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Biomedical subjects

P A Wackym

Publications and source records attributed to P A Wackym.

At least 19 recordsLinked to original sources
PubMed1992

In situ hybridization for the localization of gene products in the auditory system.

In this article, an overview of the technique and examples of our data demonstrate that our protocols produce excellent, reliable in situ hybridization of mRNAs and immunohistochemical localization of mRNA translation products in the temporal bone and brain stem. This in situ hybridization protocol has been used to localize eight different mRNAs, including four types of nicotinic acetylcholine receptor subunit mRNAs, actin mRNA, CGRP mRNA, and two mRNAs coding for gap junction proteins, in both paraffin-embedded and sectioned (6 microns) and cryostat sectioned (15 microns) temporal bones (unpublished data). In conclusion, in situ hybridization of mRNAs with single-stranded RNA probes allows screening of brain stem and temporal bone sections for the expression of specific genes, with cellular resolution, and this protocol gives reliable results for all mRNAs studied. Of great potential significance is the ability to assess variations in gene expression by in situ hybridization of specific mRNAs. It is expected that such variations accompany changes in the physiologic state of an organism, such as developmental, pathologic or experimentally induced, and that these variations in gene expression are important in understanding basic auditory processes at the cellular level.

Auditory Pathways↗
PubMed1992

Differential diagnosis of virus-like particles in the human inner ear.

The entire endolymphatic duct and sac as well as the vestibular epithelia were obtained from four patients with Meniere's disease during translabyrinthine (TL) eighth nerve section and from 12 patients undergoing TL resection of acoustic schwannomas. After these specimens were processed for routine transmission electron microscopy (TEM), they were studied for morphologic evidence of viral infection. Although no virus particles were identified, numerous regularly occurring cell components and artifacts were found to morphologically mimic viruses. An atlas of these structures is presented.

Diagnosis, Differential↗
PubMed1991

Technique for transmission and immunoelectron microscopy of the human endolymphatic sac and vestibular epithelia.

A surgical technique is presented to obtain the entire vestibular aqueduct, containing the endolymphatic duct and sac as well as the vestibular epithelia, from the maculae and cristae during labyrinthectomy. The inner ear tissue was fixed in 3% glutaraldehyde, decalcified in 0.1 mol/L Na-EDTA, and routinely processed for transmission electron microscopy, including post-fixation with osmium tetroxide. Postembedding protein A-colloidal gold immunoelectron microscopy was performed after exposure of cellular antigenic sites by sodium metaperiodate. To validate this technique, thin sections from the crista ampullaris and the endolymphatic sac were incubated with antibodies raised against S-100, a protein present in specific types of neural tissue. Specific immunoreactivity was observed in the crista ampullaris, but not in the endolymphatic sac. The surgical biopsy technique described provides a means for the neuro-otologist to collect inner ear tissue from surgical patients that can be used for pathological studies using immunohistochemistry and electron microscopy.

Biopsy↗
PubMed1991

Cell and molecular anatomy of nicotinic acetylcholine receptor subunits and calcitonin gene-related peptide in the rat vestibular system.

In this report we demonstrate the pattern of calcitonin gene-related peptide (CGRP) mRNA and immunoreactivity in the central and peripheral vestibular system of the rat, using a CGRP cRNA probe and a polyclonal CGRP antiserum. We present evidence that somata in all regions of efferent vestibular neurons contain CGRP based on the correspondence between in situ hybridization (mRNA) and immunohistochemistry (mRNA translation product). CGRP immunohistochemistry (CGRPi) and in situ hybridization confirm that CGRPi axons and terminals present in the vestibular neuroepithelium are efferent in origin. Immunoelectron microscopy revealed an extensive innervation of the afferent vestibular pathway by CGRPi terminals that was not limited to the primary afferent chalice, as previously reported by Tanaka et al. (Brain Res 1989;504:31-5). An efferent neuromodulatory role of CGRP can be inferred from the distribution of terminals found on the primary afferent fibers, and type I and type II hair cells. In addition, we present evidence that nicotinic acetylcholine receptor (nAChR) subunit mRNA is expressed by primary afferent cell bodies. On the basis of these data, a hypothetical molecular mechanism of vestibular efferent modulation of the primary afferent pathway is proposed.

Animals↗
PubMed1991

Molecular biology of the vestibular system.

Molecular biology of the vestibular system has been limited by a number of technical difficulties including fixation, decalcification of the temporal bone and the small size of specific structures relative to their surroundings. In situ hybridization histochemistry and immunoelectron microscopy allow the subcellular study of gene expression and gene products, respectively. We developed the methodologies necessary to apply these techniques to the central and peripheral vestibular systems. The central and temporal bone distributions of the neuropeptide calcitonin gene-related peptide (CGRP) mRNA and two genes coding for gap junction proteins, connexin C32 and C43 mRNA, were studied. The cellular distributions of these mRNAs are presented. In addition, examples of pre-embedding and postembedding immunoelectron microscopy are presented demonstrating the usefulness of these techniques in studying the subcellular localization of specific antigens. The ultrastructural innervation of the vestibular periphery by the efferent neuropeptide CGRP and ultrastructural evidence of glycoprotein secretion by the human endolymphatic sac is presented.

Animals↗
PubMed1990

Subdural empyema of otorhinological origin.

The UCLA experience with subdural empyema (SDE) of otorhinological origin was reviewed. All cases of SDE with additional intracranial complications were excluded. Thirteen cases of SDE were identified with the predisposing factors being sinusitis (ten), mastoiditis (two), and otitis media (one). Based on data obtained from this review and from studies previously published in the literature, the keys to optimal outcome are rapid diagnosis, craniotomy with complete evacuation of the purulent collection followed by immediate surgical management of the otorhinological source of the SDE, and appropriate antibiotic therapy. Computed tomography is nearly always diagnostic but can be equivocal and magnetic resonance imaging may become the diagnostic study of choice. Of the five patients initially treated with burr holes. None of the patients initially managed with craniotomy were worse or died, whereas of the four patients initially managed with burr holes, two were worse (25 per cent) and two died (25 per cent). Antibiotic therapy is guided by the organisms found in the empyema and the site of origin of the infection. The otolaryngologist must remain aware of the clinical features and management of SDE and work closely with his neurosurgical colleagues to provide early, decisive surgical treatment.

Adolescent↗
PubMed1990

Re-evaluation of the role of the human endolymphatic sac in Menière's disease.

The role of endolymphatic sac (ES) dysfunction in the etiopathogenesis of Meniere's disease has remained controversial since the early 1900s. The first reports of the ultrastructural (transmission electron microscopy, TEM) pathology of the human ES in Meniere's disease have been published only in the last decade. These studies have been based on biopsies of the extraosseous (intradural) ES and in no cases has the TEM appearance of the intraosseous ES been described. Likewise the control material used has been from biopsies of extraosseous ES taken from patients with acoustic schwannomas. To date, no reports have compared the ultrastructure of the intrasosseous ES from normal control patients to patients with Meniere's disease. Since the intraosseous ES is believed to be the most active portion of the entire ES, studies were made of the ultrastructure of ten normal interosseous human ESs fixed immediately after death and obtained at autopsy (control material). Fourteen patients undergoing translabyrinthine (TL) neurotologic procedures (10, TL resection of acoustic schwannoma; 4, TL eighth cranial nerve section for Meniere's disease) had the entire vestibular aqueduct, containing the endolymphatic duct and the intraosseous ES, removed and processed for TEM. The roles of the epithelium, subepithelial space, and vasculature were morphologically studied to evaluate possible ES pathology in Meniere's disease and in patients with acoustic schwannoma. Wide anatomic variation in the distribution and density of the subepithelial connective tissue was observed in all groups. There was no difference in the TEM appearance of the intraosseous ES from normal controls and patients with eighth nerve schwannoma, nor was there any difference in the ES collagen deposition in patients with Meniere's disease. The ESs from two patients with Meniere's disease showed evidence of abnormal glycoprotein metabolism; one with possible hypersecretion and one with possible alteration of degradation of resorbed glycoprotein. The results of this preliminary study suggest that "perisaccular fibrosis" of the intraosseous ES was not a pathologic feature in these four cases of Meniere's disease and that alteration of ES glycoprotein secretion/resorption may be of etiopathologic significance.

Adult↗
PubMed1990

In situ hybridization for the study of gene expression in neuro-otologic research.

In situ hybridization histochemistry technology was developed for future application to neuro-otologic research. This method allowed the detection of cellular mRNA in tissue sections from the temporal bone or brainstem after cRNA/mRNA hybridization. To produce specific cRNA, single-stranded 35S-labeled cRNA (complimentary to target mRNA) is transcribed from commercially available plasmid vectors. These vectors contain promotor sequences for specific synthesis of RNA, and polylinker regions that will accept cloned DNA inserts for virtually any target nucleic acid sequence of interest. The protocol used in this research was optimized for studies that included concomitant immunohistochemical evaluation. The combination of in situ hybridization and immunohistochemistry provides the only method to correlate molecular information (gene expression) with biochemical or molecular markers, such as peptides or proteins (mRNA translation products) on individual cells in the temporal bone or brainstem. Using these techniques, we examined the distribution of the neuropeptide calcitonin gene-related peptide in rat temporal bone and brainstem sections using calcitonin gene-related peptide (CGRP) antisera and CGRP cRNA probes. We used in situ hybridization histochemistry with a cRNA probe complementary to the 3'-end noncoding sequence of the alpha CGRP mRNA and immunohistochemistry with a polyclonal antibody to the (TYR)CGRP23-37 to study the distribution of CGRP mRNA and CGRP-like immunoreactivity in the central and peripheral facial nerve. Numerous motoneuron cell bodies in the facial nucleus and accessory seventh nucleus and cell bodies in the gustatory geniculate ganglion were found to contain CGRP mRNA and the CGRP peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Academies and Institutes↗
PubMed1990

A technique for reembedding celloidin sections for electron microscopy.

A new technique for reembedding celloidin sections of human temporal bones for transmission electron microscopy is described. It consists of four steps: 1. loosening of celloidin sections from glass slides with use of xylene and dissection of the area of interest, 2. removal of celloidin with use of clove oil, 3. staining with 1% osmium tetroxide and 1% tannic acid, and 4. embedding in epoxy resin. Autolytic changes were seen due to poor fixation. TEM of reembedded celloidin sections of optimally fixed tissue revealed that the celloidin-embedding procedure affected ultrastructural preservation to some degree. This included less well-preserved cell membranes and some increased electron density of the cytosol decreasing the EM resolution of intracytoplasmic organelles. The technique allows TEM analysis of the intact labyrinth at all regions in the same specimen without dissection of the fragile tissue components of the membranous labyrinth. This might make the technique useful for processing freshly fixed human inner ear tissue and temporal bones for ultrastructural histopathological analysis.

Animals↗
PubMed1990

Size variations in the lateral intercellular spaces of the endolymphatic sac induced by dietary factors.

Since much evidence suggests that the endolymphatic sac is responsible for endolymph resorption, and that the endolymphatic sac lateral intercellular spaces which are lined by the energy-dependent transport complex NA+,K(+)-ATPase are important in this process, we sought to evaluate the effects of dietary salt and a food extract that inhibits the activity of Na+,K(+)-ATPase on lateral intercellular space size. Animals fed this food factor and a high-sodium diet had significantly smaller endolymphatic sac lateral intercellular spaces than those animals fed only a high-sodium diet (analysis of variance with Scheffe's multiple comparison test, P less than 0.001). Animals fed a high-sodium diet had significantly larger endolymphatic sac lateral intercellular spaces than those animals fed a control diet only (analysis of variance with Scheffe's multiple comparison test, P less than 0.001). Results of this study suggest that dietary sodium affects endolymphatic sac fluid dynamics and that other food factors may regulate sodium metabolism, and therefore endolymphatic sac function.

Animals↗
PubMed1990

Immunoelectron microscopy of the human inner ear.

The immediate fixation required for satisfactory morphologic preservation of the human inner ear has not only limited the ultrastructural study of this region, but has also limited the application of immunohistochemistry. The technique of postembedding protein A-colloidal gold immunoelectron microscopy was used on human inner ear tissue taken at operation and on celloidin-embedded temporal bone sections from a traditional temporal bone bank. We describe the utility of postembedding immunoelectron microscopy for studying the localization of a wide variety of antigens including type I collagen, S-100, and calcitonin gene-related peptide. The use of this method in material available in temporal bone banks or that has been routinely processed for transmission electron microscopy provides the potential for broad application to collections of otologic material.

Acoustic Maculae↗
PubMed1990

Exposed endoprosthesis. Management protocol using muscle and myocutaneous flap coverage.

A tenet in the orthopedic community is that dehiscent wounds overlying exposed prostheses should be treated by implant removal and delayed reconstruction. A management protocol using thorough debridement and irrigation and muscle flap coverage was accomplished in four patients with exposed endoprostheses after total arthroplasty or limb salvage surgery. Predisposing factors for late wound dehiscence in the four oncology patients were preoperative radiation and chemotherapy as well as multiple subsequent reoperations. In this study, all four prostheses and extremities were retained without the need for prosthetic removal or exchange. No infections developed. Late aseptic wound dehiscence with exposed conventional or tumor endoprosthesis need not be managed with prosthetic removal, arthrodesis, or amputation. This one-stage procedure avoided infection, allowed early mobilization, shortened hospitalization and, most important, avoided amputations.

Adult↗
PubMed1989

Management of vein of Galen aneurysms. Combined surgical and endovascular approach.

The extremely high morbidity and mortality associated with all forms of management of aneurysms of the vein of Galen continues to pose a tremendous challenge. Recent advances in neuroradiologic endovascular embolization techniques have offered some encouraging early results in the treatment of these vascular lesions. Our experience in managing two neonates and a 10-year-old boy with vein of Galen aneurysms is presented. The neonates underwent transtorcular embolization, while the boy was treated by standard transarterial balloon embolization. All patients had satisfactory outcomes. The combined surgical and radiologic approaches are simple when compared to previous surgical techniques, and early results suggest that they are preferable alternatives. The natural history of vein of Galen aneurysms and the various therapeutic modalities available are discussed.

Cerebral Angiography↗
PubMed1989

Craniotomy improves outcome in subdural empyema.

Renewed uncertainty regarding the best technique of surgical therapy (burr holes versus craniotomy) for subdural empyema prompted a review of the experience with this disease entity at UCLA. Based on data obtained from this review and from studies previously published in the literature, the keys to optimal outcome are rapid diagnosis, craniotomy with total removal of pus, and appropriate antibiotic therapy. The clinical syndrome of subdural empyema can overlap that of other intracranial infections; infants and young children present with a different syndrome than older children and adults. Computed tomography scanning is nearly always diagnostic but may be equivocal or normal; magnetic resonance imaging may become the diagnostic study of choice. Multiple organisms are often cultured, which requires multiple drug therapy.

Adolescent↗
PubMed1989

Malignant hyperthermia: experience in the prospective management of eight children.

Malignant hyperthermia (MH) is a seemingly rare genetic myopathy. Hypermetabolic crisis accompanied by a rise in body temperature to as high as 44 degrees C, is its hallmark. Malignant hyperthermia is usually triggered by potent inhalation anesthetics and/or depolarizing muscle relaxants. Because of the extraordinary incidence of death in patients who are at risk, pediatric surgeons may be reluctant to operate on these patients. Eight such patients were referred to the Pediatric Surgery Service and the UCLA Malignant Hyperthermia Center following pediatric surgical procedures aborted for first episodes of malignant hyperthermia (five) or for a strong family history of malignant hyperthermia (three). They were anesthetized with nitrous oxide, barbiturates, opiates, tranquilizers, and nondepolarizing muscle relaxants. The patients were not treated prophylactically with dantrolene. Cardiac monitoring, end-tidal PCO2, and rectal temperatures were monitored. After completion of their pediatric surgical procedures, all eight patients had a vastus lateralis muscle biopsy performed and subsequent caffeine/halothane contracture studies completed. The contracture study result was positive in all patients studied. No anesthetic or surgical complications were encountered. This study shows that patients at risk for developing MH crisis can have pediatric surgical procedures performed safely with appropriately selected general anesthesia.

Adolescent↗
PubMed1989

Muscle-flap coverage of exposed endoprostheses.

A well-entrenched tenet in the orthopedic community is that dehiscent wounds overlying exposed endoprostheses should be treated by implant removal and delayed reconstruction. A new management protocol utilizing thorough soft-tissue debridement and myocutaneous or muscle-flap coverage was evaluated in four patients at the UCLA Medical Center who presented with exposed endoprostheses. These prostheses were placed for total-joint replacement or limb salvage surgery. All four prostheses and extremities were salvaged without the need for endoprosthesis removal or exchange, and no infections developed. The results suggest that late aseptic wound dehiscence with an exposed endoprosthesis need not be managed with prosthetic removal, arthrodesis, or amputation. This one-stage procedure avoided infection, allowed early mobilization, and shortened hospitalization.

Adult↗
PubMed1988

Immunohistochemical localization of Na+, K+-ATPase in the human endolymphatic sac.

The presence and distribution of the transport protein complex Na+,K+-ATPase in the human endolymphatic sac (ES) was demonstrated immunohistochemically using a monoclonal antibody directed toward the denatured catalytic subunit of Na+,K+-ATPase from lamb kidney medulla. Our findings support an active transcellular ion exchange by the ES epithelium with subsequent passive transcellular and paracellular outflow of water. The possible role of the lateral intercellular spaces in the outflow of endolymph at the level of the ES is discussed.

Antibodies, Monoclonal↗