Network remodeling of intramural coronary resistance arteries in the aged rat: A statistical analysis of geometry

Mechanisms of Ageing and Development 134 (2013) 307–313

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Network remodeling of intramural coronary resistance arteries in the aged rat: A statistical analysis of geometry Edina A. Wappler a,*, Pe´ter Antal b, Szabolcs Va´rbı´ro´ c, Be´la Sze´ka´cs d, Andrea Simon b, Zolta´n Nagy e, Emil Monos b, Gyo¨rgy L. Na´dasy b a

Department of Anesthesiology and Intensive Therapy, Semmelweis University, Budapest, Hungary Experimental Research Department and Department of Human Physiology, Semmelweis University, Budapest, Hungary Second Clinics of Gynecology and Obstetrics, Semmelweis University, Budapest, Hungary d Second Clinics of Medicine, Geriatry Group, Semmelweis University, Budapest, Hungary e Department Section of Vascular Neurology, Semmelweis University Heart Center, Budapest, Hungary b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 October 2012 Received in revised form 17 February 2013 Accepted 9 March 2013 Available online 21 March 2013

Aims: To identify the geometrical alterations in the age-remodeled rat coronary artery network and to develop a useful technique to analyze network properties in the rat heart. Methods and results: We analyzed the networks of the left anterior descendent coronary arteries on in situ perfused hearts of young (3 months) and old (18 months) male rats. All segments and branching over >80 mm diameter were analyzed using 50 mm long cylindrical ring units of the networks. Arterial widening and paucity, increased tortuosity were typical features in the old network. In addition, axis angles deviated more from the mother branches in the old, whereas the diameters of daughter branches fit the Murray law in both groups. The detected changes in the old network resulted in a longer blood flow route for the same direct distance. Conclusion: We developed a useful method to investigate arterial network property changes in the rat heart. Ageing resulted in longer, more tortuous flow route in the LAD network that might be hemodynamically disadvantageous. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Coronary Resistance arteries Network Ageing

1. Introduction Age related alteration in tissue morphology (Cooper et al., 2012; Fontana et al., 2012; Han, 2012), functionality (de Freitas et al., 2012; Fontana et al., 2012; Grady, 2012; Mieno et al., 2006), vulnerability (Meier et al., 2012; Wappler et al., 2010), regenerative potential (Wappler et al., 2012), and vessel remodeling (Charifi et al., 2004) is widely investigated in different organs. Decreased lumen diameter (Dumont et al., 2008; Na´dasy et al., 2010), thickened wall (Auerbach et al., 1971; Dumont et al., 2008; Moreau et al., 1998; Na´dasy et al., 2010), increased wall rigidity (Gardner and Parker, 2011; Na´dasy et al., 2010), alteration of the branching angles (Bearden et al., 2004), tortuosity (Bearden et al., 2004; Charifi et al., 2004; de Margerie and Boyd, 1961; Thore et al., 2007), and rarefaction (Bearden et al., 2004; Behnke et al., 2006; Riddle et al., 2003) are the most common changes in the vessels with

* Corresponding author at: Department of Anesthesiology and Intensive Therapy, Semmelweis University, Kutvolgyi Street 4, Budapest H-1125, Hungary. Tel.: +36 13556565. E-mail address: [email protected] (E.A. Wappler). 0047-6374/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mad.2013.03.002

ageing. Ventricular tissue perfusion deteriorates in the aged as well (Friberg et al., 1985; Hachamovitch et al., 1989; Susic et al., 1998; Tomanek et al., 1993). In addition, previous studies showed impaired coronary resistance artery contractility and reduced vasodilatory reserve (Shipley and Muller-Delp, 2005; Susic et al., 1998; Tomanek et al., 1993), impaired endothelium-dependent dilation (Csiszar et al., 2002; Kang et al., 2009; LeBlanc et al., 2008; Muller-Delp, 2006; Shipley and Muller-Delp, 2005), arterial wall thickening (Auerbach et al., 1971; Rakusan et al., 1994; Rakusan and Nagai, 1994), and microcirculatory network rarefaction (Rakusan et al., 1994; Rakusan and Nagai, 1994; Vitullo et al., 1993) in the heart with ageing. Network properties are just as important in determining blood blow as vessel reactivity and functionality. Because of methodical difficulties, however, there is limited number of publications in the literature studying alteration in small coronary artery network in ageing. Previously, diameter frequency of intramural coronary arterioles in young and aged mice was analyzed in fixed histological sections on non-pressurized system by Rakusan and Nagai (1994). They found a decreased arteriole density with an elevated external diameter and wall thickness in the elderly. This is, however, in contrast with an earlier report that used the same

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technique (Vitullo et al., 1993), where no change in arteriolar density was found between the 1.5 and the 22 months old rats. The limitations of these histological studies are the non-physiological intra-arterial pressure; and the inability to measure important network properties, such as segmental lengths and branching angles. The left anterior descendent (LAD) coronary artery of the rat and its main branches run mainly intramurally, mostly parallel with the surface. On the other hand, smaller side and end branches in the small arteriolar range (<80 mm) penetrate deeper layers of the wall (Na´dasy et al., 2001; Zamir et al., 1984). Using our preparation technique we can visualize the entire LAD network on in situ rat heart down to 80 mm size branches while maintaining the physiological pressure with continuous perfusion through the orifice/coronary ostium (Na´dasy et al., 2001). The artery network can be then recorded and analyzed. The specific geometry of the LAD network with its location on a cylindrical surface makes it possible to use two-dimensional evaluation. That study, however, described the preparation technique without discussing the possible ways to examine how the network can be analyzed. The goal of our study now was to investigate the age-induced remodeling process in the rat LAD system. Understanding the pathological changes in the ageing heart is crucial to develop new strategies in fighting the heart diseases in the old. It is necessary in our ageing society. In clinical practice the percentage of patients affected by heart diseases increase with advancing age; whereas, preclinical data are mainly generated using young animals. In addition, the secondary goal of our study was to find the most useful methods to analyze coronary network differences. Routine parameters of microvascular network analysis such as segmental lengths (lengths between two consecutive branching points), segmental tortuosities, statistical distribution of segmental diameters, mother/daughter branch diameter characteristics, and branching angles were measured and compared of the >80 mm diameter arteries. These arteries are known to be the most important determinant of an artery network (Anversa and Capasso, 1991; Auerbach et al., 1971; Bearden et al., 2004; Chapman et al., 2002; Charifi et al., 2004; de Margerie and Boyd, 1961; Dumont et al., 2008; Hutchins et al., 1976; Kaimovitz et al., 2010; Molloi and Wong, 2007; Moreau et al., 1998; Oka and Nakai, 1987; Rakusan et al., 1994; Rakusan and Nagai, 1994; Rossitti and Lo¨fgren, 1993; Schreiner et al., 1994; Smith et al., 2000; Stanton

et al., 1995; Thore et al., 2007; VanBavel and Spaan, 1992; Vitullo et al., 1993; Zamir et al., 1983, 1984; Zamir and Phipps, 1988). The limitations of our study, however, are the lack of data on vessel reactivity and the difference in the size of the heart and arteries in the rat compared to human. Although there can be similarities in the rat and human coronary network remodeling, larger arteries and systems usually have different methods to react to a new environment as well. With the intention of better describing the network we theoretically divided the segments into 50 mm length ring units. 2. Methods 2.1. Micropreparation and in situ videomicroscopic recording of whole coronary networks Eight young (3 month-old) and seven old (18 month-old) male Sprague–Dawley rats were included in this study. Their heart was removed under deep pentobarbital (50 mg/kg, i.p.) anesthesia. Animal use was approved by the Animal Examination Ethical Council of the Animal Protection Advisory Board at the Semmelweis University, Budapest, which meets the European Commission guidelines. For LAD network preparation we used a Wild M3Z preparation microscope as described previously (Na´dasy et al., 2001). Briefly, the preparation process made the arterial segments visible that were larger than 80 mm leaving the penetrating arteries intact. After preparation the orifice was cannulated and the network was perfused with Krebs–Ringer solution (bubbled with a gas mixture of 75% N2, 20% O2, and 5% CO2) at 100 mmHg pressure. The outer surface of the vessels was also continuously superfused with the same solution. Arteries remain intact with this technique as it was shown with a vasoreactivity (Na´dasy et al., 2001) study. After vessel diameter equilibration without any added vasoactive substance the arterial network was recorded with our videomicroscope using small and large magnifications (Fig. 1a). For precise geometrical reconstruction, the optical angle of the microscope tube was placed perpendicular with the tangential plane of the network which was expected to have a cylindrical shape. Small and large, magnified photos (pixel sizes from 5.21 to 31.04 mm) were selected for further analysis. 2.2. Geometric analysis All resistance arteries in LAD coronary networks with outer diameters over 80 mm at the time of preparation and their branchings have been identified. A twodimensional Cartesian coordinate system was constructed for each heart with the bottom rim of the left orifice serving as the origin and a straight line was leading from it to the apex as X axis. Outer vessel diameters at several points along the segmental axis (diameter typically did not change along the segment, however, more points were measured), distances of typical branch points from origin (direct distances) and from each other (segmental lengths in case of two consecutive branching points), angles of distances and of segmental axes with the coordinate system and with each other were measured using the Leica QWin image analysis software. Direct distance between two points can be defined as the shortest distance between those points, whereas actual distance is equal to the flow route.

Fig. 1. Typical network pictures. (a) Picture of young network with segment identification, characteristic trigonometric points, line of the orifice-apex coordinate system drawn. X and Y coordinates are shown in micrometers. (b) Old network, broken course of main branch, triple branching. (c) Old network, triple branching, rarely seen in normal young network.

E.A. Wappler et al. / Mechanisms of Ageing and Development 134 (2013) 307–313 The latter was not measured but calculated. Trigonometric methods were used to compute coordinate positions. Finally, pixel (bmp) coordinates along all segmental axes were read and transformed into orifice-apex coordinates. A detailed twodimensional map of each network was thus constructed and the plotted maps were compared with the original pictures at low magnification for control. Direct distances and actual vessel lengths of 255 segments were determined. In order to keep the information given by the exact location of a network element, we theoretically divided the arterial segments into 50 mm long ring units, where each ring units were located in the two-dimensional Cartesian coordinate system. The 50 mm length was chosen as being comparable with the range of segmental diameters studied (80–1000 mm). Ring unit analysis can give more precise data on the network properties, such as diameter distribution in the network, flow route, or the rarely seen altered diameter along a segment, than the use of the a segment in the comparison. Each ring unit was characterized by its diameter, location of its mid-point in the orifice-apex coordinate system, and angle of its axis with the X axis. Direct and actual distances from the orifice along the flow route were also computed for all the 9797 ring units identified. The 125 branching units were characterized by diameters of the mother and daughter branches, by the intersecting angles of the mother and daughter branches with each other, and with the coordinate system X axis, as well as by their location in the XY coordinate system of the given heart. 2.3. Statistical analysis Parametric ANOVA test was used to compare body weights, and heart weights and lengths. Frequencies of segments and ring units in different diameter ranges in young and old networks were compared with the x2 test. Non-parametric tests (Kruskal–Wallis with Dunn’s pairwise comparisons) were used to compare segmental lengths, tortuosities of segments, direct and flow distances of ring units from the orifice and ring axis angles. The significance level of the Pearson correlation between branch angle and branch diameter was also computed. p < 0.05 was considered as statistically significant difference.

3. Results The body weight of the older animals was 861  151 g, which was more than twice as high as that of the younger ones (366  18 g); and the old animals had significantly larger hearts than the young ones (p < 0.05; 2.06  0.24 g vs. 1.27  0.06 g). The orifice to apex distances increased in the old compared to the young from 17,132  1404 mm to 14,419  941 mm (p < 0.05) as well. 3.1. Segmental analysis

increased in number while 150–250 mm diameter segments diminished (both p < 0.05 vs. young). Mean segmental lengths (curved ‘‘flow’’ lengths) otherwise were similar (not significant) between young and old (at 150–250 mm: 1.49  0.21 in the young, 1.11  0.14 mm in the old; 250–350 mm: 1.71  0.24 mm in the young, 1.77  0.25 mm in the old; and 350–450 mm: 2.24  0.24 mm in the young, 2.08  0.24 mm in the old). Despite the similar mean segmental ‘‘flow lengths’’, these segments spanned lesser distances and were more tortuous in the old. The tortuosity index (ratio of curved length per direct length, being 1.00 for the straight segment (Charifi et al., 2004; de Margerie and Boyd, 1961; Thore et al., 2007) was significantly higher in the old segments than in the young ones in the <450 mm diameter ranges (p < 0.05). In the young population of these vessels there were 94 segments practically straight (tortuosity index below 2%), 19 were moderately, and 4 segments were seriously tortuous (indexes 2–8% and over 8%, respectively). The same values for the old population were 52, 33, and 7, respectively (p < 0.05 using x2 test for the difference between measured and expected frequencies). 3.2. Vascular ring unit analysis Diameter distribution of 50 mm length ring units of a network is shown in Fig. 3. We used ring units instead of segments in our analysis, because of the variability of the segment lengths makes it impossible to correctly analyze network properties, such as diameter frequency, direct and actual distances. These network properties then are essential in how the resistance alters in the system. The two ring unit distributions are substantially different (p < 0.01 young vs. old). Whereas the smaller (>300 mm) ring units are the most frequent in young networks, older networks mainly have larger (<300 mm) artery rings. The old network has two entirely new peaks (500–550 and 800–850 mm sizes) as well. It is also remarkable that there is a peak both in the young and in the old network around 400 mm diameter. The steric distribution of ring units in the coordinate system is shown on a three dimensional plot (Fig. 4) where the diameter frequency plots are shown color coded for direct distances of ring units from the orifice. This diagram clearly demonstrates that in addition to alterations in ring unit frequency, there is a substantial alteration in the steric distribution of such ring units. An entirely new population of large rings (700–1000 mm) developed close to the orifice. Another typical transformation in the old was the development of a secondary 400 mm (300–500 mm) peak into a much wider (up to 600 mm) and larger peak than what the young network has between a much narrower peak of 200–400 mm

Number of ring units per heart

An entirely new population of vessel segments with larger diameters (>600 mm) appeared in the older, larger hearts. They were located in a separate section of the length–diameter plot (outlined with an ellipsis in Fig. 2). Those in the 450–550 mm range

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Fig. 2. Segmental analysis. Length–diameter scatter plot of young and old segments. Typical position outlined with triangles. An additional population, completely missing in youngsters appears in the old networks (outlined by the ellipsis).

Fig. 3. Frequency of ring units per heart with different diameters. Note shared 400– 450 mm population maximum, drift of 200–250 population up to 300–350 mm, two newly formed peaks at 500–550 and 800–850 mm in the elderly (p < 0.01 using x2 test). Data shown were calculated as follows: pooled data from every hearts in a group were normalized to a single heart.

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Fig. 4. Direct distance frequency of ring units, color coded for diameter. Note newly formed isolated population of thickened vessels, an upward widening of the 400 mm population, overall elevation in color code (increased distance from orifice) and the upward movement (widening) of the 200 mm population leaving a rarefaction in its original place (distances different, p < 0.01 Kruskal–Wallis in the 100–700 mm ranges). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

(narrower both in the sense of lumen diameter and peak width). Among the homogenous segmental widening (upward translocation on plot) and axial elongation (overall elevation in color coded frequencies of distance) in the old, there was a characteristic reduction in the 200 mm ring population (rarefaction) as well. A similar picture is obtained when not the direct distance, but the flow-distance from the orifice is color-coded (not shown). The young and old networks were significantly different at all comparable diameter sizes (p < 0.01). The transformation of resistance artery populations into each other shows up even in a more demonstrative manner in Fig. 5, where the diameter range-flow distance range plot is color coded for ring unit frequency. At 3 months of age, the microvasculature is organized around two centers (clusters), a 300–450 diameter population in a distance of 3–8 mm, and another, and a 150–350

diameter range population at a distance of 7–13 mm from the orifice. The aged network has a more diverse distribution with 3 main and 4–5 additional population clusters. Probable transformation routes (due to age induced network geometry alterations) are shown by white arrows. In addition to frequency and steric distribution, young and old ring units are also different in their angles formed with the X (orifice-apex) axis. Higher absolute angle values (pooling together leftward and rightward angles) were observed for the old networks (p < 0.01) in the 100–400 mm range. The newly developed thick vessels (where such comparison would be irrational as they are limited in numbers or non-existing in the young networks) are uncertain in their direction. In summary, the effect was that blood needs to cover longer distances for the same direct distance in the old network than in the young (diagram not shown, p < 0.01).

Fig. 5. Flow distance frequency of ring units, color coded for diameter. Two newly developed populations can be identified, a large diameter vessels (>700 mm) conducting blood up to 15 mm and an isolated new population of 300–700 mm vessels developed 10–15 mm far from the orifice (flow distances different, p < 0.01 Kruskal–Wallis in the 100–700 mm ranges). (d) Diameter-flow distance plots, color coded for ring unit frequency. Note two basic clusters in young network, visibly given rise to multiple clusters ‘‘a’’ through ‘‘d’’ and ‘‘e’’ through ‘‘g’’ clusters, respectively, by variable combinations of diameter elevation, axial elongation and axial displacement (due to elongation in more proximal parts of the network). A weaker cluster ‘‘h’’ representing thin vessels close to orifice, seems to remain in place. (e) Deviation of ring unit axes from orifice-apex line. Rightward (negative) and leftward (positive) angles are shown. Frequency of ring units with higher deviation (absolute value) than marked with the color code for different diameter ranges are shown (statistically higher for the aged, p < 0.01 Kruskal–Wallis in the 100–400 mm ranges). Also note substantial deviation even toward the right in newly developed large vessels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 6. Analysis of branchings. Interconnection between mother (Dm) and daughter (D1 and D2) branch diameters. D31 þ D32 was plotted against D3m , these are predicted to be equal according to the Murray law (line of identity). Double logarithmic scatter plot, mother diameters are also shown. Note fairly good adherence in aged networks, too.

3.3. Analysis of branchings Considering the diameters of branching vessels, both young and old networks kept the Murray law, describing the interconnection between mother (Dm) and daughter (Dd1 and Dd2) branch diameters as Dm3 = Dd13 + Dd23 (Fig. 6). Branch angles are interpreted as deviation of the angle of the daughter branch from the axis of the mother branch measured in degrees, with absolute value computed (no leftward-rightward distinctions) and negatively correlated with the Dd/Dm ratio (p < 0.02 both for young and old branchings). This means that larger branches tended to keep the original direction of the mother branch, while smaller branches, as a rule, deviated more from its axis. In addition to segmental tortuosity, the ‘‘broken’’ appearance of the course of old networks can partially be explained by the increased frequency the large daughter branches the deviated more from the course of the mother branch than in the young. Using a feasible definition of Dd/ Dm > 0.794, Dm > 300 mm, and an axis deviation >58, there was a significantly higher number of such branchings in the old, than in the young networks (6.3 vs. 3.6 per heart, p < 0.02 with the x2 test). 4. Discussion This is the first publication to demonstrate age-induced network remodeling in the arterial network using segment analysis instead of histological techniques. This approach leads to a better understanding of the mechanisms underlying arterial remodeling processes, such as axial lengthening, diameter enlargement, diameter frequency alterations, axial displacement, and changes in branching angle pattern. 4.1. What altered by ageing, and what did not change Significant geometrical alterations occurred in the left anterior descendent coronary resistance artery network of the rat during the process of ageing as it was revealed by us on in situ perfused whole networks. The network should accommodate to an increased size of the heart: an almost 2.5fold elevation in body weight was accompanied by a 62% elevation of heart weight and a 19% (close to the expected linear elongation of 3H1.62 = 1.175) elevation in the orifice-apex length. We did not observe an overall linear enlargement of the network, but larger branches in the neighborhood of the orifice radially dilated and axially extended inducing an axial displacement of the more distal parts of the network increasing their distance from the orifice. There were alterations among the smaller vessels either: a population of

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originally 150–250 mm vessels moved into the 250–350 mm population, leaving a rarefaction in the original 150–250 mm population in the subsurface network. Tortuosity of individual segments increased, scatter of angle of vascular elements in the old network was elevated, causing an elevated flow route compared to direct distance traveled. Certain optimizing processes, however, were unaffected by age. Mean segment lengths were identical in the young and old in several diameter ranges, suggesting that diameter rise and axial elongation were to some degree in concert. The Murray’s law describing interconnection of mother–daughter diameters was fairly kept in both populations, suggesting a maintenance of flow and endothelial shear governed morphological dilation. While larger daughter branches deviated more from the course of the mother branch in the old than in the young networks (contributing this way to a more broken course), the tendency of smaller branches to deviate more from the mother’s axis than larger ones was generally kept in the old network, too. 4.2. Age-induced rarefaction There is a limited data available on coronary resistance artery rarefaction and network analysis with ageing in normotensive animals. Most of the studies investigated the age factor in genetically, surgically, or pharmacologically induced hypertensive animals. In addition, most of the former publications on ageingrelated coronary network remodeling used non-pressurized vessels. Previous studies, however, have already used pressurized vessels for network analysis swine (Huo and Kassab, 2012a,b). In previous studies, where tissue fixation was performed on zeroarterial pressure, both diminished (Rakusan and Nagai, 1994) and unchanged (Vitullo et al., 1993) arteriolar density was reported with ageing in the heart. In our study the rarefaction of the 150– 250 mm vessels were seen both at the segmental and at the ring unit levels in the present study. Furthermore, our frequency plots convincingly showed that this population is moved to 300 mm size in the elderly animals, whereas the height of 400 mm peak in the young was not affected by the age. Our studies have proven an age induced rarefaction in the 150–250 mm diameter range arteries both when comparing the number of affected segments and total segmental length. We believe that the population of such vessels simply enlarged to the 250–350 mm diameter size, whereas their number was not replaced from a pool of smaller vessels. These new observations shed a new light on the arterial rarefaction process (Behnke et al., 2006; Rakusan and Nagai, 1994), where diminishment of the 200 mm diameter elements is caused by a shift in this diameter range to a higher one, emptying this population group. 4.3. Segmental lengths Segment length is an important functional parameter of the coronary artery network. Our report is the first to show elevated tortuosity in aged intramural coronary resistance arteries, which reached significance in the 150–350 mm diameter range. In a previous study a linear relationship between segment length and diameter was described in the 10–1000 mm diameter range in the porcine coronary resistance arteriole network (VanBavel and Spaan, 1992). Our results on segmental lengths showed similar statistical regularity in the 150–450 mm range, however, in the young group we found typical triangle-type distribution of the length–diameter plots relative short lengths of the largest diameter vessels. Otherwise, mean segmental lengths were not different in our groups, suggesting some determination by a control mechanism in both groups. This is in accordance with an earlier report (Bearden et al., 2004), where no age induced alteration was found in segment lengths in the mouse skeletal muscle arteriolar casts (10–50 mm). For example flow simulation

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studies by Molloi and Wong (2007) predicted a linear relationship between maximum coronary blood flow and the sum of its arterial branch lengths. Segmental tortuosity determined as the ratio of curved length/per direct length is thought to be a key factor in the microvascular ageing process (Bearden et al., 2004; Charifi et al., 2004; de Margerie and Boyd, 1961; Thore et al., 2007). 4.4. Age induced remodeling on the diameter-flow distance frequency plot We believe that our image-analyzing technique gives a better resolution for visualizing and analyzing the frequency and spacedistribution of different vascular elements on the heart tissue, than histological cross-sections. It is more likely the reason why a previous study on dog (Tomanek et al., 1991) did not find any diameter alteration in coronary arterioles with ageing. In addition, our diameter-flow distance plot color coded for ring unit frequency (Fig. 5) gave us the possibility to determine probable routes of ageinduced network remodeling of the rat LAD network as well. We detected both a vertical movement of a cluster, meaning diameter enlargement without axial displacement; and a horizontal movement, meaning axial replacement without diameter alteration. Axial displacement can be simply the result of elongation of upstream elements (horizontal replacement toward the right without alteration in color coded frequency) or axial elongation without diameter alteration (continuous ‘‘growth’’ toward the left and elevation of color coded frequency). There were originally two clusters of ring units (a 300–450 diameter population in a distance of 3–8 mm, and a 150–350 diameter population at 7–13 mm from the orifice; white arrows in Fig. 5) that characteristically disintegrated during the ageing process. The aged network had a much more diverse distribution, with 3 main and 4–5 additional population clusters. 4.5. Age induced alterations of branchings Multiple branching was more frequent in aged networks, however, this increment did not reach the level of statistical significance. Representative pictures of triple branching are shown in Fig. 1a and b. In addition, our data confirmed that the Murray law is maintained in the aged coronary network as it was shown previously in the heart and other organs (Aharinejad et al., 1998; Hutchins et al., 1976; Kamiya et al., 1974; Lo´ra´nt et al., 2003; Murray, 1927; Rossitti, 1995; Stanton et al., 1995; Zamir et al., 1983, 1984; Zhou et al., 1999). The basis for Murray law is thought to be the sensitivity of the endothelium to shear stress (Rodbard, 1975), meaning that aged endothelium retains its (proportional) sensitivity to shear stress, or that there is a larger flow in the old LAD system that maintains the given diameter despite of the less sensitive endothelium. In our study, however, vessel reactivity was not studied. In agreement with the latter explanation, some previous publications showed substantially reduced endothelial dilatation in the aged (Csiszar et al., 2002; Kang et al., 2009; LeBlanc et al., 2008; Muller-Delp, 2006; Shipley and Muller-Delp, 2005). In former publications, branching angles, which are defined as deviation of axis from that of mother branch, increased with decreasing diameter of the daughter branch (Zamir et al., 1983), a characteristics also described in rat coronary casts (Zamir et al., 1984), but missing in human coronary angiographies of mostly larger vessels (Hutchins et al., 1976). A coronary model constructed by Schreiner (Schreiner et al., 1994) supposing endothelial shear stabilization and minimization of vascular volume predicted a similar behavior of branch angles. Bearden and colleagues (Bearden et al., 2004) have found increased inter-daughter branch angles in older mice. Difference in this respect, did not reach the level of statistical significance between young and old networks in

our studies. However, in case of asymmetric larger branches, deviation from the maternal course was significantly more frequent contributing to the broken flow course. Such alterations can be formed by asymmetric diameter alterations of daughter branches. The existence of a lateral displacement of certain elements of the network has been proven, however by the larger deviation of ring unit axis angles from the orifice-apex axis. Another possibility suggested by our material is that enlarged daughter branches will tend to drag mother branches toward them at points of their insertions. These phenomena may explain the more broken course of the segments and the larger overall angle deviation in the elderly. 5. Conclusion We conclude that a combined statistical analysis of elements with advanced image-analyzing techniques is needed to get a full picture on age-induced remodeling processes in microvascular networks. Such techniques are needed to evaluate the processes of axial elongation, radial dilation, as well as axial and lateral displacement, acting either separately or in concert in different microvascular populations. In addition, we found a disproportional enlargement of the LAD network in the enlarged heart of the aged rats. A new, isolated population of large-diameter (>500 mm) vessels appeared with ageing as well, as a combined result of axial and circumferential growth of originally 400 mm vessels. Overall, elongated flow length increases vascular resistance. Additionally, a population of 200 mm vessels was enlarged, enriching the 300 mm population while not being replaced from below (rarefaction). A large population of 400 mm vessels was simply displaced from the orifice by axial growth to more proximal segments. The overall scatter of axis directions increased, which, together with increasing segmental tortuosity, increased the overall length the blood travels to span the given direct distance from the orifice. This is a hemodynamically disadvantageous characteristic. Other networkbuilding, long-term control processes seem to work in youngsters and aged alike: mean segment lengths were almost identical in comparable diameter ranges, the Murray law governs the diameters of daughter branches at bifurcations, and smaller daughter branches deviate at larger angles from the course of the mother branch both in the young and in the aged. Funding This work was supported by the Hungarian National Scientific Foundation (OTKA TO 32019, OTKA TO 42670), the Ministry of Health of Hungary (ETT 128/2006), the Hungarian Hypertension Society, and the Hungarian Kidney Foundation. Conflict of interests None declared. Acknowledgements The authors would like to thank Mrs. Ildiko Oravecz for her expert technical assistance. References Aharinejad, S., Schreiner, W., Neumann, F., 1998. Morphometry of human coronary arterial trees. The Anatomical Record 251, 50–59. Anversa, P., Capasso, J.M., 1991. Loss of intermediate-sized coronary arteries and capillary proliferation after left ventricular failure in rats. American Journal of Physiology 260, H1552–H1560.

E.A. Wappler et al. / Mechanisms of Ageing and Development 134 (2013) 307–313 Auerbach, O., Hammond, E.C., Garfinkel, L., Kirman, D., 1971. Thickness of walls of myocardial arterioles in relation to smoking and age. Findings in men and dogs. Archives of Environmental Health 22, 20–27. Bearden, S.E., Payne, G.W., Chisty, A., Segal, S.S., 2004. Arteriolar network architecture and vasomotor function with ageing in mouse gluteus maximus muscle. The Journal of Physiology 561, 535–545. Behnke, B.J., Prisby, R.D., Lesniewski, L.A., Donato, A.J., Olin, H.M., Delp, M.D., 2006. Influence of ageing and physical activity on vascular morphology in rat skeletal muscle. The Journal of Physiology 575, 617–626. Chapman, N., Dell’omo, G., Sartini, M.S., Witt, N., Hughes, A., Thom, S., Pedrinelli, R., 2002. Peripheral vascular disease is associated with abnormal arteriolar diameter relationships at bifurcations in the human retina. Clinical Science (London) 103, 111–116. Charifi, N., Kadi, F., Fe´asson, L., Costes, F., Geyssant, A., Denis, C., 2004. Enhancement of microvessel tortuosity in the vastus lateralis muscle of old men in response to endurance training. The Journal of Physiology 554, 559–569. Cooper, L.L., Odening, K.E., Hwang, M.S., Chaves, L., Schofield, L., Taylor, C.A., Gemignani, A.S., Mitchell, G.F., Forder, J.R., Choi, B.R., Koren, G., 2012. Electromechanical and structural alterations in the aging rabbit heart and aorta. American Journal of Physiology. Heart and Circulatory Physiology 302, H1625–H1635. Csiszar, A., Ungvari, Z., Edwards, J.G., Kaminski, P., Wolin, M.S., Koller, A., Kaley, G., 2002. Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function. Circulation Research 90, 1159–1166. de Freitas, E.V., Batlouni, M., Gamarsky, R., 2012. Heart failure in the elderly. Journal of Geriatric Cardiology 9, 101–107. de Margerie, J., Boyd, T.A., 1961. A statistical investigation of the correlation of retinal arteriolar tortuosity with blood pressure and age. Transactions of the Canadian Ophthalmological Society 24, 6–17. Dumont, O., Pinaud, F., Guihot, A.L., Baufreton, C., Loufrani, L., Henrion, D., 2008. Alteration in flow (shear stress)-induced remodelling in rat resistance arteries with aging: improvement by a treatment with hydralazine. Cardiovascular Research 77, 600–608. Fontana, L., Vinciguerra, M., Longo, V.D., 2012. Growth factors, nutrient signaling, and cardiovascular aging. Circulation Research 110, 1139–1150. Friberg, P., Nordlander, M., Lundin, S., Folkow, B., 1985. Effects of ageing on cardiac performance and coronary flow in spontaneously hypertensive and normotensive rats. Acta Physiologica Scandinavica 125, 1–11. Gardner, A.W., Parker, D.E., 2011. Predictors of large and small artery elasticity in healthy subjects from 9 to 89 years old. American Journal of Hypertension 24, 599–605. Grady, C., 2012. The cognitive neuroscience of ageing. Nature Reviews. Neuroscience 13, 491–505. Hachamovitch, R., Wicker, P., Capasso, J.M., Anversa, P., 1989. Alterations of coronary blood flow and reserve with aging in Fischer 344 rats. American Journal of Physiology 256, H66–H73. Han, H.C., 2012. Twisted blood vessels: symptoms, etiology and biomechanical mechanisms. Journal of Vascular Research 49, 185–197. Huo, Y., Kassab, G.S., 2012a. Intraspecific scaling laws of vascular trees. Journal of the Royal Society, Interface 9, 190–200. Huo, Y., Kassab, G.S., 2012b. Compensatory remodeling of coronary microvasculature maintains shear stress in porcine left-ventricular hypertrophy. Journal of Hypertension 30, 608–616. Hutchins, G.M., Miner, M.M., Boitnott, J.K., 1976. Vessel caliber and branch-angle of human coronary artery branch-points. Circulation Research 38, 572–576. Kaimovitz, B., Lanir, Y., Kassab, G.S., 2010. A full 3-D reconstruction of the entire porcine coronary vasculature. American Journal of Physiology. Heart and Circulatory Physiology 299, H1064–H1076. Kamiya, A., Togawa, T., Yamamoto, A., 1974. Theoretical relationship between the optimal models of the vascular tree. Bulletin of Mathematical Biology 36, 311–323. Kang, L.S., Reyes, R.A., Muller-Delp, J.M., 2009. Aging impairs flow-induced dilation in coronary arterioles: role of NO and H(2)O(2). American Journal of Physiology. Heart and Circulatory Physiology 297, H1087–H1095. LeBlanc, A.J., Shipley, R.D., Kang, L.S., Muller-Delp, J.M., 2008. Age impairs Flk-1 signaling and NO-mediated vasodilation in coronary arterioles. American Journal of Physiology. Heart and Circulatory Physiology 295, H2280–H2288. Lo´ra´nt, M., Na´dasy, G.L., Raffai, G., Monos, E., 2003. Remodeling of the rat saphenous vein network in response to long-term gravitational load. Physiological Research 52, 525–531. Meier, S., Demirakca, T., Brusniak, W., Wolf, I., Liebsch, K., Tunc-Skarka, N., Nieratschker, V., Witt, S.H., Mattha¨us, F., Ende, G., Flor, H., Rietschel, M., Diener, C., Schulze, T.G., 2012. SCN1A affects brain structure and the neural activity of the aging brain. Biological Psychiatry. Mieno, S., Boodhwani, M., Clements, R.T., Ramlawi, B., Sodha, N.R., Li, J., Sellke, F.W., 2006. Aging is associated with an impaired coronary microvascular response to

313

vascular endothelial growth factor in patients. The Journal of Thoracic and Cardiovascular Surgery 132, 1348–1355. Molloi, S., Wong, J.T., 2007. Regional blood flow analysis and its relationship with arterial branch lengths and lumen volume in the coronary arterial tree. Physics in Medicine and Biology 52, 1495–1503. Moreau, P., d’Uscio, L.V., Lu¨scher, T.F., 1998. Structure and reactivity of small arteries in aging. Cardiovascular Research 37, 247–253. Muller-Delp, J.M., 2006. Aging-induced adaptations of microvascular reactivity. Microcirculation 13, 301–314. Murray, C.D., 1927. A relationship between circumference and weight in trees and its bearing on branching angles. The Journal of General Physiology 10, 725–729. Na´dasy, G.L., Szekeres, M., De´zsi, L., Va´rbiro´, S., Sze´ka´cs, B., Monos, E., 2001. Preparation of intramural small coronary artery and arteriole segments and resistance artery networks from the rat heart for microarteriography and for in situ perfusion video mapping. Microvascular Research 61, 282–286. Na´dasy, G.L., Va´rbı´ro´, S., Szekeres, M., Kocsis, A., Sze´ka´cs, B., Monos, E., Kollai, M., 2010. Biomechanics of resistance artery wall remodeling in angiotensin – II Hypertension and subsequent recovery. Kidney and Blood Pressure Research 33, 37–47. Oka, S., Nakai, M., 1987. Optimality principle in vascular bifurcation. Biorheology 24, 737–751. Rakusan, K., Cicutti, N., Flanagan, M.F., 1994. Changes in the microvascular network during cardiac growth, development, and aging. Cellular and Molecular Biology Research 40, 117–122. Rakusan, K., Nagai, J., 1994. Morphometry of arterioles and capillaries in hearts of senescent mice. Cardiovascular Research 28, 969–972. Riddle, D.R., Sonntag, W.E., Lichtenwalner, R.J., 2003. Microvascular plasticity in aging. Ageing Research Reviews 2, 149–168. Rodbard, S., 1975. Vascular caliber. Cardiology 60, 4–49. Rossitti, S., Lo¨fgren, J., 1993. Optimality principles and flow orderliness at the branching points of cerebral arteries. Stroke 24, 1029–1032. Rossitti, S., 1995. Energetic and spatial constraints of arterial networks. Arquivos de Neuro-psiquiatria 53, 333–341. Schreiner, W., Neumann, M., Neumann, F., Roedler, S.M., End, A., Buxbaum, P., Mu¨ller, M.R., Spieckermann, P., 1994. The branching angles in computer-generated optimized models of arterial trees. The Journal of General Physiology 103, 975–989. Shipley, R.D., Muller-Delp, J.M., 2005. Aging decreases vasoconstrictor responses of coronary resistance arterioles through endothelium-dependent mechanisms. Cardiovascular Research 66, 374–383. Smith, N.P., Pullan, A.J., Hunter, P.J., 2000. Generation of an anatomically based geometric coronary model. Annals of Biomedical Engineering 28, 14–25. Stanton, A.V., Wasan, B., Cerutti, A., Ford, S., Marsh, R., Sever, P.P., Thom, S.A., Hughes, A.D., 1995. Vascular network changes in the retina with age and hypertension. Journal of Hypertension 13, 1724–1728. Susic, D., Nunez, E., Hosoya, K., Frohlich, E.D., 1998. Coronary hemodynamics in aging spontaneously hypertensive and normotensive Wistar–Kyoto rats. Journal of Hypertension 16, 231–237. Thore, C.R., Anstrom, J.A., Moody, D.M., Challa, V.R., Marion, M.C., Brown, W.R., 2007. Morphometric analysis of arteriolar tortuosity in human cerebral white matter of preterm, young, and aged subjects. Journal of Neuropathology and Experimental Neurology 66, 337–345. Tomanek, R.J., Aydelotte, M.R., Torry, R.J., 1991. Remodeling of coronary vessels during aging in purebred beagles. Circulation Research 69, 1068–1074. Tomanek, R.J., Aydelotte, M.R., Anderson, K.E., Torry, R.J., 1993. Coronary blood flow in senescent rats with late-onset hypertension. American Journal of Physiology 264, H1854–H1860. VanBavel, E., Spaan, J.A., 1992. Branching patterns in the porcine coronary arterial tree. Estimation of flow heterogeneity. Circulation Research 71, 1200–1212. Vitullo, J.C., Penn, M.S., Rakusan, K., Wicker, P., 1993. Effects of hypertension and aging on coronary arteriolar density. Hypertension 21, 406–414. Wappler, E.A., Felszeghy, K., Szila´gyi, G., Ga´l, A., Skopa´l, J., Mehra, R.D., Nyakas, C., Nagy, Z., 2010. Neuroprotective effects of estrogen treatment on ischemiainduced behavioural deficits in ovariectomized gerbils at different ages. Behavioural Brain Research 209, 42–48. Wappler, E.A., Felszeghy, K., Varshney, M., Mehra, R.D., Nyakas, C., Nagy, Z., 2012. Brain plasticity following ischemia: effect of estrogen and other cerebroprotective drugs. Advances in the Treatment of Ischemic Stroke 89–114. Zamir, M., Wrigley, S.M., Langille, B.L., 1983. Arterial bifurcations in the cardiovascular system of a rat. The Journal of General Physiology 81, 325–335. Zamir, M., Phipps, S., Langille, B.L., Wonnacott, T.H., 1984. Branching characteristics of coronary arteries in rats. Canadian Journal of Physiology and Pharmacology 62, 1453–1459. Zamir, M., Phipps, S., 1988. Network analysis of an arterial tree. Journal of Biomechanics 21, 25–34. Zhou, Y., Kassab, G.S., Molloi, S., 1999. On the design of the coronary arterial tree: a generalization of Murray’s law. Physics in Medicine and Biology 44, 2929–2945.