Point counting on the Macintosh. A semiautomated image analysis technique.

In image analysis, point counting is used to estimate three-dimensional quantitative parameters from sets of measurements made on two-dimensional images. Point counting is normally conducted either by hand only or manually through a planimeter. We developed a semiautomated, Macintosh-based method of point counting. This technique could be useful for any point counting application in which the image can be digitized. We utilized this technique to demonstrate increased vacuolation in white matter tracts of rat brains, but it could be used on many other types of tissue. Volume fractions of vacuoles within the corpus callosum of rat brains were determined by analyzing images of histologic sections. A stereologic grid was constructed using the Claris MacDraw II software. The grid was modified for optimum line density and size in Adobe Photoshop, electronically superimposed onto the images and sampled using version 1.37 of NIH Image public domain software. This technique was further automated by the creation of a macro (small program) to create the grid, overlay the grid on a predetermined image, threshold the objects of interest and count thresholded objects at intersections of the grid lines. This method is expected to significantly reduce the amount of time required to conduct point counting and to improve the consistency of counts.

Quantitation of smooth muscle proliferation in cultured aorta. A color image analysis method for the Macintosh.

Color image analysis was used to assess proliferative changes in smooth muscle cells from cultured segments of rabbit aortas. Proliferating cells were labeled with bromodeoxyuridine (BrdU) and visualized by immunohistochemical staining of histologic sections. A Macintosh IIfx computer with a Data Translation digitizer board, Javelin color camera and a color-enhanced version of National Institutes of Health Image 1.31 image analysis software (ColorImage 1.31) was used to acquire red, green and blue (RGB)-filtered grayscale images from microscopic slides of control and treated aortas. The BrdU-labeled (brown) and nonlabeled, hematoxylin (blue)-stained nuclei were identified on the RGB gray-scale images using a thresholding technique and sampled for nuclear number and area. An increase in the number of BrdU-labeled nuclei in the region of experimental perturbation was demonstrated by this semiautomated method. Thus, this Macintosh-based color image analysis method proved to be effective in rapidly quantitating immunohistochemically defined smooth muscle proliferation in microscopic tissues.

Artificial intelligence in automated classification of rat vaginal smear cells.

Microscopic examination of vaginal smears has been used routinely to determine the stage of the estrous cycle of female rats in reproductive research. The stage of the estrous cycle is based on relative counts of nucleated epithelial cells, cornified epithelial cells and leukocytes. The purpose of this project was to explore automation of vaginal smear analysis using image processing and artificial intelligence techniques. A fully connected back-propagation neural network was used to locate all potential objects in a digitized scene. A unique algorithm was then employed to center a subsequent sampling box to collect pixel intensity values from the red and green components of each image. A final neural network was used in the classification of cell type. Neural networks were used because of their ability to generalize among input patterns and to tolerate extraneous noise due to variations in staining artifacts and aberrant illumination of the microscope field. This preliminary cell diagnosing system not only provides the basis for the fully automated system but also provides a method by which many other cytologic image processing problems can be automated.

Effect of aging on concentrations of D2-receptor-containing neurons in the rat striatum.

Striatal D2 dopamine receptor concentrations were shown to decrease 30-35% during the lifespan of Wistar rats as assessed both radiochemically and autoradiographically. Binding densities and degree of age-change varied within the striatum; the latter ranging from 17 to 44% in 4 different regions. Overall neuronal loss during aging was 19%, and also varied considerably within the different striatal regions. Thus, it appears that neuronal loss may account for up to roughly half of the striatal D2 receptor loss during aging.

Modification of ATP-dependent Ca2+ transport in rat parotid basolateral membranes during aging.

Previous experiments have shown that senescent rat parotid acinar cells display marked reductions in Ca2+ release following alpha 1-adrenoreceptor stimulation. We report here, that in this naturally occurring perturbation of exocrine secretion, ATP-dependent Ca2+ transport in the parotid basolateral plasma membrane, the principal Ca2+ extrusion pathway in the parotid, is also modified. ATP-dependent Ca2+ transport in membrane vesicles isolated from senescent rats (approx. 24 months) is decreased approximately 30-50% as compared to that in vesicles isolated from younger rats (approx. 4 months). This alteration in Ca2+ pump activity is not due to (i) non-specific effects of vesicle preparation in the two animal groups, (ii) increased leakiness to Ca2+, or (iii) any apparent alteration in permeability of the membrane to K+ and Cl-. Kinetic studies demonstrate that the ATP-dependent Ca2+ transport activity in vesicles from senescent rats has similar maximal velocity to that of vesicles from young adult rats (27 vs. 31 nmol Ca2+/mg protein per min), however, it exhibits an approximately 50% increase in Km for Ca2+ (91 nM vs. 60 nM). Cytosolic free Ca2+, measured by Quin 2 fluorescence, in parotid acini following alpha 1-adrenoreceptor stimulation was much less elevated in preparations from senescent rats. These results may account, at least in part, for the previously reported physiological alteration in Ca2+ efflux seen in senescent rat parotid cells.

Alpha 1-adrenergic receptors in the neural lobe of the rat pituitary: autoradiographic identification and localization.

alpha 1-Adrenergic receptors were identified, characterized, and localized in rat pituitary gland by quantitative light microscopic autoradiography. Autoradiographic studies were carried out in slide-mounted rat pituitary sections using both [125I]2-[beta-(4-hydroxy-3-iodophenyl)ethyl-aminomethyl]tetralone ([125I]HEAT) and [3H]prazosin to localize alpha 1-adrenergic receptors. Data analysis by densitometry showed that [125I]HEAT binding in the rat neural lobe was saturable and of high affinity, with an apparent dissociation constant (Kd) of about 5 pM. Data from competition studies using a variety of compounds demonstrated an alpha 1-adrenergic receptor profile for [125I]HEAT-binding sites in the rat pituitary. A high density of alpha 1-adrenergic receptors (1 microM prazosin-displaceable [125I]HEAT binding or 10 microM phentolamine-displaceable [3H]prazosin binding) was found present only in the neural lobe, with negligible concentrations in the anterior and intermediate lobes. The regulation of [125I]HEAT-binding sites in the neural lobe was examined in pituitary stalk-transected and superior cervical ganglionectomized rats. Significant increases in [125I]HEAT-binding sites were observed after superior cervical ganglionectomy, but no changes in [125I]HEAT binding were found in pituitary stalk-transected rats compared to that in sham-operated controls. These data provide the first identification of alpha 1-adrenergic receptors in the neural lobe of the rat pituitary and suggest that these receptors may be localized primarily in blood vessels. In addition, a primary role for the peripheral sympathetic nervous system in regulating the neurohypophyseal vasculature is suggested. The precise function of alpha 1-adrenergic receptors in the neural lobe in regulating posterior lobe hormone secretion remains to be demonstrated.

Autoradiographic localization of 3H-paroxetine-labeled serotonin uptake sites in rat brain.

Paroxetine is a potent and selective inhibitor of serotonin uptake into neurons. Serotonin uptake sites have been identified, localized, and quantified in rat brain by autoradiography with 3H-paroxetine; 3H-paroxetine binding in slide-mounted sections of rat forebrain was of high affinity (KD = 10 pM) and the inhibition affinity constant (Ki) values of various drugs in competing 3H-paroxetine binding significantly correlated with their reported potencies in inhibiting synaptosomal serotonin uptake. Serotonin uptake sites labeled by 3H-paroxetine were highly concentrated in the dorsal and median raphe nuclei, central gray, superficial layer of the superior colliculus, lateral septal nucleus, paraventricular nucleus of the thalamus, and the islands of Calleja. High concentrations of 3H-paroxetine binding sites were found in brainstem areas containing dopamine (substantia nigra and ventral tegmental area) and norepinephrine (locus coeruleus) cell bodies. Moderate concentrations of 3H-paroxetine binding sites were present in laminae I and IV of the frontal parietal cortex, primary olfactory cortex, olfactory tubercle, regions of the basal ganglia, septum, amygdala, thalamus, hypothalamus, hippocampus, and some brainstem areas including the interpeduncular, trigeminal, and parabrachial nuclei. Lower densities of 3H-paroxetine binding sites were found in other regions of the neocortex and very low to nonsignificant levels of binding were present in white matter tracts and in the cerebellum. Lesioning of serotonin neurons with 3,4-methylenedioxyamphetamine caused large decreases in 3H-paroxetine binding. The autoradiographic distribution of 3H-paroxetine binding sites in rat brain corresponds extremely well to the distribution of serotonin terminals and cell bodies as well as with the pharmacological sites of action of serotonin.

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles. Modulation by agents which elevate cyclic AMP.

ATP-dependent Ca2+ transport was studied in basolateral membrane vesicles prepared from rat parotid gland slices incubated without or with agents which increase cyclic AMP. Isoproterenol (10(-5) M), forskolin (2 X 10(-6) M) and 8-bromocyclic AMP (2 X 10(-3) M) all increased ATP-dependent 45Ca2+ uptake 1.5- to 3-fold. The effect of isoproterenol was concentration-dependent and blocked by the beta-adrenergic antagonist propranolol. Enhanced uptake did not appear an artifact of vesicle preparation as apparent vesicle sidedness, 45Ca2+ efflux rates, specific activity of marker enzymes and equilibrium Ca2+ content were identical in vesicle preparations from control and 8-bromocyclic AMP-treated slices. Kinetic studies showed the ATP-dependent Ca2+ transport system in vesicles from 8-bromocyclic AMP-treated slices displayed a approximately 50% increase in Vmax and in Km Ca2+, compared to controls. The data suggest that physiological secretory stimuli to rat parotid acinar cells, which involve cyclic AMP, result in a readjustment of the basolateral membrane ATP-dependent Ca2+ pump.

Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland.

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any 'uphill' transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited 'uphill' transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.

Alpha 1-adrenergic responsiveness of young adult and aged rat submandibular cells in vitro.

The present study has evaluated, in vitro, alpha 1-adrenergic receptor mediated responses in submandibular cells from young adult and aged rats. Submandibular glands from different aged rats possess a similar number of alpha 1-adrenergic receptors that display comparable binding characteristics. Following alpha 1-adrenergic stimulation, cells from both groups of rats show a similar ability to mobilize intracellular Ca2+ (45Ca2+ time course, agonist dose-response) and to elicit a functional response (inhibition of protein synthesis by epinephrine) which reflects Ca2+ mobilization.

Stimulation of glucose oxidation in rat submandibular gland cells in vitro by analogues of cyclic AMP.

Secretion from salivary glands, following autonomic stimulation, is energy-dependent. Rat submandibular gland cells, when treated in vitro with both alpha- and beta-adrenergic agonists, showed increased glucose oxidation. The effects of alpha-adrenergic agents on glucose metabolism can be mimicked by non-receptor mobilization of Ca2+. Analogues of cyclic AMP were capable of elevating glucose metabolism to nearly the same extent as beta-adrenergic agonists.

Effects of hypothyroidism on the DNA, carbohydrate, soluble protein and sialic acid contents of rat submandibular glands.

Submandibular glands are a target organ of thyroid hormones. This study examined the effects of hypothyroidism on the biochemical characteristics of these glands in the rat. There were no effects on the neutral sugar and DNA contents. However, soluble protein concentrations (micrograms/mg wet weight) were significantly decreased and sialic acid concentrations micrograms/mg soluble protein) were significantly elevated.

Alpha 1-adrenergic inhibition of protein synthesis in rat submandibular cells.

The incorporation of [14C]leucine into protein in dispersed cells from rat submandibular gland was inhibited by epinephrine. The dose required for a half-maximal effect was approximately 10(-6) M. This sensitivity is similar to that of mucin and potassium secretion induced by epinephrine from these cells. The inhibitory effect of epinephrine was mediated by the activation of alpha 1-adrenergic receptors and was partially independent of extracellular calcium. The calcium ionophore A23187 also decreased protein synthesis in the presence and absence of extracellular calcium. Epinephrine diminished alpha-[14C]aminoisobutyric acid (AIB) influx, but the extent of altered AIB uptake was much less than the extent of decrease in protein synthesis. Analysis of newly synthesized protein by gel electrophoresis and autofluorography showed that epinephrine selectively inhibited the synthesis of specific submandibular proteins. In aggregate these results suggest that activation of alpha 1-adrenergic receptors in rat submandibular cells influences the regulation of protein synthesis by a mechanism that uses intracellular calcium.

Rat parotid cell function in vitro following X irradiation in vivo.

The effect of X irradiation on rat parotid acinar cell function was evaluated in vitro 1, 3, and 7 days following in vivo exposure to 2000 R. Several cellular functions were followed: protein secretion (amylase release), ion movement (K+ efflux and reuptake), amino acid transport (alpha-amino[14C]isobutyric acid), and an intermediary metabolic response ( [14C]glucose oxidation). In addition both the morphologic appearance and in vivo saliva secretory ability of parotid cells were assessed. Our results demonstrate that surviving rat parotid acinar cells, isolated and studied in vitro 1-7 days following 2000 R, remain functionally intact despite in vivo diminution of secretory function.

Adrenergic regulation of protein synthesis in rat submandibular salivary gland cells.

Epinephrine, a mixed alpha- and beta-adrenergic agonist, modulated new protein synthesis in dispersed rat submandibular cells. Both adrenoreceptors separately could increase protein synthesis after cells were exposed to [14C]-leucine for 10 min and then with 1 mM unlabelled leucine for 10-30 min. The extent of response was dependent on the concentration of epinephrine to which cells were exposed. Alteration in the sequence of exposure of cells to tracer and agonist changed the direction of agonist response.

Protein production and processing in young adult and aged rat submandibular gland cells in vitro.

Protein production and processing were evaluated in vitro in dispersed submandibular gland cells from young adult (4-6 months) and aged (24 month) rats. A modest decrease (approx. 25%) in protein production (incorporation of radiolabeled amino acids into 10% CCl3COOH-insoluble material) was found with old cells in both continuous-pulse and pulse-chase studies. Also new protein processing, followed by gel electrophoresis and autofluorography of radiolabeled samples, showed specific, marked alterations in old cells. In particular a significant difference in the processing of a 225 000 molecular weight glycoprotein (likely the major rat submandibular mucin) was detected.

Rat parotid gland amylase: evidence for alterations in an exocrine protein with increased age.

The content of alpha-amylase, the major exocrine secretory protein from rat parotid glands, was studied in young adult and aged rat tissue. alpha-Amylase protein was determined with an enzyme-linked immunosorbent assay. This employed antisera, produced against alpha-amylase purified from young adult rats, but which recognized and precipitated alpha-amylase enzyme activity equally well from both age groups. The parotid gland content of alpha-amylase was reduced about 50% in aged rats. Furthermore, the percentage of total gland protein which was alpha-amylase was decreased about 40% in aged animals. The data suggest that a somewhat specific alteration in alpha-amylase production (synthesis and/or degradation) occurs in parotid glands from aged rats. In addition, alpha-amylase functional activity was followed. The specific enzyme activity (U amylase activity per mg immunoreactive amylase) was about 35% higher in extracts from aged rat parotid glands compared to that of young adult glands.

Characteristics of submandibular glands from young and aged rats.

Submandibular glands from young adult and aged male and female rats have been examined and various descriptive parameters obtained. Several sex differences were observed with tissue from young and old animals. Sex differences were found in the gland contents of DNA, protein, and sialic acid and in some protein components resolved by gel electrophoresis. Significant age-related decreases in the gland contents of protein, sialic acid, and neutral sugar, but not DNA, were found with male rats. In female rats these values were constant across the animal's adult lifespan.

Histochemical evaluation of thiamine pyrophosphatase activity during first molar odontogenesis of the neonatal hairless mouse.

Localization of thiamine pyrophosphatase activity has been evaluated in the developing first molar of the neonatal hairless mouse. Postnatal animals from parturition to five days of age were decapitated and the severed heads frozen and sectioned in a frontal plane on a cryostat. 14 micron thick sections were fixed and subsequently incubated for thiamine pyrophosphatase activity according to the method of GOLDFISCHER et al. (1971). The tissue was visualized, dehydrated, cleared and mounted. Light microscopy was utilized in evaluating thiamine pyrophosphatase activity. Thiamine pyrophosphatase activity in the first molar of the hairless mouse is presented in tabular form and compared to similar data for the Swiss albino mouse. Enzyme activity increased as the metabolic activities of various cell layers increased. Thus, thiamine pyrophosphatase activity appeared to be related to the degree of differentiation and functional completency of the odontogenic tissues in the hairless mouse.