Expression of purine metabolism-related enzymes by microglial cells in the developing rat brain

The effects of elevated blood lead on semen quality were evaluated in the rabbit model and compared to published effects in humans. Mature, male rabbits were given lead acetate by subcutaneous injection in the dose range of 0 to 3.85 mg/kg on a Monday-Wednesday-Friday basis. In each of eight treatment groups, a dosing regimen was developed to produce blood lead levels of 0, 20, 40, 50, 70, 80, 90, and 110 microg/dL. A 5-week pre-exposure period was followed by a 15-week exposure testing period allowing for response through six cycles of the seminiferous epithelium. Semen analyses revealed that increased blood lead levels were associated with adverse changes in the sperm count, ejaculate volume, percent motile sperm, swimming velocities, and morphology. Hormonal responses were minimal. Testicular pathology revealed a dose-dependent inhibition of spermiation. For six measures of semen quality, threshold estimates ranged from 16 to 24 microg/dL. Using the species extrapolation factor derived in this study, a rabbit dose would have to be divided by 1.56 to obtain the equivalent human dose for an equal percentage decrease in sperm concentration; however, rabbits are 3.75 more sensitive in terms of absolute decrease in sperm count for a given blood lead level.     

A regional study of developing rat brain: the accumulation and distribution of proteolipid proteins

The accumulation and distribution of proteolipid proteins in rat brain and selected brain regions (cerebellum, cerebral cortex, basal ganglia, and hippocampus) were studied during early postnatal development. In whole brain an eightfold increase of proteolipid was observed between ten and 33 days after birth. This was reflected in the separate regions examined where the proteolipid protein content increased six- to ten-fold during the same period. The basal ganglia and cerebral cortex contributed the greatest amount to the total proteolipid present. However, at 28-33 days the greatest concentration (mg/g tissue) was observed in the basal ganglia and hippocampus. When the proteolipid protein preparations were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, distinctive, heterogeneous patterns for each brain region were obtained. Proteolipid from basal ganglia (the region richest in white matter) consisted primarily of two major protein bands with apparent molecular weights of approximately 21,500 and 26,000. Both of these bands dramatically increased in quantity during myelination, and the larger protein coelectrophoresed with isolated myelin proteolipid protein. Both bands were also found present in proteolipid preparations from the other brain regions but in varying amounts relative to the total. The data suggest that the increase in proteolipid observed during this developmental period was due in large measure to the accumulation of myelin-specific proteolipids, but also that a significant proportion of the increase was due to the accumulation of nonmyelin components.     

Regulation of kynurenic acid levels in the developing rat brain

Several brain-specific mechanisms control the formation of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the adult rat brain. Two of these, dopaminergic neurotransmission and cellular energy metabolism, were examined in the brain of immature (postnatal day 7) rats. The results indicate that during the early postnatal period cerebral KYNA synthesis is exceptionally amenable to modulation by dopaminergic mechanisms but rather insensitive to fluctuations in cellular energy status. These findings may be of relevance for the role of KYNA in the function and dysfunction of the developing brain.     

Spatiotemporal appearance of developing LHRH neurons in the rat brain

To obtain insight into the development of the heterogeneous intracerebral populations of luteinizing hormone-releasing hormone (LHRH) neurons, their spatiotemporal appearance was examined at different stages in normal rat embryos, in nasal epithelial explants in vitro, and in intrauterine nasal-operated embryos. Following the appearance of nerve cell adhesion molecule in the nasal placode at embryonic day (E) 12.5, LHRH neurons, generated in the nasal placode at E13.5, penetrated the forebrain vesicle (FV) by E14.5-15.5. After E16.5, as the FV elongated to form the olfactory bulb, the migrating neurons traversed posteriorly through the interhemispheric space to penetrate the septopreoptic (S-P) area. By E18.5, LHRH neurons were detected in the preoptic-diagonal band (P-D) area as well as in the S-P region, along with some scattered extrahypothalamic LHRH neurons. To determine the source of these neurons, we separately cultured dissected parts of E12.5 nasal pit epithelium. Neuronal generation was predominantly from the medial wall epithelium (NAP), but some LHRH neurons originated in the roof epithelium. Cocultures of the NAP (E12.5) with the FV, median eminence-arcuate complex, Rathke's pouch, mesencephalon, or medulla oblongata from E14.5 embryos revealed the ability of LHRH cells to penetrate all of these tissues. Uni- or bilateral nasal destruction was conducted at E16.5 or E15.5, respectively, and examined at E18.5 and E21.5. In the operated embryos, most LHRH neurons were present in the P-D system and some in the S-P area. This finding suggests that the neurons generated before E15.5 are primarily predisposed to form the P-D system, whereas those derived afterward form the S-P system.     

A morphological and histochemical study of a drug-induced enteropathy in the Alderley Park rat

Two experiments were devised to produce an experimental enteropathy. In Experiment I, male Alderley Park rats were dosed daily by gavage with 20 mg/kg and 60 mg/kg of an antibacterial compound ICI 17,363. Animals were killed sequentially at daily intervals up to and including Day 9 to study the development of the enteropathy. In Experiment II rats were dosed daily with 60 mg/kg of the same compound. All animals were killed on Day 5 owing to a rapid development of the enteropathic condition. The duodenum was examined histologically and histochemically. Duodenal changes included vacuolation of columnar epithelial cells and villus stunting. There were marked reductions in mitotic activity in the crypt epithelial cells from Day 7 onwards (Experiment I) and almost total loss of hydrolytic and oxidative enzyme activity. In Experiment II the changes were more severe and haemorrhage and erosion of the duodenal mucosa were observed. The development of the enteropathic lesion appears to be due largely to the antimitotic effect of the compound, although a direct toxic effect upon the intestinal mucosa cannot be ruled out.     

A quantitative pharmacological study of the cholinergic depolarization of hippocampal pyramidal cells in rat brain slices

Twenty-eight unicompartmental knee arthroplasties performed as an alternative to high tibial osteotomy or tricompartmental knee arthroplasty in patients under 60 years of age were reviewed after 2 to 6 years of follow-up. The patient's age at the time of operation averaged 52 years. Using the Knee Society Score, 90% were rated good or excellent results in terms of function and pain relief. The average flexion angle obtained was 124 degrees, and the average postoperative alignment was 4 degrees of anatomic valgus for varus deformities and 8 degrees for valgus deformities. The average activity level according to the Tegner and Lysholm score slightly improved (preoperative, 2.3; follow-up, 2.7 points). Of the 28 knees, 9 (32%) presented radiolucent lines about the tibial component and two had incomplete radiolucent lines at the bone-cement interface on the femoral side. There was no correlation between activity level and tibial radiolucent lines. Two revisions were performed because of loosening of the femoral component at the prosthesis-cement interface. One was converted to another unicompartmental arthroplasty and the other to a tricompartmental arthroplasty. One tibial component exhibited an asymptomatic slowly progressive radiolucency. Unicompartmental knee arthroplasty in middle-aged patients yields 2- to 6-year results competitive with osteotomy but inferior to tricompartmental arthroplasty in terms of revision. The specific prosthetic design used in this series appeared to be vulnerable to femoral component loosening possibly because of constrained tibial topography and smooth tapered femoral fixation lugs.     

A metabolic map of cytochrome oxidase in the rat brain: histochemical, densitometric and biochemical studies

To examine brain patterns of metabolic and functional activity, the distribution of cytochrome oxidase, a mitochondrial enzyme marker for neuronal functional activity, was mapped throughout the rat brain. Mapping was done qualitatively by enzyme histochemistry of brain sections cut in three planes (coronal, sagittal and horizontal), and quantitatively by optical densitometry of stained sections and by biochemical assays of brain tissue homogenates. Activity of the enzyme was distributed in characteristic patterns and amounts that differed among various neural pathways, brain nuclei, cerebral cortical areas and layers, and neuron types. Gray matter essentially always had higher enzyme activity than did white matter, by a factor of eight- to 12-fold. Among different neural pathways, cytochrome oxidase activity was relatively high in special sensory, somatosensory and motor systems, and was relatively low in associative, limbic, autonomic and visceral regulatory systems (though exceptional areas were present). Among 11 different neuron types, nearly a two-fold range of histochemical staining intensities was observed, with the darkest staining in neurons of the mesencephalic trigeminal nucleus. The observed patterns of cytochrome oxidase activity were mostly similar to the patterns of 2-deoxyglucose uptake seen previously [Schwartz W. J. and Sharp F. R. (1978) J. comp. Neurol. 177, 335-360; Sokoloff L. et al. (1977) J. Neurochem. 28, 897-916] in conscious, "resting" animals, though some differences were found. For example, whereas 2-deoxyglucose uptake was about three-fold higher in gray matter than in white matter [Sokoloff L. et al. (1977) J. Neurochem. 28, 897-916], cytochrome oxidase activity was about eight- to 12-fold higher. This and other discrepancies probably reflect basic technical differences between these two methods. Compared to 2-deoxyglucose, cytochrome oxidase is more specific for oxidative rather than glycolytic metabolism, and more reflective of overall neuronal functional activity occurring over longer time periods lasting hours to weeks, rather than minutes. The anatomical resolution of cytochrome oxidase histochemistry is also finer than that of 2-deoxyglucose autoradiography, extending to the electron microscopic level. The metabolic map of cytochrome oxidase activity reveals patterns of normal brain function, and may be useful as a baseline for comparison in studies of brain disease, development, ageing and plasticity.     

Migratory pattern of fetal rat brain cells and human glioma cells in the adult rat brain

The migratory behavior of two human glioma cell lines (D-54MG and GaMG) and fetal rat brain cells grafted into the adult rat brain was studied. To trace the implanted cells, they were stained with the carbocyanine vital dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate before injecting them into the white matter above the corpus callosum. The animals were sacrificed 2 h and 7 and 21 days after injection, and the brains were removed and cryosectioned. Fluorescence microscopy showed that both the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-stained fetal and tumor cells had the same migratory pattern. Implanted cells were found along myelinated fibers in the corpus callosum and in the perivascular space. After immunostaining for several extracellular matrix (ECM) components (laminin, fibronectin, collagen type IV, and chondroitin sulfate), laminin deposits were observed in the border zone between the host tissue and implanted tumor cells as well as fetal cells. By using two different types of antibodies against fibronectin, it is shown that the fibronectin expression observed in the tumor matrix may be host derived. This was further supported by the fact that tumor spheroids obtained from the two glioma cell lines were negative when immunostained for these ECM components. Several of the ECM components may be host derived. This can be caused by neovascularization and repair synthesis or by a local production of guiding substrates which are important for tumor cell locomotion. The present data suggest that the migratory patterns of fetal and glioma cells are indistinguishable when transplanted into the adult rat brain. Thus, glioma cells may be routed by the same ECM components that play a major role during brain development.     

Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF-alpha on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF-alpha in the presence of interferon-gamma (IFN-gamma) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN-gamma, no increase in Mn-SOD activity was detected. The induction of IFN-gamma-dependent Mn-SOD activity by anti-Fas antibody or TNF-alpha was concentration-dependent; the maximal effect was observed at 1-10 micrograms/ml and 5-10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF-alpha in the presence of IFN-gamma resulted in an additive increase in Mn-SOD activity. Although the addition of 12-o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN-gamma-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF-alpha. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN-gamma and that TPA augments the process through the activation of protein kinase C.     

Localization of mRNAs for Rlim-1, the rat Xlim-1 homolog, in the developing rat brain

A simple and simultaneous determination of melatonin and its precursors, serotonin (5-HT) and N-acetylserotonin, was achieved by reversed-phase high-performance liquid chromatography with electrochemical detection. The addition of an ion-pairing agent, sodium 1-octanesulfonate, to the chromatographic mobile phase caused an increase of the retention time of 5-HT, and resulted in the successful simultaneous resolution of these three indoleamines. This method was used to quantitate these indoleamines in the pineal gland of juvenile golden hamsters.     

Immunobiochemical characterization of the NMDA-receptor subunit NR1 in the developing and adult rat brain

To investigate the developmental and regional expression of the NR1-subunit of the NMDA-receptor on the protein level, two polyclonal antisera [NR1(N) and NR1(C)] were raised against fusion proteins derived from the N- and C-terminal domain of the NR1-subunit, respectively. In Western blots of rat brain membranes, both antisera specifically recognized a single protein band with an apparent molecular size of 115 kDa. The regional distribution of the NR1-subunit immunoreactivity was analyzed in the developing and adult rat brain using sections blotted onto nitrocellulose membranes for immunostaining. With the NR1(N)-antiserum, strongest signals were detected in hippocampus, followed by cortex, striatum and thalamus, and weaker staining was observed in tectum, brainstem and cerebellum of adult brain. The NR1(C)-immunoreactivity exhibited a similar distribution, except that the staining in thalamus, tectum, brainstem and cerebellum was faint or virtually absent. The distinct pattern of NR1(N)- and NR1(C)-immunoreactivity arose during postnatal development. At birth, moderate staining with both NR1-subunit antisera was observed throughout the brain increasing strongly in most brain regions until postnatal day 21. In some brain areas, however, the NR1(C)-, in contrast to the NR1(N)-staining, decreased postnatally e.g. in thalamus, tectum and brainstem. The restricted staining intensity of the NR1(C)-antiserum in particular areas of adult and developing brain appears to reflect the emergence of C-terminal splice variants of the NR1-subunit which are not recognized by the NR1(C)-antiserum.     

Use of rat microglial cells for the study of a possible mechanism of brain damage in AIDS

The long-term graft function after withdrawal of steroids from maintenance immunosuppression was analyzed in 98 kidney recipients (59 on cyclosporin monotherapy, 39 on cyclosporin plus azathioprine) who had not developed an early rejection episode when prednisolone was discontinued. Seven years after steroid withdrawal the probability of an increase in serum creatinine (> 20% of baseline levels) was 51%. The increase in creatinine was associated with sclerosing arteriopathy as a marker of chronic rejection in 29 of 43 graft biopsies. The addition of azathioprine had no effect on the stability of long-term graft function and did not influence the 7-year graft survival rate in this highly selected group of patients.     

Enzyme histochemical and immunohistochemical localization of carbonic anhydrase as a marker of ductal differentiation in the developing rat parotid gland

Whether the two earliest cortical somatosensory evoked potentials (SEPs) to tibial nerve stimulation (N37 and P40) are generated by the same dipolar source or, instead, originate from different neuronal populations is still a debated problem. We recorded the early scalp SEPs to tibial nerve stimulation in 10 healthy subjects at rest and during voluntary movement of the stimulated foot. We found that the P40, which reached its highest amplitude on the vertex at rest, changed its topography during movement, since its amplitude was reduced much more in the central than in the parietal traces. These findings suggest that two different components contribute to the centro-parietal positivity at rest: (1) the P37 response, which is parietally distributed and is not modified by movement, and (2) the 'real' P40 SEP, which is focused on the vertex and is reduced in amplitude during voluntary movement. Since, also, the N37 response did not vary its amplitude under interference condition, it is possible that the N37 and P37 potentials are generated by the same dipolar source. Other later components, namely P50 and N50 were significantly reduced in amplitude during foot movement. Lastly, the subcortical P30 far-field remained unchanged and this suggests that the phenomenon of amplitude reduction during movement (i.e. gating) occurs above the cervico-medullary junction.