Social stress alters expression of large conductance calcium-activated potassium channel subunits in mouse adrenal medulla and pituitary glands.

Large conductance calcium-activated potassium (BK) channels are very prominently expressed in adrenal chromaffin and many anterior pituitary cells, where they shape intrinsic excitability complexly. Stress- and sex-steroids regulate alternative splicing of Slo-alpha, the pore-forming subunit of BK channels, and chronic behavioural stress has been shown to alter Slo splicing in tree shrew adrenals. In the present study, we focus on mice, measuring the effects of chronic behavioural stress on total mRNA expression of the Slo-alpha gene, two key BK channel beta subunit genes (beta2 and beta4), and the 'STREX' splice variant of Slo-alpha. As a chronic stressor, males of the relatively aggressive SJL strain were housed with a different unfamiliar SJL male every 24 h for 19 days. This 'social-instability' paradigm stressed all individuals, as demonstrated by reduced weight gain and elevated corticosterone levels. Five quantitative reverse transcriptase-polymerase chain assays were performed in parallel, including beta-actin, each calibrated against a dilution series of its corresponding cDNA template. Stress-related changes in BK expression were larger in mice tested at 6 weeks than 9 weeks. In younger animals, Slo-alpha mRNA levels were elevated 44% and 116% in the adrenal medulla and pituitary, respectively, compared to individually-housed controls. beta2 and beta4 mRNAs were elevated 162% and 194% in the pituitary, but slightly reduced in the adrenals of stressed animals. In the pituitary, dominance scores of stressed animals correlated negatively with alpha and beta subunit expression, with more subordinate individuals exhibiting levels that were three- to four-fold higher than controls or dominant individuals. STREX variant representation was lower in the subordinate subset. Thus, the combination of subunits responding to stress differs markedly between adrenal and pituitary glands. These data suggest that early stress will differentially affect neuroendocrine cell excitability, and call for detailed analysis of functional consequences.

Delayed cell death in neonatal mouse hippocampus from hypoxia-ischemia is neither apoptotic nor necrotic.

Hypoxic-ischemic (HI) injury in neonatal mice is associated with significant cell loss in hippocampus, striatum and deep layers of the cortex. The pattern of cell death in hippocampus after a moderate focal ischemic-global hypoxic insult is studied through morphologic changes in dying neurons at both the light and ultrastructural levels. Light microscopy at 24 h showed a number of injured neurons, as evidenced by dark, round, condensed nuclei, primarily in CA1 through CA3. Nuclei appeared punctate and cytoplasm vacuolated. Electron microscopy revealed that the punctate appearance of the nuclei corresponded to clumped chromatin. At 7 days after HI, injured neurons were shrunken and had a uniformly dark, angular appearance. While dying cells had an appearance consistent with apoptosis on light microscopy, cells were neither necrotic nor apoptotic at the ultrastructural level.

Characterization of the microvascular glycocalyx in normal and injured spinal cord in the rat.

The glycocalyx of microvasculature in normal and injured spinal cord was characterized by using cationized ferritin to define anionic sites and the lectins concanavalin agglutinin (Con A) and Ricinus communis agglutinin I (RCA) to delineate carbohydrate moities. Binding of cationized ferritin was evaluated at the ultrastructural level in control animals and at 3 hours after spinal cord injury. Horseradish peroxidase (HRP) was administered intravenously before euthanasia. In control spinal cord, there was continuous even binding of cationized ferritin along the luminal front of microvasculature and no evidence of barrier permeability to HRP. After spinal cord injury, there was a reduction in binding of cationized ferritin in those regions of spinal cord that exhibited barrier breakdown to HRP. Lectin binding in the spinal cord was evaluated at 3 hours and 3 days postinjury. At the light microscopic level, there appeared to be increased binding of Con A and RCA in microvessels by 3 days postinjury as compared with the control spinal cord. At the ultrastructural level, a significant increase in RCA binding was noted along luminal fronts in the injured spinal cord. This increased binding coincided with a significant elaboration of the endothelial glycocalyx. These findings demonstrate that the charge, structure, and carbohydrate composition of the endothelial glycocalyx in microvessels in the spinal cord may be dramatically altered after spinal cord injury. Furthermore, there is an association between the loss of charge and disruption of the barrier, suggesting that anionic sites may contribute to maintenance of the blood-spinal cord barrier.

Ultrasound imaging of the radial artery following its use for cardiac catheterization.

Our experience suggests transradial arterial access with 5Fr catheters can be used for cardiac angiography with a low incidence of clinical complications, and supports the findings of previous investigators. Subclinical complications at the catheterization site were infrequent in this study (1 patient with asymptomatic radial artery occlusion). The presence of a palpable radial pulse may not be a reliable estimate of artery patency as evidenced by our patient with a palpable pulse due to retrograde flow. The theoretical advantage of the procedure is derived from the dual vascular supply to the hand. Radial artery occlusion, while uncommon, results in no ischemic sequelae in the setting of a patent ulnar artery.

Immunolocalization of endothelin in the traumatized spinal cord: relationship to blood-spinal cord barrier breakdown.

The purpose of this study was to determine the relationship between endothelin-1 (ET-1), a prominent vasoactive agent, and the breakdown of the blood-spinal cord barrier along the axis of the cord after a moderate spinal cord injury. In the first study rats (n = 10) were euthanized 24 h after spinal cord injury and compared to sham (n = 5) and unoperated (n = 10) controls. Endothelin and immunoglobulins (IgG) were immunolocalized in adjacent sections of spinal cord using semiquantitative immunocytochemical techniques. In the second study animals were pretreated with the endothelin antagonist Bosentan (n = 6) or vehicle (n = 6) prior to spinal cord injury. Animals were euthanized at 24 h postinjury. Ten minutes prior to euthanasia animals were given horseradish peroxidase (HRP) intravenously. After perfusion fixation sections of cord were prepared for quantitative HRP histochemistry. After spinal cord injury there was enhanced staining for endothelin along the axis of the cord that correlated with the anatomical pattern of barrier breakdown to IgG. In those animals that were pretreated with Bosentan, there was a significant reduction in barrier breakdown along the axis of the injured cord as compared to those animals that received vehicle only. Taken together, this data implicate involvement of endothelin in the axial pattern of barrier breakdown after spinal cord injury.

Altered immunoexpression of microglia and macrophages after mild head injury.

In this study we examined the temporal response of microglia and macrophages to mild head injury in the rat. Microglia and macrophages were identified by their distinct morphology and by immunophenotype. With regard to the latter, antibodies to OX42 and ED1 were used to define microglia and macrophages, respectively. Although there was no change in the morphology of brain macrophages after mild head injury, the morphology of microglia was dramatically altered. Microglial cell bodies appeared larger with a more elaborate arborization of cellular processes. After head injury certain populations of macrophages and microglia were more intensely immunostained. By 3 days postinjury these intensely stained cells exhibited a characteristic distribution in the brain. Prominently stained microglia were detected in the thalamus, hippocampus, lateral and medial geniculate body, and the substantia nigra. Intensely stained macrophages were located primarily in the cortex and subarachnoid space adjacent to the site of impact. By 7 days postinjury intensely immunostained macrophages and microglia were widespread throughout the injured cortex. These results demonstrate that microglia and macrophages are sensitive to mild head injury. Early changes in the macrophage population are more directly correlated with the most damaged tissue and may reflect migration of these cells from either the subarachnoid space or across the damaged blood-brain barrier. The early widespread microglial response in regions exhibiting no overt neuronal cell damage suggests that these cells are responding to more subtle factor(s) that are expressed in the mildly traumatized brain.

Fluid-phase endocytosis of horseradish peroxidase by cerebral endothelial cells in primary culture: characterization and kinetic analysis.

Endocytosis of horseradish peroxidase (HRP) was studied in primary cultures of cerebral endothelial cells prepared from 2-week-old rats. These cultures were considered "endothelial-like" on the basis of their ability to internalize acetylated low density lipoprotein. Cellular localization of HRP protein was examined at the light and ultrastructural levels and endocytosis of the protein was evaluated by a colorimetric assay. HRP was localized in discrete cytoplasmic granules by light microscopy. At the ultrastructural level these granules corresponded to pleomorphic membrane-bound structures that were present throughout the cytoplasm. The amount of internalized HRP was directly related to the concentration of the protein in the medium, was not saturable at high concentrations of HRP, and increased with time. Endocytosis proceeded at 37 degrees C, but was abolished at 4 degrees C. In pulse-chase experiments, the quantity of internalized protein in the cells did not significantly change during the 2 hr chase period. Taken together, these findings suggest that internalization of HRP occurs by fluid-phase endocytosis, a non-receptor-mediated process, and that the protein is stable within an intracellular compartment for at least several hours.

Immunocytochemical localization of immunoglobulins in the rat brain: relationship to the blood-brain barrier.

The central nervous system has been traditionally regarded as an immunologically privileged area. This feature has been in part attributed to the blood-brain barrier, which provides a restrictive interface to circulating immunoglobulins (IgG). Recent kinetic studies suggest that the barrier to immune proteins is not absolute, but rather may be regulated by a specific transfer mechanism. In this study, we confirm the presence of IgG in the central nervous system by immunocytochemistry and demonstrate a close anatomical relationship between the distribution of this protein and the blood-brain barrier. IgG was immunolocalized in the normal rat brain by using monoclonal and polyclonal antibodies to IgG and its subclasses. On the basis of an initial evaluation, the most appropriate antibodies and dilutions were selected for subsequent analyses. In the first study, IgG and albumin were immunolocalized in adjacent sections. In the second study, horseradish peroxidase (HRP) was given intravenously prior to sacrifice, in order to examine artifacts related to perfusion fixation. The distribution of HRP and IgG was then examined in adjacent sections. In the third study, IgG was immunolocalized in sections of brain after mild traumatic head injury. A monoclonal antibody to IgG2a and a polyclonal antibody to IgG were selected on the basis of specificity and consistent, mutual localization. Distinct, patchy, perivascular staining, infrequently associated with labeled neurons, was noted throughout the brain. Electron microscopy confirmed the perivascular localization; IgG was localized along the basal lamina of microvasculature and within the adjacent parenchyma. Albumin and HRP did not exhibit a similar pattern of perivascular immunostaining. After head injury, prominent immunostaining for IgG was observed in the injured hemisphere. In summary, these data indicate that the normal rat brain contains IgG, which dramatically increases after head injury. The distinct perivascular distribution in the normal brain suggests local microvascular permeability. This permeability is selective for IgG, since albumin does not share a similar perivascular localization. The neuronal staining which is closely associated with perivascular label may reflect one intracellular route for extravasated IgG.

Improving vaccination coverage: the experience of the Expanded Programme on Immunization in Vanuatu.

This paper looks at the success of measures adopted to improve vaccination coverage of infants in the Republic of Vanuatu. In 1982 the Department of Health introduced an Expanded Programme on Immunization (EPI). Since the republic has over 80 inhabited islands, a scattered population, rough terrain and a lack of transport and communications infrastructure, achieving a high vaccination coverage rate proved difficult. Coverage of infants remained low until 1987. From that year onwards various strategies were employed to increase coverage, including 1) adopting the WHO-recommended diphtheria-pertussis-tetanus (DPT) and oral polio vaccine (OPV) vaccination schedule (6, 10 and 14 weeks) instead of the former schedule (3, 6 and 9 months); 2) improving the training, support and supervision of staff delivering maternal and child health (MCH) services; and 3) improving community involvement through social mobilization activities in areas of low coverage. Data on vaccination coverage of infants for the period 1984 to 1990 were compared. Over this period coverage of infants with 3 doses of DPT rose from 29% to 76%, with 3 doses of OPV from 29% to 78%, and with measles vaccine from 19% to 66%. These dramatic improvements have largely occurred since 1987. The results demonstrate the success of the measures adopted, and the experience of Vanuatu offers lessons in improving vaccination coverage for other countries in the region.

Dapsone syndrome in Vanuatu: a high incidence during multidrug treatment (MDT) of leprosy.

Side-effects of leprosy treatment with dapsone are said to be uncommon, with drug allergy occurring in only one of every several hundred patients treated with dapsone. The dapsone or sulphone syndrome (DDS) has been recognized since the earliest days of sulphone therapy but until recently its incidence had been decreasing. In Vanuatu, during the years 1988-1991, nine leprosy patients have developed the dapsone syndrome, four of whom have died. During the last 4 years only 37 patients were started on treatment, which is an incidence of the dapsone syndrome of 24% with a fatality rate of 11%. All the patients were being given multi-drug treatment (MDT) of daily dapsone (100 mg) and clofazimine (50 mg) and monthly rifampicin (600 mg) and clofazimine (300 mg). There has been speculation that the increased incidence of what was previously described as a rare reaction is due to the use of MDT, and the reasons for this are discussed. We feel the increase in the number of reactions in Vanuatu since starting MDT is probably due to the high starting dose of 100 mg of dapsone, possibly enhanced by the combination with clofazimine and rifampicin and a genetic susceptibility of the Melanesian population.

Morphologic changes in cultured astrocytes after exposure to glutamate.

Cultured astrocytes, exposed to glutamate at a dose that is generally neurotoxic in vitro (1 mM), exhibit transient swelling in the absence of cell death. In the present study, we further characterize this response by examining the distribution of intermediate filaments and evaluating cellular ultrastructure in primary cultures of astrocytes after exposure to 1 mM glutamate. In addition, cellular swelling was determined using the nonmetabolizable hexose 3-O-methyl [14C]-glucose (3-MG). Glial fibrillary acidic protein (GFAP) was immunolocalized at the light microscopic level to study the distribution of intermediate filaments. GFAP was immunolocalized to a fine cytoskeletal network in control cultures. Four to 24 h after exposure to glutamate, this detailed localization was replaced by a diffuse, uneven pattern of immunoreactivity. The most prominent ultrastructural changes were identified at 4 and 8 h after glutamate exposure. Nucleoli underwent transformation from a normal compact appearance to a markedly dispersed state. The cell body typically exhibited cytoplasmic lucency, swollen mitochondria, and dilated cisterns. Intermediate filaments within cellular processes appeared widely spaced in comparison to the controls. These ultrastructural changes coincided with findings of increased intracellular water space as determined with 3-MG. These findings demonstrate that astrocytes exposed to 1 mM glutamate exhibit transient morphologic changes that not only suggest cellular swelling but also define a more diverse response that is reflected in the altered immunolocalization of GFAP and in unique changes in the nucleolus.