Recent Publications by CFE Educators

This report described a new microdissection procedure to evaluate the regional distribution of neuromediators in the rat spinal cord. Different segments are first divided into sagittal slices. From these, different grey and white matter regions can be microdissected. This permits selective biochemical measurements in different laminae, including the area around the central canal. White and grey matter can also be differentially analysed. Using HPLC with electrochemical detection, we report on the regional analysis of biogenic amines as well as uric acid. An increase in 5-hydroxytryptamine (5-HT) levels was observed from cervical to lumbar segments. 5-HT levels were highest in the motoneurone samples (lamina IX) of the lumbar cord. The next highest levels were found in lamina X, followed by the intermediate grey matter laminae and the dorsal horn. Small amounts of 5-HT were detected in the white matter. Interestingly, the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT was greatest in the white matter and least in the motoneurone sample. Norepinephrine (NE) levels were higher in the thoracic than in the cervical or the lumbar cord. The highest levels were found in the lamina X in thoracic segments. With this exception, no marked laminar difference in NE levels was observed. Dopamine (DA) levels were highest in the dorsal horn of the cervical and the thoracic cord, the next highest levels were found in the intermediate grey matter and lamina X in the same segments. The distribution of uric acid (UA) was comparable to that of NE: UA levels were highest in the thoracic cord, but no marked laminar difference was observed. On the other hand, UA levels in white matter generally exceeded those in the grey matter. These data indicate the value of a sagittal regional microdissection of the spinal cord. The ability to separately analyse different laminae of the cord (as well as differentiating grey and white matter) should prove useful in future studies of experimentally evoked changes in neurotransmitters within functionally distinct regions of the spinal cord.

Albumin transfer across cultured porcine pulmonary artery endothelial cell monolayers was studied at different concentrations of luminal and interstitial albumin. Increased interstitial and luminal albumin concentrations increased the rates of transfer of albumin from interstitium to lumen and lumen to interstitium, respectively. Interstitial-to-luminal albumin clearance reached a maximum rate at 725 microM interstitial albumin. The clearance of dextran from interstitium to lumen also increased when interstitial-to-luminal albumin clearance increased, suggesting that albumin was not transferred alone across the endothelium. The increase in interstitial-to-luminal albumin clearance did not occur in the absence of endothelial cells, at 4 degrees C, or when dextran or dextran sulfate sodium were added to the interstitial compartment. These data demonstrate that albumin concentrations can influence the rates of endothelial albumin transfer and that albumin can be transported across the endothelium by a process mediated by endothelial cells.

Single C- and A-delta fibers were isolated from dissected filaments of the saphenous nerve in pentobarbital anesthetized rats and the corresponding cutaneous receptive fields mapped with calibrated von Frey hairs. Nociceptors were characterized by their responses to noxious mechanical, thermal and chemical stimuli, including intradermal injections of leukotriene B4, prostaglandin E2, bradykinin and capsaicin. Leukotriene B4 decreased the mean mechanical threshold by a maximum of 80% within 10 min and for more than 3 h after intradermal injection of 75 ng of leukotriene B4. The degrees of sensitization of a fiber by leukotriene B4 and prostaglandin E2 were highly correlated. A potentiation effect also was observed, in that injection of prostaglandin E2 or leukotriene B4 1 h after the other eicosanoid further lowered the mechanical threshold of a sensitized fiber, whereas fibers that were not sensitized by leukotriene B4 were unaffected by prostaglandin E2. The sensitizing action of leukotriene B4 and prostaglandin E2 was directed to multiple classes of cutaneous nociceptors including 73% of C-polymodal, 60% of C-mechano-heat, 42% of C-mechano-cold nociceptors and 70% of A-delta high-threshold mechanonociceptors. The pain-evoking substances bradykinin and capsaicin activated 81% and 88%, respectively, of the sensitized C-polymodal nociceptors, 17% and 84% of the sensitized-C-mechano-heat nociceptors, 12% and 37% of the sensitized C-mechano-cold nociceptors, and 17% and none of the sensitized A-delta high-threshold mechanociceptors. The responses of C-fibers to bradykinin and capsaicin were highly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

Some clinical features of rheumatoid arthritis (RA) (for example, preferential joint involvement and bilateral symmetry), taken together with the strong evidence of neurogenic inflammatory processes, suggest that the nervous system contributes to the inflammatory component of RA and other polyarthritides. The authors propose that the increased risk and severity of disease in particular joints reflects a greater innervation of those joints by unmyelinated afferent and sympathetic efferent fibers. Release of the proinflammatory peptide, substance P, from the peripheral terminals of nociceptive joint afferent fibers, through interactions with many nonneural cells, exacerbates the inflammatory process. Release of mediators from sympathetic efferents (including norepinephrine) also contributes to the inflammation, either through an independent mechanism or by acting in concert with the nociceptive afferent-derived substances. Therapies directed at interruption of the nervous system contribution to the pathophysiology of these diseases should offer a new direction to treatment.

In this report, we describe a new colloidal-gold-labelled retrograde tracer, wheat germ agglutinin (WGA) conjugated to enzymatically inactive horseradish peroxidase (apoHRP). This protein gold complex (WGAapoHRP-Au) is a sensitive marker for retrograde tracing of the projections of CNS neurons at the light-microscopic (LM) level when a silver-enhancement procedure is used to detect the gold in the tracer. For electron-microscopic (EM) analysis, the silver-enhanced sections undergo a further gold-toning step. This protects against rapid oxidation and dissolution of the silver precipitate during the osmication procedure. A major advantage of WGAapoHRP-Au is that it can be used in a variety of multiple-labelling studies. When the retrograde transport of the new tracer is combined with that of the fluorescent dye, True Blue, neurons that have bifurcating axons can be readily demonstrated. Simultaneous immunofluorescent detection of the cytochemistry of the double-retrogradely labelled neurons is also possible. In contrast to a WGA-HRP gold complex, the new complex has no enzymatic activity. Thus HRP-based techniques (e.g., anterograde transport of WGA-HRP or peroxidase-antiperoxidase immunocytochemistry) can be performed on tissue that contains retrogradely labelled neurons marked with WGAapoHRP-Au without having to pretreat tissue so as to destroy endogenous HRP enzyme activity. At the EM level, the gold is readily distinguished from DAB immunoreaction product. This makes both LM and EM double-labelling studies possible. The great sensitivity of the new tracer, its compatibility with a variety of aldehyde fixatives, its ease of detection, and the fact that it can be simultaneously used with several fluorescent and HRP-based immunocytochemical and tracing techniques make WGAapoHRP-Au a valuable tool for LM and EM characterization of CNS cytochemistry and connectivity.

Monoclonal antibodies raised against highly purified protein kinase C were used to localize protein kinase C in the rat brain. Using various monoclonal antibodies, at least three distinct antibody-staining patterns were found. One monoclonal antibody exclusively labeled astroglial elements, including astrocytes, tanycytes, and cerebellar radial glia. Another monoclonal antibody exclusively labeled neural cells, including cortical and hippocampal pyramidal dendrites and Purkinje cells of the cerebellum. A third monoclonal antibody (which inhibited protein kinase C activity) intensely stained more limited brain regions, particularly thalamic neurons, and also stained astroglial structures in brain, spinal cord, and cerebellum. The possibility that the three staining patterns reflect the differential regional and cellular localization of related, but distinct, enzymes of protein kinase C is discussed.

A 48-year-old woman with profound, rapidly progressive dyspnea requested a second opinion regarding the diagnosis and management of an undiagnosed interstitial process. One year prior to this evaluation, she had been placed on therapy with exogenous estrogens for the treatment of osteoporosis. During this therapy, she had a marked deterioration of her pulmonary status. Review of her open lung biopsy, which was obtained five years previously, revealed lymphangioleiomyomatosis. Discontinuation of estrogen therapy and treatment with tamoxifen were successful in stopping the progressive course. This patient's clinical course suggested an association between estrogen therapy and clinical deterioration during the menopause.

Neutrophils play a role in the development of pulmonary edema in many models of the adult respiratory distress syndrome, but the mechanism of their action is not completely understood. We asked whether two neutrophil secretory products, human neutrophil cationic protein (NCP) and human neutrophil elastase (HNE), would nonenzymatically alter the movement of albumin across a cultured endothelial monolayer. Both enzymes were inactivated by heating before use. HNE was additionally enzymatically inactivated with a chloromethylketone oligopeptide (CMK) inhibitor and with alpha 1-proteinase inhibitor (alpha 1-PI). Heated NCP, heated HNE, and CMK-complexed HNE all increased transendothelial albumin transfer. The cation protamine also increased albumin transfer across the endothelium and this increase was blocked by heparin. Alpha 1-PI and fetal bovine serum also prevented the cationic proteins from increasing albumin transfer. Using the release of lactate dehydrogenase as a marker of cytotoxicity, heated HNE was toxic to endothelial cells, heated NCP had only minimal toxicity, and protamine had no toxicity. Changes in endothelial cell shape with gap formation was seen after exposure to both heated HNE and heated NCP. Both the cytotoxicity associated with heated HNE and the cell shape changes associated with heated NCP and heated HNE could be blocked by heparin. These results suggest that in addition to neutrophil proteases and reactive O2 molecules, neutrophil-derived cationic proteins can directly and nonenzymatically contribute to edema formation during acute inflammation.

Analysis of bronchoalveolar lavage cellular constituents has been recommended as a valuable method for the characterization of the inflammatory cellular population and for studying cellular interactions in the lower respiratory tract of patients with idiopathic pulmonary fibrosis (IPF). However, the clinical relevance of the enumeration of cells in bronchoalveolar lavage fluid (BALF) from patients with IPF remains controversial. We therefore examined the correlations between BALF cellular constituents and both the histopathologic abnormalities and the subsequent clinical response to corticosteroid therapy in 26 newly diagnosed, untreated patients with IPF. The BALF lymphocytosis was associated with moderate-to-severe alveolar septal inflammation (p less than 0.0005) and with a relative lack of histologic honeycombing (p less than 0.05). On the other hand, BALF neutrophil and eosinophil contents did not significantly correlate with any of eleven particular histopathologic abnormalities, and BALF neutrophil and lymphocyte contents did not correlate with the degree of clinical impairment (quantitated by a composite score based on dyspnea, radiographic abnormalities, and physiologic impairment) upon presentation. However, BALF eosinophil content correlated significantly with the severity of clinical impairment, higher eosinophil counts being associated with more severe initial clinical impairment (p less than 0.01). Neither pretreatment BALF neutrophil nor eosinophil content was related to the frequency or magnitude of subsequent clinical change in 20 patients evaluated before and after 1 yr of corticosteroid therapy. In contrast, pretreatment BALF lymphocytosis was associated with significant subsequent clinical improvement (p less than 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)