Ovarian sympathectomy in the guinea pig

Summary The role of ovarian adrenergic nerves in follicular growth was studied in prepubertal guinea pigs by determining the effect of sympathectomy on 1) follicle populations and 2) follicular development following exogenous gonadotropin administration. Selective unilateral ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian bursa on day 20 postpartum. The contralateral surgically closed ovarian bursa was injected with the vehicle used for 6-hydroxydopamine. On day 25, animals were injected with pregnant mare serum or saline followed by human chorionic gonadotropin or saline 48 h later. All animals were laparotomized on day 28 and blood from utero-ovarian veins was collected bilaterally for androstenedione determination. Ovaries were processed for morphometric analysis of follicles. The sympathectomized ovary in saline-injected animals had a significant decrease in preantral follicles (characterized by 2 layers of granulosa cells without antrum formation), an increase in 310–500 m diameter atretic follicles and an increase in follicles 700 um compared to the contralateral control ovary. There were no differences in androstenedione levels from the two sides, ovarian weights or the total number of follicles per ovary. Neither ovary had corpora lutea. The sympathectomized ovary in animals injected with gonadotropins was not different from the contralateral ovary in any of the parameters measured. Both control and sympathectomized ovaries had newly formed corpora lutea in response to the exogenous gonadotropins. These results suggest that ovarian adrenergic nerves normally participate in follicular development in the prepubertal guinea pig. However, exogenous gonadotropins may override neural influences on the prepubertal ovary.     

Intravesical BCG administration in the guinea pig

Intravesical BCG administration is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The mechanisms of its antitumor activity are not known. The aim of this study was to characterize the histomorphological changes in various organs of the guinea pig after intravesical BCG administration. The BCG preparation used was BCG-RIVM, a Dutch BCG preparation. Instillations were performed in previously undamaged bladders weekly for 6 consecutive weeks and lasted 30 min or 1 h. Different doses were used ranging from 10³ culturable particles (c.p.) to 5 × 10⁷ c.p. of BCG. After 6 weeks, the animals were killed and postmortem examination was performed. The bladder wall, retroperitoneal lymph nodes, spleen, liver, lungs and distant lymph nodes were examined histologically. The BCG therapy, with a dose of 10₆ culturable particles and higher, induced an inflammatory reaction consisting of mononuclear infiltrates in the subepithelial tissue of the bladder wall. In approximately 50% of the animals investigated, the infiltrates were accompanied by noncaseating granulomatous lesions indicated by the presence of epithelioid cells. In general, the epithelial layer of the bladder showed no visible alterations. Similarly, a granulomatous inflammatory reaction was observed in the first retroperitoneal (iliac) lymph nodes draining the bladder. Granulomatous lesions were occasionally also present in liver and lung. In three of the 29 animals investigated, lesions were present both in liver and lungs, and in two of these three animals a granulomatous reaction was observed in the spleen and distant lymph nodes indicating a generalized inflammatory response induced by BCG. No microorganisms were detected by Ziehl-Neelsen (ZN) staining or culture in L?wenstein-Jensen medium in the first draining (iliac) lymph nodes of the bladder or in the spleen. In this study we found that BCG could induce inflammatory reactions in the bladder wall after its introduction into the previously undamaged bladder. Ulceration of the epithelium covering the mononuclear infiltrates was not observed. Occasionally a generalized inflammatory response to BCG was present in the animals investigated.     

Regeneration of the islets of Langerhans in the guinea pig

Summary The regeneration of the islets of Langerhans in the guinea pig was studied after intravenous injection of alloxan. A number of cells in the islets was destroyed within 24 h after alloxan, but after 48 h there was a rapid proliferation of the surviving cells of the islets. This depended on the dosage of the drug as well as the timing. Electron microscopy of the islet at 48 h showed that the dividing cells had small electron dense granules and resembled a subtype of normal A cells, whose function is not yet known. There were also many agranular cells in the islet. These two groups of cells seen in the regenerating islet could be precursor cells, which could differentiate into cells. There was no evidence for transformation of duct cells or acinar cells into islet cells. None of the guinea pigs became permanently diabetic. This was probably due to inadequate dosage which resulted in only partial degeneration of the cells followed by regeneration and recovery. There was also some regeneration of the liver, kidney and the adrenal cortex following alloxan.The author is grateful to Professor R. Barer for his guidance and for providing the facilities for this study. Thanks are also due to Mrs. D. Barraclough for technical assistance and to Mrs. M. Hollingsworth for assistance with the photographsThis work was financed by a grant from the Cystic Fibrosis Research Trust     

Forms and activity of high molecular weight adrenocorticotropin in guinea pig

High molecular weight forms of adrenocorticotropin (ACTH) and endorphin were identified in extracts of guinea pig anterior and intermediate/posterior pituitary. Extracts of anterior pituitary contained ACTH immunoactive material with apparent molecular weights of 36,000, 24,000 and 4,500 daltons. The highest molecular weight form the ACTH co-migrated with a peak of endorphin immunoactive material. No material the size of glycosylated ACTH(1--39) was detected. Separated forms of high molecular weight ACTH prepared from mouse tumor cell culture medium stimulated the same maximal production of steroid as ACTH(1--39) in the guinea pig adrenal cell bioassay. Pro-ACTH/endorphin and ACTH biosynthetic intermediate were two orders of magnitude less potent than synthetic human ACTH(1--39); glycosylated ACTH(1--39) was equipotent to ACTH(1--39) although no similar material was detected in guinea pig pituitary extracts. Isolated guinea pig adrenal cortical cells were incubated with the various separated form of mouse tumor cell ACTH and products synthesized from (3H)pregnenolone were analyzed by two-dimensional thin-layer chromatography. The ratio of cortisol-related to corticosterone-related products was the same in response in glycosylated and nonglycosylated ACTH.     

Involvement of cell proliferation in the process of follicular atresia in the guinea pig

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.     

The thyrotropic cells of the guinea pig pituitary

Summary Three different immunocytoenzymatic techniques were used to identify and characterize the thyrotropic cells in the pituitary of normal guinea pigs at the ultrastructural level (superimposition technique, immunocytochemical technique using P.A.P. and indirect immunohistoenzymatic method before embedding).These cells are characterized by a dark cytoplasm with granules ranging from 1500 to 2000 Å in diameter. The appearance of these granules is very variable: some display a marked electron density and are homogeneous but some have a less marked electron density with a more electron dense peripherally situated region.The TSH molecules are essentially confined to the granules but when the immunocytochemical reactions are carried out before embedding, positive staining is also seen in the cytoplasm and the outer surface of most of the rough endoplasmic reticulum membranes. These results are discussed.We thank D. Quief for technical assistance. This work was supported by a grant from U.E.R. III LilleAttaché de Recherche INSERM     

Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.     

The permeability of the guinea pig cochlear capillaries to horseradish peroxidase

Summary Guinea pigs were given horseradish peroxidase intracardially. Examination of the cochlear capillaries 2 to 10 min after the injection revealed that the capillaries of the vascular stria are permeable to the peroxidase whereas the capillaries of the basilar membrane, the spiral ligament, and the spiral prominence are impermeable.

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Zusammenfassung Meerrettich-Peroxydase wurde Meerschweinchen intracardial verabfolgt. Die Untersuchung der Kapillaren der Schnecke 2–10 min nach der Injektion zeigte, daß die Kapillaren der Stria vascularis für die Peroxydase permeabel waren, jene der Membrana basilaris, des Ligamentum spirale und der Prominentia spiralis dagegen impermeabel sind.

     

Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38°C. NT contracted the longitudinal muscle (ED₅₀, 0.3 nM), the 2–3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 μM), ≥50% of the response was blocked and the residual effect gave an ED₅₀ of 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 μM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED₅₀, 0.3 nM) and one substance P (ED₅₀, 1.4 nM). Using membrane preparations and ¹²⁵I-labeled NT, specific, high affinty receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (K_ds: 0.1 nM and 2 nM). Sodiu ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg⁺⁺ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.     

A cytofluorometric study of the myenteric plexus in the guinea pig

Summary The myenteric plexus of the guinea pig ileum was studied in stretch preparations of the longitudinal muscle layer with adherent plexuses, and in freeze-dried transverse sections from the small intestinal wall. Catecholamines and serotonin (5-HT) were visualized according to the Falck-Hillarp technique. Emission spectra from the resulting fluorophores and recordings of their rates of photodecomposition were analysed. Adrenergic nerve terminals showed a slow fluorescence fading rate and a fluorescence spectrum compatible with their known contents of noradrenaline (NA), while the enterochromaffin cells showed a rapid exponential fading and a fluorescence spectrum compatible with their known contents of 5-HT. In order to unmask any low amounts of 5-HT in the neurons of the plexus, analysis of fluorescence parameters at various time intervals after pretreatment with reserpine followed by MAO-inhibition was performed. With the methods used no evidence of the presence of 5-HT in the myenteric plexus of the guinea pig could be found.We thank Iréne Svensson and Uno Johansson for skilful technical assistance. We are also indebted to Ciba, Pfizer and Draco for generous supplies of Reserpine, Nialamide and Pheniprazine. —This work was supported by grants from the Swedish Medical Research Council (Project 14 X-2235) and Göteborgs Läkaresällskap.     

Distribution of acetyl cholinesterase in the hippocampal region of the guinea pig

Summary 1. The distribution of acetyl cholinesterase (AChE) has been described in the dentate area, a part of the hippocampal region, in the adult guinea pig. The enzyme was demonstrated histochemically with a modification of the Koelle thiocholine method applied to formaldehyde-fixed frozen sections and unfixed cryostat sections. Non-specific cholinesterase was suppressed by ethopropazine, while the staining reaction for AChE was controlled by complete specific inhibition with BW 284c51. A single brain was stained according to the method of Karnovsky and Roots.2. The abundant AChE found in the dentate area exhibited a distinctly stratified distribution pattern. In the molecular layer, strong reaction was present in the outer third and immediately above the granular cell layer, the intermediate zone being light. The granular cell bodies were unstained. In the hilus, five layers showing alternating stronger and weaker reaction for AChE were recognizable.3. In view of the opinions of Cajal, Lorente de Nó, and Blackstad criteria for the definition of the dentate area are discussed. The present results fit into a concept of a layered guinea pig hilus representative of one group of mammals (other members being rabbit, monkey, and man) differing morphologically from the non-layered hilus of rat and mouse. The distribution of metal in the guinea pig hilus supports the concept.4. Possible structural correlates to the AChE are considered and a comparison with the distribution of AChE in the rat, reported earlier, has been made. In the molecular layer, the most striking difference was the heavy activity observed in the outer third in the guinea pig, where the content is moderate in the rat. The granular cell layer appeared virtually identical in both species. In the hilus the stratified pattern in the guinea pig, contrasting with the more diffuse distribution in the rat, essentially reflects the differing structural architectonics in the hilus of the two species.I am indebted to Mrs. L. Knudsen, Mr. A. Meier, Mr. Th. Nielsen, Mrs. K. Sørensen, Miss M. Sørensen, and Miss B. Ørum for skillful technical assistance.This study was supported in part by U.S.P.H.S. Grant NS 07998.     

Diffusion barrier to horseradish peroxidase in the vascular stria of the guinea pig

Summary Guinea pigs were given horseradish peroxidase intracardially and its diffusion in the vascular stria was studied. The Peroxidase spred freely among the intermediate cells and the marginal cells, but was never found to have passed zones occupied by tight junctions. It is concluded that the zones of tight junctions bordering the vascular stria represent a diffusion barrier to horseradish peroxidase.     

Occurrence of the neurosecretory substance during the embryonic development of the guinea pig

Summary The time and place of occurrence of the neurosecretory substance in the hypothalamo-neurohypophysial system of the guinea pig during embryogenesis have been investigated. Use is made of the luminiscence of neurosecretion stained with paraldehydefuchsin when observed in a dark field. It is established that the neurosecretory material occurs first in some cells of the supraoptic nucleus about the 39th–40th day of intrauterine development. In the paraventricular nucleus it is observed about the 44th–45th day. At that time it is seen also in Eminentia mediana and in the neurohypophysis. In the latter, however, it is in a smaller amount than in the areas situated above it. These results are discussed in connection with the transport theory of Bargmann and Scharrer.     

The epithelium of the middle ear in the guinea pig

Summary An electron microscopic study of aldehyde and osmium fixed normal guinea pig middle ear epithelium was made. Numerous branching microvilli occur between the cilia of the ciliated cells. The granules of the secretory cells are always surrounded by a membrane, and they vary in their content of electron dense substance. Half desmosomes are frequent in basal cells. The squamous epithelial cells of the bulla contain few microvilli and pinocytoric invaginations. In the basal part of the squamous epithelium dilations of the intercellular clefts often occur. The luminal part of the intercellular clefts are closed by multiple tight junctions.     

Age-related changes of hypocretin in basal forebrain of guinea pig

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) have been implicated in a wide variety of functions including sleep and wakefulness as well as related behaviors. Many of these functions of the hypocretins involve the activation of cholinergic neurons in the basal forebrain (BF). These neurons have been shown to exhibit age-related changes in a variety of species. In the present experiment, in adult and aged guinea pigs, we compared hypocretin immunoreactivity in regions of the BF that include the medial septal nucleus (MS), the vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB) and the magocellular preoptic nucleus (MCPO). In adult guinea pigs (3–5 months of age), all of the preceding BF regions contained dense hypocretin fibers with varicosities. On the contrary, in old guinea pigs (27–28 months), although the MS exhibited a similar intensity of hypocretin immunoreactivity compared with the adult guinea pig, there was a significant decrease in the intensity of immunoreactivity of hypocretinergic fibers in the VDB, HDB and MCPO. These data indicate that the hypocretinergic innervation of specific nuclei of the BF is compromised during the aging process. We suggest that the reduction in hypocretinergic innervation of the BF nuclei may contribute to the age-related changes in the states of sleep and wakefulness as well as deficits in related systems that occur in old age.     

Purification and characterization of an enkephalin-degrading aminopeptidase from guinea pig serum

An enkaphalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The amino-peptidase had a pH optimum of 7.0 with K_m values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid β-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.     

Two populations of somatostatin-immunoreactive neurons in the guinea pig striatum

Summary Two populations of neurons displaying somatostatin-like immunoreactivity were detected immunohistochemically in the guinea pig striatum using a monoclonal antibody. Sparse, well-stained neurons similar to those described in other species were observed throughout the guinea pig caudate-putamen. These neurons contained both neuropeptide Y and NADPH-diaphorase in addition to somatostatin. A second large population of somatostatin immunoreactive neurons in which these other substances did not coexist was found within the putamen.     

The capillaries of the middle ear mucosa in the guinea pig

Summary The middle ear capillaries of the guinea pig have fenestrated endothelium, and the intercellular clefts are closed by tight junctions. Intracardially injected horseradish peroxidase penetrates the fenestrae of the endothelium and gains access to the extra-cellular space beneath the epithelium, and the intercellular clefts of the epithelium.     

Development of cholinephosphotransferase in guinea pig lung mitochondria and microsomes

Development of mitochondrial and microsomal choline phosphotransferase in the fetal guinea pig lung was investigated. The activity in fetal mitochondria was more than twice of that in fetal microsomes. However, in adult lung, the enzyme was distributed mostly in microsomes. In fetal lung, both the mitochondrial and microsomal enzyme activity was greatest at approx. 81% of the total gestation period (55 days). The specific activity in the microsomal fraction then declined until term, but increased again in the 24-h newborn from 1.0 to 2.3 nmol/min per mg protein. The activity in the mitochondrial fraction declined after 61 days (2.8 nmol/min per mg) to a minimal level at term (0.6 nmol/min per mg). Although the enzyme activity decreased from day 55 (1.2 nmol/min per mg), the amount of phosphatidylcholine gradually increased between day 55 and term.