Donald D. Denson

Validating three-dimensional imaging of the breast.

The potential to extrapolate accurate data from 3-dimensional (3D) images of the breast is enormous and will greatly improve our ability to qualitatively determine differences in shape, size, and contour. The validity of these calculated measurements is important and needs to be determined before any meaningful data can be evaluated. Part I:Premastectomy 3D images (3dMD patient) were obtained on 19 breasts (14 patients). The volume of the mastectomy specimen was determined intraoperatively using water displacement. Two independent raters then calculated breast volumes using the 3D images and software, and these were compared with the intraoperative volume. Inter- and intrarater reliability was determined. Part II: Surface measurements (nipple to notch) were then evaluated on 20 breasts (10 patients) by comparing the 3D image determined distance to the known measurements. Part I:The average breast volume was 500 mL, compared with 489 mL for rater 1 and 490 mL for rater 2. The relative difference between the measured volume and the calculated volume for rater 1 and rater 2 was about −2%, with a standard deviation of ± 13% to 16%. The coefficient of reproducibility for each reader was excellent, at 0.80 for rater 1 and 0.92 for rater 2. The level of agreement between the readers was also high at 0.975. Part II: The average nipple to notch measurement for each patient was 27.1 cm, compared the calculated average of 25.1 cm for rater 1 and 26.1 cm for rater 2. The mean relative difference between the measured and calculated distances for raters 1 and 2 was about −6%, with a standard deviation of ± 6% to 7%. The level of agreement between readers was high, at 0.975. The ability to objectively determine breast volume and surface measurements using 3D imaging technology is now available with consistent and reproducible accuracy. Measurements are typically underestimated, with more variability when calculating volumes. Although inherent subjectivity will always exist when evaluating breast measurements, 3D technology provides invaluable information, particularly in the longitudinal evaluation of results.

An objective evaluation of breast symmetry and shape differences using 3-dimensional images.

Background:The concept of natural breast asymmetry is well known; however, actual documentation in the literature is limited. New technology is currently available which provides 3-dimensional surface images of the breast and the ability to qualitatively determine differences in breast size, shape, and contour. The purpose of this report is to objectively determine the extent to which this natural breast asymmetry exists. Methods:Eighty-seven women without a history of breast cancer or previous breast surgery were included. Images were obtained using 3dMD technology. Data points queried included age, parity, body mass index (BMI), ethnicity, and bra size. Left/right images were superimposed and the distance between the 2 surfaces, and contour was calculated. The degree of asymmetry was determined and comparisons were made. Similar differences in nipple-to-notch measurements were calculated and compared. Subjective evaluations were included for clinical relevance. Results:The average age was 49.6 years (range: 19–77), with an average BMI of 25 (range: 18.5–36.7). The average nipple to notch on the left was 24.3 cm and 23.8 cm on the right. The nipple-to-notch asymmetry was on average 3.2%, with the left breast measurement being greater the majority of the time (62%). The mean distance between each breast demonstrated consistent breast asymmetry, with an average measurement of +0.5 mm (left breast being larger than the right). The degree of breast asymmetry was documented by a root mean square value (RMS) of 5.93 mm. This was not related to age, parity, or ethnicity; however, it was significantly higher in those patients with larger BMI, cup size, and chest-wall circumference. Only 10% of women were found to have severe breast asymmetry on subjective evaluation, which correlated objectively with the RMS in that group being significantly higher at 9.8 mm (P < 0.05). There were no predictable patterns of asymmetry; however, the most common pattern was larger laterally and smaller medially, found in 32% of the women. Conclusion:Natural breast asymmetry does exist, demonstrated objectively using 3-dimensional surfaces images. The left breast is on average larger than the right, with differences in size and shape being consistent but fairly unpredictable. It is important that we know baseline differences in breast symmetry prior to objectively analyzing results following esthetic and reconstructive breast surgery.

Results of immediate breast reconstruction after skin-sparing mastectomy.

Skin sparing mastectomy (SSM) removes the breast, nipple–areolar complex, previous biopsy incisions, and skin overlying superficial tumors. Preservation of the native skin envelope facilitates immediate breast reconstruction. The procedure has been adopted for the treatment of breast cancer. All cases of SSM and immediate breast reconstruction performed by the senior author (G.W.C.) from January 1, 1993, through December 12, 1997, were reviewed. Patient demographics, cancer staging, treatment, types of surgery performed, and postoperative outcomes were examined. Aesthetic outcomes were measured using four 3-point subscales. A total of 100 patients underwent 118 SSMs during the study period. The American Joint Committee on Cancer staging was as follows: stage 0, 27 patients; stage I, 25 patients; stage II, 39 patients; stage III, 7 patients; stage IV, 3 patients; recurrent, 2 patients; and cystosarcoma phylloides, 1 patient. The mean follow-up was 42.7 months. Local recurrence occurred in 2 patients (2.7%). Reconstructive methods included the transverse rectus abdominis musculocutaneous flap (N = 82; pedicled, 73; free, 9), the latissimus flap (N = 18), and tissue expansion (N = 20). Two patients underwent contralateral delayed reconstruction. The aesthetic results achievable with the three methods were similar. The failure rate was higher for expander reconstruction (10%) than those observed for transverse rectus abdominis musculocutaneous (4.9%) and latissimus (5.6%) flaps. SSM can be used in the treatment of invasive breast cancer without compromising local control. The aesthetic results of the three methods were similar, but tissue expander reconstruction had a higher failure rate.

Steady-State Serum Concentrations of Progesterone Following Continuous Intravenous Infusion in Patients With Acute Moderate to Severe Traumatic Brain Injury

Progesterone (PG) has been shown to provide substantial neuroprotection after traumatic brain injury (TBI) in multiple animal models. As a first step in assessing applicability to humans, the authors examined the effects of acute TBI and extracranial trauma on the pharmacokinetics of PG given by intravenous infusion. Multiple blood samples were obtained from 11 female and 21 male trauma patients receiving PG and 1 female and 3 male patients receiving placebo infusions for 72 hours. Values for CSS, CL, t1/2, and Vd were obtained using AUC(0–72) and postinfusion blood samples. CSS values were 337 ± 135 ng/mL, which were significantly lower than the target concentration of 450 ± 100 ng/mL. The lower CSS is attributed to the CL, which was higher than anticipated. In addition, t1/2 was longer and Vd was higher than anticipated. These results demonstrate that stable PG concentrations can be rapidly achieved following TBI.

WNK4 kinase inhibits Maxi K channel activity by a kinase-dependent mechanism

WNK [with no lysine (k)] kinase is a serine/threonine kinase subfamily. Mutations in two of the WNK kinases result in pseudohypoaldosteronism type II (PHA II) characterized by hypertension, hyperkalemia, and metabolic acidosis. Recent studies showed that both WNK1 and WNK4 inhibit ROMK activity. However, little is known about the effect of WNK kinases on Maxi K, a large-conductance Ca(2+) and voltage-activated potassium (K) channel. Here, we report that WNK4 wild-type (WT) significantly inhibits Maxi K channel activity in HEK αBK stable cell lines compared with the control group. However, a WNK4 dead-kinase mutant, D321A, has no inhibitory effect on Maxi K activity. We further found that WNK4 inhibits total and cell surface protein expression of Maxi K equally compared with control groups. A dominant-negative dynamin mutant, K44A, did not alter the WNK4-mediated inhibitory effect on Maxi K surface expression. Treatment with bafilomycin A1 (a proton pump inhibitor) and leupeptin (a lysosomal inhibitor) reversed WNK4 WT-mediated inhibition of Maxi K total protein expression. These findings suggest that WNK4 WT inhibits Maxi K activity by reducing Maxi K protein at the membrane, but that the inhibition is not due to an increase in clathrin-mediated endocytosis of Maxi K, but likely due to enhancing its lysosomal degradation. Also, WNK4s inhibitory effect on Maxi K activity is dependent on its kinase activity.

The effect of racemic ketamine on the large conductance Ca+2-activated potassium (BK) channels in GH3 cells

Recently, inhalation anesthetics have been reported to block BK channels in adrenal chromaffin cells. To determine if BK block was characteristic only of inhalation anesthetics or was also a property of other general anesthetics we examined the effects of ketamine, an intravenous general anesthetic which is structurally different than inhalation anesthetics. Cell-attached and excised patch single channel and standard whole cell recording techniques were used to examine the effect of racemic ketamine on the BK channel activity in GH3 cells. When solutions containing 150 mM KCl are used in both the pipette and bath, the BK channels are characterized as a voltage-dependent channel with a unit conductance of 150-300 pS. Racemic ketamine (at clinically relevant concentrations; 2-500 microM) selectively blocked BK channels in a dose-dependent, reversible manner as evidenced by decreases in NPo (number of channels x open probability). This decrease was due to both a decrease in mean open time and an increase in the mean closed time but without a decrease in single-channel current amplitude. Ketamine shifts the Po vs voltage curve to higher potentials without a change in the slope of the voltage dependence. Ketamine also shifts the Po vs [Ca+2] relationship to higher Ca+2 concentrations. The IC50 for the single-channel block by ketamine is 20.3 +/- 15.9 microM. In an effort to confirm that the effect of ketamine was predominantly due to a block of the BK channels, standard whole cell techniques were utilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic Phospholipase A2 Is Required for Optimal ATP Activation of BK Channels in GH3 Cells

To test the hypothesis that ATP activation of BK channels in GH3 cells involves cytosolic phospholipase A2 (cPLA2) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA2 by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA2, phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5′-adenylyl-β,γ-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP. Experiments with an activator and inhibitor of protein kinase C (PKC) support the hypothesis that PKC can be involved in the activation of BK channels by ATP; and in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity. Our data point to cPLA2-α (but not cPLA2-γ) as one target protein for phosphorylation that is intimately associated with the BK channel protein.

Ethanol stimulates epithelial sodium channels by elevating reactive oxygen species

Alcohol affects total body sodium balance, but the molecular mechanism of its effect remains unclear. We used single-channel methods to examine how ethanol affects epithelial sodium channels (ENaC) in A6 distal nephron cells. The data showed that ethanol significantly increased both ENaC open probability (P(o)) and the number of active ENaC in patches (N). 1-Propanol and 1-butanol also increased ENaC activity, but iso-alcohols did not. The effects of ethanol were mimicked by acetaldehyde, the first metabolic product of ethanol, but not by acetone, the metabolic product of 2-propanol. Besides increasing open probability and apparent density of active channels, confocal microscopy and surface biotinylation showed that ethanol significantly increased α-ENaC protein in the apical membrane. The effects of ethanol on ENaC P(o) and N were abolished by a superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) and blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Consistent with an effect of ethanol-induced reactive oxygen species (ROS) on ENaC, primary alcohols and acetaldehyde elevated intracellular ROS, but secondary alcohols did not. Taken together with our previous finding that ROS stimulate ENaC, the current results suggest that ethanol stimulates ENaC by elevating intracellular ROS probably via its metabolic product acetaldehyde.

Protein-protein interaction between cPLA2 and splice variants of α-subunit of BK channels

Altering the splice variant composition of large-conductance Ca(2+)-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca(2+) and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK alpha-subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A(2) (cPLA(2)). Here we used the Mammalian Matchmaker two-hybrid assay and two BK alpha-subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA(2) associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA(2) might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA(2) is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA(2) to the COOH terminus of the rSlo(0) alpha-subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.

Subcutaneous bupivacaine for treatment of spasticity: a case report.

In a previous report, we described heretofore undiscovered possibilities that neuropathic pain and spasticity may share some common pathophysiological mechanisms. Currently, systemically delivered local anesthetics are being used for the evaluation and treatment of neuropathic pain. We present a case describing the treatment of spasticity of spinal origin with continuous subcutaneous infusion of 0.75% bupivacaine in a patient who did not respond to traditional treatments and has become tolerant to intrathecal baclofen.