Adult rodent double skeletal stain

Rodent embryo double skeletal staining has long played a role in toxicological studies and is now an important part of selected genetic studies involving knockout or transgenic animals. However, phenotypic changes are sometimes not seen until animals reach adulthood. This study expands a previously developed embryonic staining method for use with adult mice.     

Effects of orthodontic tooth movement on the Alcian blue staining patterns of rat alveolar bone: an histochemical study

There is little information available concerning the effects of orthodontic forces on glycosaminoglycans (GAG) of alveolar bone. The present study identifies changes in Alcian blue staining intensity in rat alveolar bone undergoing resorption resulting from a heavy (25g) tipping force applied to the adjacent teeth by a separating spring. One day after force application, bone from treated animals (internal control and experimental sides) demonstrated more intense staining with Alcian blue, pH 1.0 (p less than 0.005) and pH 2.5 (p less than 0.05) than external controls (untreated animals). By day 3, the intensity of Alcian blue staining of treated alveolar bone was similar to untreated. Chondroitinase AC, ABC and testicular hyaluronidase predigestion did not completely block the staining reaction, suggesting that both GAG and noncollagenous proteins were demonstrated. Mean cross-sectional areas of the interdental septum of the experimental side were nearly 44% less than that of the internal control side after 3 days and nearly 62% less after 5 days. The study suggested that alterations in bone GAG levels occurred prior to tooth movement as histochemical changes occurred after force application but before initiation of significant septal resorption. A precise appraisal of the types of macromolecules effected awaits future biochemical analysis. The results of the present work strongly suggest the use of an external control group for future studies, as Alcian blue staining reactions of the internal control side of treated animals were not similar to those of external controls.     

Immunocytochemical localization of a folicle stimulating hormone-like molecule in the testis.

A follicle-stimulating hormone (FSH)-like molecule was localized in normal adult rat testes as well as testosterone-treated hypophysectomized rat tests with an unlabeled antibody (anti-FSH), peroxidase-antiperoxidase complex technique. Anti-FSH bound specifically to ultrathin sections of acrosomes of spermatids and intranuclear bodies of early spermatids. Quantitation of staining intensity demonstrated that FSH, used as an absorbing antigen, would significantly reduce this binding. There was less anti-FSH binding to the acrosomes of spermatozoa in the body and tail of the epididymis as compared to the less mature germ cells located in the testis and head of the epididymis. The acrosomal and nuclear staining of spermatids taken from hypophysectomized animals was similar to staining observed in sham injected animals. Taken together, these results suggest that there is a molecule within the acrosome that is immunologically similar to FSH. Most importantly, these results emphasize the importance of conducting physiologic experiments in conjunction with immunocytochemical studies.     

A Common Marking Technique Affects Tadpole Behavior and Risk of Predation

In many studies, it is necessary for researchers to mark individual animals for later identification. It is often assumed in the interpretation of these studies that marks have no effects on the biology of the animals. This assumption is insufficiently tested, and, when it is, coarse assessments of negative effects are often used, such as survival and growth under simplified laboratory conditions. We examined the consequences of a common larval amphibian marking technique (staining with methylene blue) for wood frog tadpole behavior and survival in an ecologically realistic scenario (predation). We measured a number of tadpole behavioral variables, under baseline conditions and in the presence of olfactory cues of a predator, for marked and unmarked tadpoles. We then exposed pairs of tadpoles (one marked and one unmarked) to one of two predators and tested for the effects of marking on the susceptibility of tadpoles to predation. We found that marking suppressed the increase in movement rate that typically occurred in (unmarked) tadpoles in the presence of predator cues. Marked tadpoles were significantly more likely to be captured by predators, an effect that could not be attributed to this difference in movement rate. These results raise concern about the use of this staining method and highlight the need for studies involving marked animals to thoroughly address any relevant effects the marks may have on the biology of the subjects.     

Agonistic behavior in naïve juvenile lobsters depleted of serotonin by 5,7-dihydroxytryptamine

We have been exploring the role of serotonin in fighting behavior in lobsters using a specific model of agonistic behavior, the establishment of hierarchical relationships between pairs of socially naive juvenile lobsters. We selected this model because the behavior is easily evoked, readily quantifiable, and the effects of experience are eleminated by using socially naive animals. In these studies we injected a specific neurotoxin, 5,7-dihydroxytryptamine, into juvenile lobsters over a 4-week period and then measured the effects on fighting behavior. This treatment reduces the levels of serotonin in the nervous system and immunocytochemical studies show a dramatic reduction in neuropil staining for the amine. Control animals received vehicle injection alone. All injected animals were paired against larger or smaller non-injected opponents, and three successive 30-min fights were carried out and statistically analyzed. The results were surprising: As with elevations of serotonin, reduced levels of serotonin increased the amount of time animals engaged in fighting behavior. No significant effects were seen on who initiated encounters, who retreated first, or who the eventual winner would be. Thus, in this model, elevation or reduction of serotonergic function increases the tendency of animals to engage in agonistic encounters.     

A new histochemical method to assess and evaluate potential bone marrow damage from therapeutic substances

Summary A new model is presented for assessing and evaluating the influence of bone-marrow-damaging substances in mice. Qualitative and quantitative results of histological, histochemical and enzyme histochemical studies facilitate the assessment of bone marrow damage in terms of extent and intensity. Bone marrow taken from the right femur of treated animals was embedded in renal tissue of controls for subsequent work-up in different techniques. From each of the experimental groups specimens from 10 animals were frozen in liquid nitrogen, specimens from another 10 animals were fixed in buffered formalin. Assessment and evaluation of changes was performed after the required histologic and histochemical staining (nucleic acid). Results were correlated with the cytology of bone marrow smears sampled from the left femur of each respective animal. Damage was visualized, in smear cytology or in histologic and histochemical preparations, and quantified by microphotometry and special staining for cytochrome oxidase activity.     

A neuron-specific antigen in C. elegans allows visualization of the entire nervous system.

In the present work, I describe an antiserum that specifically stains all neurons in C. elegans. This probe should facilitate developmental studies in this organism in the same way as anti-horseradish peroxidase has in Drosophila. The antiserum was raised against an 8 amino acid peptide representing part of an insect neuropeptide precursor, but there are no indications that this cross-reactivity reflects evolutionary homology. The antiserum allows the whole nervous system of C. elegans, including all cell bodies and processes, to be brightly stained. The subcellular staining pattern suggests that a cytoskeletal component is recognized. I have also isolated a mutation (e2481) that abolishes staining in all but 7 neurons, the 6 microtubule cells and 1 cell in the tail. Finally, I show that the pattern of staining in the nematode P. redivivus is similar to that seen in animals carrying the e2481 mutation.     

Histochemical localization of cholinesterase in a rodent,a sub-primate and a primate brain

Summary The distribution of acetylcholinesterase (AChE) was studied in the various rhinencephalic structures of the brains of the coypu rat, the galago and the Grivet monkey. Serial sections of the brains of these animals, stained by the classical neurohistological methods, were available and were used for the identification of the nuclear arrangements. Although there are minor differences there seems to be a general similarity between the coypu, galago and monkey in so far as the distribution of cholinesterase is concerned. The intensity of staining is nearly the same in the coypu and galago brains. Although the staining is less intense in the brain of the monkey, yet one must be fairly cautious before drawing any conclusions simply from the intensity of staining. So much may depend on the method, for example the optimum pH for one animal may not be appropriate for another. In all these animals the most intense reaction is observed in the olfactory tubercle, amygdala and the caudate-putamen. The fibres of the anterior commissure and lateral olfactory tract stain only very faintly. These comparative histochemical studies suggest that cholinesterase — containing fibres in general are better developed, in the phylogenetically older parts of the brain.     

CXCR2 regulates respiratory syncytial virus-induced airway hyperreactivity and mucus overproduction

Severe inflammation and mucus overproduction are partially responsible for respiratory syncytial virus (RSV)-induced disease in infants. Using a murine model, we characterized the virally induced chemokine receptors responsible for mediating the pathophysiological response to RSV infection, we found that CXCR2 mRNA was induced at 4 days after RSV infection. Immunohistochemical staining demonstrated that CXCR2 protein was expressed on alveolar macrophages. Immunoneutralization of CXCR2 resulted in decreased airway hyperreactivity relative to the RSV-infected controls. In addition, there was decreased mucus in the bronchoalveolar lavage fluid, decreased periodic-acid Schiff staining, and significantly less mucus-associated gob-5 mRNA and protein in anti-CXCR2-treated mice. The effects of anti-CXCR2 treatment were not a result of differences in viral clearance or neutrophil influx, as these parameters were comparable in both groups of animals. To confirm our immunoneutralization studies, we performed experiments in CXCR2(-/-) mice. Results in CXCR2(-/-) mice recapitulated results from our immunoneutralization studies. However, CXCR2(-/-) mice also showed a statistically significant decrease in muc5ac, relative to RSV-infected wild-type animals. Thus, CXCR2 may be a relevant target in the pathogenesis of RSV bronchiolitis, since it contributes to mucus production and airway hyperreactivity in our model of RSV infection.     

Effect of progesterone administration on the distribution of oviductal carbohydrates in <Emphasis Type="Italic">Rana tigrina</Emphasis>

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed. This research was supported by Thailand Research Funds (to W.W.), a Research Initiate Grant from Kasetsart University (to A.T.), and Mahidol University.     

A Procedure for Staining Cartilage and Bone of Whole Vertebrate Larvae While Rendering All Other Tissues Transparent

A new procedure for clearing and differentially staining skeletons of vertebrate larvae is described. Body pigments of fixed specimens are bleached by treatment with alkaline H₂O₂. This process markedly enhances the transparency of alcian blue-alizarine red S stained whole animals without damaging the specimens. This procedure should be useful for analyzing skeletal structures in studies of embryology, comparative anatomy and teratology of small vertebrates.     

Heterogeneity of keratin distribution in the oral mucosa and skin of mammals as determined using monoclonal antibodies

The immunohistochemical localization of keratins in the oral epithelia of several mammals was investigated using the monoclonal antibodies to keratins, PKK1 (41-56 kilodaltons) and KL1 (55-57 kilodaltons). The staining patterns obtained in different locations of the oral mucosa and of the skin epidermis were compared. In the papillae on the dorsal surface of the tongue, some areas exhibited marked PKK1 staining, while other area were PKK1 negative. In general, rodent oral epithelia were negative for PKK1 in the basal layer, while comparatively strong PKK1 staining was observed in cells of the upper spinous layer. In the epidermis, positive PKK1 reactions were confined to the basal layer, while KL1 staining was occasionally seen in the basal layer of oral epithelia. In cats, dogs, and monkeys, different PKK1 and KL1 binding patterns were observed in oral epithelia. Also, the distribution in oral epithelia differed from that seen in the epidermis of these animals. In the epidermis, the distribution of PKK1 and KL1 was regular, with PKK1 usually being confined to the basal layer, while KL1 binding was found in the spinous and granular cell layers, and was dependent on the degree of keratinization. In the animals studies, keratin expression--as detected by PKK1 and KL1--was different in the skin epidermis and oral epithelia, and the localization of these keratins differed in the various types of oral mucosa.     

Ultrastructural localization of thyrotropin (TSH)-like immunoreactivity in specific secretory cells of the hypophyseal pars tuberalis in the Djungarian hamster,Phodopus sungorus

Summary Specific secretory cells in the hypophyseal pars tuberalis of Djungarian hamsters maintained under different photoperiods were investigated immunocytochemically by means of the colloidal gold technique using antibodies against rat thyrotropin (TSH). Secretory cells of animals kept under long photoperiods (LD16:8) showed positive staining of secretory granules (diameters 90–130 nm), whereas other intracellular structures were free of immunoreactivity. In animals kept under short photoperiods (LD8:16) secretory cells displayed increased numbers of secretory granules, but these organelles were devoid of immunoreactivity. In contrast, immunoreactivity of thyrotropes in the pars distalis did not differ between the two groups of animals investigated. The present results confirm earlier light-microscopical studies that in the pars tuberalis specific secretory cells show TSH-like immunoreactivity; however, they differ in their reactivity pattern from classical thyrotropes in the pars distalis.     

The immunocytochemical localization of a cat uterine protein that is estrogen dependent (CUPED)

An estrogen-dependent polypeptide (CUPED), which was purified from uterine flushings of estrogen-treated cats, was localized in endometrial epithelial cells of cats using the peroxidase-antiperoxidase immunocytochemical staining procedure. Epithelial cells from animals treated with estradiol for 4, 7, or 14 days and estrogen-primed animals treated with progesterone for 2 days showed positive immunostaining. Staining was absent in untreated ovariectomized animals and in estrogen-primed animals treated with progesterone for 4 days. Specific cytoplasmic staining was confined to apical secretory granules in nonciliated cells of deep uterine glands. Staining was also commonly observed in the lumen of deep glands. Immunostaining was absent in the cells of the surface epithelium, stroma, and myometrium. In addition, other organs such as the oviduct, kidney, liver, pancreas, and lung showed no evidence of specific immunocytochemical staining. Therefore, the estrogen-dependent polypeptide obtained from uterine flushings of estrogen-treated ovariectomized cats is a uterine-specific secretory product that is packaged in apical cytoplasmic granules of uterine epithelial gland cells before being released into the uterine lumen.     

Use of prostaglandin inhibitor, 2-(6-methoxy-2-naphthyl) propionic acid,with regard to morphological and enzymatic changes of gastric mucosa

2-(6-methoxy-2-naphthyl) propionic acid is introduced to treatment as non-steroid antiinflammatory drug (NSAID) under the trade-mark of Naprosyn. Inhibition of prostaglandin synthesis is regarded as the most likely mechanism of its action. In some patients, its side-effects include gastritis, reactivation of ulcerous niche, and upper gastrointestinal haemorrhage. The absence of complex studies addressed to the question of morphological and histochemical changes in gastric mucosa after oral administration of Naprosyn prompted our undertaking. In experimental animals, with varying doses (10 mg/kg and 50 mg/kg) and variously long administrations (1 week and 3 weeks), a trial has been reported here upon. In the frozen-sectioned preparations, the histochemical reaction for acid phosphatase activity, according to Gomori, was made. The paraffin sections were subjected to the HE staining, PAS staining according to McManus and with the Masson's method. Our results of the morphological and histochemical studies in rats support the clinical observations of mucosal destruction in stomach in patients after oral administration of Naprosyn.     

Alterations in the distribution of glycoproteins in epithelial cells of murine colon after injection of tunicamycin

Summary Glycoproteins are associated with several structures of colonic absorptive cells of the mouse. These include the cell coat, Golgi apparatus and vesicles that transport the glycoproteins from the apparatus to the cell surface (Michaels and Leblond 1976). In many in vitro systems, the antibiotic tunicamycin inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins. In the present in vivo study, tunicamycin was injected into mice. The murine colonic epithelial cells were prepared routinely for electron microscopy and cytochemistry. Cells from the experimental and control animals were similar morphologically. However, staining by the periodic acid-chromic acid-silver methenamine technique, revealed differences in the distribution of glycoproteins. In animals that received the higher dosages of tunicamycin there was a substantial reduction in silver staining in both the Golgi apparatus and the vesicles of colonic epithelial cells compared to these structures in cells of identically treated control tissues, whereas the staining over the cell coat was not significantly altered. Possible explanations for the staining of the cell coat in the treated animals were provided in the text. This report demonstrates the feasibility of using tunicamycin in vivo and detection of the changes obtained by the silver methenamine method.     

Patterns of inhibin and FSH localization in endometrium of baboon (Papio anubis) during menstrual cycle and early pregnancy.

Immunoreactive 10.5 KDa moiety of inhibin and hFSH was present in the baboon endometrium during menstrual cycle, early pregnancy and in castrated animals treated with steroid hormones, estrogen and/or progesterone. Endometrial differences during the menstrual cycle altered the intensity of immunostaining of inhibin and FSH. Maximum staining was observed in late luteal phase for both the hormones. In early pregnancy (35th day), the conceptus increased the staining for inhibin in the adjoining endometrial glands. Treatment of castrated animals with steroids for 14 days caused increased staining for inhibin. Maximum staining was observed when treated with estradiol or progesterone, whereas combination of estrogen and progesterone treatment decreased the staining reaction. In conclusion, both inhibin and FSH were localized in baboon endometrium and were under the influence of estrogen and progesterone.     

Sister chromatid exchange in the Mongolian gerbil, Meriones unguiculatus

Sister chromatid exchange (SCE) studies have been performed in a variety of animals, both in vitro and in vivo, as well as in plants. To date, no such studies have been performed in any member of the gerbil sub-family (Gerbillinae). A new sister chromatid differential staining method was used with mice in vivo and has now been applied to the Mongolian gerbil, Meriones unguiculatus. With some modifications, this in vivo method was found to be highly reproducible in gerbils, and had consistently produced many metaphase cells with clear sister chromatid differentiation. The method involves subcutaneous implantation of a 50 mg slow-release BrdU pellet, the fluorochrome Hoechst 33258, phosphate buffering at pH 6.8, and incandescent light exposure.     

Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS

When collection of ejaculated sperm samples is not possible, as is the case with wild species, the epididymides of sacrificed wild males become the only possible source of spermatozoa. Mature cauda epididymal spermatozoa display characteristics similar to those of ejaculated sperm cells. The present work proposes a sperm staining technique suitable for the morphometric evaluation of red deer epididymal sperm using a new computerized system. Epididymides from wild animals were extracted no later than 2h post mortem. After epididymal sectioning, sperm samples were collected, cooled to and equilibrated at 5 degrees C, and frozen in liquid nitrogen. Before staining, sperm samples were thawed for 20s at 37 degrees C, and used for the preparation of slides. Three different sperm stains were tested: Hemacolor, Diff-Quik, and Harris' Hematoxylin. Morphometric analyses of sperm samples were performed using the morphologic module of the ISAS. Two hundred spermatozoa per sample and stain were captured at random and analyzed. Sperm morphometric values were significantly affected by the staining technique used. Moreover, significant differences were observed between animals. In our study, Diff-Quik could be considered to be the best sperm staining method, as it provided the highest percentage of well automatically analyzed cells by the ISAS, and discriminates better between animals. This sperm staining technique also proved to be a useful method for characterizing and discriminating between sperm samples of different animals.