The present investigation is the first to quantitatively characterize IEL fenestration along multiple segments of the skeletal muscle arterial network. Consistent with others 
, we observe modest IEL fenestration (≤5%) in multiple locations along the mesenteric vasculature. However, while the skeletal muscle conduit (popliteal) arteries reveal a somewhat similar IEL fenestration to mesenteric arteries, we show for the first time that skeletal muscle feed arteries have significantly large number of IEL holes, and that a vastly different and significantly greater total IEL fenestration area occurs within the intramuscular 1st
order and 2nd
order resistance arterioles (~15% and ~55%, respectively) compared to their respective upstream arteries or the mesenteric arterial network. Specifically, these smaller skeletal muscle resistance vessels have an apparent plexus or mesh-like IEL as opposed to distinct holes that are typically observed in other vascular beds. We demonstrate that this alteration in IEL fenestration does not correlate with vessel diameter per se
, but appears to be a unique property of intramuscular resistance arterioles. To expand on these observations, we further aimed to determine whether proteins involved in EDH, such as, KCa
R, and TRPC3, were also incongruent along the skeletal muscle arterial tree. The presence of these EDH proteins is similar in both large skeletal muscle conduit arteries (popliteal) as well as small first order arterioles, as evidenced by our immunostaining data in –. However, due to the significant lack of IEL in the resistance arterioles, a noticeably larger area of channel protein is observed (see smooth muscle face of –), and in theory, could offer less barrier to intercellular vasomotor communication. Additionally, an enhanced view of cross-sectional images suggests the possible presence of larger myoendothelial projections in the small intramuscular arterioles vs. the larger skeletal muscle conduit arteries. This raises the point that anatomy rather than simply EDH ‘machinery’ per se, may also have an impact on observed differences in vasodilatory signaling between small and large arteries. Importantly however, such a postulate has yet to be definitively tested. Collectively, these observations highlight the robust area of fenestration within intramuscular arterioles and indicate that the anatomical architecture and EDH apparatus for distinct vasodilatory signaling is potentially present within skeletal muscle of the rat hindlimb.
The rationale for the present investigation stems from the massive vasodilatory capacity within active skeletal muscle 
, and from the understanding that rapid and precisely controlled regulation of blood flow is needed to match the ever changing metabolic demand for oxygen within this tissue 
. From this foundation, we reasoned that the vascular anatomical architecture associated with vasomotor tone 
could be divergent between skeletal muscle and a bed of lower maximal blood flow capacity, such as the mesenteric circulation. In agreement with this underlying principle, we observe a considerably different pattern of IEL fenestration within skeletal muscle arterial network compared to that of the mesenteric, whereby along the vascular tree from the conduit artery to the 2nd
order arteriole, distinct holes transition into a more plexus like appearance. This was not observed in the mesenteric network, as small holes in the IEL were seen in all segments of this vascular bed. Similarly, small IEL holes are also detected in the cerebral vasculature 
; a vascular bed recognized for relatively steady flow. These data are supported by evidence indicating that acute interventions aimed to elevate blood flow in non-resistance vessels (carotid) stimulate in increased IEL fenestration, while reducing blood flow accelerates a decrease in IEL fenestrae size 
. Evidence suggests that high shear stress may stimulate changes in IEL fenestration by way of or repressing adhesion protein synthesis, lowering cell-matrix interactions, and thus consequently elevating IEL fenestration for means of increasing macromolecule exchange 
. Regardless of the underlying mechanism, our data make clear that under normal (non-manipulated) physiological conditions the intramuscular resistance vasculature is replete with large IEL fenestrae.
IEL fenestration could in some circumstances act as an index for potential myoendothelial communication but it must be emphasized that the majority of data indicate that IEL holes sites and myoendothelial projections are not 1
. Indeed, perusal of the cross-sectional images (–) would support this concept as identification of penetrating protein within the IEL was not observed for every IEL hole-site. The IEL tissue layer parts the vascular endothelium from nearby vascular smooth muscle cells and in theory, less IEL could presumably allow for greater communication between cell layers resultant from the open IEL space (>50% Total IEL fenestration in the 2nd
order skeletal muscle arterioles; ). In this respect, prior data demonstrate lack of lamina allows for greater molecule diffusion 
, thus aiding in the propensity for agents such as K+
ions, epoxyeicosatrienoic acids (EETs), nitric oxide or prostaglandins to act more readily between cell layers. In addition to diffusible myoendothelial crosstalk, direct physical interface through endothelial cell plasma membrane projections with vascular smooth muscle cells takes place through these fenestrae in the IEL 
. These projections house ER that spans throughout the length of the extension, of which is closely coupled as a microdomain with KCa2.3, KCa3.1, IP3
R, and gap-junction forming connexin proteins, thus precisely placing the Ca2+
signaling and responsive apparatus in a location that would enable rapid cell-to-cell communication. Importantly, it is now clear that electrical events involving such an activation cascade are essential to initiate and sustain endothelial and vascular smooth muscle cell hyperpolarization and subsequent vasodilation 
Our observations confirm the presence of endothelial KCa2.3, KCa3.1, and IP3R located within the IEL fenestrae of skeletal muscle conduit and small resistance vessels and cross-sectional analysis indicates the likely presence of penetrating projections (–). Furthermore, we extend these findings with the demonstration of endothelial TRPC3 embedded within the myoendothelial contact space (), thought to be an important upstream signaling component participating in the increase in intracellular endothelial cell calcium levels necessary to activate KCa2.3 and KCa3.1 channels. Quantitative analysis of fluorescence intensity as an index of the amount of protein is problematic with immunostaining procedures. Nevertheless, as a result of less physical IEL barrier, a more obvious presence of endothelial cell protein is clearly observed (–; smooth muscle face), highlighting the potential importance of anatomical differences. Therefore, future study is warranted to determine whether our observations in skeletal muscle artery morphology are associated with EDH and divergent control of vessel tone.
We observed KCa2.3 channel expression in popliteal and skeletal muscle 1st
order arterioles that appears localized to the IEL fenestrae. Functional evidence suggests a crucial role for KCa2.3 in skeletal muscle hyperemia via hyperpolarizing mechanisms 
. Our data corroborate these observations and demonstrate its localization to the myoendothelial space (). While previous demonstration of KCa3.1 in these regions has been published 
, KCa2.3 localization has often thought to predominate at the inter-endothelial junctions as evidenced by less discrete staining that outlines the endothelial cell body 
. Why these other studies did not observe KCa2.3 in the MEC space is not overly clear, however it should be emphasized that the present study is unique to skeletal muscle of the rat hindlimb; a region of muscle engagement during daily locomotion. Alternatively, antibody specificity could perhaps explain such observations. However, we did not observe staining in our negative control experiments indicating the selectivity of the compound (–, bottom images). Additionally, we have previously shown no presence of KCa2.3 in cerebral arteries using the same antibody; hence we believe an authentic regional difference is involved. We speculate that KCa2.3 and KCa3.1 act in tandem to regulate the electrical activity of the endothelium within the skeletal muscle resistance vasculature 
Previous investigation has hinted at greater myoendothelial cell projections as a function of decreasing vessel diameter within mesenteric arteries 
. Though IEL fenestrae and endothelial cell projections are not necessarily one in the same, we did not observe that vessel diameter per se
was explanatory for our observations of greater IEL fenestrae, as descending vessel diameter did not occur concomitantly with greater fenestration in mesenteric arteries. Further, while 1st
order skeletal muscle arterioles and 3rd
order mesenteric arteries were of the same diameter, total fenestration area was significantly greater in the 1st
order skeletal muscle arterioles ().
Protein content of each EDH protein assessed was observed in both skeletal muscle artery segments (popliteal and 1st
order). Moreover, cross-sectional viewing of this (Panels B of –) suggests that EDH protein is observed penetrating within the IEL of both vessel segments. In spite of this, given the greater total fenestration area of muscle arterioles ( & ), a larger projection could be present as evidenced by protein staining as an index (–). Further study is needed to substantiate such notions as our data do not provide ultimate evidence of such projections. These types of studies require finite experimentation and ultrastructural images obtained through the use of transmission electron microscopy, and are of interest to us for future study 
In the present study, we examined IEL fenestration in longitudinally opened and pinned arteries. It has been suggested by others that IEL properties should ideally be assessed under maximally dilated conditions to account for any differences in tone on the IEL 
, thus our data is open to limitation. Nonetheless, all vessel segments studied in the present investigation were handled and examined in a consistent and uniform manner to allow for relative comparison.
Lastly, it should be noted that EDH-mediated dilation is observed in both mesenteric arteries 
as well as arteries from skeletal muscle 
. This makes the case that anatomical differences between arterial beds cannot be a sole determinant underlying vasodilatory signaling.
Our morphological assessment and characterization of ion channel presence in skeletal muscle are important initial observations. Future understanding of the physiological and functional role these differences in IEL fenestrae poses equally critical. Our working hypothesis () is that the plexus like IEL found in intramuscular arterioles may provide less of a diffusional barrier to vasoregulatory molecules, and furthermore may allow a greater size or number 
of endothelial cell projections may make direct contact with the vascular smooth muscle. Housed along the membrane of these endothelial cell protrusion lies TRPC3 channels that respond to G-coupled receptor stimulation and aid in raising intracellular Ca2+
through activation of IP3 receptors located on the ER. Following intracellular Ca2+
elevation, endothelial KCa channels are activated to hyperpolarize the plasma membrane and evoke a local and spreading vasodilation upstream from the intramuscular arterioles 
. We speculate that the large fenestration area in small resistance arterioles may in some manner aid in optimizing blood flow and oxygen delivery to skeletal muscle; a vascular bed that undergoes tremendous vasodilation during conditions such as exercise and therefore necessitating rapid and robust intra- and intercellular communication.
“Working hypothesis” schematic for electrical communication within large and small skeletal muscle arteries.
The IEL fenestration within skeletal muscle arteries appears distinct from low-flow vascular beds such as the mesentery and is characterized by plexus like IEL (very high fenestration) in intramuscular arterioles. Given the presence of proteins involved in EDH, such as, KCa2.3, KCa3.1, IP3R, and TRPC3, throughout the entire skeletal muscle vascular network, and a greater area of exposure of these proteins due to less IEL barrier, these observations are suggestive that the anatomical architecture for distinct vasodilatory signaling is potentially present within skeletal muscle arteries.