in addition , vascular regeneration and repair are essential for the survival of blood vessels .
there are still uncharacterized and less characterized cell types in vascular adventitia , which include vascular stem / progenitor cells [ 110 ] and adventitial neuronal somata ( annies ) .
the vascular adventitia is a complicated tissue , which is found to be the most active layer in terms of cell turnover .
nerva vasorum  . using fluorescence confocal microscopy ( fcm ) to visualize vascular wall 3d organization of different cellular and extracellular elements of the intact artery with minimal 3d distortion
[ 11 , 13 ] , we recently demonstrated annies coexpressing neural cell adhesion molecule ( ncam ) and calcitonin gene - related peptide ( cgrp ) in the adult rat mesenteric branch artery ( mba ) .
the present study was designed to further characterize annies in adult rat mba as cells with axonal and neurite growth markers , such as growth - associated protein-43 ( gap-43 ) and palladin , and to determine whether they express the calcium sensing receptor ( casr ) .
recently , casr mrna and protein expression has been demonstrated in rat whole mba and other vascular tissue extracts , also shown in vascular smooth muscle cells in culutre [ 15 , 16 ] .
none of these studies show the specific cell(s ) in the artery in situ expressing the casr .
we demonstrate here that nucleated annies cells together with nerve processes clearly express palladin , casr , and coexpress gap-43 protein in the whole mount mba by immunofluorescent staining followed by laser confocal analysis .
all procedures using laboratory animals were reviewed and approved by the institutional animal care and use committee of north carolina central university .
male wistar rats , 1012 weeks of age , were obtained from harlan sprague dawley ( indianapolis , usa ) .
all animals were continually monitored , and upon arrival , they were maintained in colony rooms with fixed light : dark cycles and constant temperature and humidity and provided with purina rodent chow ( harlan teklad , madison , wis , usa ) and water ad libitum .
mesenteric arteries were dissected from rats ( n = 10 ) as previously described . in brief , rats were deeply anesthetized with 2% isoflurane and then sacrificed by open chest cardiac puncture .
the small intestine and all vessels feeding it were removed in block and placed in cold physiological salt solution ( pss ) .
branch i and ii arteries were carefully dissected from the surrounding fat and mesenterium , taking care to leave a portion of the omental membrane attached to the vessel .
a 12  m - diameter stainless steel wire was inserted into the lumen to remove blood and to serve as a handle for moving the vessel segment between solutions .
vessels of mba were fixed in buffered formalin for 20  min and washed three times in tbs ( tris - buffered saline ) .
the vessels were kept in blocking solution containing 8% bsa in tbs and incubated with primary antibody such as polyclonal anti - gap-43 , anti - casr ( molecular probes ) , anti - palladin ( gift from dr .
otey , unc - chapel hill ) overnight at 4c . for dual immunofluorescence staining anti - cgrp ( phoenix pharmaceuticals , calif ) , and anti - gap-43 ( molecular probes ) were incubated for 2  h at room temperature ( rt ) . after incubation
, the vessels were washed thrice in tbs and incubated with appropriate secondary antibody tagged with alexa fluor 647 alone , and with 488 in case of co - staining for 1  h at rt .
after washing thrice in tbs , vessels were incubated with the nuclear stain sytox ( petticoat junction , or ) to identify the nuclei of annies .
vessels were mounted on glass slides in a glycerol - based antifade medium ( molecular probes ) after washing .
segments were viewed with a zeiss lsm 510 confocal microscope ( zeiss instruments ) with 100x , 40x , and 20x oil immersion objectives .
we considered the number of cgrp staining cells as 100% of annies , and calculated the percentage of annies expressing the other markers ( gap-43 , casr and palladin ) in comparison to the cgrp expressing cells .
protein was extracted from minced mba ( n = 5 ) by homogenizing in a ground - glass homogenizer ( mba ) in buffer containing 10  mm tris , ph 7.5 , 0.25  m sucrose , 3  mm mgcl2 containing 1% ( v / v ) triton x-100 , dithiothreitol ( 1  mm ) , pe - fabloc ( 1  mm ) , leupeptin ( 10  m ) , bestatin ( 130  m ) , pepstatin ( 1  m ) , and calpain inhibitor ii ( 10  g / ml )
. the homogenate was then centrifuged at 16,000  g for 10  min and the pellet was used for ncam , gap-43 and palladin .
the pellet was dissolved in buffer containing 10  mm tris , ph 7.5 , and 1% triton x-100 , size separated using 8% sds - page , and electroblotted onto nitrocellulose membrane ( bio - rad laboratories , ca ) as described .
, calif ) and visualized using an hrp - conjugated secondary antibody , and the chemiluminescence method ( amersham pharmacia biotech , nj ) .
we used the whole mounts of the adult rat mba and immunofluorescence confocal microscopy imaging to further characterize annies in arterial adventitia .
the use of whole - mount mba from rats enabled us to have an accurate reconstruction of the cellular and nerve interrelationships and innervation patterns of perivascular adventia and vasa nervorum within the mba .
this also facilitates the ability to study the expression pattern of the neurochemical markers in situ , without the need to cut them as with conventional histology .
furthermore , this approach allows us to localize and visualize the morphology / phenotype and quantify these cells ( annies ) and nerve structures in situ coexpressing vasodilator trophic factor cgrp and ncam , and axonal growth markers gap-43 ( figure 3 ) as well as casr ( figure 1 ) .
casr is shown to play significant role in blood pressure regulation by releasing an unknown vasodilator .
more recently , weston et al .   reported that this vasodilator is still unidentified .
we hypothesize that the vasodilator released by casr , which is still unidentified , might be the potent vasodilator neuropeptide , cgrp .
our present finding demonstrates that 94  6% ( n = 3 ) annies cell bodies express the casr ( figure 1 ) , compared with the annies identified by the expression of cgrp as previously demonstrated .
the casr is shown to regulate the production of cgrp   that is involved in the regulation of the release of the neurotransmitter in the synaptic space .
our laboratory has demonstrated that the casr mediates ca2 + induced relaxation of isolated mesenteric arteries   via an unknown vasodilator substance , which is independent of endothelium and no .
these previous findings support our hypothesis that this putative vasodilator released by the casr might be the cgrp expressed in annies and perivascular nerve fibers .
we demonstrate here that nucleated annies cells and the nerve processes strongly express the casr .
it is shown to locate in the growth cones of growing neurites , where it interacts with f - actin associated adhesion molecule and/or extracellular matrix complexes to promote neurite extension .
in addition , it has been shown that the phosphorylated gap-43 stabilizes long actin filaments and has the ability to directly influence the structure of the actin cytoskeleton response to extracellular signals .
similarly , palladin has been shown to express in the growth cone and colocalize with focal adhesion   and respond to vascular   and dermal injury .
confocal analysis of the whole mount mba in our experiment showed that gap-43 is strongly distributed in 37  4% of the perivascular nerve fibers ( figure 2 ,
n = 3 ) , and coexpressing cgrp ( figure 3 ) , also identified by the expression of casr ( figure 1 ) .
palladin is present in both smc   and annies in the rat mba ( figure 1 ) .
palladin expression is apparent in a mixed population of fibroblasts and in cells with nerve processes , annies ( figure 1 ) that were shown to coexpress cgrp and ncam .
the casr and palladin were present in annies about 94  6% and 80  10% ( n
strong immunofluorescence for ncam , and axonal growth markers such as gap-43 as reported in this paper , revealed the presence of many neuronal cell bodies within the vasa nervorum of rat mba .
in addition , the presence of gap-43 in annies indicates that annies shows features of sensory neurons of the drg , undergoing responses to stress / injury , given that the expression of gap-43 in drg cells is increased in response to injury [ 27 , 28 ] .
in addition , cgrp induces schwann cell proliferation , and thus thought to be involved in peripheral nerve injury and repair .
immunoblots from the rat mba also confirmed the presence of gap-43 ( figure 4 ) and palladin as two isoforms of 9092 and 140  kd ( n = 3 ) .
this protein pattern is different from other adult tissues such as brain that expresses three isoforms ( 9092 , 140 , and 200  kd ) or smc where only one isoform ( 9092  kda ) is expressed .
casr protein expression by western blot in the protein extract of the whole mesenteric artery was reported .
we previously demonstrated beta - cgrp expression in rat mba by rt - pcr , cloning , and sequence analysis .
cells that are not with nerve fibers , probably fibroblasts , were also stained for palladin as it has been reported by other investigators .
there is no nerve like structures around some of the fibroblast like cells that express palladin .
in addition , ncam , cgrp , and gap-43 are expressed in annies , but these markers are not shown to express in the other adventitial fibroblasts , smooth muscle , and endothelial cells .
this is the first report that cells in the peripheral vasculature with a neuronal phenotype express markers of active neurite growth .
the presence of cgrp - containing neural cells in the vascular adventitia may participate in response to injury and vasodilator mechanisms as part of a perivascular sensory neural network .
annies cell morphology with nerve fibers and expression of neural and neurite growth markers reveal that these cells are distinct from the other known cells in blood vessel .
the additional finding that the casr is associated with annies suggests that these cells may participate in the regulation of myogenic tone .
 a novel perivascular adventitial cell termed , adventitial neuronal somata ( annies ) expressing the neural cell adhesion molecule ( ncam ) and the vasodilator neuropeptide , calcitonin gene - related peptide ( cgrp ) , exists in the adult rat mesenteric branch artery ( mba ) in situ . 
 in addition , we have previously shown that annies coexpress cgrp and ncam . 
 we now show that annies express the neurite growth marker , growth associated protein-43(gap-43 ) , palladin , and the calcium sensing receptor ( casr ) , that senses changes in extracellular ca(2 + ) and participates in vasodilator mechanisms . 
 thus , a previously characterized vasodilator , calcium sensing autocrine / paracrine system , exists in the perivascular adventitia associated with neural - vascular interface . 
 images of the whole mount mba segments were analyzed under scanning confocal microscopy . 
 confocal analysis showed that the gap-43 , casr , and palladin were present in annies about 37  4% , 94  6% , and 80  10% 
 respectively , comparable to cgrp ( 100% ) . 
 immunoblots from mba confirmed the presence of gap-43 ( 48  kd ) , ncam ( 120 and 140  kd ) , and palladin ( 9092 and 140 kd ) . in summary , 
 cgrp , and ncam - containing neural cells in the perivascular adventitia also express palladin and casr , and coexpress gap-43 which may participate in response to stress / injury and vasodilator mechanisms as part of a perivascular sensory neural network . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion