of lost synaptic contacts followed by a dramatic decrease in the rate of replacement. By 60 days after the lesion both groups of animals showed synapse replacement equivalent to young adult controls while significant amounts of degenerating synapses still remained in the denervated neuropil. The results demonstrate that circulating glucocorticoids can exert a marked influence on lesion-induced synaptic replacement in the hippocampal dentate gyrus.

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To provide high resolution information on the subcellular localization of the phenothiazines and tetracycline, we have developed a new histochemical method that circumvents the difficulties inherent in classical electron microscopic tissue preparatory procedures. Specific and reliable localizations of these drugs were accomplished by their rapid precipitation with phosphotungstic acid (PTA) at pH 7. A cell suspension of Ehrlich ascites carcinoma cells was incubated with a given drug (2.5 x 10(-4) M) and then briefly cross-linked with 1% glutaraldehyde at 4 degrees C. After washing, the cells were exposed to 2% PTA (pH 7) to precipitate the drug at its binding sites. Then the samples were rapidly dehydrates in 80% ethylene glycol (4 degrees C) and embedded in the polyester, Vestopal W. This protocol provides a low denaturation, low extraction approach to tissue preparation. Control samples (without drug) demonstrated an amorphous distribution of PTA throughout the cell and no specific dense precipitates. Those cells treated with the phenothiazines (chlorpromazine or fluphenazine) or tetracycline demonstrated very discrete (4-8 nm), electron-dense drug-PTA reaction products associated with different nuclear components as well as several cytoplasmic organelles. These subcellular localizations verify the binding sites reported by the biochemical literature. In addition, several previously unresolvable binding sites are reported. The rationale and limitations of this procedure are presented. This new histochemical methodology may have broad applications in the study of drug distribution, receptors, and drug-induced pathology and toxicity that may provide new information regarding drug action and design.

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Synapse replacement following injury is delayed in aged animals, but once underway it proceeds at the same rate as in younger animals. Thus, aged animals have a similar capacity to support synapse formation but appear incapable of initiating it as rapidly. A unilateral lesion results in the loss and return of normal synaptic density not only in denervated zones but also in nondenervated zones of the hippocampus [17, 19]. In young adult animals this occurs on both sides of the hippocampus, whereas in aged animals it occurs only on the same side as the lesion. It has long been thought that the plasticity of neuronal circuitry includes its major remodeling, which is probably sensitive to outside influences which produce disturbances in the circuitry. Our electron microscopic study provides quantitative evidence that the CNS is capable of synaptic turnover and major remodeling in the absence of a morphologically demonstrable degenerative process. We suggest this process is not just restricted to damage, but it is utilized by the CNS during ongoing natural remodeling of neuronal circuitry throughout the animal's life. We have also demonstrated that circuitry remodeling within damaged zones was linked in some manner to the degeneration clearing process, which was markedly reduced in aged animals. This age-related problem appears to be related to the effects of the elevated blood levels of corticosteroids. It is evident that the dentate gyrus provides a useful model system in which the synaptic turnover process can be readily manipulated and accurately studied.

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The molecular layer of the dentate gyrus of normal rats shows a large incidence of perforated postsynaptic densities (PSDs). The perforations or discontinuities occur almost exclusively in PSDs located in spines showing a U- or W-shaped junctional profile (complex PSDs). Perforated PSDs account for 16-25% of the total complex PSD profiles in young adult rats and 12-29% of those in aged animals. The frequency of perforations in the inner molecular layer of the dentate gyrus undergoes significant changes during a cycle of nondegenerative synapse turnover induced by ipsilateral ablation of the entorhinal cortex. During the first 2 days postlesion nonperforated PSDs (simple PSDs) decrease sharply, whereas perforated PSDs change little. However, at later times (4-10 days) there is a significant increase in the number of perforated PSDs that balances the number of simple PSDs lost. Beyond 10 days postlesion the proportion of both types of PSD is restored slowly to normal--i.e., nonperforated PSDs increase in number and perforated PSDs decrease, returning to the values in unoperated animals by 120 days postlesion. This inverse relationship between small nonperforated PSDs and large perforated PSDs suggests a precursor-product relationship between them. We propose that perforated PSDs are intermediates in an ongoing cycle of synapse turnover that is a part of the normal maintenance and adaptation of the nervous system.

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Previously we reported that a delayed onset in the reinnervation of the outer two-thirds of the dentate molecular layer occurred in aged rats after an entorhinal lesion. Several factors associated with formation of new synaptic contacts and removal of degenerative debris may affect the reinnervation process. In this study the appearance and removal of degeneration was analyzed and evaluated with respect to the delayed reinnervation process in aged rats. After a complete lesion of the entorhinal cortex, 85-90% of the input to the outer two-thirds of the ipsilateral molecular layer is lost. Electron-dense and electron-lucent degeneration are present throughout the outer two-thirds of the denervated molecular layer. In both aged and young adult rats, the electron-lucent degeneration disappears by 10 days postlesion. The predominant electron dense degeneration, however, is removed at a different rate by young adult and aged rats. Young adults demonstrate a biphasic degeneration removal process, with almost half of this degeneration rapidly lost by 10 days postlesion, and nearly all by 60 days postlesion. Aged animals in contrast, have lost only 16% of the dense degeneration at 10 days postlesion, with about 30% of the degeneration remaining at 60 days postlesion. The impaired removal of the degeneration from the denervated zone appears to be reciprocally related to the reinnervation response in both age groups and may be related to the normal astrocyte hypertrophy and elevated corticosteroid levels in aged rats.

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Quantitative electron microscopy was used to examine the ability of aged (2-year-old) and young adult (90-day-old) rats to replace those synapses lost (85-90%) in the outer two-thirds of the molecular layer of the dentate gyrus after a complete unilateral lesion of the entorhinal cortex. In aged rats the synaptic density is significantly lower than that of young adults at 10 days postlesion. Synaptic replacement begins between 2 and 4 days postlesion in young adults, whereas there is a delay until after 10 days postlesion in aged rats. Once synapse replacement begins in aged rats, the rate of synapse reappearance is about equal that of young adults. Thus the initial 10 days postlesion appears critical to growth of responding afferents and reformation of synaptic contacts. Analysis of synapses in terms of noncomplex and complex synaptic types shows that the noncomplex type accounts for the significant synaptic density difference between the two age groups. Replacement of complex synapses is nearly indistinguishable between age groups and is complete by 60 days postlesion. In contrast the initial replacement rate of noncomplex synapses in aged rats is much slower than young adults, though the control synaptic density is achieved by the end of the time course.

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It is well established that partial denervation causes the formation of new synapses within denervated areas. It is also possible that synapse formation and remodeling occurs outside denervated zones. In this study we evaluate this possibility by examining the effect of a unilateral entorhinal lesion on the number and characteristics of synapses in non-denervated zones of the dentate gyrus within the hippocampal formation. A unilateral entorhinal lesion massively denervates the outer two-thirds of the ipsilateral dentate molecular layer and also causes a minor loss of synapses in the outer two-thirds of the contralateral dentate molecular layer. The inner one-third of the molecular layer is not denervated on either side. In the ipsilateral inner molecular layer the number of synapses rapidly decreases by about 20% and recovers by 10 days post-lesion. Similarly, in the contralateral inner molecular layer, synapses are lost and replaced, but the time course is slower. Loss is maximal at 60 days post-lesion and this recovers by 180 days post-lesion. Thus, a complete cycle of turnover occurs in both of the inner molecular layers. No degenerating terminals of any type were seen throughout the time course in these layers. Small synapses with non-complex synaptic junctions appear to account for most of the changes. Also the outer two-thirds of the contralateral molecular layer, which has lost less than 5% of its input, loses about 37% of its synapses and replaces the majority of them over time. However, the total number of synapses in the contralateral molecular layer never fully attains the value of unoperated animals. The total synaptic population reaches a value such that the ipsilateral and contralateral molecular layers are nearly equivalent. These changes, achieved through synaptic turnover, may represent a homeostatic response to nearby denervation which may facilitate restoration of bilateral function in the dentate gyrus.

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Partial denervation of the dentate molecular layer causes sprouting and reinnervation by undamaged afferents within the denervated zones of young adult animals and to a lesser extent in aged animals. We have previously reported a non-degenerative remodeling of the dentate molecular layer in areas outside the primary denervated zone of young adult rats after a unilateral entorhinal lesion. In this study, we evaluate the response of aged rats under the same conditions, to see if aged animals also respond to injury in non-denervated zones. After a unilateral entorhinal lesion, the outer two-thirds of the ipsilateral dentate molecular layer loses about 85% of its input, while the outer two-thirds of the contralateral molecular layer loses less than 5% of its input (crossed temporo-dentate path). Denervation does not occur in the inner one-third of the molecular layer on either side. Within the ipsilateral inner molecular layer, the synaptic density rapidly drops 21% in the absence of degeneration and then recovers by 10 days post-lesion, as is the case in young adult animals. On the contralateral side, young adult animals show synapse turnover similar to the ipsilateral inner molecular layer. In contrast, no significant response in the total synaptic density was observed in the non-denervated contralateral inner molecular layer or the partially denervated outer two-thirds of the contralateral molecular layer. Thus, in aged animals, synaptic turnover is restricted to the massively denervated ipsilateral side. The small loss of input to the contralateral side apparently is not sufficient to initiate quantifiable turnover of synaptic contacts. This steady-state situation may be the result of an on-going stabilization of neuronal circuitry, which may limit restoration of function after injury in aged animals.

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The in vitro elution of penicillin and gentamicin from polymethylmethacrylate was studied qualitatively and quantitatively. Penicillin eluted poorly from Simplex P. Higher levels of penicillin eluted in sustained fashion from Palacos; concentrations of one microgram or more per gram of cement were recorded during the thirteen weeks of observation. Although the in vitro leaching of gentamicin from Palacos cement was similar to that of penicillin, there were two important differences: (1) most of the gentamicin leaching from the Palacos did so within the first twenty-four hours, and (2) the concentrations decreased to less than one microgram per gram of cement after the sixth week of observation. The in vivo elutions of penicillin and gentamicin from Palacos were studied in canine femora. The concentrations of penicillin and gentamicin in the endosteal bone at the bone-cement interface exceeded the concentrations found after intravenous administration of either agent. Bactericidal concentrations of gentamicin in osseous tissue persisted for seven months after implantation. Peak concentrations in serum following the depot administration of either penicillin or gentamicin occurred within thirty minutes of implantation. Concentrations of gentamicin in serum did not approach toxic levels. These data suggest that a high concentration of either penicillin or gentamicin can be obtained at the bone-cement interface--one of the vulnerable sites in total joint arthroplasty--for a prolonged period with the depot administration of these agents in acrylic bone cement. These osseous concentrations can be achieved without exposing the patient to elevated levels in serum and their potential toxic side effects.

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