Malignant astrocytoma with binucleated granular cells in a Sprague-Dawley rat.

A 2-year-old Sprague-Dawley rat with hindlimb paralysis was diagnosed with a cerebral malignant astrocytoma. The distinctive feature of this astrocytoma was the presence of scattered binucleated cells that contained hypereosinophilic, 1-2 micro m in diameter, cytoplasmic granules. The neoplastic astrocytes stained positively for vimentin (VIM), lysozyme, and phosphotungstic acid hematoxylin (PTAH). Within the binucleated cells, granules stained with PTAH and periodic acid-Schiff (PAS) before and after diastase digestion. Ultrastructurally, neoplastic astrocytes were characterized by cytoplasmic aggregates of electron-dense intermediate filaments consistent with VIM and desmin. The cytoplasm of binucleated cells contained numerous phagolysosomes enlarged by myelin figures and glycoprotein or glycolipid. Intermediate filaments were not present. This is the first description, in the rat, of a neoplasm with features resembling the human granular cell astrocytoma. Our findings suggest that an astrocytic origin should be considered for the binucleated cells in this neoplasm.

Radiation exposure to the spine surgeon during fluoroscopically assisted pedicle screw insertion.

STUDY DESIGN: In vitro study to determine occupational radiation exposure during lumbar fluoroscopy. OBJECTIVES: To assess radiation exposure to the spine surgeon during fluoroscopically assisted thoracolumbar pedicle screw placement. SUMMARY OF BACKGROUND DATA: Occupational radiation exposure during a variety of fluoroscopically assisted musculoskeletal procedures has been previously evaluated. No prior study has assessed fluoroscopy-related radiation exposure to the spine surgeon. METHODS: Bilateral pedicle screw placement (T11-S1) was performed in six cadavers using lateral fluoroscopic imaging. Radiation dose rates to the surgeon's neck, torso, and dominant hand were measured with dosimeter badges and thermolucent dosimeter (TLD) rings. Radiation levels were also quantified at various distances from the dorsal lumbar surface using an ion chamber radiation survey meter. RESULTS: The mean dose rate to the neck was 8.3 mrem/min. The dose rate to the torso was greatest when the surgeon was positioned ipsilateral to the beam source (53.3 mrem/min, compared with 2.2 mrem/min on the contralateral side). The average hand dose rate was 58.2 mrem/min. A significant increase in hand dose rate was associated with placement of screws ipsilateral to the beam source (P = 0.0005) and larger specimens (P = 0.0007). Radiation levels significantly decreased as distance from the beam source and dorsal body surface increased. The greatest levels of radiation were noted on the side where the primary radiograph beam entered the cadaver. CONCLUSION: Fluoroscopically assisted thoracolumbar pedicle screw placement exposes the spine surgeon to significantly greater radiation levels than other, nonspinal musculoskeletal procedures that involve the use of a fluoroscope. In fact, dose rates are up to 10-12 times greater. Spine surgeons performing fluoroscopically assisted thoracolumbar procedures should monitor their annual radiation exposure. Measures to reduce radiation exposure and surgeon awareness of high-exposure body and hand positions are certainly called for.

Ossification of the posterior longitudinal ligament of the spine: a case report.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common cause of severe myelopathy and radiculopathy in Oriental populations. It typically involves the cervical spine. We present a 60-year-old Asian male with OPLL who developed progressively worsening cervical myopathy. The diagnosis and management are discussed.

The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle.

The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T-tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma.

Frog cardiac calsequestrin. Identification, characterization, and subcellular distribution in two structurally distinct regions of peripheral sarcoplasmic reticulum in frog ventricular myocardium.

Calsequestrin is a calcium-binding protein known to sequester calcium accumulated in the sarcoplasmic reticulum (SR) of muscle cells during relaxation. In the present study, we used affinity-purified antibodies to chicken cardiac calsequestrin to identify a 60,000-Da calsequestrin in frog myocardium. Like previously identified cardiac calsequestrins, it is enriched in cardiac microsomes, it is enriched by biochemical procedures previously used to purify cardiac and skeletal calsequestrins, and it exhibits a pH-dependent shift in its apparent Mr on a two-dimensional gel system. Finally, the NH2-terminal amino acid sequence of this 60,000-Da immunoreactive protein purified by fast protein liquid chromatography was identical to that of rabbit skeletal and canine cardiac calsequestrin. Thus, we conclude that this protein corresponds to the calsequestrin isoform in frog ventricular muscle. Frog calsequestrin was localized in discrete foci present at the periphery but absent from the central regions of frog ventricular myocytes as determined by immunofluorescence labeling. Immunoelectron microscopic labeling demonstrated that calsequestrin was confined to the lumen of two structurally distinct regions of the SR, where it was localized in the subsarcolemmal region of the myofibers. One of these appeared to correspond to the terminal SR previously reported to be closely apposed to the sarcolemma of frog myofibers. The other region, although close to the sarcolemma, was not physically joined to it and appeared to correspond to corbular SR. It generally is believed that frog cardiac SR does not provide activator Ca2+ required for excitation-contraction coupling. However, the identification of a calsequestrin isoform very similar to mammalian cardiac calsequestrin that is confined to specialized regions of frog cardiac SR lends support to the idea that frog cardiac SR has the ability to store Ca2+ and thus function in some capacity in frog cardiac muscle contraction.

Identification of novel proteins unique to either transverse tubules (TS28) or the sarcolemma (SL50) in rabbit skeletal muscle.

Novel proteins unique to either transverse tubules (TS28) or the sarcolemma (SL50) have been identified and characterized, and their in situ distribution in rabbit skeletal muscle has been determined using monoclonal antibodies. TS28, defined by mAb IXE112, was shown to have an apparent relative molecular mass of 28,000 D. Biochemical studies showed that TS28 is a minor membrane protein in isolated transverse tubular vesicles. Immunofluorescence and immunoelectron microscopical studies showed that TS28 is localized to the transverse tubules and in some subsarcolemmal vesicles possibly corresponding to the subgroup of caveolae connecting the transverse tubules with the sarcolemma. In contrast, TS28 is absent from the lateral portion of the sarcolemma. Immunofluorescence studies also showed that TS28 is more densely distributed in type II (fast) than in type I (slow) myofibers. Although TS28 and the 1,4-dihydropyridine receptor are both localized to transverse tubules and subsarcolemmal vesicles, TS28 is not a wheat germ agglutinin (WGA)-binding glycoprotein and does not appear to copurify with the 1,4-dihydropyridine receptor after detergent solubilization of transverse tubular membranes. SL50, defined by mAb IVD31, was shown to have an apparent relative molecular mass of 50,000 D. Biochemical studies showed that SL50 is not related to the 52,000-D (beta subunit) of the dihydropyridine receptor but does bind to WGA-Sepharose. Immunofluorescence labeling imaged by standard and confocal microscopy showed that SL50 is associated with the sarcolemma but apparently absent from the transverse tubules. Immunofluorescence labeling also showed that the density of SL50 in type II (fast) myofibers is indistinguishable from that of type I (slow) myofibers. The functions of TS28 and SL50 are presently unknown. However, the distinct distribution of TS28 to the transverse tubules and subsarcolemmal vesicles as determined by immunocytochemical labeling suggests that TS28 may be directly involved in excitation-contraction coupling. Our results demonstrate that, although transverse tubules are continuous with the sarcolemma, each of these membranes contain one or more unique proteins, thus supporting the idea that they each have a distinct protein composition.

Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold-labeling study.

The subcellular distribution of the 1,4-dihydropyridine receptor was determined in rabbit skeletal muscle in situ by immunofluorescence and immunoelectron microscopy. Longitudinal and transverse cryosections (5-8 microns) of rabbit gracilis muscle were labeled with monoclonal antibodies specific against either the alpha 1-subunit (170,000-D polypeptide) or the beta-subunit (52,000-D polypeptide) of the 1,4-dihydropyridine receptor by immunofluorescence labeling. In longitudinal sections, specific labeling was present only near the interface between the A- and I-band regions of the sarcomeres. In transverse sections, specific labeling showed a hexagonal staining pattern within each myofiber however, the relative staining intensity of the type II (fast) fibers was judged to be three- to fourfold higher than that of the type I (slow) fibers. Specific immunofluorescence labeling of the sarcolemma was not observed in either longitudinal or transverse sections. These results are consistent with the idea that the alpha 1-subunit and the beta-subunit of the purified 1,4-dihydropyridine receptor are densely distributed in the transverse tubular membrane. Immunoelectron microscopical localization with a monoclonal antibody to the alpha 1-subunit of the 1,4-dihydropyridine receptor showed that the 1,4-dihydropyridine receptor is densely distributed in the transverse tubular membrane. Approximately half of these were distributed in close proximity to the junctional region between the transverse tubules and the terminal cisternae. Specific labeling was also present in discrete foci in the subsarcolemmal region of the myofibers. The size and the nonrandom distribution of these foci in the subsarcolemmal region support the possibility that they correspond to invaginations from the sarcolemma called caveolae. In conclusion, our results demonstrate that the 1,4-dihydropyridine receptor in skeletal muscle is localized to the transverse tubular membrane and discrete foci in the subsarcolemmal region, possibly caveolae but absent from the lateral portion of the sarcolemma.

Ultrastructural localization of calsequestrin in adult rat atrial and ventricular muscle cells.

The distribution of calsequestrin in rat atrial and ventricular myocardial cells was determined by indirect immunocolloidal gold labeling of ultrathin frozen sections. The results presented show that calsequestrin is confined to the sarcoplasmic reticulum where it is localized in the lumen of the peripheral and the interior junctional sarcoplasmic reticulum as well as in the lumen of the corbular sarcoplasmic reticulum, but absent from the lumen of the network sarcoplasmic reticulum. Comparison of these results with our previous studies on the distribution of the Ca2+ + Mg2+-dependent ATPase of the cardiac sarcoplasmic reticulum show directly that the Ca2+ + Mg2+-dependent ATPase and calsequestrin are confined to distinct regions within the continuous sarcoplasmic reticulum membrane. Assuming that calsequestrin provides the major site of Ca2+ sequestration in the lumen of the sarcoplasmic reticulum, the results presented support the idea that both junctional (interior and peripheral) and specialized nonjunctional (corbular) regions of the sarcoplasmic reticulum are involved in Ca2+ storage and possibly release. Furthermore, the structural differences between the junctional and the corbular sarcoplasmic reticulum support the possibility that Ca2+ storage and/or release from the lumen of the junctional and the corbular sarcoplasmic reticulum are regulated by different physiological signals.

Ultrastructural localization of calsequestrin in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections.

The ultrastructural localization of calsequestrin in rat skeletal muscle (gracilis) was determined by indirect immunoferritin labeling of ultrathin frozen sections. Calsequestrin was found in the lumen of transversely and longitudinally oriented terminal cisternae but was absent from most of the longitudinal sarcotubules and the fenestrated sarcoplasmic reticulum. Calsequestrin was occasionally observed in vesicular structures found in the central region of the I band. Since calsequestrin is believed to provide the major site of Ca2+ sequestration in the sarcoplasmic reticulum, the present results support the view that Ca2+, transported to the lumen of the sarcoplasmic reticulum, is preferentially sequestered in the terminal cisternae, but they also suggest that additional Ca2+ sequestration may occur near the center of the I band.

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle.

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.

Ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections.

The ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat gracilis muscle was determined by indirect immunoferritin labeling of ultrathin frozen sections. Simultaneous visualization of ferritin particles and of adsorption-stained cellular membranes showed that the Ca2+ + Mg2+-ATPase was concentrated in the longitudinal sarcoplasmic reticulum and in the nonjunctional regions of the terminal cisternae membrane but was virtually absent from mitochondria, plasma membranes, transverse tubules, and junctional sarcoplasmic reticulum. Ferritin particles were found preponderantly on the cytoplasmic surface of the membrane, in agreement with published data showing an asymmetry of the Ca2+ + Mg2+-ATPase within the sarcoplasmic reticulum membrane. Comparison of the density of ferritin particles in fast and slow myofibers suggested that the density of the Ca2+ + Mg2+-ATPase in the sarcoplasmic reticulum membrane in a fast myofiber is approximately two times higher than in a slow myofiber.

Histogenesis of the pituitary in rainbow trout (Salmo gairdneri) in different ambient salinities with particular reference to the rostral pars distalis.

The pituitary gland is first evident in rainbow trout two weeks before hatching. Differentiation of prolactin and ACTH cells is not marked until 3-4 days after hatching when the follicular arrangement of the prolactin cells become apparent. There is no difference in the time of development of either prolactin or ACTH cells in larvae reared in different ambient salinities despite marked changes in tissue ion and water content. This suggests that prolactin and ACTH do not play the osmo- and iono-regulatory roles in larval rainbow trout that they are considered to play in adult salmonids.

Myocardial calcium and magnesium in acute ischemic injury.

The effect of ischemic injury on calcium and magnesium distribution in dog myocardial cells was investigated in tissue damaged by occlusion of the circumflex branch of the left coronary artery for 60 minutes or for 40 minutes followed by 20 minutes of reperfusion of the damaged tissue by arterial blood. No significant change in the concentration of these cations was noted in permanently ischemic, irreversibly injured myocardial cells, but tissue calcium was markedly increased in cells killed by an episode of transient ischemia. Tissue water and sodium also were increased and magnesium was decreased significantly in the transient ischemia model. Investigation of the localization of the increased Ca(--) by cellular fractionation and chemical analysis as well as by electron miscroscopy and microincineration showed that much of it was localized in dense bodies within the mitochondria. Within the intramitochondrial dense bodies, the calcium appeared to be a precipitate of an as yet undefined form of calcium phosphate.

Kinetics of calcium accumulation in acute myocardial ischemic injury.

The effect of ischemic injury on calcium uptake by dog myocardial cells was investigated in tissue damaged by transient or permanent occlusion of the circumflex branch of the left coronary artery. Tracer doses of (45)CaCl(2) were given at selected intervals before or after occlusion, and tissue uptake was measured in damaged and control left ventricular myocardium. No significant uptake of (45)Ca occurred after 60 minutes of ischemia produced by permanent occlusion of a coronary artery. However, 40 minutes of ischemia followed by 10 minutes of arterial reflow resulted in an 18-fold increase in Ca uptake in the injured tissue. Tissue (45)Ca increased linearly up through 10 minutes of arterial reflow but did not increase further with an additional 10 minutes of reflow. Myocardium reversibly injured by 10 minutes of ischemia followed by 20 minutes of arterial reflow did not accumulate excess (45)Ca. Calcium uptake is assumed to be an active process associated with mitochondrial accumulation of calcium into dense intramitochondrial granules of calcium phosphate. The uptake is a feature of irreversible cellular injury, but occurs only when arterial blood flow is present. The mechanism of the uptake has not been established. It appears to be related to defects in cellular permeability or mitochondrial function.