An automated cell-counting algorithm for fluorescently-stained cells in migration assays

A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting

Pre-diabetes augments neuropeptide Y1- and α1-receptor control of basal hindlimb vascular tone in young ZDF rats.

Peripheral vascular disease in pre-diabetes may involve altered sympathetically-mediated vascular control. Thus, we investigated if pre-diabetes modifies baseline sympathetic Y(1)-receptor (Y(1)R) and α(1)-receptor (α(1)R) control of hindlimb blood flow (Q(fem)) and vascular conductance (VC).Q(fem) and VC were measured in pre-diabetic ZDF rats (PD) and lean controls (CTRL) under infusion of BIBP3226 (Y(1)R antagonist), prazosin (α(1)R antagonist) and BIBP3226+prazosin. Neuropeptide Y (NPY) concentration and Y(1)R and α(1)R expression were determined from hindlimb skeletal muscle samples.Baseline Q(fem) and VC were similar between groups. Independent infusions of BIBP3226 and prazosin led to increases in Q(fem) and VC in CTRL and PD, where responses were greater in PD (p<0.05). The percent change in VC following both drugs was also greater in PD compared to CTRL (p<0.05). As well, Q(fem) and VC responses to combined blockade (BIBP3226+prazosin) were greater in PD compared to CTRL (p<0.05). Interestingly, an absence of synergistic effects was observed within groups, as the sum of the VC responses to independent drug infusions was similar to responses following combined blockade. Finally, white and red vastus skeletal muscle NPY concentration, Y(1)R expression and α(1)R expression were greater in PD compared to CTRL.For the first time, we report heightened baseline Y(1)R and α(1)R sympathetic control of Q(fem) and VC in pre-diabetic ZDF rats. In support, our data suggest that augmented sympathetic ligand and receptor expression in pre-diabetes may contribute to vascular dysregulation

Blood pressure and heart rate responses associated with each condition.

<p>Values are mean ± SE. CTRL, control, n = 8; PD, pre-diabetic, n = 9.</p>*<p><i>p</i><0.05 vs. Baseline.</p>†<p><i>p</i><0.025 vs. CTRL.</p

α₁R expression is augmented in PD.

<p>Western blot analysis of α₁R expression (∼42 kDa) in hindlimb muscle homogenate of CTRL (n = 6 per muscle group) and PD (n = 6 per muscle group). PD had greater α₁R expression in red vastus muscle, compared to CTRL. * Indicates different from CTRL (<i>p</i><0.05).</p

Percent change in hindlimb vascular conductance (VC) following Y₁R and α₁R blockade.

<p>The percent increase in VC following BIBP3226, prazosin and BIBP3226+prazosin treatments was greater in PD (n = 9) compared to CTRL (n = 8). *Indicates different from CTRL (<i>p</i><0.05).</p

Sympathetic receptor blockade elicits greater vascular responses in PD.

<p>Panel A: Change in hindlimb blood flow (Q_fem) and Panel B: vascular conductance (VC) from baseline following Y₁R and α₁R blockade. With Y₁R blockade, the increase in Q_fem and VC was greater in PD (n = 9) <i>versus</i> CTRL (n = 8) (<i>p</i><0.05). α₁R blockade elicited an increase in Q_fem, as well as an increase in VC that was greater in PD compared to CTRL (<i>p</i><0.05). * Indicates different from CTRL (<i>p</i><0.05).</p

Y₁R expression is augmented in PD.

<p>Western blot analysis of Y₁R expression (∼43 kDa) in hindlimb muscle homogenate of CTRL (n = 6 per muscle group) and PD (n = 6 per muscle group). PD had greater overall expression of Y₁R in both white and red vastus muscles. * Indicates different from CTRL (<i>p</i><0.05).</p

Skeletal muscle NPY concentration is elevated in PD.

<p>NPY concentration normalized to total protein for whole muscle homogenate of white vastus (WV) and red vastus (RV). PD (n = 6 per muscle group) tissue had greater NPY concentration compared to CTRL (n = 6 per muscle group). * Indicates different from CTRL (<i>p</i><0.05).</p