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At least 991 records · Page 55Linked to original sources

PubMed2000

Permanent and functional male-to-female sex reversal in d-rR strain medaka (Oryzias latipes) following egg microinjection of o,p'-DDT.

Complete sex reversal of fish is accomplished routinely in aquaculture practices by exposing fish to exogenous sex steroids during gonadal differentiation. A variety of environmental chemicals are also active at sex steroid receptors and theoretically possess the potential to alter normal sexual differentiation in fish. However, in controlled environmental chemical exposures to date, only partial alterations of fish sexual phenotype have been observed. Here we report complete, permanent, and functional male-to-female sex reversal in the Japanese medaka (Oryzias latipes, d-rR strain) after a onetime embryonic exposure to the xenoestrogen o, p'-DDT. d-rR strain medaka are strict gonochorists that possesses both sex-linked pigmentation, which distinguishes genotypic sex, and sexually dimorphic external secondary sexual characteristics, which distinguish phenotypic sex. We directly microinjected the xenoestrogen o, p'-DDT into the egg yolks of medaka at fertilization to parallel the maternal transfer of lipophilic contaminants to the embryo. At 10 weeks of age, microinjected medaka were examined for mortality and sex reversal. A calculated embryonic dose of 511 +/- 22 ng/egg o, p'-DDT (mean +/- standard error) resulted in 50% mortality. An embryonic exposure of 227 +/- 22 ng/egg o, p'-DDT resulted in 86% (6 of 7) sex reversal of genetic males to a female phenotype (XY females). XY females were distinguished by sex-linked male pigmentation accompanying female secondary sexual characteristics. Histologic examination of the gonads confirmed active ovaries in 100% of the XY females. In 10-day breeding trials in which XY females were paired with normal XY males, 50% of the XY females produced fertilized embryos; this represents a comparable breeding success rate to normal XX females. Fertilized eggs produced from XY females hatched to viable larvae. These results clearly indicate that a weakly estrogenic pesticide, o, p'-DDT, when presented during the critical period of gonadal development, can profoundly alter sexual differentiation.

Animals↗

PubMed1992

Transport of particles of colloidal gold within and from rat lung after local deposition by alveolar microinjection.

Because inhalation and intratracheal instillation deposit particles throughout the respiratory tract, these methods of administration give little information on the movement of particles within the lung and no direct information on the clearance kinetics from locally defined sites within alveolar tissue. Approximately 0.05 microL of 195Au-labeled gold colloid was administered to 32 rats by microinjection into a small volume of subpleural alveoli. Its fate was studied by whole-body counting and serial sacrifice over 15 months. The kinetics of clearance from the subpleural deposition site showed that there was no rapid removal of particles, and the main clearance process was defined by an exponential term with a half-time averaging 583 days. There was a wide variation between individual animals. The distribution of 195Au at sacrifice showed that the gold colloid was nearly all retained within the respiratory tract. The particles were not appreciably redistributed throughout the lung volume, so most of the material not cleared from the lung remained close to the deposition site. At the later times after microinjection, much of the gold colloid was associated with thickened pleura and adjoining septae.

Animals↗

PubMed2004

Expression of G-protein signaling components in adult mammalian neurons by microinjection.

Although methods for expressing foreign proteins in clonal cell lines are well established, mature neurons remain a difficult preparation for the introduction of foreign genes. Microinjection is a reliable method for producing robust targeted expression in neurons that has advantages over conventional transfection/infection methodologies. Here, I describe procedures for expressing signaling proteins in adult rat sympathetic neurons by direct microinjection of cRNA and cDNA into the cytoplasm and nucleus, respectively. The methods are applicable to a wide variety of peripheral and central neuron preparations, as well as clonal cell lines.

Animals↗

PubMed2006

Mammalian cell microinjection assay to study the function of Rho family guanosine triphosphatases.

Microinjection is an excellent technique for studying the acute responses of cells to proteins and can be used to investigate the effects of mutations in proteins on their activity. It has been used widely to study the responses to Rho family guanosine triphosphatases and is particularly useful for cell types that are difficult to transfect. Here, we describe the procedure for microinjecting cells with purified recombinant proteins or with expression vectors encoding proteins, and for analyzing the cells after injection.

3T3 Cells↗

PubMed1976

Effects of microinjection of various prostaglandins into the 3rd ventricle, median eminence and pituitary on plasma LH in rats.

The effects of microinjection of PG'S (PGE1, E2, F2a) into the 3rd ventricle, median eminence (ME) and anterior pituitary on plasma LH in rats were investigated. Blood samples were obtained by jugular puncture before, and 10 and 45 min after the injection of PGS (50 or 100 mug), plasma LH was measured by radioimmunoassay. In the 3rd ventricle microinjection, PGE2 prodiced a significant rise in plasma LH. PGE1 and F2a did not significantly after plasma LH levels. In the median eminence, PGE2 and E1 produced a significant rise in plasma LH. PGF2a did not alter plasma LH levels. In the pituitary, PGE2 and E1 produced a significant rise in plasma LH. PGF2a did not alter plasma LH levels. These observations indicate that PGs act directly on the hypothalamic-pituitary axis, and that particular PG may be involved in the release of particular horomones from the hypothalamus and pituitary.

Animals↗

PubMed1985

The vasopressin response to microinjections of norepinephrine into the brain of the conscious pig.

The possibility that the amygdala influences the release of vasopressin has been raised by the observation that electrical stimulation of this area results in vasopressin release in the conscious animal. Therefore the effect on vasopressin concentrations has been studied by microinjections of norepinephrine into the amygdala of the conscious miniature pig. The studies were performed on castrated male and female pigs with chronically implanted cannulae in the brain and in the external jugular vein. Lysine vasopressin concentrations were determined using a specific sensitive radioimmunoassay. Microinjections of 1 microliter 0.12 M sodium chloride were without effect. Injections of norepinephrine in the dose range 10(-4)-10(-10) M had no effect in the male animal but a significant vasopressin response could be obtained in the females.

Amygdala↗

PubMed2004

Effective production of microinjectable blastocysts for germ-line transmission of embryonic stem cells.

Blastocysts of C57BL/6 mice obtained either 2.5 or 3.5 days post-coitum (dpc) were examined for efficient microinjection after overnight in vitro culture. Incidences of zona-free embryos were much higher at 3.5 dpc after natural mating (1.05/mouse) and superovulation (2.83/mouse) than at 2.5 dpc after natural mating (0.05/mouse) and superovulation (0.01/mouse). By testing germ-line competency of gene-targeted J1 embryonic stem cells, superovulation and/or in vitro culture should be recommended for producing microinjectable blastocysts for production of high germ-line chimeras.

Animals↗

PubMed1997

Behavioral effects of "vehicle" microinjected into the dorsal periaqueductal grey of rats tested in the elevated plus maze.

To investigate the behavioral effects of different vehicles microinjected into the dorsal periaqueductal grey (DPAG) of male Wistar rats, weighing 200-250 g, tested in the elevated plus maze, animals were implanted with cannulas aimed at this structure. One week after surgery the animals received microinjections into the DPAG of 0.9% (w/v) saline, 10% (v/v) dimethyl sulfoxide (DMSO), 2% (v/v) Tween-80, 10% (v/v) propylene glycol, or synthetic cerebrospinal fluid (CSF). Ten min after the injection (0.5 microliters) the animals (N = 8-13/group) were submitted to the elevated plus maze test. DMSO significantly increased the number of entries into both the open and enclosed arms when compared to 0.9% saline (2.7 +/- 0.8 and 8.7 +/- 1.3 vs 0.8 +/- 0.3 and 5.1 +/- 0.9, respectively. Duncan test, P < 0.05), and tended to increase enclosed arm entries as compared to 2% Tween-80 (8.7 +/- 1.3 vs 5.7 +/- 0.9, Duncan test, P < 0.10). In a second experiment no difference in plus maze exploration was found between 0.9% saline-or sham-injected animals (N = 11-13/group). These results indicate that intra-DPAG injection of some commonly used vehicles such as DMSO, saline or Tween-80 affects the exploratory activity of rats exposed to the elevated plus maze in statistically different manners.

Animals↗

PubMed1998

Cardiovascular responses to microinjection of trans-(+/-)-ACPD into the NTS were similar in conscious and chloralose-anesthetized rats.

The changes in mean arterial pressure (MAP) and heart rate (HR) in response to the activation of metabotropic receptors in the nucleus tractus solitarii (NTS) with trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-(+/-)-ACPD) were evaluated in conscious and anesthetized Wistar, male rats weighing 240-260 g (N = 8). The responses obtained with trans-(+/-)-ACPD were compared with the responses to L-glutamate (1 nmol/100 nl), since in a previous study we showed that anesthesia converted a pressor response to L-glutamate microinjected into the NTS of conscious rats to a depressor response in the same rats under urethane or chloralose anesthesia. Microinjection of 3 doses of trans-(+/-)-ACPD (100, 500 and 1000 pmol/100 nl) produced a dose-dependent fall in MAP (range, -20 to -50 mmHg) and HR (range, -30 to -170 bpm) under both conscious and chloralose anesthesia conditions. These data indicate that the cardiovascular responses to the activation of metabotropic receptors by trans-(+/-)-ACPD are not affected by chloralose anesthesia while the cardiovascular responses to the activation of excitatory amino acid (EAA) receptors by L-glutamate are significantly altered.

Analysis of Variance↗

PubMed1999

Anxiolytic effect of methylene blue microinjected into the dorsal periaqueductal gray matter.

The dorsal periaqueductal gray (DPAG) has been implicated in the behavioral and autonomic expression of defensive reactions. Several results suggest that, along with GABA, glutamate and serotonin, nitric oxide (NO) may play a role in defense reactions mediated by this region. To further investigate this possibility we microinjected methylene blue (MB; 10, 30 or 100 nmol/0.5 microl) into the DPAG of rats submitted to the elevated plus-maze test, an animal model of anxiety. MB has been used as an inhibitor of soluble guanylate cyclase (sGC) to demonstrate cGMP-mediated processes, and there is evidence that NO may exert its biological effects by binding to the heme part of guanylate cyclase, causing an increase in cGMP levels. The results showed that MB (30 nmol) significantly increased the percent of time spent in the open arms (saline = 11.57 +/- 1.54, MB = 18.5 +/- 2.45, P<0.05) and tended to do the same with the percentage of open arm entries (saline = 25.8 +/- 1.97, MB = 33. 77 +/- 3.07, P<0.10), but did not change the number of enclosed arm entries. The dose-response curve, however, had an inverted U shape. These results indicate that MB, within a limited dose range, has anxiolytic properties when microinjected into the DPAG.

Animals↗

PubMed1999

Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats.

We studied the effects of microinjection of leptin into the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) on glucose uptake in peripheral tissues in unanesthetized rats. The rate of glucose uptake was assessed in vivo by 2-[3H]deoxyglucose incorporation. Single injection of leptin into VMH increased glucose uptake in brown adipose tissue (BAT), heart, skeletal muscles, and spleen but not in white adipose tissue or skin. On the other hand, microinjection of leptin into LH had little effect on glucose uptake in those tissues. The plasma concentrations of glucose and insulin were unaltered by intrahypothalamic injection of leptin into either VMH or LH. Among skeletal muscles, the increase in glucose uptake induced by intrahypothalamic injection of leptin was greater in the soleus than in the extensor digitorum longus. Likewise, the increased glucose uptake in the gastrocnemius in response to leptin was more prominent in the red part than in the white part of the tissue. When surgical sympathetic denervation of the interscapular BAT was performed, the enhanced glucose uptake by BAT in response to intrahypothalamic leptin was completely suppressed. These findings suggest that intrahypothalamic injection of leptin preferentially increases glucose uptake by some peripheral tissues through activation of the VMH-sympathetic (or its neighboring medial hypothalamus-sympathetic) nervous system, thereby contributing to the maintenance of energy balance.

Adipose Tissue, Brown↗

PubMed1988

Microinjection of DNA into Aedes triseriatus ova and detection of integration.

A technique was developed for microinjection and transformation of Aedes triseriatus mosquitoes. Mosquito ova can be readily microinjected with approximately 0.9 nl of inoculum if desiccated and inoculated before hardening of the chorion. Ova were injected with molecular constructs that conferred antibiotic (G418) resistance to larvae. Insertion of the antibiotic resistance construct into the mosquito genome was demonstrated by Southern blot analysis.

Aedes↗

PubMed1998

Modulation of intrathecal morphine-induced immunosuppression by microinjection of naloxone into periaqueductal gray.

AIM: To study the involvement of opioid receptor of periaqueductal gray (PAG) and hypothalamic-pituitary-adrenal (HPA) axis in the effect of intrathecal morphine on immune function. METHODS: Rat splenic natural killer (NK) cell activity was determined by a europium release assay; the concanavalin A-induced splenic IL-2 production, TNF-beta activity, and serum TNF-alpha level were determined by colorimetric thiazolyl blue tetrazolium bromide (MTT) and gentian violet assay, and serum corticotrophin (ACTH) level by radio-immunological method after intrathecal injection of morphine and PAG microinjection of naloxone. RESULTS: Intrathecal morphine inhibited splenic NK cell activity, IL-2 production, TNF-beta activity, and increased in serum ACTH level. Microinjection of naloxone 1 microgram into PAG partially antagonized the inhibition of NK cell activity and the elevation of serum ACTH level by morphine. CONCLUSION: The opioid receptor of PAG involved in the suppression of NK cell activity by intrathecal morphine, which was accompanied by an activation of HPA axis.

Adrenocorticotropic Hormone↗

PubMed1978

[Role of the Raphe Magnus nucleus in morphine analgesia : studies with intracerebral microinjections in the rat].

Microinjections of low concentration of morphine (5 micrograms) into the nucleus Raphé Magnus of the Rat produce a strong analgesia that can be reversed by systemic naloxone, an opiate antagonist. The administration of naloxone (5 micrograms) into the Raphé Magnus considerably reduces the effects of intravenous morphine. The effects of microinjections of morphine are strongly reduced by Cinanserin, suggesting a role for serotoninergic mechanisms in morphine analgesia.

Analgesia↗

PubMed2000

Effect of microinjections of 5-hydroxytryptamine and adrenaline in central grey on pain responsiveness during acute food deprivation in conscious rats.

To study the effects of microinjections of 5 hydroxytryptamine and adrenaline in central grey on pain responsiveness during acute food deprivation, experiments were conducted in nine male rats. Microinjections of 5 HT (10 micrograms/microliter) and adrenaline (10 micrograms/microliter) were given in central grey before and at the end of 6, 12, 18 and 24 hr food deprivation and the effects on pain threshold, cardiorespiratory parameters and body temperature were noted. Observations showed that 5 HT increased the pain threshold (antinociception) significantly (P < 0.05) with no change in cardiorespiratory response and body temperature, adrenaline did not alter pain threshold with no change in cardiorespiratory response and body temperature. The observations suggest the possible existence of two types of monoaminergic receptors or pathways in the central grey.

Animals↗

PubMed2000

Selective and protracted apoptosis in human primary neurons microinjected with active caspase-3, -6, -7, and -8.

We have shown previously that caspase-6 is activated in serum deprivation-mediated human neuronal cell death and correlates with increased production of Alzheimer's disease (AD) amyloid beta peptide (Abeta). Here, we show by direct microinjection of recombinant active enzymes that caspase-6 (>0.5 pg/cell) induces a protracted course of apoptosis in neurons in a caspase-specific, dose- and time-dependent manner in the presence of serum. Only transient activation of caspase-6 is required to initiate apoptosis. Caspase-6 induces apoptosis directly without the activation of other caspase effectors. Doses of caspase-6 of <0.25 pg/cell induce only 20% cell death within 16 d but render neurons vulnerable to oxidative stress, indicating that caspase activation affects neurons despite the absence of cell death. Caspase-3 induces neuronal apoptosis in 20% of the cells, whereas caspase-7 or -8 do not induce apoptosis. In contrast, astrocytes undergo apoptosis within 24 hr when microinjected with caspase-3 but not caspase-6, -7, or -8. These results show cell type-specific vulnerability to caspases in the CNS. The results suggest that activation of caspases in human neurons does not lead to an immediate and rapid process of cell death but provokes a protracted form of apoptosis. Activation of caspases in human neurons may participate in the long-term overproduction of Abeta and other potential toxic fragments resulting from caspase-mediated proteolysis. These results are consistent with the protracted and age-dependent nature of AD.

Alzheimer Disease↗

PubMed2000

Germ line transformation of mammals by pronuclear microinjection.

The most popular approach for generating transgenic mammals is the direct injection of transgenes into one pronucleus of a fertilized oocyte. In the past 15 years microinjection has been successfully applied in laboratory as well as in farm animals. The frequency of transgenic founders, although highly different between the species, is efficient enough to render this technique applicable to a wide range of mammals. The expression levels and patterns of a transgene are initially influenced by the construction of the transgene. However, the overall phenotype of a transgenic organism is influenced by several genetic and environmental factors. Due to the features of this technique not all of the genetic factors can be experimentally controlled by the scientist. In this article we will emphasize some peculiarities which have to be taken into account for the successful performance of transgenesis by pronuclear microinjection.

Animals↗

PubMedDate not supplied

[In vitro development of murine embryos using different types of microinjections].

We studied the effects of different types of microinjections, such as the mechanical damage to cytoplasmic and nuclear membranes of the zygote and the injection of various gene-engineering constructs or buffer solutions into the cytoplasm or the pronucleus, on the preimplantation of murine embryos (CBA x x C57BL)F1. The survival rate of the embryos was estimated by their capacity to develop in vitro to the blastocyst or hatched blastocyst stages. Puncture of the cytoplasm using a microneedle and injection of buff or foreign DNA did not affect the zygotes capacity for further in vitro development. But, the puncture of the pronucleus and microinjection of gene-engineering constructs or buffer into it reliably decreased the survival rate of embryos, as compared to the control. The differences were found in the capacity of murine zygotes for in vitro development after injection with gene-engineering constructs.

Animals↗