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A new method for continuous measurement of regional cerebral blood flow using laser Doppler flowmetry in a conscious rat
ELSEVIER
Neuroscience Letters 175 (1994) 149-152
NEUROSCItNCi [HTERS ,
A new method for continuous measurement of regional cerebral blood flow using laser Doppler flowmetry in a conscious rat Akio Sato*, Sae Uchida, Yoshiko Yamauchi** Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan
Received 16 May 1994; Accepted 20 May 1994
Abstract We have developed a new system to continuously measure regional cerebral blood flow (rCBF) in the cortex of a conscious animal. For this purpose, we used rats and laser Doppler flowmetry. Under pentobarbital anesthesia, the animal'~ skull was opened making a small square hole 3 mm x 3 mm in size. A transparent acrylic plate was placed over the hole in the skull. A polyethylene cannula (inner diameter 1.0 mm, length 5.0 mm) was fixed on the plate as a guide for the laser Doppler flowmeter (LDF) probe (outer diameter 1.0 mm, length 5.5 mm). Both the plate and guide cannula were fixed to the skull by dental cement. Every day for the following two weeks after surgery, the conscious animal was placed in a hammock for recording rCBF. A LDF probe was freely attachable to the plate above the cortex via the guide cannula during measurement of rCBF. The rats were kept in a hammock with their legs firmly touching the floor during measurement of rCBF. It was possible to measure rCBF every day for about two weeks, and rCBF responded consistently to inhalation of 7% CO: when the responses were expressed as percentages of the prestimulus control rCBF values. This system is recommended for the continuous measurement of rCBF in a conscious animal.
Key words: Regional cerebral blood flow; Laser Doppler flowmetry; Conscious rat
In 1945, Kety and Schmidt developed a method that permitted the measurement of the total cerebral blood flow of humans using N20 [8]. In 1961, Ingvar and Lassen succeeded in measuring regional cerebral blood flow (rCBF) in the human cerebral cortex using 133Xe injected into an internal carotid artery [7]. Recently, it became possible to measure rCBF in the deep brain regions using Xenon-computer tomography (Xe-CT), single photon emission CT (SPECT), and positron emission tomography (PET) in humans. In animals, more inexpensive methods such as the hydrogen gas clearance method, the [~4C]iodoantipyrine method, the microsphere method and the laser Doppler technique have been used for measuring rCBF. Each method has its advantages and disadvantages in practical usage. Most of the quantitative measuring methods *Corresponding author. Fax: (81) (3) 3964-1415. ** Present address: Department of Electrical and Information Engineering, Faculty of Engineering, Yamagata University, Yonezawa, 992, Japan.
0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00407-2
used so far, such as hydrogen gas clearance, microspheres, or the [14C]iodoantipyrine method, have provided data on rCBF at a certain time stage, but not continuously recorded data of rCBF. The possibility of continuous measurement of rCBF emerged with the development of laser Doppler flowmetry. This technique, originally proposed by Riva et al. [11] and Stern [16], is based on the principle of frequency shift in the spectrum of scattered laser light reflected from moving blood cells in microcirculation. The laser Doppler technique continuously measures the total directional movement of blood cells in a limited region, so that this method is not suitable for measuring absolute flow. However, the response of rCBF qualitatively measured by this method in rats, correlates well with the result quantitatively measured by the hydrogen clearance method [12,15] or by the [~4C]iodoantipyrine method [6]. Laser Doppler flowmetry has recently been used to analyze continuous changes in rCBF in the cortex [1--4], hippocampus [5], thalamus [9] and striatum [17] of anesthetized rats [13]. The main reason using anesthe-
150
.4. Sato et al./Neuroscience Letters 175 (1994) 149-152
tized rats for measuring rCBF with laser Doppler flowmetry was to attach the laser Doppler flowmeter (LDF) probe tightly on a specific region of the brain and avoid animal's movement which produces a mechanical artifact on the recording system. The present study aimed to develop a new experimental method to continuously measure rCBF in the cerebral cortex of a conscious rat using laser Doppler flowmetry by attaching a LDF probe chronically to the cortex with the help of a guide cannula that was fixed on the opened skull. Using this technique, the response of rCBF to inhalation of 7% CO2 was examined to determine whether the response was stable and reliable at different days after the LDF probe was attached to the cortex. Four adult male Wistar rats (290-375 g, 3-5 months old) were used for the experiments. Aseptic operation for the recording of rCBE Animals were anesthetized with pentobarbital (50 mg/kg, i.p.). The animal was fixed on a stereotaxic instrument (SR-5, Narishige, Tokyo) in the prone position. Through a midline incision of the skin of about 2 cm in length, two pieces of the skull (3 x 3 ram) were removed bilaterally. The stereotaxic coordinates, as per Paxinos and Watson [10], for the craniotomy areas were: AP = -0.5 to -3.5 mm, L -- 1.5 to 4.5 ram. Two l-ram thick acrylic plates of about 6 x 6 mm were placed over the holes in the skull (Fig. 1A). On each acrylic plate a guide cannula (outer diameter 2.0 mm, inner diameter 1.0 mm, length 5.0 mm) for a LDF probe (outer diameter 1.0 mm, length 5.5 mm) (ALF 2100, Advance Co. Ltd., Tokyo) was fixed perpendicularly. The guide cannula was positioned above the surface of the cortex, avoiding visible blood vessels. Each acrylic plate and guide cannula was fixed to the skull using dental cement. Two small screws were placed in the
skull to anchor the dental cement. The probe was attached to the acrylic plate via the guide cannula whenever recordings of rCBF were to be taken. After surgery, antibiotics (viccillin 50 mg/kg, i.m.) was administered. After awakening, the animals were housed at an ambient temperature of 22 + 2°C under artificial illumination (light on between 08.00 and 20.00 h), and fed on laboratory food with water ad libitum. Placement of rats in the hammock. The animals were anesthetized in the box with 3% halothane for 2-3 rain. Immediately after removal of halothane the animals were placed in a cloth hammock suspended from a metallic frame (Fig. 1B). The height of the frame was adjusted to allow the animals' legs to support their weight on the floor. Usually, the animals became calm when their legs were firmly on the floor. The recordings of rCBF and inhalation of COe. The recording of rCBF using LDF began 30 min after the end of halothane anesthesia. The recording LDF probe was inserted in the guide cannula and the output (in mV) was recorded on a polygraph (RM-6000, Nihon Kohden, Tokyo). CO2 was supplied to the animals as follows. The CO2 concentration of the gas in the frame box (Fig. 1B) was measured using a gas monitor (1H26, NEC San-ei, Tokyo). Inhalation of CO2 was started by the infusion of 100% CO2 into the box for a few seconds to increase the CO2 concentration as quickly as possible, and then, the box was perfused with a mixture of 7% CO> 20-25% 02, and 68-73% N: for 3 min. Finally, the gas mixture in the box was replaced within several seconds by air by opening the side plates of the box. Fig. 2 shows a typical continuous recording of rCBF in the parietal cortex of a conscious rat in response to
laser DoPl
flowmeter
i:? monitor
~ ~ : . ~ rz:=: . a.::~Sr.~-.ss, ? "
fl(~r
"~
--fra b oex m
, ~tllv _ v
Fig. 1. Illustration of the present experimental procedure. A: fixation of the guide cannula of the laser Doppler flowmeter (LDF) probe on the skull. B: the conscious rat was placed in a hammock (a) suspended from' a metallic frame (b) for recording rCBF, and 7% CO2 was inhaled.
A. Sato et al./Neuroscience Letters 175 (1994) 149-152
of rCBF and the response of rCBF to inhalation of 7% CO2 were almost constant. When the recordings of rCBF were performed on both sides of the cortex on the same day, or the recordings of rCBF were repeated on different days, the LDF baseline values and responses of rCBF were variable, when expressed in absolute terms. Fig. 3A summarizes rCBF of the left cortex measured every day by the present method in conscious state for 15 days after the guide cannula for the LDF probe was attached on the skull. The LDF baseline values (mV) of rCBF and increases in rCBF during 7% CO2 inhalation varied remarkably from day to day when presented in absolute terms as demonstrated in Fig. 3A. However, the responses to inhalation of 7% CO2 were quite stable when presented as percentages of the prestimulus control values as shown in Fig. 3B. The increases were within 110% and 140% of the controls for 15 days, indicating that in the conscious state the responses of rCBF to CO2 inhalation, were meaningful, if expressed as percentages, up to almost two weeks after the guide cannula for the LDF probe was attached to the skull. The present response of rCBF in the parietal cortex to inhalation of CO: was in agreement with the increased response of rCBF in anesthetized rats reported previously [1,3,14]. It has been generally accepted that absolute measures of rCBF by LDF are not reliable, but relative values between two recordings measured at different times under the same recording conditions can be comparable [6,12,15]. Using the present new method, it was possible to continuously record rCBF in the cortex of conscious animals without any serious mechanical artifact. This successful recording depends firstly on the rigid attachment of the LDF probe to the cortex with a help of a guide cannula and acrylic plate fixed on the skull, and
mV
-] 700
1 ,o0 "15oo
rCBF
l'Z
"J 400 %
Fco~ 7%C02, 3min
Fig. 2. A typical response of rCBF in the parietal cortex recorded by LDF during 7% CO2 inhalation for 3 min in a conscious rat and CO2 concentration in the frame box.
inhalation of 7% CO2. The concentration of CO2 in the box was recorded as shown in the lower trace. The concentration of CO2 fluctuated for about the first 10 s and then remained at about 7% for a 3 min period. The rCBF started to increase several seconds after the onset of inhalation of CO2, continued to increase for the following 10 s (aprrox.) and then plateaued until the end of inhalation of CO2 after 3 min. After the end of inhalation of 7% CO2, the rCBF decreased to the control level within another 10 s. The animal was quiet during the inhalation of 7% CO2 up to 3 min. Sometimes animals moved their heads and bodies spontaneously, but their movements did not cause any artifacts in the recording of rCBF, indicating that this attachment of the LDF probe on rat's head is reliable for continuously measuring rCBF without any mechanical artifact in a conscious rat. When rCBF was recorded for 1-2 h on the same cortex on the same day, the baseline (or absolute) value
A
8O0
151
[ ] base line value
]
" 60014.
m 40020001
B
150]
2
3
4
5
~
6
7
8
9
10 11 12 13 14 15 days
,
,
, -
.
1
.
2
.
.
3
.
4
.
5
.
6
.
7
.
8
.
.
9
10 11 12 13 14 15 days
.
.
.
.
Fig. 3. A: rCBF in the left parietal cortex measured in conscious state on 15 consecutive days after attachment of the LDF probe before 7% CO2 inhalation (baseline value) and during 7% CO2 inhalation. Each column and vertical bar indicates the mean + S.E.M. (n = 4). rCBF during 7% CO2 inhalation increased significantly from baseline value by paired t-test (P < 0.05). B: summary of the responses to 7% CO2 inhalation for 15 consecutive days expressed as % of prestimulus control flow. Each circle and vertical bar indicates the mean + S.E.M. (n = 4)
152
A. Sato et al./Neuroscience Letters 175 (1994) 149-152
secondly the placement of the animals in a hammock, keeping the animals' legs touching on the floor. This placement of animals seems to be effective for avoiding mechanical artifacts in recording due to animals' movements, and lessens the stress of the situation by keeping their legs touching the floor. Once the LDF probe was attached to the cortex via the guide cannula, the recordings of rCBF were stable. Even when the attachment of the probe was changed, particularly on different days; the responses of rCBF to 7% CO2 were stable when expressed as percentages of the prestimulus control values. In conclusion, the present new method is useful for continuously detecting responses of rCBF to certain kinds of stimuli in conscious animals, particularly for responses changing in a time range of seconds or minutes. This work was supported by a Grant-in Aid for Developmental Scientific Research from the Ministry of Education, Science, and Culture, a research grant from the Ministry of Health and Welfare of Japan and an SRF Grant for Biomedical Research. [1] Adachi, T., Baramidze, D.G. and Sato, A., Stimulation of the nucleus basalis of Meynert increases cortical cerebral blood flow without influencing diameter of the pial artery in rats, Neurosci. Lett., 143 (1992) 173-176. [2] Adachi, T., Biesold, D., Inanami, O. and Sato, A., Stimulation of the nucleus basalis of Meynert and substantia innominata produces widespread increases in cerebral blood flow in the frontal, parietal and occipital cortices, Brain Res., 514 (1990) 163-166. [3] Adachi, T., Inanami, O. and Sato, A., Nitric oxide (NO) is involved in increased cerebral cortical blood flow following stimulation of the nucleus basalis of Meynert in anesthetized rats, Neurosci. Lett., 139 (1992) 201-204. [4] Biesold, D., Inanami, O., Sato, A. and Sato, Y., Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats, Neurosci. Lett., 98 (1989) 39-44.
[5] Cao, W.-H., Inanami, O., Sato, A. and Sato, Y., Stimulation of the septal complex increases local cerebral blood flow in the hippocampus in anesthetized rats, Neurosci. Lett., 107 (1989) 135140. [6] Dirnagl, U., Kaplan, B., Jacewicz, M. and Pulsinelli, W., Continuous measurement of cerebral cortical blood flow by laser-Doppler flowmetry in a rat stroke model, J. Cereb. Blood Flow Metab., 9 (1989) 589-596. [7] Ingvar, D.H. and Lassen, N.A., Quantitative determination of regional cerebral blood flow in man, Lancet, ii (1961) 806-807. [8] Kety, S.S. and Schmidt, C.F., The determination of cerebral blood flow in man by the use of nitrous oxide in low concentrations, Am. J. Physiol., 143 (1945) 53--66. [9] Koyama, Y., Toga, T., Kayama, Y. and Sato, A., Regulation of regional blood flow in the laterodorsal thalamus by ascending cholinergic nerve fibers from the laterodorsal tegmental nucleus, Neurosci. Res., in press. [10] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. [11] Riva, C., Ross, B. and Benedek, G.B., Laser Doppler measurements of blood flow in capillary tubes and retinal arteries, Invest. Ophthalmol., 11 (1972)936-944. [12] Saeki, Y., Sato, A., Sato, Y. and Trzebski, A., Effects of stimulation of cervical sympathetic trunks with various frequencies on the local cortical cerebral blood flow measured by laser Doppler flowmetry in the rat, Jpn. J. Physiol., 40 (1990) 15-32. [13] Sato, A. and Sato, Y., Regulation of regional cerebral blood flow by cholinergic fibers originating in the basal forebrain, Neurosci. Res., 14 (1992) 242-274. [14] Sato, A., Trzebski, A. and Zhou, W., Local cerebral blood flow responses in rats to hypercapnea and hypoxia in the rostral ventrolateral medulla and in the cortex, J. Auton. Nerv. Syst., 41 (1992) 79-86. [15] Skarphedinsson, J.O., Harding, H. and Thoren, P., Repeated measurements of cerebral blood flow in rats. Comparisons between the hydrogen clearance method and laser Doppler flowmetry, Acta Physiol. Scand., 134 (1988) 133-142. [16] Stern, M.D., In vivo evaluation of microcirculation by coherent light scattering, Nature, 254 (1975) 56-58. [17] Wada, H., Iijima, S., Orimo, H., Ito, H. and Sato, A., Stimulation of the substantia nigra increases striatal blood flow in rats, Biogenic Amines, 9 (1992) 29-39.
Neuroscience Letters 175 (1994) 149-152
NEUROSCItNCi [HTERS ,
A new method for continuous measurement of regional cerebral blood flow using laser Doppler flowmetry in a conscious rat Akio Sato*, Sae Uchida, Yoshiko Yamauchi** Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan
Received 16 May 1994; Accepted 20 May 1994
Abstract We have developed a new system to continuously measure regional cerebral blood flow (rCBF) in the cortex of a conscious animal. For this purpose, we used rats and laser Doppler flowmetry. Under pentobarbital anesthesia, the animal'~ skull was opened making a small square hole 3 mm x 3 mm in size. A transparent acrylic plate was placed over the hole in the skull. A polyethylene cannula (inner diameter 1.0 mm, length 5.0 mm) was fixed on the plate as a guide for the laser Doppler flowmeter (LDF) probe (outer diameter 1.0 mm, length 5.5 mm). Both the plate and guide cannula were fixed to the skull by dental cement. Every day for the following two weeks after surgery, the conscious animal was placed in a hammock for recording rCBF. A LDF probe was freely attachable to the plate above the cortex via the guide cannula during measurement of rCBF. The rats were kept in a hammock with their legs firmly touching the floor during measurement of rCBF. It was possible to measure rCBF every day for about two weeks, and rCBF responded consistently to inhalation of 7% CO: when the responses were expressed as percentages of the prestimulus control rCBF values. This system is recommended for the continuous measurement of rCBF in a conscious animal.
Key words: Regional cerebral blood flow; Laser Doppler flowmetry; Conscious rat
In 1945, Kety and Schmidt developed a method that permitted the measurement of the total cerebral blood flow of humans using N20 [8]. In 1961, Ingvar and Lassen succeeded in measuring regional cerebral blood flow (rCBF) in the human cerebral cortex using 133Xe injected into an internal carotid artery [7]. Recently, it became possible to measure rCBF in the deep brain regions using Xenon-computer tomography (Xe-CT), single photon emission CT (SPECT), and positron emission tomography (PET) in humans. In animals, more inexpensive methods such as the hydrogen gas clearance method, the [~4C]iodoantipyrine method, the microsphere method and the laser Doppler technique have been used for measuring rCBF. Each method has its advantages and disadvantages in practical usage. Most of the quantitative measuring methods *Corresponding author. Fax: (81) (3) 3964-1415. ** Present address: Department of Electrical and Information Engineering, Faculty of Engineering, Yamagata University, Yonezawa, 992, Japan.
0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00407-2
used so far, such as hydrogen gas clearance, microspheres, or the [14C]iodoantipyrine method, have provided data on rCBF at a certain time stage, but not continuously recorded data of rCBF. The possibility of continuous measurement of rCBF emerged with the development of laser Doppler flowmetry. This technique, originally proposed by Riva et al. [11] and Stern [16], is based on the principle of frequency shift in the spectrum of scattered laser light reflected from moving blood cells in microcirculation. The laser Doppler technique continuously measures the total directional movement of blood cells in a limited region, so that this method is not suitable for measuring absolute flow. However, the response of rCBF qualitatively measured by this method in rats, correlates well with the result quantitatively measured by the hydrogen clearance method [12,15] or by the [~4C]iodoantipyrine method [6]. Laser Doppler flowmetry has recently been used to analyze continuous changes in rCBF in the cortex [1--4], hippocampus [5], thalamus [9] and striatum [17] of anesthetized rats [13]. The main reason using anesthe-
150
.4. Sato et al./Neuroscience Letters 175 (1994) 149-152
tized rats for measuring rCBF with laser Doppler flowmetry was to attach the laser Doppler flowmeter (LDF) probe tightly on a specific region of the brain and avoid animal's movement which produces a mechanical artifact on the recording system. The present study aimed to develop a new experimental method to continuously measure rCBF in the cerebral cortex of a conscious rat using laser Doppler flowmetry by attaching a LDF probe chronically to the cortex with the help of a guide cannula that was fixed on the opened skull. Using this technique, the response of rCBF to inhalation of 7% CO2 was examined to determine whether the response was stable and reliable at different days after the LDF probe was attached to the cortex. Four adult male Wistar rats (290-375 g, 3-5 months old) were used for the experiments. Aseptic operation for the recording of rCBE Animals were anesthetized with pentobarbital (50 mg/kg, i.p.). The animal was fixed on a stereotaxic instrument (SR-5, Narishige, Tokyo) in the prone position. Through a midline incision of the skin of about 2 cm in length, two pieces of the skull (3 x 3 ram) were removed bilaterally. The stereotaxic coordinates, as per Paxinos and Watson [10], for the craniotomy areas were: AP = -0.5 to -3.5 mm, L -- 1.5 to 4.5 ram. Two l-ram thick acrylic plates of about 6 x 6 mm were placed over the holes in the skull (Fig. 1A). On each acrylic plate a guide cannula (outer diameter 2.0 mm, inner diameter 1.0 mm, length 5.0 mm) for a LDF probe (outer diameter 1.0 mm, length 5.5 mm) (ALF 2100, Advance Co. Ltd., Tokyo) was fixed perpendicularly. The guide cannula was positioned above the surface of the cortex, avoiding visible blood vessels. Each acrylic plate and guide cannula was fixed to the skull using dental cement. Two small screws were placed in the
skull to anchor the dental cement. The probe was attached to the acrylic plate via the guide cannula whenever recordings of rCBF were to be taken. After surgery, antibiotics (viccillin 50 mg/kg, i.m.) was administered. After awakening, the animals were housed at an ambient temperature of 22 + 2°C under artificial illumination (light on between 08.00 and 20.00 h), and fed on laboratory food with water ad libitum. Placement of rats in the hammock. The animals were anesthetized in the box with 3% halothane for 2-3 rain. Immediately after removal of halothane the animals were placed in a cloth hammock suspended from a metallic frame (Fig. 1B). The height of the frame was adjusted to allow the animals' legs to support their weight on the floor. Usually, the animals became calm when their legs were firmly on the floor. The recordings of rCBF and inhalation of COe. The recording of rCBF using LDF began 30 min after the end of halothane anesthesia. The recording LDF probe was inserted in the guide cannula and the output (in mV) was recorded on a polygraph (RM-6000, Nihon Kohden, Tokyo). CO2 was supplied to the animals as follows. The CO2 concentration of the gas in the frame box (Fig. 1B) was measured using a gas monitor (1H26, NEC San-ei, Tokyo). Inhalation of CO2 was started by the infusion of 100% CO2 into the box for a few seconds to increase the CO2 concentration as quickly as possible, and then, the box was perfused with a mixture of 7% CO> 20-25% 02, and 68-73% N: for 3 min. Finally, the gas mixture in the box was replaced within several seconds by air by opening the side plates of the box. Fig. 2 shows a typical continuous recording of rCBF in the parietal cortex of a conscious rat in response to
laser DoPl
flowmeter
i:? monitor
~ ~ : . ~ rz:=: . a.::~Sr.~-.ss, ? "
fl(~r
"~
--fra b oex m
, ~tllv _ v
Fig. 1. Illustration of the present experimental procedure. A: fixation of the guide cannula of the laser Doppler flowmeter (LDF) probe on the skull. B: the conscious rat was placed in a hammock (a) suspended from' a metallic frame (b) for recording rCBF, and 7% CO2 was inhaled.
A. Sato et al./Neuroscience Letters 175 (1994) 149-152
of rCBF and the response of rCBF to inhalation of 7% CO2 were almost constant. When the recordings of rCBF were performed on both sides of the cortex on the same day, or the recordings of rCBF were repeated on different days, the LDF baseline values and responses of rCBF were variable, when expressed in absolute terms. Fig. 3A summarizes rCBF of the left cortex measured every day by the present method in conscious state for 15 days after the guide cannula for the LDF probe was attached on the skull. The LDF baseline values (mV) of rCBF and increases in rCBF during 7% CO2 inhalation varied remarkably from day to day when presented in absolute terms as demonstrated in Fig. 3A. However, the responses to inhalation of 7% CO2 were quite stable when presented as percentages of the prestimulus control values as shown in Fig. 3B. The increases were within 110% and 140% of the controls for 15 days, indicating that in the conscious state the responses of rCBF to CO2 inhalation, were meaningful, if expressed as percentages, up to almost two weeks after the guide cannula for the LDF probe was attached to the skull. The present response of rCBF in the parietal cortex to inhalation of CO: was in agreement with the increased response of rCBF in anesthetized rats reported previously [1,3,14]. It has been generally accepted that absolute measures of rCBF by LDF are not reliable, but relative values between two recordings measured at different times under the same recording conditions can be comparable [6,12,15]. Using the present new method, it was possible to continuously record rCBF in the cortex of conscious animals without any serious mechanical artifact. This successful recording depends firstly on the rigid attachment of the LDF probe to the cortex with a help of a guide cannula and acrylic plate fixed on the skull, and
mV
-] 700
1 ,o0 "15oo
rCBF
l'Z
"J 400 %
Fco~ 7%C02, 3min
Fig. 2. A typical response of rCBF in the parietal cortex recorded by LDF during 7% CO2 inhalation for 3 min in a conscious rat and CO2 concentration in the frame box.
inhalation of 7% CO2. The concentration of CO2 in the box was recorded as shown in the lower trace. The concentration of CO2 fluctuated for about the first 10 s and then remained at about 7% for a 3 min period. The rCBF started to increase several seconds after the onset of inhalation of CO2, continued to increase for the following 10 s (aprrox.) and then plateaued until the end of inhalation of CO2 after 3 min. After the end of inhalation of 7% CO2, the rCBF decreased to the control level within another 10 s. The animal was quiet during the inhalation of 7% CO2 up to 3 min. Sometimes animals moved their heads and bodies spontaneously, but their movements did not cause any artifacts in the recording of rCBF, indicating that this attachment of the LDF probe on rat's head is reliable for continuously measuring rCBF without any mechanical artifact in a conscious rat. When rCBF was recorded for 1-2 h on the same cortex on the same day, the baseline (or absolute) value
A
8O0
151
[ ] base line value
]
" 60014.
m 40020001
B
150]
2
3
4
5
~
6
7
8
9
10 11 12 13 14 15 days
,
,
, -
.
1
.
2
.
.
3
.
4
.
5
.
6
.
7
.
8
.
.
9
10 11 12 13 14 15 days
.
.
.
.
Fig. 3. A: rCBF in the left parietal cortex measured in conscious state on 15 consecutive days after attachment of the LDF probe before 7% CO2 inhalation (baseline value) and during 7% CO2 inhalation. Each column and vertical bar indicates the mean + S.E.M. (n = 4). rCBF during 7% CO2 inhalation increased significantly from baseline value by paired t-test (P < 0.05). B: summary of the responses to 7% CO2 inhalation for 15 consecutive days expressed as % of prestimulus control flow. Each circle and vertical bar indicates the mean + S.E.M. (n = 4)
152
A. Sato et al./Neuroscience Letters 175 (1994) 149-152
secondly the placement of the animals in a hammock, keeping the animals' legs touching on the floor. This placement of animals seems to be effective for avoiding mechanical artifacts in recording due to animals' movements, and lessens the stress of the situation by keeping their legs touching the floor. Once the LDF probe was attached to the cortex via the guide cannula, the recordings of rCBF were stable. Even when the attachment of the probe was changed, particularly on different days; the responses of rCBF to 7% CO2 were stable when expressed as percentages of the prestimulus control values. In conclusion, the present new method is useful for continuously detecting responses of rCBF to certain kinds of stimuli in conscious animals, particularly for responses changing in a time range of seconds or minutes. This work was supported by a Grant-in Aid for Developmental Scientific Research from the Ministry of Education, Science, and Culture, a research grant from the Ministry of Health and Welfare of Japan and an SRF Grant for Biomedical Research. [1] Adachi, T., Baramidze, D.G. and Sato, A., Stimulation of the nucleus basalis of Meynert increases cortical cerebral blood flow without influencing diameter of the pial artery in rats, Neurosci. Lett., 143 (1992) 173-176. [2] Adachi, T., Biesold, D., Inanami, O. and Sato, A., Stimulation of the nucleus basalis of Meynert and substantia innominata produces widespread increases in cerebral blood flow in the frontal, parietal and occipital cortices, Brain Res., 514 (1990) 163-166. [3] Adachi, T., Inanami, O. and Sato, A., Nitric oxide (NO) is involved in increased cerebral cortical blood flow following stimulation of the nucleus basalis of Meynert in anesthetized rats, Neurosci. Lett., 139 (1992) 201-204. [4] Biesold, D., Inanami, O., Sato, A. and Sato, Y., Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats, Neurosci. Lett., 98 (1989) 39-44.
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