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1 Laboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands 2 Department of Physiology, University of Maastricht, The Netherlands 3 Medicine and Therapeutics, University of Aberdeen, Scotland, UK 4 School of Pharmacy, Robert Gordon University, Aberdeen, Scotland, UK 5 Departments of Chemistry and Molecular Cell Physiology, Vrije Universiteit, Amsterdam, The Netherlands
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Abstract |
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, in µmol min–1 (g dry wt)–1) in the same location, calculated from the 13C spectrum of tissue extracts after intracoronary infusion of 3–13C-lactate. Since both innervated and denervated regions are subject to the same arterial pressure, lower blood flow indicates higher resistance. Mean MBF was 5.56 ml min–1 (g dry wt)–1 (heterogeneity of 3.47 ml min–1 (g dry wt)–1) innervated, 7.48 ml min–1 (g dry wt)–1 (heterogeneity of 3.62 ml min–1 (g dry wt)–1) denervated (n.s.). Significant linear relations were found between MBF and 
of individual samples within the innervated and denervated regions. The slopes of these relations were not significantly different, but the adjusted mean was significantly higher in the denervated regions (+1.92 ml min–1 (g dry wt)–1, an increase of 38% of the mean MBF at the pooled mean 
, P
= 0.028, ANCOVA). The ratio 
(in ml µmol–1) was significantly higher, being 0.296 ± 0.167 ml µmol–1 in the denervated region compared with the innervated region, 0.216 ± 0.126 ml µmol–1, P
= 0.0182, Mann–Whitney U test. These results indicate that sympathetic tone under chloralose anaesthesia imposes a moderate vasoconstrictive effect in the myocardium that is not detected by comparison of the mean blood flow or resistance.
(Received 15 November 2006;
accepted after revision 9 February 2007; first published online 15 February 2007)
Corresponding author J. H. G. M. van Beek: Departments of Chemistry and Molecular Cell Physiology, Vrije Universiteit, Amsterdam, The Netherlands. Email: mimnoble{at}abdn.ac.uk
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Introduction |
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, also averaged over the whole left ventricle (Tune et al. 2004). However, effects of sympathetic vasoconstriction could be concealed because of the considerable spatial heterogeneity of MBF whether measured with microspheres (Bassingthwaighte et al. 2001; van Oosterhout et al. 2002) or positron emission tomography (Blanksma et al. 1995; Visser et al. 1998). In addition, one must take into account local metabolism as indicated by glucose uptake (Blanksma et al. 1995) or the primary determinant of MBF, the myocardial oxygen consumption (Alders et al. 2004). There is also considerable point-to-point spatial heterogeneity of 
(Bassingthwaighte et al. 2001). Local differences in MBF and/or 
could be related to local differences in adrenergic innervation. Higher local noradrenaline levels could increase local contractility and so oxygen demand; conversely, higher adrenergic vasoconstriction could reduce MBF at a given 
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Here we investigate the contribution of sympathetic innervation to blood flow heterogeneity and the blood flow–tricarboxylic acid (TCA) cycle flux relation. To investigate whether such an effect is detectable when MBF is related to TCA flux heterogeneity, regional denervation experiments were performed in anaesthetized dogs. After 3 weeks of regional myocardial denervation, MBF and 
were determined in samples within innervated and denervated regions. In the same tissue sample, MBF was measured by the standard microsphere method (Domenech et al. 1969), and 
by magnetic resonance spectroscopic measurements of metabolites labelled with 13C via the TCA cycle (van Beek et al. 1999; Alders et al. 2004) after intracoronary infusion of 13C-labelled lactate.
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Methods |
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Experiments were performed in seven intact dogs (25–30 kg body weight), after approval by the Advisory Boards for the Use of Experimental Animals of Maastricht University & Home Office (1986) Procedures at Imperial College, London. These Boards conform to the European Convention for the Protection of Vertebrate Animals used for Experimental Purposes (Council of Europe no. 123, Strasbourg, 1985). The housing, health and husbandry of the dogs were under independent professional veterinary supervision. Three of the seven experiments were performed in London at Imperial College and four in Maastricht. Regional denervation of the left ventricle was performed using anaesthesia induced with thiopentone, 15 mg kg–1 I.V. The trachea was intubated and the lungs were mechanically ventilated. During surgery, anaesthesia was continued with halothane (1%) in a mixture of oxygen and nitrous oxide (40%:60%). A left thoracotomy was performed, and the heart exposed by opening of the pericardium. The circumflex branch of the left coronary artery (LCx) was dissected free. Phenol (6%) was applied to the circumflex artery and surrounding tissue to destroy the nerves accompanying the artery. The thorax was closed and the animal was allowed to recover. Postoperative analgesia was provided using buprenorphine (15 µg kg–1 I.M.) immediately and 12 h after ending the surgical procedure. A period of 3–4 weeks was allowed to elapse for myocardial catecholamines to deplete.
Final acute experiment
Initial anaesthesia was identical to that described for the regional denervation but halothane used during the surgical procedure was replaced by 
-chloralose (60 mg kg–1
I.V.) prior to the start of the experimental protocol (Lawrence et al. 1996). Mechanical ventilation was adjusted to obtain optimal arterial blood gas values throughout. Rectal temperature was monitored throughout the experiment, and body temperature maintained at 38 ± 1.5°C, if necessary using a heating blanket. A left thoracotomy was performed to expose the heart. A catheter-tip manometer was introduced into the left ventricular cavity via the carotid artery for measurement of left ventricular pressure (LVP) and maximum rate of rise of left ventricular pressure (LVdP/dt); catheters were introduced into a femoral artery for withdrawal of arterial blood, into the left atrium for injection of microspheres, and into the coronary sinus for withdrawal of coronary venous blood. A small cannula was inserted into the left main coronary artery, in order to infuse the 13C-enriched lactate (to measure aerobic metabolism; see below) to the tissue supplied by the left anterior descending artery (LAD) and the left circumflex artery (LCx). Boundaries of the LAD and LCx were delineated by small ligatures sewn into the surface of the heart. Lactate was chosen as a convenient physiological substrate, known to be preferentially taken up (Drake et al. 1979), that is available with a 13C label. After finishing all instrumentation, the animal was allowed to stabilize for 30 min.
Arterial and coronary venous blood samples were taken and haemodynamic measurements made every 15–30 min during the whole experiment. In Maastricht (n = 4), fluorescent microspheres (orange, crimson, blue-green; 3 x 106 per injection) and in London (n = 3) radioactive microspheres (141Ce, 103Ru, 113Sn; 3 x 106 per injection) were injected over 20 s into the left atrium to measure blood flow; simultaneously, an arterial reference blood sample was withdrawn over 1.5 min at a rate of 11 ml min–1. Two of the three available labels were used in random order. Injection of 3 x 106 microspheres allows a distribution of more than 400 spheres per tissue sample, as required for adequate accuracy. The first microsphere injection was just after the stabilization period of the final acute experiment, and the second one just before 13C-labelled lactate infusion, i.e. 30 min later. After the first microsphere injection, unlabelled sodium lactate was infused directly into the left main stem (intracoronary concentration, 1.5 mmol l–1 for 30 min), in order to achieve a metabolic steady state. At the end of 30 min, a quick switch was made from unlabelled lactate to 3–13C-lactate at unchanged concentration. After infusion of this labelled lactate for exactly 8 min, the heart was rapidly excised, frozen in isopentane (cooled to –120°C in liquid nitrogen) and stored at –80°C until further analysis. Rapid excision of the heart under anaesthesia was the humane method employed for killing the experimental animals.
The use of either fluorescent or radioactive microspheres in the same study was necessitated by logistic conditions at the two institutions (moving the experiments from London to Maastricht was necessitated by the unforeseen closure of the London laboratory); the comparability of the methods is justified by previous studies in which we showed close correlation between the MBF results obtained from the two types of microsphere label (Van Oosterhout et al. 1995).
Tissue preparation and magnetic resonance spectroscopy (MRS) measurements
All tissue analysis was carried out in Amsterdam. The left ventricle was cut from the rest of the heart at –20°C and freeze-dried for 3 days (Modulyo freeze-dryer; Edwards). After freeze-drying, the ventricle was divided into two parts, i.e. that supplied by the LAD (innervated) and that supplied by the LCx (denervated). Both parts were cut into five or six slices of approximately 1 cm from apex to base. To determine the profile of oxygen consumption, central contiguous pieces of approximately 0.5 ml volume of the denervated and innervated regions were homogenized. Potentially overlapping areas of LAD and LCx were discarded. Tissue samples were weighed and homogenized in 4.0 ml ice-cold perchloric acid (0.6 mol l–1) for 1 min, and then centrifuged (10 min at 4000g). The pellet of each homogenate was used for microsphere label identification and the supernatant for MRS, so that colocalization was perfect.
Blood flow was determined according to the standard formula: Fl = (Il/Ia) x Fa, with Fl being local blood flow, Il tissue microsphere counts, Ia arterial reference microsphere counts and Fa arterial reference flow (Raab et al. 1999), and expressed in milliltres per minute per gram dry weight.
The supernatant was neutralized to pH 7.0 with buffer containing 3 mol l–1 KOH and 0.3 mol l–1 imidazole. The sample was centrifuged again (10 min at 4000g), and the supernatant was freeze-dried for 48 h. Freeze-dried supernatants were re-dissolved in tri-distilled H2O and deuterium oxide (0.5 ml total). 13C-MRS spectra were obtained at 100.62 MHz with a Bruker Avance400 spectrometer. The samples were studied in a 5 mm probe at 27°C with a WALTZ-16 broad-band 1H decoupling pulse sequence, 13C-pulse angle 45 deg, repetition time 7.3 s, 32 k data points, sweep width 100 p.p.m. with 1470 scans accumulated. Spectra were analysed in the time domain with the MRUI/AMARES software package after conversion to Magnetic Resonance User Interface format (European Union Human Capital and Mobility Project). Individual glutamate multiplet areas were quantified with reference to a standard spectrum of a 50 mmol l–1 unlabelled glutamate solution (13C present at the natural abundance of 1.1%), and converted to micromoles per gram dry weight.
Model analysis of glutamate multiplets
The glutamate multiplet peaks were analysed with a previously published and validated computer model (van Beek et al. 1999; Alders et al. 2004), resulting in quantification of the TCA cycle flux. We have previously used 2–13C-acetate in isolated hearts as our label in MRS studies (Alders et al. 2004). In the present study, we used 3–13C-lactate, thus avoiding the haemodynamic effects of infused acetate (Yamada et al. 1986); these were also discovered in unpublished experiments in which acetate, at the unphysiological concentrations needed when using the 13C isotope, was infused into anaesthetized dogs. In both cases, 
determined with MRS corresponds to 
determined from blood gases and MBF (Fig. 1). Labelling of glutamate with 13C after infusion of 13C-labelled lactate leads to the appearance of a maximum of seven different multiplets for the 4-, 3-, and 2-carbon atoms of glutamate. The multiplet labelling of glutamate after 8 min infusion is dependent on the fractional enrichment of acetyl-CoA (FC2) with 13C and the turnover rate of the TCA cycle (JTCA). The model parameters for the isotope distribution are adjusted to fit the multiplet data by non-linear least-squares parameter optimization using the XSIM simulation software package (National Simulation Resource on Cardiovascular Mass Transport and Exchange, Seattle, WA, USA). In 17 tissue samples, we were able to estimate FC2 (0.188 ± 0.12 (S.D.)) simultaneously with JTCA using the full multiplet model. Since, in other cases, the multiplet intensity-to-noise ratio of the MR spectra was sufficient for only a small subset of the multiplets, we used the ratio method (Alders et al. 2004), which estimates JTCA by fitting the sum of the G2 and G3 multiplets, divided by the total of the G4 peaks, with FC2 assumed to be 0.2. The value of glutamate was fixed at the biochemical assay value. The ratio method shows excellent correspondence with the multiplet method in those cases when both can be applied (Alders et al. 2004).
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is derived from the model parameter JTCA, using the formula: 
, where JTCA is the oxygen consumed for one carbon atom of substrate, multiplied by three because there are three oxygen molecules consumed per acetyl-CoA molecule entering the TCA cycle. This figure is exactly true for lactate and other carbohydrates and is a good approximation for fatty acids (Alders et al. 2004). Biochemical and blood gas analysis
Noradrenaline was measured in samples from all hearts by HPLC (Martin et al. 1983) with dihydroxylbenzylamine (DHBA) as internal standard. Global oxygen consumption was measured according to standard methods, in that blood gases in arterial and coronary sinus blood, haemoglobin (Hb) and oxygen saturation (%) were measured with a blood gas analyser (ABL, Radiometer, Copenhagen, Denmark). Myocardial oxygen delivery 
was computed as the product of mean flow measured in the tissue samples of the particular heart and arterial O2 content. Myocardial oxygen consumption (
, in ml min–1) in the left ventricle was calculated as the product of average MBF and the difference in O2 content between the arterial and coronary venous blood.
Analysis of data
From the individual data on MBF and 
within an experiment, the index of heterogeneity was calculated (HI = 100 x standard deviation/mean). In order to take heterogeneity and dependence upon local 
into account when comparing innervated and denervated MBF values, we divided MBF by 
. To eliminate errors resulting from between-experiment variation, measurements of blood flow and oxygen consumption were normalized by dividing the values by the mean values of MBF and 
of each experiment.
Statistics
Results are given as means ±
S.D. Methodological comparison of 
measurements by the MRS and Fick methods was carried out by linear regression and Bland–Altman comparison of mean differences versus average values (Bland & Altman, 1999). Comparison of MBF and 
between innervated and denervated tissue was carried out by Mann–Whitney U test, because the data were not normally distributed. Analyses of covariance were performed on the linear regressions of MBF upon 
. Statistical analyses were performed using InStat or Prism software (Graph Pad Inc., San Diego, CA, USA). A value of P < 0.05 was considered statistically significant.
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Heart rate and ECG did not change significantly over the course of the experiment, nor did left ventricular pressures or maximum LVdP/dt; all non-significant (n.s.) on repeated measures testing (Table 1). None of the blood gas values changed significantly during the experiment. Myocardial oxygen delivery and global oxygen consumption did not change during the course of the experiment (n.s., repeated measures testing). Body temperature was 38 ± 1.5°C throughout the procedures. All comparisons of local MBF and local 
between the denervated and innervated regions in the same heart were made immediately prior to and at the time of heart excision, respectively.
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Noradrenaline content (in pmol (mg dry wt)–1) was very much lower in denervated areas (0.26 ± 0.05) compared to innervated areas (5.85 ± 0.40). These levels are comparable to a separate closed-chest choralose-anæsthetized series of experiments (n = 13) in which regional denervation was also used (Rimoldi et al. 2006), where tissue noradrenaline was 0.34 ± 0.25 (denervated) and 5.87 ± 3.7 (innervated; P = 0.03).
Blood flow measurements
When pooling all samples per region, mean MBF (in ml min–1 (g dry wt)–1, ±
S.D.) was 5.56 ± 3.47 innervated and 7.48 ± 3.62 denervated (n
= 7). This large difference is not statistically significant because of the large standard deviations, which resulted from heterogeneity of values from different locations. The heterogeneity index for MBF was 31.5 ± 19.5% in innervated and 31.7 ± 20.9% in denervated areas (n.s.). Heterogeneity of MBF and 
are illustrated in Fig. 2A and B, in which the spread of values about the mean for the experiment can be appreciated. There were no significant differences in MBF heterogeneity in the periods before and after lactate infusion.
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A total of 59 samples from the seven hearts were used for measurement of oxygen consumption with the MRS method. When pooling all samples per region, oxygen consumption (in µmol min–1 (g dry wt)–1) derived from the MRS method was 28.23 ± 11.39 in innervated and 28.22 ± 13.09 in denervated areas (n.s.). Again, there was no significant difference in the heterogeneity index between the innervated (35.8%) and denervated areas (34.7%). Comparison of mean oxygen consumption values derived from blood gas versus values derived from the MRS method showed a good correspondence between these measurements (n.s., Fig. 1). In the Bland–Altman analysis, the mean difference was 4.6 ± 5.5 µmol min–1 (g dry wt)–1 (mean ± S.D.), confidence limits: –0.02 to +9.2 (mean difference not significantly different from zero).
Perfusion–metabolism correlation
Using quantitative raw (not normalized) data, total regional MBF (in ml min–1) was correlated with regional 
(in µmol min–1, Fig. 3A): innervated regions, 
; and denervated regions, 
. Using analysis of covariance (i.e. comparing these two linear regression relationships), the adjusted mean MBFs for the denervated data set (i.e. the mean MBF at the pooled mean 
calculated from the regression equations) was significantly higher than for the innervated data set (P
= 0.028), whereas the slopes of the regression lines were not significantly different.
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values to the mean values within each heart. (Fig. 3B): innervated regions, 
; and denervated regions, 
. Analysis of covariance to compare the two relationships resulted in an F variance ratio for comparison of slopes of 1.004 (P = 0.32, n.s.) and an F variance ratio for difference of elevations (adjusted means) of 4.810 (P = 0.032). (Adjusted mean MBF innervated = 0.82, denervated = 1.09, i.e. an increase in denervated tissue of 27%; the intercept increase was 37%.)
Ratio of MBF to M
O2
This ratio reflects the inverse of oxygen extraction and thereby shows how matching of blood flow to local oxygen consumption is affected by innervation. The 
ratio was, on average, 36.4% higher for denervated than innervated tissue (innervated, 0.22 ± 0.13 ml µmol–1; denervated, 0.30 ± 0.17 ml µmol–1; P
= 0.0182, Mann–Whitney U test, Fig. 4), i.e. innervated tissue extracted more oxygen from the blood than denervated tissue. The Mann–Whitney U test was used because these data do not have a Gaussian distribution (Fig. 4).
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Discussion |
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, this study shows that under the conditions of sympathetic tone induced by chloralose anaesthesia, there is no change in means or degree of heterogeneity between chronically denervated and innervated tissue. If one looks closely at the relation between MBF and 
in each location within the denervated region, there is a small, but significant increase in blood flow for each level of oxygen consumption, when compared with the same analyses within the innervated region. However, the spread of myocardial blood flow around the regression line is not clearly influenced by denervation. This suggests that sympathetic innervation leads to slightly lower blood flow in this animal model for the same local oxygen consumption. The increase in blood flow after denervation is very modest relative to the scatter in blood flow and is easily masked by the natural spatial variation in the blood flow, which is considerably larger than mere measurement error. We conclude that: (i) sympathetic innervation, in this animal model, leads to a small decrease in blood flow (implying vasoconstriction because the perfusion pressure is the same for all sites) at constant local oxygen consumption; (ii) this vasoconstriction is not detectable from measurements of mean MBF and resistance; and (iii) the main determinant of MBF heterogeneity, even in innervated myocardium under chloralose anaesthesia, is 
heterogeneity.
A study on myocardial flow and regional denervation in conscious dogs also showed no difference in mean MBF between innervated and denervated regions (Chilian & Ackell, 1988), but would a difference have been found in such experiments at a local level if heterogeneity of MBF and 
had been measured? Unfortunately, heterogeneity of 
can only be measured once in each heart by rapid excision and immediate freezing, requiring an open-chest anaesthetized preparation, so it is not possible to carry out our type of analysis in conscious animals, as used by Chilian & Ackell (1988). The fact that the MBF values of 5.56 ml min–1 (g dry wt)–1 (equivalent to 1.1 ml min–1 (g wet wt)–1) are the same as for baseline closed-chest dogs is compatible with the idea that sympathetic stimulation in our preparation was low, even though chloralose anaesthesia is known to preserve cardiovascular reflexes. Surgical procedures cause a release of catecholamines, mainly adrenaline, which has ß-adrenergic vasodilating properties that might mask -adrenergic vasoconstriction by sympathetic nerves. If there is an effect of adrenaline carried in the bloodstream, this would be the same for the innervated and denervated regions, since denervated myocardium is only supersensitive to noradrenaline, not adrenaline (Donald & Shepherd, 1965). The larger proportion of catecholamine in the myocardium is overwhelmingly noradrenaline, released and taken up again by sympathetic postganglionic nerve endings.
The observation that heterogeneity of blood flow and oxygen consumption is not abolished by sympathetic denervation indicates that such heterogeneity is not solely caused by regional differences in sympathetic vasoconstrictor tone. We cannot, however, exclude the possibility that such heterogeneity is related to vascular anatomy (Bassingthwaighte, 2001).
It has been shown that chronic ventricular pacing can change low-flow regions into high-flow regions and vice versa (van Oosterhout et al. 2002). Although vascular changes with chronic pacing could occur, we prefer to postulate that the distribution of blood flow is strongly dependent on the distribution of 
(Bassingthwaighte, 2001), which would be altered by the changed distribution of mechanics in the pacing situation. Matching of MBF to 
is well recognized, although the mechanism of the matching, according to the latest authoritative opinion from Feigl's group, is still unknown (Tune et al. 2004). From this standpoint, it seems reasonable to deduce that in the present case of regional denervation, the distribution of MBF heterogeneity in these hearts is also determined, to a large extent, by the distribution of 
heterogeneity, as is implied by the correlations found between these two variables.
The reliability of lactate as a source of the 13C label for TCA cycle intermediates can be explained by the fact that in the normal heart lactate is a favoured substrate for oxidative metabolism (Drake, 1979; Laughlin et al. 1993). Pyruvate infusion, at the unphysiological levels required when using the 13C isotope, changes energy-rich phosphate levels (Laughlin et al. 1993). High-concentration acetate infusion causes haemodynamic changes (A.J. Drake Holland & M.I.M. Noble, unpublished preliminary obervations). These normoxic hearts are different from ischaemic hearts, where regions with high lactate production can be anticipated (Gertz et al. 1981; Wisneski et al. 1985). However, the route by which 13C enters the TCA cycle (and whether some of the label does not enter the cycle) does not affect the determination of 
, which depends on the relative labelling of the different carbon atoms in the glutamate chain.
The accuracy of the 
measurements in individual locations was previously examined (Alders et al. 2004). The model, previously used for acetate, was extended to account for carboxylation reactions of pyruvate via pyruvate carboxylase and malic enzyme, leading to the formation of oxaloacetate and malate from labelled and unlabelled pyruvate (Panchal et al. 2000). Pyruvate carboxylation usually amounts to 3–6% of the TCA cycle flux in myocardium (Comte et al. 1997). We predicted from the results of global denervation, that an increase in MBF and 
(Drake et al. 1978) would be found (in association with a decrease in lactate utilization) owing to the fact that, in global denervation, 
is increased, and pyruvate dehydrogenase is downregulated (Van der Vusse et al. 1998) with inhibition of glucose oxidation (Drake et al. 1980) and increased fatty acid utilization (Drake-Holland et al. 2001). We adopted regional denervation as a possibly more accurate way of studying such changes, but regionally denervated myocardium behaves quite differently. For instance, there is no increase in mean MBF in the denervated regions (Rimoldi et al. 2006). In the present study, we calculated the fractional enrichment of 13C intermediate metabolites and found no difference between innervated and denervated tissue, whereas global denervation experiments predicted lower lactate utilization owing to pyruvate hydrogenase downregulation. This is a problem to be discussed in relation to global denervation rather than to the present study. For present consideration, we must recognize that there is no evidence to date of metabolic abnormalities in myocardium subjected to sympathetic denervation only.
The relief of vasoconstrictor tone could not be deduced from measurements of mean MBF over the whole denervated region compared with the whole innervated region (Vatner et al. 1970; Chilian et al. 1981; Chilian & Ackell, 1988; Shen et al. 1988; Rimoldi et al. 2006). In the present study, we have shown that within such whole innervated and denervated regions, there are such wide standard deviations caused by spatial heterogeneity that considerable overlap of data must occur, potentially resulting in non-significant statistical comparisons of mean values. It is only by taking this problem into account, and relating MBF to the 
in each location, that it is possible to detect an increased MBF caused by denervation.
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