Journal of the neurological Sciences, 1975, 26:529-544
529
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Spinal Cord Blood Flow measured by a Hydrogen Clearance Technique IAN R. GRIFFITHS, J. O. ROWAN* AND R. A. CRAWFORD Department of Veterinery Surgery (LR.G. and R.A.C.), University of Glasgow Veterinary School, Bearsden Road, Glasgow, G62 1QH and Institute of Neurolo#ical Sciences (J.O.R.) Southern General Hospital, Glasgow (Great Britain) (Received 30 April, 1975)
INTRODUCTION
Hydrogen gas has been used by many workers to measure cerebral blood flow (CBF) and blood flow in other organs. Aukland, Bower and Berliner (1964) described the theoretical considerations of the method where the partial pressure of hydrogen in the tissue is measured by a polarised platinum electrode. The current generated by the oxidation of the hydrogen is proportional to the partial pressure. After administration of the hydrogen the desaturation process can be monitored and a clearance curve, similar to those obtained in the inert radioactive gas techniques, is produced. In the measurement of CBF hydrogen has been given by inhalation (Bozzao, Fieschi, Angoli and Nardini 1968; Haining, Turner and Pantall 1968; Pasztor, Symon, Dorsch and Branston 1973) or injection (Shinohara, Meyer, Kitamura, Toyoda and Ryu 1969; Meyer, Fukuuchi, Kanda, Shimazu and Hashi 1972). One major attraction of the method is that microregional CBF (rCBF) can be measured with very little damage to the cerebral tissues because of the small diameter of the electrodes. The spinal cord blood flow (SCBF) has been measured by a number of techniques; --heat clearance (Wiillenweber 1968), autoradiography (Landau, Freygang, Roland, Sokoloff and Kety 1955), particle distribution (Flohr, P611 and Brock 1971) and xa3Xe clearance (Smith, Pender and Alexander 1969; Griffiths 1973a, b). All these methods have certain disadvantages such as, lack of quantitative results with heat clearance, single measurements, and repeated trauma due to injection of 133Xe. A single injection of 13axe yields good results but repeated studies at the same injection site causes unacceptable tissue damage. Kobrine, Doyle and Martins (1974) have described the use of hydrogen clearance in the measurement of SCBF in rhesus monkeys. It was hoped that the hydrogen clearance technique would provide an improved method for detecting SCBF in both physiological and pathological studies. This present publication deals with the methodology and the response to CO 2. This study was supported by the Wellcome Trust.
530
I. R. GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD METHODS
Surgicalprocedures Forty unselected dogs were used weighing between 12 and 20 kg. Twenty-eight dogs were anaesthetised with pentobarbitone (25 mg/kg) and maintained on a semiclosed system with 50~o N20/O 2 mixture. Muscle paralysis was produced with gallamine (1 mg/kg) and the gas mixture delivered through a pump adjusted to maintain normocarbia during surgical preparation. A small dose of additional pentobarbitone was given following surgery, if necessary. One femoral artery was cannulated for the continuous recording of arterial blood pressure and the analysis of arterial CO 2 (PaCOa) , 0 2 (PaO2) and pH. Any significant base deficit was corrected with 8.4~o sodium bicarbonate. The other femoral artery was used for the introduction of the arterial (aortic) hydrogen electrode. One jugular vein was cannulated lbr the measurement of central venous pressure (CVP) and administration of drugs. Heating lamps were used to maintain the pharyngeal temperature between 37 and 38°C. Hypercarbia was produced by adding CO 2 to the inspired gas mixture and hypocarbia by increasing the pump volume. Ten min were allowed for stabilization of PaCOa before a flow measurement was made. Twelve dogs were anaesthetised with a sleep dose of thipentone sodium and surgically prepared under N 2 0 / O 2 and 0.5~ halothane. One hour before flow measurements commenced the halothane was discontinued and a-chloralose (60 mg/kg) was injected intravenously. In 9 dogs the flow in the spinal grey matter at L6 and L7 segments was investigated using 0.2 mm platinum electrodes. In the other 3 dogs SCBF was investigated as described later and in addition 0.2 mm platinum electrodes were inserted, through burr holes, into the cerebral cortex and 0.4 mm electrodes into the subcortical white matter so that simultaneous cord and brain recordings could be made. To substantiate further the results found in dogs, 2 baboons were investigated. They were anaesthetised with phencyclidine and N20/02 mixture. The baboon was used as the CBF for both white and grey matter has been extensively investigated by both 133Xe and hydrogen clearance in this animal. CBF from numerous sites and SCBF from the grey and white matter of the lumbar enlargement were simultaneously measured. Dorsal laminectomies were performed to expose the required spinal segments, usually T13, L2 and L4. In the 9 dogs previously mentioned the L6 and L7 segments were exposed. The bone was sealed with bone wax and perspex laminae were inserted to replace the removed bone. These contained a small hole through which the electrodes could be placed in the cord. The laminae were held in position by dental cement (model Kryptex)*. The dog was then suspended by 4 Steinman intramedullary pins placed through appropriate dorsal spines and held by retort stands fastened on the operating table. The elevation was such that no respiratory movements were transmitted to the exposed spine. The epidural fat was removed and dural incisions were made with watchmakers' scissors at the sites of intended electrode placement. The canine dura is a tough fibrous membrane and even sharp electrodes would not penetrate easily. They first compressed the cord and then penetrated the dura rapidly *Model Kryptex powder and liquid : SS White Dental Mfg. Co., Harrow, Middlesex.
SPINAL CORD BLOOD FLOW MEASURED BY HYDROGEN CLEARANCE TECHNIQUE
531
so preventing accurate placement. Dural incision was therefore found to be essential. A maximum of 2 electrodes were placed at any one transverse area of the cord although more than 2 could be placed in any given segment. The electrodes were held in a micromanipulator which was itself attached to a more coarse manipulator. The penetration of the cord was made obliquely which decreased any tendency for cord compression by the electrodes. It was found useful to connect the electrodes to the recording circuit during placement. The initial polarisation could then be monitored before cementing the electrodes to the lamine with SS white zinc cement. This was found to be very suitable for holding the electrodes as it hardened rapidly and was not effected by any blood which subsequently accumulated. The vertebral canal was completely sealed with cement.
Electrodes and recordin9 circuit The arterial electrode was a 1 cm long, 0.4 mm diameter platinum wire projecting from a polythene cannula which contained the lead, soldered to the platinum. The exposed tip was covered with a small ball of araldite. The tissue electrodes were made from 0.3 or 0.2 mm grade 2 platinum wire*. They were insulated with araldite** and the terminal 1 mm scraped bare. Particular attention was paid to making a smooth junction between the recording tip and the araldite and in providing a sharp tip to the electrode. These procedures were performed under a dissecting microscope and the insulation was checked in saline using a Multimeter. The electrode tips were then cathodised in 5~o platinum chloride solutions. The design of the electrodes was very important and the majority of failures or poor recordings could be attributed to imperfect electrodes. A silver/silver chloride EEG electrode, placed subcutaneously in the animal's back, was used as the reference electrode and a positive polarising voltage of 700 mV was employed. The small current obtained from the platinum electrodes were amplified using a 6-channel system employing Analog Devices amplifiers type 233K which have a low electrode 6 5
Ao.A~ d
A Fig. 1A. Schematic diagram of 6-channel hydrogen detection system.
*Johnson Matthey Metals Limited, London. **Ciba Geigy.
532
I . R . GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD + 15v 1M.~7-
/
)
l
recorder
~ "
lOM~-
lOpF
50 K.~. " ~ l a n c e - 15V
Fig. 1B. Circuit diagram of hydrogen electrode current amplifier.
input bias current drift of less than 1 pAp C. The system is shown schematically in Fig. 1A and the amplifier circuit diagram is shown in Fig. lB. Three dual channel Servoscribe chart recorders were used to display the output from each amplifier. Every amplifier channel had an input balance control so that the output to each recorder could be set to zero to correspond with zero hydrogen concentration in tissue. The gain of each amplifier was set to give an output of 1V for an input of 1 #A, while each recorder channel had an independent variable gain control so that the hydrogen concentration equilibrium plateau level could be set to full scale deflection without affecting the original zero concentration setting. The amplifier bandwidth was restricted to 0-1 Hz to limit the effect of high frequency noise.
Technique of SCBF measurement Hydrogen gas was introduced into the anaesthetic circuit before the respiratory pump. During inhalation of hydrogen the N20 was turned off and the 02 increased so that approximately a 60/40~o mixture of O2/H2 was delivered. The inhalation time varied but approximately 5-6 min was necessary to achieve a plateau. The hydrogen was then turned off, the N20/O2 returned to their former concentrations and the clearance curve recorded. The curve was transposed on to semilogarithmic paper and the T½ was measured. Biexponentiel clearances were separated into fast and slow components for calculation of T½. SCBF was calculated from the formula" SCBF =
2 x log~ x 60
T½
x 100 ml/100g/min
2 (tissue/blood partition coefficient) was taken to be 1 (Aukland et al. 1964). RESULTS
(1) Effect of inhalation of hydrogen gas About 20 sec after introducing the H 2 into the anaesthetic circuit the aortic electrode recorded a rapidly-increasing current. This reached a plateau which usually decreased gradually over the inhalation period as previously reported (Pasztor et al. 1973). Following the end of hydrogen inhalation the arterial concentration fell rapidly with a T½ of 25-40 sec. The tissue electrodes followed the rise and fall of the arterial electrode with delay periods of 5 sec to 2 rain (Figs. 2A, 3A). As the inspired concentrations of hydrogen were calculated to be higher than those
SPINAL CORD BLOOD FLOW MEASURED BY HYDROGEN CLEARANCE TECHNIQUE
533
J
Fig. 2A. Clearance curves from 2 electrodes placed in grey matter of dog anaesthetised with ¢-chloralose. Above: the clearances at PaCO2 34 mm Hg, and below: at PaCO2 52.3 mm Hg. There is virtually no lag phas~ between the cessation of hydrogen administration (arrow at top of curves) and the onset of desaturation.
104
104
26"7
[]
109 pCO 2 - 34
[]
24"8
35"9
[]
pCO 2- 52.z
[]
Fig. 2B. The semilogarithmic plots of the clearence curves in Fig. 2A. above: PaCO2 34 mm Hg, below: PaCO 2 52.3 mm Hg. The enclosed numbers refer to the identity of the clearance curve and the figures on the lines give the SCBF in ml/100g/min. Note that electrode 2 has become monoexponential at hypercarbia and the flow of the fast component in electrode 1 has not increased with the change of PaCO2.
534
I. R. GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD
8 ~ -100 - -
,O
A Fig. 3A. Clearance curves recorded from the brain and cord of a baboon at normocarbla. Curve 2 was recorded from an electrode placed in cerebral deep white matter, 3 from parietal cortex, 5 from frontal cortex, 7 from spinal ~ey matter, and 8 from spinal white matter. The arrowhead shows the point of cessation of inhalation and the time marker is 1 min.
Fig. 3B. The semilogarithmic plots of the curves shown in Fig. 3A. The enclosed nttmhers relate the identity of the curves shown in Fig. 3A. The other figures give the flows in ml/100g/min. Note curves 2 and 5 from the cortex are biexponential.
in previously-reported studies we carefully studied its effect on all measured parameters. Usually no change was observed in the following pressures; arterial blood pressure measured by femoral and aortic cannulae, CVP and the femoral venous pressure. PaO2 and PaCo2 decreased slightly but not significantly during inhalation but returned to steady state values within 2 min of the end of inhalation.
(2) Analysis of the clearance curves In the 28 dogs anaesthetised with pentobarbitone a total of 82 clearances were recorded at normocarbia from electrodes placed in the white matter of various cord
SPINAL CORD BLOOD F L O W MEASURED BY HYDROGEN CLEARANCE TECHNIQUE
535
Fig. 4. Clearance curve from electrode placed in peripheral white matter of L4 segment. The curve transposes into a biexponential clearance. Arrow denotes cessation of inhalation. Time marker is 1 min.
segments, 74 from electrodes in the grey matter, 20 from the dorsal horn and 9 from electrodes at the grey/white junction. Sixty-four percent of the clearances recorded from the white matter at normocarbia were monoexponential, the remainder being biexponential. In the grey matter 8 9 ~ were monoexponential, the remainder being biexponential. The weight of the fast component in the grey matter was always small, often less than 10~o. At hypercarbia the monoexponentialfoiexponential percentages were 68/32~ for white matter and 75/25 ~o for grey matter. Most of the white matter electrodes which recorded biexponential clearances were placed towards the periphery of the cord, especially at the L4 segments (Fig. 4). Flows in the various compartments The SCBF calculated from the fast and slow components and the weighted mean flow were compared in grey and white matter of dogs under pentobarbitone anaesthesia in which both biexponential and monoexponential clearances were recorded. The resuits are shown in Table 1A. There is no significant difference between the SCBF calculated from the monoexponential clearance and the slow component of the biexponential clearance in either grey or white matter. To study the apparently low flows recorded from monoexponential clearances in the grey matter at T13, L2 and L4 segments, 9 dogs were investigated at the L6 and L7 segments. The grey matter has a greater area and 0t-chloralose has a less depressant effect upon neurones than barbiturates. Fifty-eight (38~o) recordings were biexponential and the fast component of these was studied. The flows and weighting factors for the fast component were calculated. The results are presented in Table lB. The flows are significantly greater than those recorded from the fast component in the grey matter under barbiturate anaesthesia (Table 1A). The weighting of the fast comT A B L E 1A Tim SCBF
C A L C U L A T E D F R O M T H E VARIOUS C O M P O N E N T S IN D O G S W H E R E B O T H BIEXFONENTIAL A N D M O N O -
EXPONENTIAL CLEARANCESWERE RECORDED ( PENTOBARBITONEANAESTHESIA)
l:.st
69_____11
Grey matter (ml/lOOo/min) Slow mono.
11.1+1.7 L___N.S.
10.9+4.0
mean
18.0_____3.1
fast
97+15
I
In all tables the values shown are m e a n + 1 standard deviation.
White matter (ml/lOOg/min) slow mono.
13.2+3.2 I
14.8+4.9
,N.S.--~
mean
25.5_____7.3
536
I. R. GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD TABLE 1B
FLOWS AND WEIGHTING FACTORS OBTAINED FROM THE FAST COMPONENT OF ELECTRODES PLACED IN GREY MATTER AT
L6 AND L7
SEGMENTS. COMBINED RESULTS FROM
9
DOGS (Ot-CHLORALOSE ANAESTHESIA)
n
SCBF (ml/l OOQ/min)
Weight of fast component ( 5~,~)
58
97.5 +_32.9
10.7 +_7.4
ponent was small, the maximum value being 25%. The effect of C O 2 o n the fast component was also studied (see later). For reasons outlined in the discussion it was felt that in the grey matter the flow derived from the slow component of biexponential clearances was a valid and more consistent measurement than the fast component. In the remainder of the results the grey flows are derived from the slow component of biexponential clearances.
Flow values in the grey and white matter of various segments of the spinal cord at normocarbia There was a considerable variation in mean flow from electrodes placed in both grey and white matter between different dogs. In both structures the mean flow varied from 7 to 30 ml/100 g/min in different dogs. In individual dogs the flow at one electrode site was more constant (see section on repeatability). The SCBF was compared at electrodes in grey and white matter at segments T13, L2 and L4 of different dogs anaesthetised with pentobarbitone. Usually the flows at the L2 segment were compared with those at either the L4 or T13 segments, so that 4 areas were examined in each dog. The results are shown in Table 2, Insufficient results are available for analysis of flows at T 13 grey matter. There is a significant difference between the flows in the white matter at T13 and L2 but not between L2 and L4. There are no differences between the flows at L2 and L4 in grey or white matter. Surprisingly the flow recorded from electrocLes placed in the white matter is higher than that from electrodes placed in grey matter at both L2 and L4. The respective blood flows from electrodes placed in the white matter, grey matter, dorsal horn TABLE 2 SCBF
(ml/100 g/min)
IN GREY AND WHITE MATTER OF 3 SPINAL CORD SEGMENTS (PENTOBARBITONE ANAESTHESIA)
Segment T13 N White matter N Grey matter
L2
17 42 N 10.9 --+l4.4. P < 0.01 14.0 +l 4.1 P < 0.025
--
I_,4 30 15.0 + 5 . 1 1 ~
[ P < 0.05 39 I 35 S 11.8+5.4 S 12.2+5.3 i -e < 0.025l-
SPINAL CORD BLOODFLOW MEASUREDBY HYDROGENCLEARANCETECHNIQUE 537 TABLE 3 FLOWSINVARIOUSAREASOFSPINALCORD.COMBINEDRESULTSOFALLDOGSFROMSEGMENTST13, L2 ANDL4 (PENTOBARBITONEANAESTHESIS)
N SCBF (ml/100g/min)
White m a t t e r
White/grey junction
Grey matter
Dorsal horn
82 13.75-4.5
9 12.3+ 2.9
74 12.05-4.5
20 11.35-4.0
TABLE 4 COMPARISON OF S C B F IN GREY AND WHITE MATTER IN THE SAME SEGMENT OF THE SAME DOG (PENTOBARBITONE ANAESTHESIA)
Dog
Segment
N
Grey electrode
A
L4
9
B
L4
5
C D E F
L4 L4 L2 L2
5 5 3 3
9.4+0.1 16.2+ 1.2 11.1+1.6 22.1 +4.8 16.6+ 1.7 15.0_ 1.3
White electrode
12.9+2.7 17.1 + 2 . 4 11.8+4.1 21.9+6.7 17.7+ 2,1 16.2 _+0.8
P
529
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Spinal Cord Blood Flow measured by a Hydrogen Clearance Technique IAN R. GRIFFITHS, J. O. ROWAN* AND R. A. CRAWFORD Department of Veterinery Surgery (LR.G. and R.A.C.), University of Glasgow Veterinary School, Bearsden Road, Glasgow, G62 1QH and Institute of Neurolo#ical Sciences (J.O.R.) Southern General Hospital, Glasgow (Great Britain) (Received 30 April, 1975)
INTRODUCTION
Hydrogen gas has been used by many workers to measure cerebral blood flow (CBF) and blood flow in other organs. Aukland, Bower and Berliner (1964) described the theoretical considerations of the method where the partial pressure of hydrogen in the tissue is measured by a polarised platinum electrode. The current generated by the oxidation of the hydrogen is proportional to the partial pressure. After administration of the hydrogen the desaturation process can be monitored and a clearance curve, similar to those obtained in the inert radioactive gas techniques, is produced. In the measurement of CBF hydrogen has been given by inhalation (Bozzao, Fieschi, Angoli and Nardini 1968; Haining, Turner and Pantall 1968; Pasztor, Symon, Dorsch and Branston 1973) or injection (Shinohara, Meyer, Kitamura, Toyoda and Ryu 1969; Meyer, Fukuuchi, Kanda, Shimazu and Hashi 1972). One major attraction of the method is that microregional CBF (rCBF) can be measured with very little damage to the cerebral tissues because of the small diameter of the electrodes. The spinal cord blood flow (SCBF) has been measured by a number of techniques; --heat clearance (Wiillenweber 1968), autoradiography (Landau, Freygang, Roland, Sokoloff and Kety 1955), particle distribution (Flohr, P611 and Brock 1971) and xa3Xe clearance (Smith, Pender and Alexander 1969; Griffiths 1973a, b). All these methods have certain disadvantages such as, lack of quantitative results with heat clearance, single measurements, and repeated trauma due to injection of 133Xe. A single injection of 13axe yields good results but repeated studies at the same injection site causes unacceptable tissue damage. Kobrine, Doyle and Martins (1974) have described the use of hydrogen clearance in the measurement of SCBF in rhesus monkeys. It was hoped that the hydrogen clearance technique would provide an improved method for detecting SCBF in both physiological and pathological studies. This present publication deals with the methodology and the response to CO 2. This study was supported by the Wellcome Trust.
530
I. R. GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD METHODS
Surgicalprocedures Forty unselected dogs were used weighing between 12 and 20 kg. Twenty-eight dogs were anaesthetised with pentobarbitone (25 mg/kg) and maintained on a semiclosed system with 50~o N20/O 2 mixture. Muscle paralysis was produced with gallamine (1 mg/kg) and the gas mixture delivered through a pump adjusted to maintain normocarbia during surgical preparation. A small dose of additional pentobarbitone was given following surgery, if necessary. One femoral artery was cannulated for the continuous recording of arterial blood pressure and the analysis of arterial CO 2 (PaCOa) , 0 2 (PaO2) and pH. Any significant base deficit was corrected with 8.4~o sodium bicarbonate. The other femoral artery was used for the introduction of the arterial (aortic) hydrogen electrode. One jugular vein was cannulated lbr the measurement of central venous pressure (CVP) and administration of drugs. Heating lamps were used to maintain the pharyngeal temperature between 37 and 38°C. Hypercarbia was produced by adding CO 2 to the inspired gas mixture and hypocarbia by increasing the pump volume. Ten min were allowed for stabilization of PaCOa before a flow measurement was made. Twelve dogs were anaesthetised with a sleep dose of thipentone sodium and surgically prepared under N 2 0 / O 2 and 0.5~ halothane. One hour before flow measurements commenced the halothane was discontinued and a-chloralose (60 mg/kg) was injected intravenously. In 9 dogs the flow in the spinal grey matter at L6 and L7 segments was investigated using 0.2 mm platinum electrodes. In the other 3 dogs SCBF was investigated as described later and in addition 0.2 mm platinum electrodes were inserted, through burr holes, into the cerebral cortex and 0.4 mm electrodes into the subcortical white matter so that simultaneous cord and brain recordings could be made. To substantiate further the results found in dogs, 2 baboons were investigated. They were anaesthetised with phencyclidine and N20/02 mixture. The baboon was used as the CBF for both white and grey matter has been extensively investigated by both 133Xe and hydrogen clearance in this animal. CBF from numerous sites and SCBF from the grey and white matter of the lumbar enlargement were simultaneously measured. Dorsal laminectomies were performed to expose the required spinal segments, usually T13, L2 and L4. In the 9 dogs previously mentioned the L6 and L7 segments were exposed. The bone was sealed with bone wax and perspex laminae were inserted to replace the removed bone. These contained a small hole through which the electrodes could be placed in the cord. The laminae were held in position by dental cement (model Kryptex)*. The dog was then suspended by 4 Steinman intramedullary pins placed through appropriate dorsal spines and held by retort stands fastened on the operating table. The elevation was such that no respiratory movements were transmitted to the exposed spine. The epidural fat was removed and dural incisions were made with watchmakers' scissors at the sites of intended electrode placement. The canine dura is a tough fibrous membrane and even sharp electrodes would not penetrate easily. They first compressed the cord and then penetrated the dura rapidly *Model Kryptex powder and liquid : SS White Dental Mfg. Co., Harrow, Middlesex.
SPINAL CORD BLOOD FLOW MEASURED BY HYDROGEN CLEARANCE TECHNIQUE
531
so preventing accurate placement. Dural incision was therefore found to be essential. A maximum of 2 electrodes were placed at any one transverse area of the cord although more than 2 could be placed in any given segment. The electrodes were held in a micromanipulator which was itself attached to a more coarse manipulator. The penetration of the cord was made obliquely which decreased any tendency for cord compression by the electrodes. It was found useful to connect the electrodes to the recording circuit during placement. The initial polarisation could then be monitored before cementing the electrodes to the lamine with SS white zinc cement. This was found to be very suitable for holding the electrodes as it hardened rapidly and was not effected by any blood which subsequently accumulated. The vertebral canal was completely sealed with cement.
Electrodes and recordin9 circuit The arterial electrode was a 1 cm long, 0.4 mm diameter platinum wire projecting from a polythene cannula which contained the lead, soldered to the platinum. The exposed tip was covered with a small ball of araldite. The tissue electrodes were made from 0.3 or 0.2 mm grade 2 platinum wire*. They were insulated with araldite** and the terminal 1 mm scraped bare. Particular attention was paid to making a smooth junction between the recording tip and the araldite and in providing a sharp tip to the electrode. These procedures were performed under a dissecting microscope and the insulation was checked in saline using a Multimeter. The electrode tips were then cathodised in 5~o platinum chloride solutions. The design of the electrodes was very important and the majority of failures or poor recordings could be attributed to imperfect electrodes. A silver/silver chloride EEG electrode, placed subcutaneously in the animal's back, was used as the reference electrode and a positive polarising voltage of 700 mV was employed. The small current obtained from the platinum electrodes were amplified using a 6-channel system employing Analog Devices amplifiers type 233K which have a low electrode 6 5
Ao.A~ d
A Fig. 1A. Schematic diagram of 6-channel hydrogen detection system.
*Johnson Matthey Metals Limited, London. **Ciba Geigy.
532
I . R . GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD + 15v 1M.~7-
/
)
l
recorder
~ "
lOM~-
lOpF
50 K.~. " ~ l a n c e - 15V
Fig. 1B. Circuit diagram of hydrogen electrode current amplifier.
input bias current drift of less than 1 pAp C. The system is shown schematically in Fig. 1A and the amplifier circuit diagram is shown in Fig. lB. Three dual channel Servoscribe chart recorders were used to display the output from each amplifier. Every amplifier channel had an input balance control so that the output to each recorder could be set to zero to correspond with zero hydrogen concentration in tissue. The gain of each amplifier was set to give an output of 1V for an input of 1 #A, while each recorder channel had an independent variable gain control so that the hydrogen concentration equilibrium plateau level could be set to full scale deflection without affecting the original zero concentration setting. The amplifier bandwidth was restricted to 0-1 Hz to limit the effect of high frequency noise.
Technique of SCBF measurement Hydrogen gas was introduced into the anaesthetic circuit before the respiratory pump. During inhalation of hydrogen the N20 was turned off and the 02 increased so that approximately a 60/40~o mixture of O2/H2 was delivered. The inhalation time varied but approximately 5-6 min was necessary to achieve a plateau. The hydrogen was then turned off, the N20/O2 returned to their former concentrations and the clearance curve recorded. The curve was transposed on to semilogarithmic paper and the T½ was measured. Biexponentiel clearances were separated into fast and slow components for calculation of T½. SCBF was calculated from the formula" SCBF =
2 x log~ x 60
T½
x 100 ml/100g/min
2 (tissue/blood partition coefficient) was taken to be 1 (Aukland et al. 1964). RESULTS
(1) Effect of inhalation of hydrogen gas About 20 sec after introducing the H 2 into the anaesthetic circuit the aortic electrode recorded a rapidly-increasing current. This reached a plateau which usually decreased gradually over the inhalation period as previously reported (Pasztor et al. 1973). Following the end of hydrogen inhalation the arterial concentration fell rapidly with a T½ of 25-40 sec. The tissue electrodes followed the rise and fall of the arterial electrode with delay periods of 5 sec to 2 rain (Figs. 2A, 3A). As the inspired concentrations of hydrogen were calculated to be higher than those
SPINAL CORD BLOOD FLOW MEASURED BY HYDROGEN CLEARANCE TECHNIQUE
533
J
Fig. 2A. Clearance curves from 2 electrodes placed in grey matter of dog anaesthetised with ¢-chloralose. Above: the clearances at PaCO2 34 mm Hg, and below: at PaCO2 52.3 mm Hg. There is virtually no lag phas~ between the cessation of hydrogen administration (arrow at top of curves) and the onset of desaturation.
104
104
26"7
[]
109 pCO 2 - 34
[]
24"8
35"9
[]
pCO 2- 52.z
[]
Fig. 2B. The semilogarithmic plots of the clearence curves in Fig. 2A. above: PaCO2 34 mm Hg, below: PaCO 2 52.3 mm Hg. The enclosed numbers refer to the identity of the clearance curve and the figures on the lines give the SCBF in ml/100g/min. Note that electrode 2 has become monoexponential at hypercarbia and the flow of the fast component in electrode 1 has not increased with the change of PaCO2.
534
I. R. GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD
8 ~ -100 - -
,O
A Fig. 3A. Clearance curves recorded from the brain and cord of a baboon at normocarbla. Curve 2 was recorded from an electrode placed in cerebral deep white matter, 3 from parietal cortex, 5 from frontal cortex, 7 from spinal ~ey matter, and 8 from spinal white matter. The arrowhead shows the point of cessation of inhalation and the time marker is 1 min.
Fig. 3B. The semilogarithmic plots of the curves shown in Fig. 3A. The enclosed nttmhers relate the identity of the curves shown in Fig. 3A. The other figures give the flows in ml/100g/min. Note curves 2 and 5 from the cortex are biexponential.
in previously-reported studies we carefully studied its effect on all measured parameters. Usually no change was observed in the following pressures; arterial blood pressure measured by femoral and aortic cannulae, CVP and the femoral venous pressure. PaO2 and PaCo2 decreased slightly but not significantly during inhalation but returned to steady state values within 2 min of the end of inhalation.
(2) Analysis of the clearance curves In the 28 dogs anaesthetised with pentobarbitone a total of 82 clearances were recorded at normocarbia from electrodes placed in the white matter of various cord
SPINAL CORD BLOOD F L O W MEASURED BY HYDROGEN CLEARANCE TECHNIQUE
535
Fig. 4. Clearance curve from electrode placed in peripheral white matter of L4 segment. The curve transposes into a biexponential clearance. Arrow denotes cessation of inhalation. Time marker is 1 min.
segments, 74 from electrodes in the grey matter, 20 from the dorsal horn and 9 from electrodes at the grey/white junction. Sixty-four percent of the clearances recorded from the white matter at normocarbia were monoexponential, the remainder being biexponential. In the grey matter 8 9 ~ were monoexponential, the remainder being biexponential. The weight of the fast component in the grey matter was always small, often less than 10~o. At hypercarbia the monoexponentialfoiexponential percentages were 68/32~ for white matter and 75/25 ~o for grey matter. Most of the white matter electrodes which recorded biexponential clearances were placed towards the periphery of the cord, especially at the L4 segments (Fig. 4). Flows in the various compartments The SCBF calculated from the fast and slow components and the weighted mean flow were compared in grey and white matter of dogs under pentobarbitone anaesthesia in which both biexponential and monoexponential clearances were recorded. The resuits are shown in Table 1A. There is no significant difference between the SCBF calculated from the monoexponential clearance and the slow component of the biexponential clearance in either grey or white matter. To study the apparently low flows recorded from monoexponential clearances in the grey matter at T13, L2 and L4 segments, 9 dogs were investigated at the L6 and L7 segments. The grey matter has a greater area and 0t-chloralose has a less depressant effect upon neurones than barbiturates. Fifty-eight (38~o) recordings were biexponential and the fast component of these was studied. The flows and weighting factors for the fast component were calculated. The results are presented in Table lB. The flows are significantly greater than those recorded from the fast component in the grey matter under barbiturate anaesthesia (Table 1A). The weighting of the fast comT A B L E 1A Tim SCBF
C A L C U L A T E D F R O M T H E VARIOUS C O M P O N E N T S IN D O G S W H E R E B O T H BIEXFONENTIAL A N D M O N O -
EXPONENTIAL CLEARANCESWERE RECORDED ( PENTOBARBITONEANAESTHESIA)
l:.st
69_____11
Grey matter (ml/lOOo/min) Slow mono.
11.1+1.7 L___N.S.
10.9+4.0
mean
18.0_____3.1
fast
97+15
I
In all tables the values shown are m e a n + 1 standard deviation.
White matter (ml/lOOg/min) slow mono.
13.2+3.2 I
14.8+4.9
,N.S.--~
mean
25.5_____7.3
536
I. R. GRIFFITHS, J. O. ROWAN, R. A. CRAWFORD TABLE 1B
FLOWS AND WEIGHTING FACTORS OBTAINED FROM THE FAST COMPONENT OF ELECTRODES PLACED IN GREY MATTER AT
L6 AND L7
SEGMENTS. COMBINED RESULTS FROM
9
DOGS (Ot-CHLORALOSE ANAESTHESIA)
n
SCBF (ml/l OOQ/min)
Weight of fast component ( 5~,~)
58
97.5 +_32.9
10.7 +_7.4
ponent was small, the maximum value being 25%. The effect of C O 2 o n the fast component was also studied (see later). For reasons outlined in the discussion it was felt that in the grey matter the flow derived from the slow component of biexponential clearances was a valid and more consistent measurement than the fast component. In the remainder of the results the grey flows are derived from the slow component of biexponential clearances.
Flow values in the grey and white matter of various segments of the spinal cord at normocarbia There was a considerable variation in mean flow from electrodes placed in both grey and white matter between different dogs. In both structures the mean flow varied from 7 to 30 ml/100 g/min in different dogs. In individual dogs the flow at one electrode site was more constant (see section on repeatability). The SCBF was compared at electrodes in grey and white matter at segments T13, L2 and L4 of different dogs anaesthetised with pentobarbitone. Usually the flows at the L2 segment were compared with those at either the L4 or T13 segments, so that 4 areas were examined in each dog. The results are shown in Table 2, Insufficient results are available for analysis of flows at T 13 grey matter. There is a significant difference between the flows in the white matter at T13 and L2 but not between L2 and L4. There are no differences between the flows at L2 and L4 in grey or white matter. Surprisingly the flow recorded from electrocLes placed in the white matter is higher than that from electrodes placed in grey matter at both L2 and L4. The respective blood flows from electrodes placed in the white matter, grey matter, dorsal horn TABLE 2 SCBF
(ml/100 g/min)
IN GREY AND WHITE MATTER OF 3 SPINAL CORD SEGMENTS (PENTOBARBITONE ANAESTHESIA)
Segment T13 N White matter N Grey matter
L2
17 42 N 10.9 --+l4.4. P < 0.01 14.0 +l 4.1 P < 0.025
--
I_,4 30 15.0 + 5 . 1 1 ~
[ P < 0.05 39 I 35 S 11.8+5.4 S 12.2+5.3 i -e < 0.025l-
SPINAL CORD BLOODFLOW MEASUREDBY HYDROGENCLEARANCETECHNIQUE 537 TABLE 3 FLOWSINVARIOUSAREASOFSPINALCORD.COMBINEDRESULTSOFALLDOGSFROMSEGMENTST13, L2 ANDL4 (PENTOBARBITONEANAESTHESIS)
N SCBF (ml/100g/min)
White m a t t e r
White/grey junction
Grey matter
Dorsal horn
82 13.75-4.5
9 12.3+ 2.9
74 12.05-4.5
20 11.35-4.0
TABLE 4 COMPARISON OF S C B F IN GREY AND WHITE MATTER IN THE SAME SEGMENT OF THE SAME DOG (PENTOBARBITONE ANAESTHESIA)
Dog
Segment
N
Grey electrode
A
L4
9
B
L4
5
C D E F
L4 L4 L2 L2
5 5 3 3
9.4+0.1 16.2+ 1.2 11.1+1.6 22.1 +4.8 16.6+ 1.7 15.0_ 1.3
White electrode
12.9+2.7 17.1 + 2 . 4 11.8+4.1 21.9+6.7 17.7+ 2,1 16.2 _+0.8
P
