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Arch Orthop Trauma SurgDOI 10.1007/s00402-013-1801-y

OrThOpAeDIc Surgery

Evidence of an autoregulatory mechanism of regional bone blood flow at hypotension

Stephan Vogt · Arne J. Venjakob · Klaus Stöckl · Thomas Tischer · Philipp J. Jost · Andreas B. Imhoff · Eckart Thein · Hermann Anetzberger

received: 25 June 2012 © Springer-Verlag Berlin heidelberg 2013

Methods The fluorescent microsphere reference sam-ple method was used to determine blood flow within the bone and kidneys. eight anesthetized female New Zealand rabbits received left ventricular injections of fluorescent microspheres over a wide range of arterial pressure levels prior to removal of kidney, femur and tibia. Blood flow values were calculated by measurement of fluorescence intensity in kidney and bone and correlated to fluorescence intensity in the peripheral blood (reference sample).Results Despite a reduction of mean arterial pressure from 100 to 80 mmhg bone blood flow remained constant. Further reduction of mean arterial pressure results in a lin-ear decrease in bone blood flow.Conclusion The correlation between arterial pressure and organ perfusion in the bone is similar to blood flow within the kidney, indicating the presence of an autoregulated blood flow mechanism within the bone tissue.

Keywords Autoregulation · Blood pressure · Bone blood flow · hypotension · Microspheres

Introduction

Blood flow in muscle and skin mainly depends on systemic blood pressure. In contrast, vital organs are equipped with an autoregulatory mechanism, which guarantees constant blood flow in an organ in spite of systemic blood pressure fluctuations. This mechanism is best known for the kidney and occurs at the preglomerular microcirculation [1]. It is mediated by two mechanisms. The faster myogenic mecha-nism and the slower tubuloglomerular feedback contrib-ute both directly and interactively to keep the renal blood flow and glomerular capillary pressure constant over a wide range of blood pressure [1]. This mechanism helps to

Abstract Background Blood flow in various organs is determined by an autoregulatory mechanism that guarantees constant organ perfusion over a wide range of arterial blood pressure changes. This physiological principle has been proven for the kidney, brain and intestinal tract, but so far not for bone. This study was carried out to determine whether there is an autoregulatory mechanism of bone or not.

S. Vogt and A.J. Venjakob equally contributed.

S. Vogt · A. J. Venjakob · K. Stöckl · A. B. Imhoff · h. Anetzberger Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar, Technical university of Munich, Ismaninger Straße 22, 81675 Munich, germany

S. Vogt (*) Department of Orthopaedic Sports Medicine, hessing Stiftung Augsburg, hessing Straße 17, 86119 Augsburg, germanye-mail: stephan-vogt@web.de

A. J. Venjakob Department of Trauma Surgery, Klinikum rechts der Isar, Technical university of Munich, Ismaninger Straße 22, 81675 Munich, germany

T. Tischer Department of Orthopaedics, university rostock, Doberaner Straße 142, 18057 rostock, germany

p. J. Jost Third Medical Department, Klinikum rechts der Isar, Technical university of Munich, Ismaninger Straße 22, 81675 Munich, germany

e. Thein Walter-Brendel-centre of experimental Medicine Munich, Ludwig-Maximilians-university of Munich, Marchionini Straße 27, 81377 Munich, germany

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maintain vital organ function in beginning shock and was later also proven for the brain and intestinal tract [1–3]. An autoregulatory mechanism for bone has not been shown so far. This protective mechanism will be useful because bones provide home to the important bone marrow, which is responsible for blood cell production. Different studies have shown bone marrow damage in hemorrhagic shock [4, 5]. It has been shown that hemorrhagic shock leads to apoptosis and morphologic alterations in bone marrow mononuclear cells [4], and therefore hematopoietic cells need to be protected by an autoregulatory mechanism of the bone tissue itself.

Few studies which state about bone blood flow and its correlation to bone metabolism are available. Decreased bone blood flow may be one aspect of steroid-induced femoral head osteonecrosis. Fat cell hypertrophy and acti-vation of adipogenesis may lead to increased intraosseous pressure and therefore decreased bone blood flow [6]. up to now, only few studies which analyze bone blood flow quantitatively are available. This may be due to the fact that measurement of bone blood flow is difficult because of the rigid structure of the bone tissue. Application of ultrasound-based blood flow measurements or intra-vital microscopy is limited. In contrast to other methods, the microsphere reference sample technique using fluorescent microspheres (FM) allows the exact measurement of bone blood flow [7–9]. This method is based on the principle that microspheres are distributed homogenously in the arterial blood after injection into the left ventricle because of tur-bulent flow [7]. Due to its greater diameter, microspheres get caught in the capillary bed. During and after the injec-tion of microspheres, arterial reference blood is sampled by means of a harvard pump with a known withdrawal rate. Blood flow in an organ can then be calculated from the number of spheres contained in the organ and in the arte-rial reference sample. The number of spheres is indirectly determined by measurement of fluorescence intensity of the tissue and reference sample. The advantages of the FM method are that repetitive measurements without surgical manipulation of the bone are possible. Furthermore, cardiac output and blood flow in other organs can be determined within the same experimental setup. In this study, we meas-ured blood flow of both kidneys to control autoregulation in our experimental setup. The methodical error of the FM method using sample-processing unit (Spu) and the robotic system for automated fluorescent measurement is very low compared to other methods [10, 11]. Due to the advantages, the FM method appears ideally suited to investigate bone blood flow during controlled hypotension.

We hypothesize that due to its important function for cell production, bone tissue is equipped with an autoregula-tory blood pressure mechanism similar to that seen in the kidneys.

Materials and methods

Animal model

eight adult female New Zealand white rabbits with a mean body weight of 3.5 ± 0.2 kg were used in this study which was authorized by the animal care committee at the Bavar-ian state authority (approval code: 55.2-1-54-2532-12-2005). The animals were anesthetized by an intramuscular injection of ketamine (15 mg/kg body weight, Ketavet®, BW, pharmacia & upjohn, erlangen, germany) and xyla-zine (2 mg/kg body weight, rompun®, BW, Bayer, Lev-erkusen, germany). Anesthesia was maintained by con-tinuous infusion of ketamine (10 mg/kg body weight/h) and xylazine (2.4 mg/kg body weight/h) through a catheter placed in an ear vein. The animals were placed in the supine position. After exposure of the trachea, an endotracheal tube was inserted and the animals were ventilated using a respirator (ventilation pressure, 12 mmhg; air to O2 ratio, 0.21–0.40; respiratory rate, 25–30 breaths per min with an inspiratory to expiratory ratio of 1:2) (Sechrist IV-100 B Infant Ventilator, Anaheim, cA). The right common carotid artery was isolated and a catheter (cavafix® MT, B. Braun Melsungen Ag, Melsungen, germany) with a maximum flow rate of 10 ml/min was introduced. The catheter served to control the arterial blood pressure and was used for the injection of microspheres. The catheter was connected to an online pressure monitoring system (Sirecust 304 D, Sie-mens, Munich, germany) and advanced into the left ven-tricle. correct position of the catheter’s tip was established by observing the typical waveform of a left ventricular pressure curve. For collection of the arterial blood sam-ple, a second catheter (cavafix® MT, B. Braun Melsungen Ag, Melsungen, germany) was introduced into the left carotid artery and advanced into the aorta. Blood pressure and heart rate were monitored continuously throughout the experiment. The first measurement was at 100 mmhg mean arterial blood pressure (MAp).Therefore if required, the mean arterial blood pressure was raised to 100 mmhg MAp by intravenous infusion of 6 % haemofusin (Baxter, unterschleißheim, germany). After injection of spheres, MAp was adjusted to 90, 80, 60, 40 and 30 mmhg by withdrawal of venous blood. MAp was maintained at the predetermined level for 20 min until the injection of micro-spheres was performed. Before each injection, arterial paO2, pacO2 and ph were determined [7].

Microsphere methodology

Six types of FM (blue, blue-green, yellow-green, orange, red and crimson, Molecular probes, eugene, Or; 15.5 ± 0.3 μm diameter, in 0.15 mol/l Nacl containing 0.05 % Tween 20) were used for the experiments. FM

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were protected from long-term exposure of light to prevent loss of fluorescence intensity. 106 FM per kilogram body weight were aspirated into a syringe after 3 min of vortex mixing, 5 min of sonication, followed again by 3 min of vortex mixing, and diluted to a total volume of 10 ml with 0.9 % Nacl. For calculating cardiac output, a 20 μl ali-quot of the suspension was stored for later determination of the total number of spheres administered. Before each injection, the correct position of the injection catheter was verified by checking left ventricular pressure curve. Blood pressure and heart rate were recorded. Arterial reference blood sampling was started 15 s before injection of the FM and was continued for 2 min. The withdrawal rate of the harvard pump (‘33’Syringe pump, FMI, engelsbach, germany) was 3.54 ml/min. The same person throughout all the experiments injected FM manually during a period of 1 min. The interval between two consecutive injections was approximately 30 min (microsphere preparation and stabilization of MAp for 20 min). After the last injection of microspheres, the animals were killed by intravenous administration of a lethal dose of pentobarbital (Nar-coren®, rhone Merieux gmbh, Laupheim, germany) and both kidneys, humeri, femora and tibias were retrieved. After removing connective and fatty tissues, each kid-ney was dissected into eight samples. Furthermore, both femurs and tibias were cleaned carefully from soft tis-sues and cartilage and dissected according to a standard-ized scheme [7]. After dissection, each tissue sample was weighed.

During the experiment, manipulation of the bone was not necessary due to indirect blood flow measurement. After the experiment, processing of the probes was essen-tial to regain fluorescence intensity and calculate bone blood flow.

FM were isolated from arterial reference blood and kid-ney samples immediately after the experiment and fluores-cence intensity was measured. Fluorescence intensity of the bone samples was determined after dissolving the crystal-line matrix for 3 weeks in hydrochloric acid (1 mol/l). Flu-orescence of tissue samples was measured automatically in our institute according to Anetzberger et al. [7] by means of an automated system [9] using a sample-processing unit (gaiser, Kappel-grafenhausen, germany) [12]. Fluo-rescent data were used to calculate blood flow values (ml/min/100 g).

where Fsample is the blood flow in the sample in ml/min, Freference the withdrawal rate of the harvard pump (3.54 ml/min), Isample the fluorescence intensity of individual tissue sample and Ireference the fluorescence intensity of the refer-ence blood sample.

Fsample = Freference × Isample × Ireference−1

To allow the comparison of different samples, the cal-culated blood flow value of the individual tissue sample (Fsample) was divided by the sample weight and normalized to 100 g. cardiac output (cO) was determined as follows:

where cO is the cardiac output in ml/min/kg, Freference the withdrawal rate of the harvard pump (3.54 ml/min), Iinjected the fluorescence intensity of totally injected FM, Ireference the fluorescence intensity of the reference blood sample and bwt the body weight. Vascular resistance:

Statistical analysis and calculations

The statistical analysis was done using the SpSS software package for Windows (Version 19, SpSS Inc., chicago, uSA).

All data are reported as mean ± standard error of mean. By using the nonparametric Friedman test for related samples, variance on ranks was tested to determine sig-nificant differences between repeated measurements. Wil-coxon signed rank test was used to evaluate the differences between single measurements and left and right specimens of bones. probability values less than 0.05 were considered to be significant.

Results

physiological parameters

MAp could be adjusted exactly (± 1 mmhg) to the level required. cO was 219 ± 64 ml/min/kg at 100 mmhg, 202 ± 46 ml/min/kg at 90 mmhg and 169 ± 47 ml/min/kg at 80 mmhg, respectively. cO then significantly decreased to139 ± 47 ml/min/kg at 60 mmhg, 96 ± 24 ml/min/kg at 40 mmhg and 65 ± 42 ml/min/kg at 30 mmhg. heart rate was constant between 100 and 80 mmhg MAp (174 ± 14 to 164 ± 11 min−1) and decreased significantly at 60 mmhg MAp to 156 ± 21 min−1. Further statistically significant decrease of heart rate was observed at 40 mmhg MAp (153 ± 16 min−1) and 30 mmhg(138 ± 20 min−1) (p < 0.05; Friedman test, post hoc test: Wilcoxon test).

Arterial blood gases remained constant throughout the experiment (Friedman test, p > 0.05). All physiological parameters are listed in Table 1.

Blood flow of the kidneys and long bones

comparison of blood flow of the right and left kid-neys resulted in identical values at each blood pressure

CO = Freference × Iinjected × Ireference−1

× bwt−1

Rorgan = MAP/Blood flowOrgan (ml/min/100 g)

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level. The mean blood flow of both kidneys showed a minor decrease between 100 and 80 mmhg MAp. At 100 mmhg, the blood flow of the right kidney was 299 ± 43 ml/min/100 g (left: 295 ± 39 ml/min/100 g); at 90 mmhg, the blood flow was 305 ± 36 ml/min/100 g (left: 298 ± 35 ml/min/100 g); at 80 mmhg, the blood flow was 329 ± 39 ml/min/100 g (left: 323 ± 35 ml/min/100 g). At 60 mmhg MAp, a decrease of blood flow was found to 228 ± 25 ml/min/100 g (left: 233 ± 26 ml/min/100 g). At 40 mmhg MAp, a statistically signifi-cant decrease of blood flow to 153 ± 18 ml/min/100 g (left: 146 ± 18 ml/min/100 g) and at 30 mmhg MAp to 91 ± 19 ml/min/100 g (left: 85 ± 19 ml/min/100 g) was

observed (Friedman test < 0.05, post hoc test: Wilcoxon test). Blood flow values of the kidney are illustrated in Fig. 1 and shown in Table 2. Values in percent of blood flow are listed in Table 3.

Mean blood flow of both femurs at 100, 90 and 80 mmhg MAp showed a minor decrease from 12.2 ± 0.9 to 10.9 ± 1.0 ml/min/100 g (left: 13 ± 1.2 to 11.3 ± 1.1 ml/min/100 g). The decrease was not statistically signifi-cant (p > 0.05 Friedman test). At 60 mmhg MAp, a statisti-cally significant decrease to 7.6 ± 0.7 ml/min/100 g (left: 7.9 ± 0.8 ml/min/100 g) was found (p < 0.05 Friedman test, post hoc test: Wilcoxon test). Blood flow at 40 mmhg was 5.6 ± 0.6 ml/min/100 g (left: 5.5 ± 0.6 ml/min/100 g) and at 30 mmhg 3.8 ± 0.5 ml/min/100 g (left: 3.6 ± 0.4 ml/min/100 g). Blood flow of the right and left femur showed minor variations (p > 0.05 Friedman test).

The mean blood flow of both tibias at 100, 90 and 80 mmhg MAp showed a slight decrease from 6.7 ± 0.8 to 6.0 ± 0.9 ml/min/100 g (left: 7.0 ± 0.8 to 6.3 ± 0.9 ml/min/100 g) The decrease of blood flow was not statisti-cally significant (p > 0.05 Friedman test). At 60 mmhg, the blood flow decreased statistically significantly to 3.7 ± 0.6 ml/min/100 g (left: 4.4 ± 0.5 ml/min/100) (p < 0.05; Friedman test, post hoc test: Wilcoxon test). A further decrease of blood flow was observed at 40 mmhg (right: 2.7 ml/min/100 g; left: 3.0 ml/min/100 g) and at 30 mmhg MAp (right: 1.7 ± 0.3 ml/min/100 g; left: 2.0 ± 0.3 ml/min/100 g). The blood flow of the right and left tibia showed minor variations that were not statistically significant (p > 0.05 Friedman test). The blood flow values of the femur and tibia are illustrated in Figs. 2a and 3.

The blood flow values and percent of blood flow of both femurs and tibias are listed in Tables 2 and 3.

Fig. 1 Blood flow of the kidney. Values of renal bloodflow (ml/min/100 g) in both kidneys at hypotension. Values are means ± SeM, (n = 8). a significantly different to 100 mmhg MAp (p < 0.05), b significantly different to 90 mmhg MAp (p < 0.05), c significantly different to 80 mmhg MAp (p < 0.05), d significantly different to 60 mmhg MAp (p < 0.05), e significantly different to 40 mmhg MAp (p < 0.05); bold right extremity, italic left extremity

Table 1 physiologic parameters

Values are means ± SeM, (n = 8)

MAP mean arterial blood pressure, HR heart rate, CO cardiac output, Hb hemoglobin, Hct hematocrit, PaO2 arterial partial pressure of O2, PaCO2 arterial partial pressure of cO2a Significantly different to 100 mmhg MAp (p < 0.05)b Significantly different to 90 mmhg MAp (p < 0.05)c Significantly different to 80 mmhg MAp (p < 0.05)d Significantly different to 60 mmhg MAp (p < 0.05)e Significantly different to 40 mmhg MAp (p < 0.05)

parameter MAp 100 mmhg MAp 90 mmhg MAp 80 mmhg MAp 60 mmhg MAp 40 mmhg MAp 30 mmhg

MAp (mmhg) 97 ± 3 89 ± 1a 80 ± 0a,b 60 ± 1a,b,c 40 ± 0a,b,c 30 ± 1a,b,c

hr (min−1) 164 ± 11 174 ± 14 166 ± 17 156 ± 21b 153 ± 16b,c 138 ± 20b,c,d,e

cO (ml x min−1 × kg−1) 219 ± 64 202 ± 46 169 ± 47 139 ± 47b 96 ± 24b,c,d 65 ± 42b,c,d

hb (g/dl) 9.2 ± 2.1 8.3 ± 2.1 7.9 ± 1.7 7.9 ± 2.0c 7.5 ± 1.6d 6.9 ± 1.4c,d,e

hct (%) 26 ± 6 22 ± 6 22 ± 6 23 ± 7c 21 ± 5d 19 ± 4c,d,e

paO2 (mmhg) 122 ± 27 110 ± 26a 127 ± 26 111 ± 21 112 ± 15 112 ± 17

pacO2 (mmhg) 24 ± 6 29 ± 7 26 ± 7 29 ± 6 29 ± 7 30 ± 5

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Vascular resistance

Decreasing blood pressure level from 100 to 80 mmhg MAp leads to a decrease in vascular resistance of the right kidneys from 0.33 ± 0.04 mmhg/(ml/min/100 g) (left: 0.34 ± 0.03 mmhg/(ml/min/100 g)) to 0.24 ± 0.025 mmhg/(ml/min/100 g) (left: 0.25 ± 0.024/(ml/min/100 g)) (p < 0.05; Friedman test, post hoc test: Wilcoxon test). Vascular resistance of the right kid-ney at 60 mmhg MAp was 0.26 ± 0.036 mmhg/(ml/min/100 g) (left: 0.26 ± 0.031 mmhg/(ml/min/100 g)), at 40 mmhg MAp was 0.26 ± 0.024 mmhg/(ml/min/100 g) (left: 0.27 ± 0.031 mmhg/(ml/min/100 g)). At 30 mmhg MAp, an increase of vascular resistance of the right kidney to 0.33 ± 0.07 mmhg/(ml/min/100 g) (left: 0.35 ± 0.09 mmhg/(ml/min/100 g)) (p < 0.05 Friedman test, post hoc test: Wilcoxon test) was found.

Vascular resistance of the femur and tibia showed the same disposition. Between 100 and 80 mmhg MAp, a

decrease of vascular resistance was found. At 30 mmhg MAp, an increase of vascular resistance was established, whereas changes were not statistically significant (p > 0.05 Friedman test). The values of vascular resistance of both kidneys, femurs and tibias are specified in Table 4.

Blood flow distribution within long bones

The blood flow of the anatomic regions of the femur and tibia showed minor variations between 100 and 80 mmhg MAp not being statistically significant (p > 0.05 Fried-man test). At 60 mmhg MAp, most regions showed a sta-tistically significant decrease of regional bone blood flow (rBBF), as shown in Fig. 2b (p < 0.05 Friedman test, post hoc test: Wilcoxon test).

regarding femur, the decrease of blood flow at 30 mmhg MAp was between −62 and −74 % compared to the initial value at 100 mmhg MAp. The greatest decrease of rBBF of the femur was found in the distal metaphysis

Table 2 Blood flow values

Values of blood flow (ml/min/100 g) in different organs at hypotension. Values are means ± SeM (n = 8)a Significantly different to 100 mmhg MAp (p < 0.05)b Significantly different to 90 mmhg MAp (p < 0.05)c Significantly different to 80 mmhg MAp (p < 0.05)d Significantly different to 60 mmhg MAp (p < 0.05)e Significantly different to 40 mmhg MAp (p < 0.05)

Organ MAp 100 mmhg MAp 90 mmhg MAp 80 mmhg MAp 60 mmhg MAp 40 mmhg MAp 30 mmhg

Kidney right 299 ± 43 305 ± 36 329 ± 39 228 ± 25 153 ± 18a,b 91 ± 19a,b

Kidney left 295 ± 39 298 ± 35 323 ± 35 233 ± 26 146 ± 18a,b 85 ± 19a,b

Femur right 12.2 ± 0.9 11.6 ± 1 10.9 ± 1.0 7.6 ± 0.7a,b,c 5.6 ± 0.6a,b,c,d 3.8 ± 0.5a,b,c,d,e

Femur left 13 ± 1.2 12.2 ± 1.2 11.3 ± 1.1 7.9 ± 0.8a,b,c 5.5 ± 0.6a,b,c,d 3.6 ± 0.4a.b.c.d.e

Tibia right 6.7 ± 0.8 6.4 ± 0.9 6.0 ± 0.9 3.7 ± 0.6a,b,c 2.7 ± 0.3a,b,c,d 1.7 ± 0.3a,b,c,d,e

Tibia left 7.0 ± 0.8 6.4 ± 0.7 6.3 ± 0.9 4.4 ± 0.5a,b,c 3 ± 0.4a,b,c,d 2 ± 0.3a,b,c,d,e

Table 3 percent values of bloodflow

Values of blood flow in percent in different organs at hypotensiona Significantly different to 100 mmhg MAp (p < 0.05)b Significantly different to 90 mmhg MAp (p < 0.05)c Significantly different to 80 mmhg MAp (p < 0.05)d Significantly different to 60 mmhg MAp (p < 0.05)e Significantly different to 40 mmhg MAp (p < 0.05)

Organ MAp 100 mmhg MAp 90 mmhg MAp 80 mmhg MAp 60 mmhg MAp 40 mmhg MAp 30 mmhg

Kidney right 100 % 102 % 110 % 76 % 51 %a,b, 30 %a,b

Kidney left 100 % 101 % 109 % 79 % 49 %a,b, 29 %a,b,

Femur right 100 % 95 % 89 % 62 %a,b,c 46 %a,b,c,d 31 %a,b,c,d,e

Femur left 100 % 94 % 87 % 61 %a,b,c 42 %a,b,c,d 28 %a,b,c,d,e

Tibia right 100 % 96 % 90 % 55 %a,b,c 40 %a,b,c,d 25 %a,b,c,d,e

Tibia left 100 % 91 % 90 % 63 %a,b,c 43 %a,b,c,d 29 %a,b,c,d,e

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(−74 %), femoral head (−73 %) and both condyles (−72 %, respectively, −71 % of the initial value).

regarding tibia, the decrease of rBBF at 30 mmhg MAp varied between −72 and −81 %.The great-est decrease of rBBF was found at the distal diaphysis (−81 %) and distal metaphysis (−79 %). The values of rBBF of all anatomical regions are specified in Tables 5 and 6.

Discussion

Autoregulation is known as the maintenance of constant blood flow within a vascular system at hypotension. previ-ous data from animal studies indicate that bone blood flow is dependent on hemodynamic parameters and is altered by neural, hormonal and metabolic mechanisms [13–18]. Due to the presence of smooth muscle cells in arteries and small arteries, regulation of blood flow due to either vasodilata-tion or vasoconstriction is possible [19, 20]. In this study

we first demonstrated that an autoregulatory mechanism of bone blood flow is present in long bones ensuring constant blood flow despite hypotension.

Although bone cells (osteoblasts and osteoclasts) are very resistant to ischemia [21], regional bone blood flow regulation by a bone intrinsic mechanism might be impor-tant. By adequate bone marrow perfusion [4, 5], protection of vulnerable hematopoietic stem cells can be guaranteed. It is well known that severe hemorrhagic shock leads to apoptosis of bone marrow mononuclear cells [4].

In our experimental setup, the model of acute blood loss was used representing a common model to induce hypoten-sion as well as hypovolemic shock [22]. We found equal blood flow values in the right and left kidney and bone samples within femurs and tibias, which is consistent with values reported in previous studies [23–28]revealing that our study protocol was correct.The curve shape of organ blood flow during hypotension, characteristic for autoregu-latory mechanism, can be described as sigmoidal [29].

regional bone blood flow of the kidneys was stable between 100 and 60 mmhg MAp and then decreased. This sigmoidal type of curve is typical for autoregulation and could also be observed for femurs and tibias. Identical curve shapes could be observed for all regions of the femur and tibia. rBBF remained constant at moderate changes of arterial pressure. From 60 mmhg MAp downward, the decrease in rBBF was linearly dependent on MAp. This demonstrates that during acute hypovolemic shock, the autoregulatory mechanism can no longer be maintained and rBBF directly depends on the perfusion pressure.

The heterogeneity of perfusion within a long bone did not change during moderate and severe hypoten-sion. This observation supports the assumption that

Fig. 2 Blood flow of the femur. a Blood flow of the whole femur; b regional bone blood flow of the collum femoris. Values of bone blood flow (ml/min/100 g) at hypotension. Values are means ± SeM, (n = 8). a Significantly different to 100 mmhg MAp (p < 0.05), b significantly different to 90 mmhg MAp (p < 0.05), c significantly different to 80 mmhg MAp (p < 0.05), d significantly different to 60 mmhg MAp (p < 0.05), e significantly different to 40 mmhg MAp (p < 0.05); bold: right extremity, italic: left extremity

Fig. 3 Blood flow of the whole tibia. Values of bone blood flow (ml/min/100 g) at hypotension. Values are means ± SeM, (n = 8). a Sig-nificantly different to 100 mmhg MAp (p < 0.05), b significantly different to 90 mmhg MAp (p < 0.05), c significantly different to 80 mmhg MAp (p < 0.05), d significantly different to 60 mmhg MAp (p < 0.05), e significantly different to 40 mmhg MAp (p < 0.05); bold: right extremity, italic: left extremity

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different bone regions have a common mechanism for control and maintenance of blood flow. cardiac output remained unchanged at moderate hypotension, which can be explained by a modification of the peripheral vascular resistance of other organs. Decrease of MAp from 100 to 40 mmhg resulted in a decrease in vascular resistance in bone and kidney, indicating that blood vessels may play a part in the autoregulatory mechanism by alteration of its diameter. Vasodilatation is probably one physiologi-cal mechanism that increases blood flow. At 30 mmhg, an

increase in vascular resistance in long bones as well as in kidneys was observed representing the response to hypo-volemic shock, the consequence being that in the trauma situation low blood loss may be compensated by physi-ologic mechanisms whereas severe blood loss cannot be compensated for.

Anetzberger et al. [7] validated the FM method for bone blood flow measurement during hypotension at 90, 70 and 50 mmhg MAp. They observed that blood flow in kidney sand bone was constant between MAp of 90 and 70 mmhg

Table 4 Vascular resistance

Values of vascular resistance, mmhg ml−1 min 100 g−1 of different organs at hypotension. Values are means ± SeM, (n = 8)a Significantly different to 100 mmhg MAp (p < 0.05)b Significantly different to 90 mmhg MAp (p < 0.05)c Significantly different to 80 mmhg MAp (p < 0.05)d Significantly different to 60 mmhg MAp (p < 0.05)e Significantly different to 40 mmhg MAp (p < 0.05)

Organ MAp 100 mmhg MAp 90 mmhg MAp 80 mmhg MAp 60 mmhg MAp 40 mmhg MAp 30 mmhg

Kidney right 0.33 ± 0.038 0.30 ± 0.026 0.24 ± 0.025a 0.26 ± 0.036a 0.26 ± 0.024a 0.33 ± 0.071c,d,e

Kidney left 0.34 ± 0.034 0.30 ± 0.028 0.25 ± 0.024a 0.26 ± 0.031a 0.27 ± 0.031a 0.35 ± 0.091c,d,e

Femur right 8.2 ± 1.0 7.8 ± 1.8 7.3 ± 1.8 7.9 ± 1.6 7.1 ± 2.8 7.9 ± 2.6

Femur left 7.7 ± 1.3 7.4 ± 2.0 7.1 ± 2.5 7.6 ± 1.8 7.3 ± 2.4 8.3 ± 2.7

Tibia right 14.9 ± 2.4 14.0 ± 1.5 13.2 ± 2.2 16.2 ± 2.3 14.8 ± 2.4 17.6 ± 2.9

Tibia left 14.3 ± 2.3 14.1 ± 1.9 12.7 ± 2.0 13.6 ± 1.9 13.3 ± 2.0 15.0 ± 2.3

Table 5 Bone blood flow values of different regions of the femur

Values of blood flow (ml/min/100 g) of the femur. Values are mean ± SeM, (n = 8); upper value: right extremity, lower value: left extremitya Significantly different to 100 mmhg MAp (p < 0.05)b Significantly different to 90 mmhg MAp (p < 0.05)c Significantly different to 80 mmhg MAp (p < 0.05)d Significantly different to 60 mmhg MAp (p < 0.05)e Significantly different to 40 mmhg MAp (p < 0.05)

region MAp 100 mmhg MAp 90 mmhg MAp 80 mmhg MAp 60 mmhg MAp 40 mmhg MAp 30 mmhg

caput femoris 7.4 ± 1.96.3 ± 1.4

9.3 ± 4.710.4 ± 4.3

7.9 ± 2.76 ± 2.2

5.9 ± 2.4b

5 ± 2.2b1.8 ± 1.2a,b,c,d

2.3 ± 1a,b,c,d1.8 ± 1.1a,b,c,d

1.9 ± 0.9a,b,c,d

collum femoris 13.0 ± 2.213.9 ± 3.6

12.7 ± 3.114.4 ± 4.5

15.1 ± 4.515.9 ± 5.1

7.9 ± 2.6a,b,c

9.5 ± 3a,b,c5.3 ± 1.3a,b,c,d

5.5 ± 1.5a,b,c,d3.9 ± 1a,b,c,d,e

3.3 ± 0.9a,b,c,d,e

Trochanter major 18.1 ± 2.419.6 ± 1.9

16.2 ± 4.419.8 ± 3.9

16.1 ± 4.319.0 ± 4

11.6 ± 2.7a,b,c

13.4 ± 2.5a,b,c8.5 ± 1.6a,b,c,d

10 ± 1.8a,b,c,d4.9 ± 1.5a,b,c,d,e

6.7 ± 1.1a,b,c,d,e

Metaphysis proximalis

17.1 ± 314.3 ± 2.9

14.6 ± 2.713.2 ± 1.9

14.3 ± 2.611.3 ± 1

9.6 ± 1.6a,b,c

9.3 ± 1a,b,c9 ± 2.5a,b,c

8.2 ± 1.5a,b,c7.5 ± 1.9a,b,c,d,e

4.5 ± 1.3a,b,c,d,e

Diaphysis 10.9 ± 1.410.5 ± 1.3

10 ± 2.38.3 ± 2.4

8.7 ± 28.6 ± 2.4

6.5 ± 1.2a,b,c

4.7 ± 1.5a,b,c5 ± 0.9a,b,c,d

3.2 ± 0.8a,b,c,d3.3 ± 1.2a,b,c,d,e

2 ± 0.8a,b,c,d,e

Metaphysis distalis 13.7 ± 2.715.6 ± 4.3

11.2 ± 1.911.7 ± 2.8

10.7 ± 211.6 ± 2.9

7.7 ± 1.7a,b,c

7.8 ± 1.4a,b,c6.3 ± 1.9a,b,c,d

5.1 ± 1.1a,b,c,d4.1 ± 1.1a,b,c,d,e

3.6 ± 0.8a,b,c,d,e

condylus medialis 10.3 ± 1.69.7 ± 1.5

10.1 ± 2.18.6 ± 2.3

8 ± 28.6 ± 2.3

6.2 ± 1.5a,b,c

5.5 ± 1.5a,b,c4.8 ± 0.7a,b,c

4.1 ± 0.8a,b,c2.9 ± 0.9a,b,c,d,e

2.8 ± 0.8a,b,c,d,e

condylus lateralis 7.6 ± 1.813.7 ± 5.9

9 ± 1.411 ± 3.6

6.8 ± 1.58.4 ± 2.3

5.1 ± 0.6a,b

7.3 ± 2.6a,b3.7 ± 0.4a,b,c,d

5.6 ± 2.5a,b,c,d2.2 ± 0.5a,b,c,d,e

3.9 ± 2a,b,c,d,e

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MAp. Decreasing blood pressure to 50 mmhg MAp was followed by decreased bone and kidney blood flow values.

Taking the results of Anetzberger et al. [7] into account, bone vessels have a mechanism for autoregulating flow between 100 and 70 mmhg MAp. The decrease in bone blood flow followed by reduction of MAp to 60 mmhg indicates the response to acute blood loss. These findings are similar to those of gross [16] and yu et al. [30]. yu et al. [30] were the first to describe a gradual fall of sys-temic blood pressure and central venous pressure due to acute hemorrhage.

This indicates that perfusion of bones and bone marrow in severe hypotension, e.g., induced by acute hemorrhage mainly depends on the perfusion pressure in contrast to moderate hypotension.

Conclusion

using the FM method, we can show that bone blood flow during hypotension is very similar to blood flow observed in the kidney. This finding indicates that blood flow within bone tissue underlies autoregulation and for the first time provides evidence that the physiological concept of autoregulated organ perfusion also applies to bone.

Acknowledgments This work was supported by KKF 8745155 Tu Munich.

Conflict of interest There are no conflicts of interest.

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Table 6 Bone blood flow values of different regions of the tibia

Values of blood flow (ml/min/100 g) of the tibia. Values are means ± SeM, (n = 8); upper value: right extremity, lower value: left extremitya Significantly different to 100 mmhg MAp (p < 0.05)b Significantly different to 90 mmhg MAp (p < 0.05)c Significantly different to 80 mmhg MAp (p < 0.05)d Significantly different to 60 mmhg MAp (p < 0.05)e Significantly different to 40 mmhg MAp (p < 0.05)

region MAp 100 mmhg MAp 90 mmhg MAp 80 mmhg MAp 60 mmhg MAp 40 mmhg MAp 30 mmhg

condylus medialis 9.9 ± 1.88.3 ± 2.3

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11.1 ± 1.814.4 ± 2.3

8.8 ± 2.211.2 ± 2.4

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3.9 ± 0.7a,b,c,d,e

Diaphysis proximalis

5.6 ± 1.86.5 ± 1.1

4.8 ± 15.4 ± 0.6

6.0 ± 1.98.7 ± 4.2

2.2 ± 0.6a,b

3.4 ± 0.7a,b1.7 ± 0.5a,b,c

2.5 ± 0.7a,b,c1 ± 0.3a,b,c,d,e

1.8 ± 0.5a,b,c,d,e

Diaphysis distalis 3.9 ± 1.13.5 ± 1.1

2.7 ± 0.94.5 ± 1.1

2.5 ± 1.12 ± 0.9

1.6 ± 0.4a

1.8 ± 0.3a1.2 ± 0.4a,b,d

0.9 ± 0.2a,b,d0.8 ± 0.6a,b,c,d

0.6 ± 0.2a,b,c,d

Metaphysis distalis 4.9 ± 1.64.6 ± 1.4

3.6 ± 1.16.1 ± 2.6

3.6 ± 1.84.4 ± 1.7

2.2 ± 0.63.1 ± 1

1.1 ± 0.4a,b,c,d

1.4 ± 0.5a,b,c,d0.7 ± 0.2a,b,c,d

1.2 ± 0.5a,b,c,d

epiphysis distalis 3.3 ± 0.93.9 ± 1.1

4.5 ± 1.55.1 ± 1.7

5.7 ± 2.83.3 ± 1.4

2.7 ± 13.3 ± 1.1

1.3 ± 0.5a,b,c,d

1.2 ± 0.4a,b,c,d0.8 ± 0.3a,b,d

1.2 ± 0.3a,b,d

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