New techniques involving Near Infrared Spectroscopy (NIRS) and imaging are rapidly evolving for a large number of new clinical applications. These techniques, based upon nearinfrared light transmission through biological tissue, aim to monitor the hemoglobin and myoglobin concentration changes due to particular physiological state. Clinical applications regard, for instance, the monitoring of muscles and cerebral oxygenation, functional brain activation studies and heart perfusion research. Recently, some works presented tissue oxygenation studies in beating or arrested isolated porcine hearts. In our work we present the design and realization of a dedicated NIRS system for the myocardial perfusion analysis of isolated, saline solution perfused beating rat hearts; in this case the absence of blood allows for unambiguous measurement of tissue myoglobin oxygenation. The presented prototype is portable, low cost, battery operated and permits the measurement of both oxy and deoxy myoglobin concentration changes during imposed regional or global ischemia and reperfusion

Benassi, Antonio

Francesconi, Raffaello

Giovannetti, Giulio

Hartwig, Valentina

Kusmic, Claudia

L\u27Abbate, Antonio

Landini, Luigi

PUblication MAnagement

The 3rd European Medical and Biological Engineering Conference November 20 – 25, 2005 
EMBEC'05  Prague, Czech Republic 
IFMBE Proc. 2005 11(1)  ISSN: 1727-1983 © 2005 IFMBE  
 MEASUREMENT OF TISSUE OXYGENATION IN ISOLATED RAT 
HEARTS USING NEAR INFRARED SPECTROSCOPY 
 
V. Hartwig*, G. Giovannetti*, R. Francesconi*, C. Kusmic*, L. Landini**, A. L’Abbate*, 
A. Benassi* 
 
* C.N.R. Institute of Clinical Physiology, Pisa, Italy 
** Department of Information Engineering, University of Pisa, Italy 
 
valeh@ifc.cnr.it 
 
 
Abstract: New techniques involving Near Infrared 
Spectroscopy (NIRS) and imaging are rapidly 
evolving for a large number of new clinical 
applications. These techniques, based upon near-
infrared light transmission through biological tissue, 
aim to monitor the hemoglobin and myoglobin 
concentration changes due to particular 
physiological state. Clinical applications regard, for 
instance, the monitoring of muscles and cerebral 
oxygenation, functional brain activation studies and 
heart perfusion research.  Recently, some works 
presented tissue oxygenation studies in beating or 
arrested isolated porcine hearts. In our work we 
present the design and realization of a dedicated 
NIRS system for the myocardial perfusion analysis 
of isolated, saline solution perfused beating rat 
hearts; in this case the absence of blood allows for 
unambiguous measurement of tissue myoglobin 
oxygenation. The presented prototype is portable, 
low cost, battery operated and permits the 
measurement of both oxy and deoxy myoglobin 
concentration changes during imposed regional or 
global ischemia and reperfusion. 
  
Introduction 
 
 Several non-invasive techniques, like positron 
emission tomography (PET), single photon emission 
computer tomography (SPECT) and magnetic resonance 
imaging (MRI), are currently used to image tissue 
damaged by an ischemia. Unfortunately, these methods 
are difficult to use under particular condition as open-
heart surgery, because of equipment size and 
characteristics. An amply used technique is X ray 
angiography, but it uses ionizing radiation and does not 
provide any information about tissue oxygenation 
depending on capillary flow.     
 In order to measure the tissue oxygenation in 
isolated hearts, the Near InfraRed Spectroscopy (NIRS) 
technique is considered. This technique has been used 
widely to measure hemoglobin (Hb) oxygenation in 
skeletal muscle and brain tissue [1]: it makes use of the 
near-IR spectra of hemoglobin/myoglobin and its 
oxygenated counterpart. 
 The more effective penetration of near infrared light 
into tissue respect to the visible light, and the very 
different in the IR absorption spectra of oxy and deoxy 
hemoglobin and myoglobin (Mb), are the reasons for 
which this technique is used in tissue oxygenation 
measurements application. In fact, IR light absorption is 
proportional to the concentration of these absorbing 
species so it is possible to have information on the tissue 
perfusion using IR light emitters and detectors. 
 Since the NIR absorption of hemoglobin and 
myoglobin is the same, the NIRS technique can be also 
used to measure Mb oxygenation in isolated heart 
perfused with saline solution [1]. The myoglobin 
oxygenation level in the beating isolated heart responds 
clearly to variations in the oxygenation perfusion and 
differences in O2 consumption are evident comparing 
the behaviour of the heart.  
 The manifestations of an ischemia, such as tissue 
deoxygenation, can be used as diagnostic tools for the 
assessment, visualization, and localization of ischemia 
and infarction. 
The aim of this work is the design and realization of 
a prototype using the NIRS technique to provide 
information regarding myocardial O2 perfusion of 
isolated, saline solution perfused beating rat hearts: in 
this report we describe the prototype and the 
experimental protocol for its preliminary test. 
 
Materials and Methods 
 
The near infrared region extended from 750 to 2000 
nm: light in the 750-1100 nm range has the 
characteristic of penetrating biological tissue for few 
centimeters. In this spectral region the major 
chromophores (pigments which absorb the light) of 
biological tissue are hemoglobin and myoglobin, which 
have a near infrared spectrum essentially identical.  
Absorption spectra of these metalloproteins are 
sensitive to oxygenation so near infrared spectroscopy 
can yield information on tissue oxygenation and 
perfusion. For this reason NIRS can be used to measure 
Mb oxygenation in isolated hearts and to obtain a map 
of perfusion areas during particular physiological state 
as regional or global ischemia and reperfusion.  
Some papers in literature report several cases of 
NIRS study on isolated arrested blood perfused porcine 
hearts [2] or saline solution perfused isolated rabbit 
hearts [3].  
We present a study on a saline solution perfused 
beating isolated rat heart using a home-made prototype.    
The 3rd European Medical and Biological Engineering Conference November 20 – 25, 2005 
EMBEC'05  Prague, Czech Republic 
IFMBE Proc. 2005 11(1)  ISSN: 1727-1983 © 2005 IFMBE  
 The designed device is constituted by two light 
emitting diodes (LED) which emit two infrared 
wavelength lights (780 nm OD 780W, Opto Diode 
Corporation and 880 nm SFH 845P, Siemens). The two 
wavelengths chosen are about the peak absorption 
wavelength of deoxy Hb/Mb and oxy Hb/Mb 
respectively (Figure 1). 
Light at wavelength lesser than 700 nm do not 
penetrate enough into the tissue and at wavelength 
greater than 900 nm the water absorption effect comes 
predominant. 
 
 
 
Figure 1: Absorption spectra of oxy and deoxy Hb and 
Mb 
 
To receive the light that passed through the tissue, 
we used a PIN photodiode (SFH 203P, Siemens) that 
converts infrared light photons in an electric current. 
The electronic part acts the light sources driving and 
the detector signal acquisition and pre processing.  
The two LEDs are powered by two square waves 
with frequencies of 27 KHz (880 nm) and 57 KHz (780 
nm), in order to recognize one signal for each light on 
the output signal from the only one detector. A mixed 
circuit splits the signal received by the detector in two 
waves relative to the two different wavelengths of 
emitted lights.  
 The whole system is battery operated and is 
provided with a plastic chassis (Figure 2). 
We designed a mechanical support for the probes in 
order to embrace the heart: the circular support is made 
of black PVC and has three holes for the two LEDs and 
the detector, respectively, placed with an angle of 120° 
one to each other. Figure 3 shows the probes support 
schematic diagram.  
The signals received to the readout electronic circuit 
are acquired by an acquisition card (DAQ6024E 
National Instruments) and visualized on a PC monitor 
using LabView software (National Instruments).  
 
 
 
 
Figure 2: System prototype 
 
 
 
 
Figure 3: Mechanical support for the NIRS probes 
(dimensions unit = mm) 
 
We created an interface in LabView for the 
visualization of the two signals on a graph relative to the 
two modulation frequencies. The interface calculates the 
concentration changes of oxy Myoglobin (MbO2) and 
deoxy Myoglobin using the modified Lambert-Beer law 
[4] for each of the two chosen wavelenght: 
 
                    GDPFlCeII +⋅⋅⋅−⋅= ε0                  (1) 
 
where I is the detected light intensity, I0 the incident 
light intensity, ε  the absorption coefficient of the 
medium, C the concentration of the medium, l the 
distance between light emitter and detector, DPF the 
differential path length factor and G the constant 
attenuation factor relative to the optical properties and 
geometry of the tissue. 
Preliminary tests were made with several substances 
with different IR absorption spectra in order to evaluate 
the prototype range of sensibility: we essentially used 
water, saline solution and human venous blood. 
Finally, we defined the experimental protocol for a 
rat isolated heart test: the animal was treated in 
accordance with the “Guiding Principles for Research 
Involving Animals and Human Beings” published by 
The American Physiological Society [5].  
The heart was isolated from Wistar rat and placed in 
a Krebs-Henseleit bicarbonate (KHB) solution bath with 
the following mmol/litre composition: 118 NaCl, 24 
NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 
0.5 EDTA, e 5 glucose. 
The 3rd European Medical and Biological Engineering Conference November 20 – 25, 2005 
EMBEC'05  Prague, Czech Republic 
IFMBE Proc. 2005 11(1)  ISSN: 1727-1983 © 2005 IFMBE  
 After foreign tissues removal, the aorta was 
cannulated and the heart was perfused according to 
Langerdoff at 37°C with a constant flow of KHB 
oxygenated with a mixture of 95% O2 and 5% CO2; the 
perfusion pressure was set at 65 mmHg. 
 
 
 
Figure 4: Langerdoff apparatus and the prototype for the 
experiment on a rat isolated heart  
 
The flow of KHB perfusion solution was measured 
and visualized on a monitor during all phases of the 
experiment.  Figure 4 shows the Langerdoff apparatus 
and the designed system: the heart chamber for the 
thermoregulation had to be removed for the placement 
of the probes support. 
The experimental protocol mimicked an ischemic 
condition followed by reperfusion by stopping and then 
restoring the oxygenation in the perfusion solution, 
respectively: the variation in oxy and deoxy Mb 
concentration was visualized on a PC monitor.  
 
  
Figure 5: Signal acquired during the venous human 
blood dilution test 
 
Results 
 
Figure 5 shows the acquired signal relative to the 
880 nm channel for a human venous blood dilution test: 
5 ml of venous human blood was diluted with 1 ml of 
physiologic solution.  
In correspondence of the dilution, the electric signal 
was quickly increased like we expected: the dilution, in 
fact, caused a decrement of Hb concentration and hence 
a decrement in O2 concentration, so the transmitted 
infrared light intensity increased. 
In the isolated rat heart experiment the output 
signals from the designed prototype are the Mb oxy and 
deoxy concentration variations regarding the whole 
heart; the two emitters and the detector are placed with 
an angle of 120 degrees between each other. Since the 
heart’s size is quite small, the infrared light penetrates 
into the myocardium and the results regard an 
integration of several contribution from all heart 
volume. In the first phase of experiment, after 1 minute 
of baseline condition (beating heart with normal 
perfusion), the oxygenation of the solution was stopped 
and a global ischemic condition was achieved. Under 
this hypoxic condition, the oxy-Mb level decreased 
quickly, with an half-time (t1/2) of about 6 sec.  
Figure 6 shows the relative level of MbO2 respect to 
the baseline condition (beating heart with normal 
perfusion): the upper graph shows the raw signal while 
the lower graph is the signal after a low pass filter 
operation (high cut frequency= 1Hz) acts by the 
implemented algorithm. 
 
 
 
 
Figure 6: MbO2 relative level during ischemic condition: 
no processed signal (up); low-pass filtered signal 
(bottom) 
 
After 100 sec of ischemic condition, the perfusion 
solution was reoxygenated. Unfortunately, the 
electronic signal did not recover as theoretically 
expected probably because the oxygen reperfusion itself 
has the potential to initiate both reversible and 
irreversible tissue injury following ischemia [6]. There 
are many mechanisms suspected of causing reperfusion 
injury, including the release of nitric oxide, reactive 
oxygen and reactive nitric oxide species. Some of them, 
namely nitric oxide and superoxide anion, react each 
other at a very fast rate (approaching the diffusion rate) 
[7] to form peroxinitrite, which causes irreversible 
The 3rd European Medical and Biological Engineering Conference November 20 – 25, 2005 
EMBEC'05  Prague, Czech Republic 
IFMBE Proc. 2005 11(1)  ISSN: 1727-1983 © 2005 IFMBE  
 inhibition of the mitochondrial respiratory chain and 
triggers apoptosis of cardiac myocytes [8]. 
In our test, the ishemia/riperfusion cycle was 
repeated two times and, finally, the perfusion solution 
was completely stopped. 
 
Discussion 
 
 The results obtained with our prototype regarding 
the MbO2 level, are coherent with the data reported on 
literature in a similar experimental case [9]. 
 In this work are reported only the results relative to 
the MbO2 variation changes in the rat isolated heart: the 
results regarding the Mb variations changes, in fact, 
were not good. Probably, this fact was due to the  
readout electronic circuit of the 780 nm channel, that is 
more affected from visible light and power line 
frequency electric noise. Moreover, changes in deoxy 
Mb concentration are much less strict. However, these 
problems can be eliminated using optical filters for the 
visible light or specific detectors, and electrical filter for 
the noise frequencies. 
 The very small size of the used heart did not permit 
a careful localization of the hypoxic perfusion in the 
myocardial area: the three NIRS probes were placed in 
contact with the heart and they covered a measurement 
area that corresponded to the whole heart zone.  
 Examining a larger heart, like a pig heart, the 
position of the probes can be chosen more accurately in 
order to obtain optimal information on the ischemic 
zone. 
   
  Conclusions 
  
 In this work-in-progress we describe a prototype for 
the measurement of oxy and deoxy Mb level in an 
isolated rat heart using NIRS technique, and the 
experimental protocol for its preliminary test.  
 Final system will be able to allow a real time 
monitoring of the changes in myocardial tissue 
oxygenation within the beating heart. 
 An  isolated rat heart perfused according to 
Langerdoff method was examined during an hypoxic/re-
oxygenation procedure: device output signal reflected 
the expected MbO2 level time course during the 
ischemia condition. 
 Future developments will regard the optimization of 
the 780 nm channel for the detection of deoxy Mb 
variation changes and a generic optimization of the 
electronic readout circuit to also measure the 
concentration changes during reperfusion condition. 
 Further optimization of the protocol could involve a 
measurement synchronism with the cardiac cycle in 
order to eliminate motion artifacts.  
 We are preparing in our laboratories a prototype for 
the 2D reconstruction of the myocardial perfusion map: 
the NIRS probes support rotates of 180° around the 
examined organ (human finger or isolated rat heart) and 
the system acquires a fixed number of measurements. A 
mathematical algorithm will have to act the 
reconstruction of the 2D organ image. 
 
References 
 
[1] SHAW R.A., MANSFIELD J.R., KUPRIYANOV 
V.V.,  MANTSCH H.H. (2000): ‘In vivo 
optical/near-infrared spectroscopy and imaging of 
metalloproteins’, Journal of Inorganic 
Biochemistry,  79, pp. 285–293 
 
[2] NIGHSWANDER-REMPEL S.P., SHAW R.A., 
MANSFIELD  J.R., HEWKO M., KUPRIYANOV 
V.V., MANTSCH H.H. (2002): ‘Regional 
variations in myocardial tissue oxygenation mapped 
by near infrared spectroscopic imaging‘, J. Mol. 
Cell. Cardiol., 34, pp. 1195-1203 
 
[3]  DE GROOT B., ZUURBIER C.J. and VAN BEEK 
J.H.G.M. (1999): ‘Dynamics of tissue oxygenation 
in isolated rabbit heart as measured with near-
infrared spectroscopy‘, Am. J. Physiol., 276, pp. 
1616-1624 
 
[4] OWEN-REECE H., SMITH M., ELWELL C.E., 
GOLDSTONE J.C. (1999): ‘Near infrared 
spectroscopy‘, British Journal of Anaesthesia, 82, 
pp. 418-426 
 
[5] THE AMERICAN PHYSIOLOGICAL SOCIETY 
(2002): ‘Guiding principles for research involving 
animals and human beings’, Am J Physiol 
Regulatory Integrative Comp Physiol,  283, pp. 
R281–R283 
 
[6] BRAUNWALD E., KLONER R.A. (1985): 
‘Myocardial reperfusion: a double-edged sword?’, 
Am J Clin Invest, 76, pp. 1713-1719 
 
[7] BECKMAN J.S., BECKMAN T.W., CHEN J, 
MARSHALL P.A., FREEMAN B.A. (1990): 
‘Apparent hydroxyl radical production by 
peroxynitrite: implications for endothelial injury 
from nitric oxide and superoxide’, Proc Natl Acad 
Sci U S A,  87, pp. 1620-1624 
 
[8] RAZAVI H.M., HAMILTON J.A., FENG Q. 
(2005): ‘Modulation of apoptosis by nitric oxide: 
implications in myocardial ischemia and heart 
failure’, Pharmacol Ther,  106, pp. 147-162 
 
[9]  KUPRIYANOV V.V., DAI G., SHAW R.A., SUN 
J., JILKINA O., LUO Z., MANTSCH H., 
DESLAURIERS R. (2000): ‘Noninvasive 
assessment of cardiac ischemic injury using 87Rb 
and 23Na MR Imaging, 31P MR, and optical 
spectroscopy’, Magn. Reson. Med., 44, pp. 899-908 
 
 
  


Measurement of tissue oxygenation in isolated rat hearts using near infrared spectroscopy

http://puma.isti.cnr.it/rmydownload.php?filename=cnr.ifc/cnr.ifc/2005-A2-010/2005-A2-010.pdf

Measurement of tissue oxygenation in isolated rat hearts using near infrared spectroscopy

Abstract

Similar works

Full text

Available Versions

PUblication MAnagement