Jonathan A. Parsons

Anatomical Mechanisms Explaining Damage to Pacemaker Leads, Defibrillator Leads, and Failure of Central Venous Catheters Adjacent to the Sternoclavicular Joint

The literature suggests that approximately 93% of all pacemaker lead fractures occur in the segment of the lead lateral to the venous entry, and costoclavicular compression has been implicated. While blood vessels can be compressed by movements of the clavicle, our research suggests that lead and catheter damage in that region is caused by soft tissue entrapment rather than bony contact. Dissection of eight cadavers with ten leads revealed that two entered the cephalic vein, and were not included in the study. Of the other eight leads, four passed through the subclavius muscle, two through the costoclavicular ligament, and two through both these structures before entering the subclavian, internal jugular, or brachiocephalic vein. Anatomical studies demonstrated that entrapment by the subclavius muscle or the costoclavicular ligament could cause repeated flexing of leads during movements of the pectoral girdle. Cineradiology of patients with position dependent catheter occlusion confirmed entrapment by the subclavius muscle. Soft tissue entrapment imposes a static load upon leads and catheters, and repeated flexure about the point of entrapment may be responsible for damage previously ottributed to cyclic costoclavicular compression.

Central and peripheral localization of somatostatin. Immunoenzyme immunocytochemical studies.

Early in 1973, Brazeau et al. (5), using monolayer cultures of rat pituitary as a bioassay system (34), reported their findings on the chemical structure and physiology of a tetradecapeptide extracted from sheep hypothalami. The new neunohonmone was named somatostatin (SRIF) because it blocked the secretion of growth hormone. Synthetic SRIF proved to be an effective hapten in conjunction with immunogenic molecules, and anti-SRIF antisera were raised in rabbits (3, 7) and guinea pigs (10). Thus, very soon after the initial description of SRIF (5), specific antibodies became available for use in quantitative immunochemical and morphologic cytoendoncninologic studies. Accordingly, Animura et al. (2) and Patel et al. (23) synthesized a tyrosine-containing analogue of SRIF that could be readily iodinated and developed radioimmunoassays (RIA). Subsequent RIA of extracts of brains and peripheral tissues of experimental animals showed that SRIF was not localized exclusively within the hypothalamus. Immunoassayable SRIF is present in significant amounts within extnahypothalamic neural tissue (6, 23, 28) and also within tissues of the gastroentenopancreatic (GEP; see Fujita and Kobayashi (13)) system of endocrine cells (3, 28). Thus SRIF may play a physiologically significant role in these tissues as well as in the hypothalamus. This prospect has become more intriguing in light of numerous reports which have demonstrated that synthetic SRIF, in addition to its inhibitory actions upon adenohypophysial function, blocks pancreatic insulin and glucagon release (see Genich et aL (14), Reichlin et at. (28) and Vale et at. (33)).

ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL LOCALIZATION OF LYSOZYME IN THE PANETH CELLS OF MAN

Human lysozyme was localized immunocytochemically at the ultrastructural level within Paneth cells of man by use of the unlabeled antibody enzyme method. Specific staining for lysozyme was observed over secretion granules in the apical cytoplasm, within the region of the Golgi apparatus and within some, but not all, lysosomes. No staining was observed within the cisternae of the rough endoplasmic reticulum or other cellular organelles. In control experiments comparing semiadjacent sections of the same Paneth cell, substitution of either normal rabbit serum for rabbit antihuman lysozyme antiserum (specificity control) or normal sheep serum for sheep antirabbit immunoglobulin G antiserum (method control) completely eliminated specific staining for lysozyme. The intensity of staining for lysozyme was related to both the titer and length of exposure to antilysozyme antiserum. Specific staining was obtained in tissue embedded in Araldite or Epon and was facilitated by etching with hydrogen peroxide. No staining was observed after prolonged fixation in glutaraldehyde or treatment with uranyl acetate in block.

Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.

Regulation of Islet β-Cell Proliferation by Prolactin in Rat Islets

This study examined the effects of prolactin on β-cell proliferation in pancreatic islet of Langerhans. Insulin secretion and β-cell proliferation were significantly increased from neonatal rat islets cultured for 4 days in the presence of either 500 ng/ml ovine prolactin (oPRL) or rat prolactin (rPRL). These effects could be prevented by including anti-oPRL serum in the culture media. Although insulin secretion and β-cell proliferation were slightly higher during the first 24 h of exposure to rPRL, maximal response was observed after 4 days for insulin secretion and 6–10 days for β-cell proliferation. The initial mitogenic response of β-cell to rPRL occurred by the limited recruitment of nondividing β-cells into the cell cycle and by most daughter cells proceeding directly into additional cell division cycles. Subsequently, the maximal effect of rPRL on β-cell proliferation was maintained by a higher rate of recruitment of previously nondividing β-cells into cell cycle with only one fourth of the daughter cells continuing to divide. These observations are difficult to reconcile with the proposal that a limited pool of β-cells capable of undergoing cell division exists in islets. Instead, these observations suggest that individual β-cells are transiently re-entering the cell cycle and dividing infrequently in response to rPRL. In this case, the majority of the β-cells would not be expected to be in an irreversible Go phase. We also demonstrated that the effects of rPRL on β-cell proliferation occur at normal serum glucose concentrations and are affected by inhibitors of polyamine metabolism. Additional studies on the effects of rPRL on β-cells should provide important information on the regulation of β-cell proliferation during conditions of increased insulin demand.

Technical parameters of immunostaining of osmicated tissue in epoxy sections.

The development of the labeled and unlabeled antibody enzyme methods and their application at the light and electron microscopic level have led to the widespread use of postembedding staining techniques of a wide variety of antigens. An important step in the postembedding staining technique is the “etching” or pretreatment of the plastic section with an agent to enhance the binding of specific primary antibody to antigen in the section. Sodium ethoxide (14) or alcoholic sodium hydroxide (8) both of which physically dissolve polymerized epoxy resin, were initially used to facilitate tinctorial staining (8, 14) and later by Lange (9, 10) to enhance the immunofluorescence for glucagon in plastic sections of pancreatic islets. Hydrogen peroxide has been used to bleach osmium from plastic sections and to enhance staining for glycoproteins at the ultrastructural level (13, 15, 19), but was first used by Nakane (18) in 1971 to enhance immunostaining at the ultrastructural level for growth hormone and prolactin in osmicated sections of rat pituitary. Using the unlabeled antibody enzyme method, Moriarty and Halmi (17) and Erlandsen et al. (5) showed that intensity of immunostaining in the postembedding technique was enhanced if sections of aldehyde fixed tissue were briefly exposed to hydrogen peroxide before application of immunoreagents. The successful use of hydrogen peroxide and alcoholic sodium hydroxide in the enhancement of immunostaining has led to their widespread adoption as an essential step in postembedding staining despite the fact that there is relatively little information available as to what effect these agents may have on the plastic resin or on the stability and extraction of antigens from the tissue sections. In this report the effect of various types of “etching” or pretreatment agents on the surface topography of the plastic sections has been investigated and has been correlated with the immunostaining for lysozyme within rat Paneth cells and with the extraction of Na’25! by these agents from cured epoxy resin.

ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL LOCALIZATION OF PROLACTIN IN RAT ANTERIOR PITUITARY BY USE OF THE UNLABELED ANTIBODY ENZYME METHOD

The National Institute of Arthritis, Metabolism and Digestive Diseases antirat prolactin antiserum, used widely for radioimmunoassay of rat prolactin, was used in the unlabeled antibody-peroxidase-antiperoxidase complex method for the ultrastructural immunocytochemical staining of rat prolactin cells. Sections were exposed to antiprolactin antiserum (1:400-1:100,000) or to antiovine luteinizing hormone antiserum (1:1,000) for 3 min or 4 or 48 hr. Specificity of prolactin staining was demonstrated by the absence of immunologic staining of other pituitary cells and by negative results with prolactin cells when normal serum or antiovine luteinizing hormone antiserum was used. Intensity of prolactin staining was directly related to the concentration and duration of exposure to antiprolactin antiserum. Prolactin secretion granules, located peripherally or within Golgi regions, were uniformly stained. Immunologic staining for prolactin was not observed within cisternae of the endoplasmic reticulum. These results show that the National Institute of Arthritis, Metabolism and Digestive Diseases antirat prolactin antiserum is specific for prolactin by ultrastructural immunocytochemical criteria and that antiserum dilutions appropriate for radioimmunoassay can be used in immunologic localization studies.

Decreased glucose stimulation threshold, enhanced insulin secretion, and increased beta cell coupling in islets of prolactin-treated rats

In order to determine the effect of lactogen on insulin secretion and junctional coupling among islet beta cells, ovine prolactin (oPRL) was infused by Alzet minipumps into female rats for 4 days. This treatment produced an oPRL level of 994 ± 122 ng/ml which, combined with residual rat PRL (rPRL) (12 ± 2 ng/ml), represented nearly a 20-fold increase from control (rPRL: 53 ± 17 ng/ml). In addition, plasma insulin was increased nearly 50% (control: 21.9 ± 3 μU/ml; experimental: 30.3 ± 3 μU/ml; p <0.05). When pancreata from lactogen-treated and control animals were perfused with linear 30–200 mg/dl glucose gradients, the apparent glucose threshold for insulin secretion in the experimental group was nearly 33% lower than that of the controls (i.e., 70 ± 4.6 mg/dl vs. 104 ± 7.5 mg/dl; p <0.01). The oPRL treatment also increased dye coupling among beta cells. Central cells in islets isolated from lactogen-treated and control animals were injected with Lucifer Yellow CH to estimate the extent of gap junctional coupling. There was nearly a twofold increase in the projected area of dye transfer per injection in the experimental vs. the controls: 4,607 ± 575 μm2 vs. 2,302 ± 474 μm2, respectively; p <0.02. The effects of oPRL decreased the apparent glucose threshold for insulin release, increased the above-threshold glucose-induced insulin secretion, and increased the extent of dye coupling among beta cells. These changes in insulin secretion and dye coupling closely resemble those observed in islets from pregnant rats.

Prolactin Enhances Cell-to-Cell Communication Among β-Cells in Pancreatic Islets

To determine the role of prolactin in increasing junctional communication among islet β-cells, we studied dye coupling in pancreatic islets exposed to elevated levels of prolactin in vivo and in vitro. Islets were isolated from rats immediately after lactation or from rats bearing mammosomatotropic tumors (MtTW15), conditions involving high levels of prolactin (either 5-fold or 1000-fold control levels, respectively). When β-cells were microinjected with the gap junction permeant dye Lucifer yellow CH, the mean number of dye-coupled cells per injection was ∼10-fold greater than in islets from virgin control rats. As a more direct test of the effects of prolactin on β-cell coupling, islets isolated from virgin rats were treated for 90 min with 500 ng/ml rat prolactin in the presence of low glucose (2.8 mM) and were microinjected with dye. The mean number of dye-coupled cells per injection increased by 6.7-fold over controls with low glucose, demonstrating a direct effect of prolactin on β-cell coupling. In vitro treatment with high glucose (16.7 mM) resulted in a 2.7-fold increase in dye-coupled cells per injection. We discuss the possible relationship between the effects of glucose and of prolactin on coupling.

Effect of MtTW15 Mammosomatotropic Tumors on Pancreatic Islet Hormones

The effects of hypersecretion of growth hormone and prolactin on islet endocrine cells have been studied by radioimmunoassays, immunocytochemistry, and morphometry in randomized samples of pancreata from MtTW15 mammosomatotropic tumor-bearing and control rats. The randomized sampling procedure, validated by immunoassays, allowed evaluation of both hormone content (immunoassay) and endocrine cell population (immunocytochemistry) on samples derived from the same origin. Hyperinsulinemia (2×) and non-fasting hypoglycemia in 10-wk-tumor rats were normalized 3 wk after tumor removal. Pancreatic weight was doubled, but proportional to body weight increases. Islet/pancreas ratio was constant (1.29 ± 0.05%) and the same in tumor, tumor-removed, and control animals, but average islet dimensions were increased by 30% and average area doubled in tumor animals. Frequency analysis showed fewer small (< 70 μm) and more large (> 140 μm) islets in tumor animals, but no change in average islet shape shown by average axis ratios of 1.4 in all groups. Pancreatic content of insulin and glucagon was doubled, while that of somatostatin was constant. These changes were not completely reversed in tumor-removed animals. Similarly, a significant doubling in islet-derived mass was mainly due to a doubling of the B-cell mass as the average proportion of endocrine cells per islet shifted from 66%, 26%, and 18% to 81%, 18%, and 3% for B-, A-, and D-cells of control and tumor-bearing rats, respectively. Immunocytochemically detectable insulin was found in duct cells of tumor animals, but not controls. Whether such cells represent a functional reserve remains to be determined.