Cervical motion assessment: a new, simple and accurate method.

Accurate assessment of head motion can be a useful tool in clinical studies. Since the head moves on a combination of axes in the cervical spine, evaluation of neck motion is difficult. Assessment of cervical mobility is further complicated because of inadequate reference points on the head from which to measure. Hence, numerous methods for approximating cervical range have been devised. These methods include visual estimation, radiographic analysis, schematography, photography, and a variety of goniometer devices. Disadvantages of these techniques are lack of accuracy and objectivity, radiation exposure, expense, time consumption, and equipment availability. To measure cervical mobility, a standard gravity goniometer with spirit level and head adapter was used, which allowed stabilization. The gravity goniometer can be obtained in a variety of sizes at most hardware stores. The head adapter consists of a wood block into which an arc is carved and elastic straps suspended for securing on the head. The reliability of this instrument was tested and compared to the universal goniometer, and correlation coefficients were determined. When two experienced examiners used the universal goniometer to assess cervical motion, significant intraclass correlation coefficients (ICC) were found with three of the six criteria measures (p less than 0.05). When one experienced and one novice examiner used the gravity goniometer with head adapter, highly significant ICC values were found for all six criteria measures (p less than 0.01). A single experienced examiner comparing both instruments on the same subjects produced significant ICC values in four of the six criterion measures (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects on mouse embryos of in utero exposure to saccharin: teratogenic and chromosome effects.

For teratogenesis studies, pregnant ICR albino mice were administered saccharin by three routes and at three different doses by each route as follows: Intraperitoneal injection of 500, 1,000, or 2,000 mg/kg saccharin on day 10 of gestation; intragastric tube delivery of 5, 10, or 25 mg/kg/day of saccharin on days 5-15 of gestation; and as drinking water containing a 5, 10, or 20% solution of saccharin from day 0 through day 17. Appropriate controls were used for each set. No increase in either resorptions or malformations was found in mouse embryos whose dams had received saccharin by any of the three routes. For chromosome studies, saccharin was administered IP to pregnant ICR albino mice on day 10 of gestation as either a 1,000 mg/kg or 2,000 mg/kg dose. To demonstrate sister chromatid exchanges (SCE), bromodeoxyuridine was administered as 16 consecutive half-hourly doses of 25 mg/kg during the 8 h prior to saccharin injection. In addition, one group received two doses of BrdU and 2,000 mg/kg saccharin 8 h apart to demonstrate SCE frequency following exposure to saccharin for two cell cycles. The mice were given colchicine (4 mg/kg) 6 h after the final injection and killed 2 h later. Embryonic cell suspensions and metaphase spreads were prepared by routine methods. Metaphase spreads were examined for breaks or gaps (after Giemsa staining), for G-banding (using the ASG technique), for C-banding (using Giemsa staining after exposure to 0.07 N NaOH and incubation in 2 X SSC at 60 degrees C), and for SCE by the Hoechst-Giemsa method. Fifty metaphase spreads were counted for each experimental condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of cyclic guanosine monophosphate (cGMP) on mouse fetal chromosomes.

In previous immunohistochemistry studies, cyclic guanosine monophosphate (cGMP) has been found in polytene chromosomes of D. melanogaster, cGMP has not been found in mammalian metaphase chromosomes, but this could be due to loss of cGMP during staining. Thus the effect of different fixation techniques on the immunohistochemically detectable cGMP associated with metaphase chromosomes from mouse fetal tissue was examined. In chromosomes from cells fixed in 2% formalin, or unfixed cells dropped on slides preheated to 60 degrees C, there was diffuse cGMP staining. When cells were fixed in methanol:glacial acetic acid, 3:1, no chromosomal cGMP immunofluorescence was observed, whereas chromosomes from cells fixed in methanol:glacial acetic acid, 6:1, had different patterns of cGMP immunofluorescence depending on the temperature of the slides onto which the fixed cells were dropped. On slides prechilled to 4 degrees C, cGMP immunofluorescence outlined the chromosomes; on room temperature slides, faint chromosomal cGMP staining was observed, and on slides preheated to 68 degrees C or room temperature slides blown dry with hot air, the chromosomes had more intense diffuse cGMP immunofluorescence or distinct symmetrical bands of cGMP immunofluorescence. We have demonstrated the presence of cGMP in mammalian metaphase chromosomes. The different patterns of cGMP immunofluorescence observed may reflect variable preservation of chromosomal proteins that have binding sites for cGMP.

Immunohistochemical studies of cyclic guanosine monophosphate and nuclear function.

In previous immunohistochemical studies, it has been found that all nuclei contain cyclic (c)GMP, which occurs in discrete aggregates and in the nucleolus. We have studied the nature of the cGMP aggregates in isolated mouse fetal nuclei using a specific immunofluorescent technique. These aggregates correspond to the areas of condensation of DNA, demonstrable by either Felugen's or acridine orange stain. Treatment with DNAase eliminated DNA and cGMP staining. Staining for RNA, with a human anti-RNA antibody, demonstrated RNA to be distributed diffusely throughout the nucleus and not preferentially in the areas of discrete cGMP aggregates. The diffuse stain for nuclear RNA was eliminated by pretreatment with RNAase but not DNAase, but aggregates of cGMP were not affected by pretreatment with RNAase. Sites of active RNA synthesis were determined by autoradiography using [3H]uridine, and did not correspond to the aggregates of cGMP. The relationship of cGMP to nucleolar function was examined in the endothelial cells of the isthmus and ampulla of the rat fallopian tube. Previous studies have shown that in proestrous, a period of increased RNA synthesis, nucleoli detectable by staining for RNA appear in the endothelial cells lining the fallopian tube. After immunofluorescent staining, we found prominent accumulation of cGMP in the nucleoli. During other phases of the cycle, there is an absence of nucleoli detectable by staining for RNA, and an absence of nucleolar cGMP. After we treated hypophysectomized or oophorectomized rats with estrogen, which is known to increase nucleolar RMA synthesis in the fallopian tube and endometrium, nucleoli in the endothelial cells of the rat fallopian tube and uterus stained strongly for cGMP. In conclusion, our studies suggest that the discrete aggregates of nuclear cGMP are associated with a fraction of DNA uninvolved in RNA synthesis. In contrast, cGMP appears in the nucleolus during a period of increased RNA synthesis, suggesting a role for cGMP in regulating nucleolar synthesis and processing of RNA.

Sister chromatid exchange frequency in mouse embryo chromosomes after in utero ethanol exposure.

The frequency of sister chromatid exchange (SCE) increased in mouse embryo chromosomes following a single maternal i.p. injection of ethanol on the tenth day of gestation, indicating a possible morphogenetic action of ethanol in the developing mouse embryo.

Detection of lateral asymmetry in the C-bands of mouse-embryo chromosomes exposed in utero to mitomycin C.

Chromosomes from 10-day mouse embryos were C-banded and examined for aymmetric bands between sister chromatids. No occurrences of lateral asymmetry (LA) were observed in this series. However, when 10-day embryos were exposed in utero to a single maternal injection of 10 mg/kg mitomycin C 4--5 h prior to metaphase arrest with colcemid, a significant level of asymmetric C-banding was demonstrated in their chromosomes. As many as 4 LAs per cell were seen in 2 of the 50 metaphase spreads examined.

Studies on the specificity of immunohistochemical techniques for cyclic AMP and cyclic GMP.

UNLABELLED: Immunohistochemical studies employing antibodies against cyclic nucleotides indicate that cyclic AMP and cyclic GMP are localized to distinct subcellular sites. These antibodies, however, cross-react weakly with noncyclic nucleotides (eg. ATP, GTP), and therefore we investigated the speficity of the immunohistochemical technique. Slides of fetal nuclei exposed to gaseous nitrous acid demonstrated reduced immunofluorescence. The slides were then incubated with cyclic and noncyclic nucleotides, and restoration of distinct cyclic AMP and cyclic GMP staining pattern was achieved only with appropriate cyclic nucleotides. Antibodies that were used have a greater affinity for acetylated derivatives of cyclic nucleotides. By using a gas phase technique, tissue slices were acetylated and immunohistochemical staining intensity was compared with the effect of acetylation on antibody affinity for various nucleotides. Acetylation greatly increased affinity of cyclic AMP antibody for cyclic AMP but not other nucleotides, and greatly intensified cyclic AMP staining. Acetylation moderately increased affinity of cyclic GMP antibody for cyclic GMP, and moderately intensified cyclic GMP staining. CONCLUSION: Both nitrous acid and acetylation studies support the specificity of the immunohistochemical method for cyclic nucleotides.

Chromosome lateral asymmetry: a sensitive assay for screening teratogenic agents.

Pregnant albino mice were administered (ip) embryotoxic doses of three individual teratogens: bromodeoxyuridine, hydroxyurea and mitomycin C on day 10 of gestation. Embryos were removed 4 hr later, a cell suspension was prepared and cultured in the presence of colcemid. Metaphase chromosome spreads were subjected to standard G-banding procedures, and the occurrence and frequency of lateral asymmetry (unequal banding of sister chromatids) was monitored. Embryotoxic levels of all three teratogens produced an increase in the number of asymmetries/karotype. For one of these (mitomycin C), the observed increase was dose-dependent.

Demonstration of lateral asymmetry in G-banded mouse embryo chromosomes.

Cells obtained from 10-day mouse embryos were cultured for 3 h in medium containing colcemid. Chromosome preparations were subjected to G-banding in either calcium-magnesium free Hank's solution or the ASG method. From one to several occurrences of lateral asymmetry (unequal banding of sister chromatids) were observed in the majority of karytotypes analyzed.