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CMPF is impaired in CVD (Araki et al., 1994; O'Brien et al., 2012; Panny et al., 2009; Recek, 2013; Shiman et al., 2009; Simka, 2007). In addition to normal venous functioning, CMPF function relies on the contraction ability of the calf muscles and in a good ankle range of motion (Cavalheri et al., 2008; de Moura et al., 2012; Panny et al., 2009; Shiman et al., 2009). Despite its importance in preventing venous stasis and hypertension, calf muscle pump is not commonly assessed in dynamic conditions. Likewise, the relationship between muscle pump efficacy and measures of muscle size and architecture has not been explored before. Hence, we hypothesized that calf muscles architecture is affected in CVD subjects and that this is related with the efficacy of the blood pumping function of this musculature, which could be assessed dynamically by means of duplex ultrasound. Despite that air- plethysmography provides a non-invasive and accurate assessment of CMP, it does not allow examining individual veins (deep and superficial) during muscle contraction or ankle motion. These measures can be gathered using duplex scanning and might be important for understanding CVD physiopathology for guiding the treatment and perhaps used as a strategy to assess efficacy of CVD treatments for CMPF.

Our results show a clear increase in venous blood flow caused by calf muscle contractions in individuals with CVD and control participants. Popliteal peak flow volume was maximal during the first contraction of the tip-toe set when the venous

reservoir is full, which has also been seen in other studies (Staubesand et al., 1995). In the CVD patients, but not in the healthy subjects, venous flow augmentation diminished during the muscle contraction set. Such apparent calf pump dysfunction might be related to weak calf muscles in CVD patients (Panny et al., 2009; Qiao et al., 2005) and is compatible with a lower ejection volume, such as has been measured before in this population with air-plethysmography (Nicolaides, 2000). In addition, abnormal venous blood reflux from deep to superficial venous system through incompetent perforator veins may blunt blood flow through the popliteal vein.

As a result of CVD, distal leg muscles may exhibit reductions in strength and power, which then might contribute to aggravate the condition (Webber et al., 2010). In fact, patients with CVD present decreased ankle muscle strength (Panny et al., 2009) with a decreased peak torque per kilogram of body weight, as well as diminished power ability (de Moura et al., 2012) and muscle resistance (van Uden et al., 2005). Accompanying muscle weakness, CVD patients are also characterized by decreased ankle range of motion (de Moura et al., 2012; Dix et al., 2003; van Uden et al., 2005), decreased gait speed (de Moura et al., 2012; van Uden et al., 2005), and impaired functional capacity and mobility (de Moura et al., 2012).

Nonetheless, it seems that calf muscle size is not a strong indicator of the efficacy of muscles to pump venous blood during contractions, at least in patients with venous ulcer (Moloney et al., 2007). In our study, gastrocnemius thickness and some other muscle architectural features were similar in patients with low to moderate severity of CVD and healthy participants and were unrelated to the severity of CVD. Despite this fact, for the medial gastrocnemius, few morphological parameters were associated with the degree of increase in peak flow velocity in the

popliteal vein during tip-toe movement. Higher muscle fascicle length and a lower pennation angle in the medial gastrocnemius, and with the ankle in dorsiflexion, were associated with larger increase in peak flow volume in the popliteal vein during tip-toe. Likewise, higher gastrocnemius muscle fascicles pennation angle with ankle in neutral position, and a larger change in pennation angle between maximal active dorsiflexion and plantarflexion are accompanied by increased venous flow volume at the end of a whole set of calf muscle contractions. The exact meaning of these findings is unclear but lower pennation angle and longer muscle fascicles at ankle dorsiflexion may indicate higher ankle joint range of motion and improved blood pumping function by the calf muscles (Duclay et al., 2009; Manal et al., 2008; Morse et al., 2007; Rassier & Herzog, 2004). Improved ankle range of motion is likely important for muscle pump function during gait and patients with CVD show notable gait alterations (de Moura et al., 2012). In addition, increased tissue hydrostatic pressure and tissue edema in the lower leg likely restricts ankle dorsiflexion and gastrocnemius muscle lengthening and can be related to less efficient calf pump function (Back et al., 1995; Cavalheri et al., 2008; Meissner et al., 2007b; Panny et al., 2009; Shiman et al., 2009).

In this study, the ultrasound measures of gastrocnemius muscle architecture were highly reproducible, which is in agreement with previous studies (Duclay et al., 2009; Narici et al., 1996). In contrast, those of popliteal venous blood flow showed quite large variation when measured within a week interval (Breen et al., 2007; Lurie et al., 2002). Such somewhat low reproducibility might be related with probe unsteadiness, combined with short sampling time of venous velocity (Breen et al., 2007; Lurie et al., 2002). We attempted to determine the error introduced by leg

movement on probe steadiness and on the measures of blood flow volume by contrasting such measures with those collected with subjects performing similar foot movement while standing supported on the opposite limb. In this case, the measures of venous blood flow collected from the popliteal vein were highly reproducible and showed good agreement with measures collected during actual tip-toe movement. This suggests that probe movement relative to the underlying vein is not a strong limitation to the use of continuous-wave Doppler ultrasound in evaluating venous hemodynamics in the lower extremity during dynamical weight-bearing conditions.

The poor test-retest reliability found for popliteal vein measures during tip- toe movements points to the need for strict standardization regarding probe positioning and movement task conditions if the use of ultrasound techniques to assess CMPF is warranted. However, the high bias of this procedure might preclude its usefulness in assessing the response to treatment interventions. Hence, this methodology was not used in our study to assess therapeutic efficacy of MLD on treatment of the impaired calf muscle pump function in this patients.