A Calibrated Method of Massage Therapy Decreases Systolic Blood Pressure Concomitant With Changes in Heart Rate Variability in Male Rats.

ObjectiveThe purpose of this study was to develop a method for applying calibrated manual massage pressures by using commonly available, inexpensive sphygmomanometer parts and validate the use of this approach as a quantitative method of applying massage therapy to rodents.MethodsMassage pressures were monitored by using a modified neonatal blood pressure (BP) cuff attached to an aneroid gauge. Lightly anesthetized rats were stroked on the ventral abdomen for 5 minutes at pressures of 20 mm Hg and 40 mm Hg. Blood pressure was monitored noninvasively for 20 minutes following massage therapy at 5-minute intervals. Interexaminer reliability was assessed by applying 20 mm Hg and 40 mm Hg pressures to a digital scale in the presence or absence of the pressure gauge.ResultsWith the use of this method, we observed good interexaminer reliability, with intraclass coefficients of 0.989 versus 0.624 in blinded controls. In Long-Evans rats, systolic BP dropped by an average of 9.86% ± 0.27% following application of 40 mm Hg massage pressure. Similar effects were seen following 20 mm Hg pressure (6.52% ± 1.7%), although latency to effect was greater than at 40 mm Hg. Sprague-Dawley rats behaved similarly to Long-Evans rats. Low-frequency/high-frequency ratio, a widely-used index of autonomic tone in cardiovascular regulation, showed a significant increase within 5 minutes after 40 mm Hg massage pressure was applied.ConclusionsThe calibrated massage method was shown to be a reproducible method for applying massage pressures in rodents and lowering BP

Macrophage gene expression associated with remodeling of the prepartum rat cervix:Microarray and pathway analyses

As the critical gatekeeper for birth, prepartum remodeling of the cervix is associated with increased resident macrophages (Mφ), proinflammatory processes, and extracellular matrix degradation. This study tested the hypothesis that expression of genes unique to Mφs characterizes the prepartum from unremodeled nonpregnant cervix. Perfused cervix from prepartum day 21 postbreeding (D21) or nonpregnant (NP) rats, with or without Mφs, had RNA extracted and whole genome microarray analysis performed. By subtractive analyses, expression of 194 and 120 genes related to Mφs in the cervix from D21 rats were increased and decreased, respectively. In both D21 and NP groups, 158 and 57 Mφ genes were also more or less up- or down-regulated, respectively. Mφ gene expression patterns were most strongly correlated within groups and in 5 major clustering patterns. In the cervix from D21 rats, functional categories and canonical pathways of increased expression by Mφ gene related to extracellular matrix, cell proliferation, differentiation, as well as cell signaling. Pathways were characteristic of inflammation and wound healing, e.g., CD163, CD206, and CCR2. Signatures of only inflammation pathways, e.g., CSF1R, EMR1, and MMP12 were common to both D21 and NP groups. Thus, a novel and complex balance of Mφ genes and clusters differentiated the degraded extracellular matrix and cellular genomic activities in the cervix before birth from the unremodeled state. Predicted Mφ activities, pathways, and networks raise the possibility that expression patterns of specific genes characterize and promote prepartum remodeling of the cervix for parturition at term and with preterm labor

Loss of progesterone receptor-mediated actions induce preterm cellular and structural remodeling of the cervix and premature birth.

A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term

A Calibrated Method of Massage Therapy Decreases Systolic Blood Pressure Concomitant With Changes in Heart Rate Variability in Male Rats.

ObjectiveThe purpose of this study was to develop a method for applying calibrated manual massage pressures by using commonly available, inexpensive sphygmomanometer parts and validate the use of this approach as a quantitative method of applying massage therapy to rodents.MethodsMassage pressures were monitored by using a modified neonatal blood pressure (BP) cuff attached to an aneroid gauge. Lightly anesthetized rats were stroked on the ventral abdomen for 5 minutes at pressures of 20 mm Hg and 40 mm Hg. Blood pressure was monitored noninvasively for 20 minutes following massage therapy at 5-minute intervals. Interexaminer reliability was assessed by applying 20 mm Hg and 40 mm Hg pressures to a digital scale in the presence or absence of the pressure gauge.ResultsWith the use of this method, we observed good interexaminer reliability, with intraclass coefficients of 0.989 versus 0.624 in blinded controls. In Long-Evans rats, systolic BP dropped by an average of 9.86% ± 0.27% following application of 40 mm Hg massage pressure. Similar effects were seen following 20 mm Hg pressure (6.52% ± 1.7%), although latency to effect was greater than at 40 mm Hg. Sprague-Dawley rats behaved similarly to Long-Evans rats. Low-frequency/high-frequency ratio, a widely-used index of autonomic tone in cardiovascular regulation, showed a significant increase within 5 minutes after 40 mm Hg massage pressure was applied.ConclusionsThe calibrated massage method was shown to be a reproducible method for applying massage pressures in rodents and lowering BP

Biolayout <i>Express</i>^3D analyses of Mφ gene expression and clustering analyses.

<p>Data were transposed in an analysis to identify correlations among groups based on individual gene expression levels (Pearson’s correlation of R≥0.9 and intensity >1 of dataset with fold >2 or <-2 and p<0.01). Markov clustering (MCL, inflation = 1.7) was used to identify clusters related to Mφ genes (microarray results from whole divided by Mφ -depleted cervix). These clusters were analyzed again to create the network schema with 1418 nodes and 69761 edges. Color of nodes represents membership in clusters. The 5 largest MCL clusters had distinct patterns that represent 80% of differentially regulated Mφ genes in cervix (histogram insets, average intensity ± SE, n = 3 rats/group).</p

Increased expression of Mφ genes exclusively in prepartum D21 rat cervix (p<0.01; n = 3/group).

<p>Function designations in rat derived from DAVID (<a href="http://david.abcc.ncifcrf.gov" target="_blank">http://david.abcc.ncifcrf.gov</a>), Biolayout <i>Express</i>^3D cluster analyses, and IPA (ECM = extracellular matrix, INFL = inflammation, SIG = Signaling).</p><p>Increased expression of Mφ genes exclusively in prepartum D21 rat cervix (p<0.01; n = 3/group).</p

Subtractive approach in which macrophages were depleted from dispersed cervix from prepartum and nonpregnant mice to identify differential gene expression by macrophages.

<p><b>A</b>. Cervix from perfused adult female Sprague-Dawley rats, on prepartum day 21 post-breeding (D21) or non-pregnant (NP) was carefully trimmed and dispersed (n = 6 each). Mφs were removed by magnetic bead separation from 3 rats in each group as described in detail in Methods. <b>B</b>. Venn diagram of differentially expressed Mφ genes in the cervix from prepartum (D21) or nonpregnant (NP) rats. Genes were exclusively increased or decreased in cervices from D21 or NP rats, except in the overlap region which indicates genes that were increased or decreased in cervices from both D21 and NP rats. No gene whose expression increased or decreased in the cervix from D21 or NP groups then decreased or increased in the cervix from NP or D21 groups, respectively. Number is the average of differentially expressed Mφ genes in whole divided by Mφ -depleted cervix/group (p<0.01, fold >2 or <-2; n = 3 rats). <b>C</b>. Pearson correlation of clustering patterns of gene expression in cervix of individual rats in NP and D21 groups with or without macrophages (Mφ-). Lines connecting microarray analysis for each individual indicates R>0.9 (group specific colors), R = 0.851–0.89 (grey), or R = 0.8–0.85 (thin grey).</p

Proposed network of pathways that reflect expression of Mφ genes in the prepartum cervix based upon Ingenuity Pathway Analysis (IPA).

<p>Network includes Mφ genes exclusively regulated in the cervix from D21 rats and those regulated in both in D21 prepartum and NP groups, as well as known key molecules. Red signifies up-regulated genes and green indicates down-regulated expression. IPA drawn lines predicted activation (orange) or inhibition (blue) of gene expression between key molecules. Other lines indicate no known relationship (black) or uncertainty about relationship (yellow). Color intensity indicates relative expression based upon p-value or prediction intensity. Groups of genes are clustered into Inflammation, Extracellular matrix, or Signaling based on annotations provided by IPA.</p

Decreased expression of genes in whole cervix from prepartum D21 postbreeding versus nonpregnant rats (p<0.01; n = 3/group).

<p>Decreased expression of genes in whole cervix from prepartum D21 postbreeding versus nonpregnant rats (p<0.01; n = 3/group).</p

Peripartum progesterone in circulation in preterm and term birth.

<p>Serum progesterone concentrations (mean±SE, n = 4–6/group) on various days postbreeding in controls, and groups of pregnant rats treated with Onapristone (Ona) or RU486 or bilaterally ovariectomized on the morning of day 17 of pregnancy (Ovx). Preterm birth (PTB) occurred on day 18 postbreeding for Ona- and RU486-treated rats, by 27 h or 26 h post-treatment, respectively (n = 5 and 6 respectively). PTB occurred by the morning of day 19 postbreeding in the Ovx group (36–44 h post-surgery, n = 5). Postpartum (PP) is the morning of the day of birth at term on day 22 postbreeding in Cons (n = 5). Typical estrous cycle serum progesterone concentrations range from 5–50 ng/ml in nonpregnant rats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#pone.0081340-Smith1" target="_blank">[12]</a>. For statistical comparisons, groups were assigned an individual letter, consecutively from ^a for Con D17.5 to ^l for Con PP groups. The letter above a bar indicates p<0.05 vs the respective treatment group (ANOVA with Tukey's test for individual comparisons). By example, ^a over bars for Ona D17.5, RU486 D17.5 and Con PP groups indicates a significant difference for each versus the Con D17.5 group. The absence of a letter, ^k for example, indicates no statistical difference for any group compared to Con D21.5. See Methods for experimental details.</p