Respiration Physiology, 90 (1992) 47-54 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0034-5687/92/$05.00
47
RESP 01943
Phrenicotomy in the rat: Acute changes in blood gases, pH and body temperature M. Maskrey, S.E. Evans, U. Mesch, N.A. Andersen and J.H. Sherrey Department of Physiology, Tasmanian School of Medicine. Universityof Tasmania at Hobart, Tasmania, Australia (Accepted 4 May 1992) Abstract. Adult male rats were used to compare blood gases, pH and body temperature (Tb) before and after acute bilateral phrenicotomy. Under anaesthesia a femoral artery was catheterised and ties were placed round the phrenic nerves of seven rats (PNX group), while in five rats the ties were placed in the vicinity of the phrenic nerves (SHAM group). Twenty-four hours after surgery arterial blood samples were collected during quiet wakefulness (QW) and grooming (G), before and I h after the ties were pulled, and analysed for Po:, Pco: and pH. No changes were detected in the SHAM samples taken before and after the ties were pulled. In the PNX group a significant decrease in Tb occurred (QW, 0.6 °C; G, 1.5 °C). Following PNX Pao, decreased by 11.2 mmHg (QW) and 10.0 mmHg (G); Paco: increased by 2.6 mmHg (QW) and 2.4 mmHg (G) and pH fell by 0.04 (QW) and 0.03 (G). All changes except in Pa¢o: (QW) were significant. It is concluded that the changes in Tb, blood gases and pH which follow phrenicotomy in the rat are due to an increase in dead space ventilation (~'D) and a small reduction in alveolar ventilation (VA) associated with a faster, shallower pattern of breathing.
Blood gases, phrenicotomy; Body temperature, phrenicotomy: Mammals, rat; Phrenicotomy, blood gases, body temperature
When a rat loses the activity of its diaphragm the frequency of respiration (f) increases and the tidal volume (VT) falls, however the minute ventilation (V) remains almost unchanged (Maskrey et al., 1990a). The maintenance of V is accomplished by reorchestrating the activity of the remaining respiratory muscles and recruiting muscles not normally involved in quiet respiration (Sherrey and Megirian, 1990). The above investigations were carried out during different states of consciousness, the former while the animals were awake and the latter during sleep. Other investigators report that phrenicotomy causes changes in ventilation and breathing pattern in the rat which depend on the preparation used. In anaesthetised, tracheostomised rats tested in the supine position the phrenicotomised rat suffers from hypoventilation due to reductions in both f and VT: Pao2 is decreased and Paco, is increased (Nachazel and Palecek, 1990). Although no blood gas data are provided, Correspondence to: M. Maskrey, Department of Physiology, University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001, Australia.
48
M. MASKREY et ai.
experiments carried out by the same workers using awake rats confirm the breathing pattern changes observed in this laboratory, i.e. a decrease in VT but an increase in f (Nachazel and Palecek, 1991). Following hemidiaphragm paralysis the awake rat's ventilatory drive is capable of overcompensating for any deficit in diaphragm movement with a consequent rise in Pao,_ (Goshgarian et al., 1986). Blood gas estimations have been carded out on both humans and other animals with a paralysed diaphragm. Newsom-Davis et al. (1976) studied seven patients with neuromuscular disease involving a paralysed diaphragm and found that three had a lowered Pao2 and a raised Paco2 while standing. Five patients showed similar changes while lying down, awake or asleep. Stradling et al. (1987) found that in the dog, the Paco., increased during rapid eye movement sleep (REMS). Sant'Ambrogio et al. (1970) have shown that Paco2 is increased in both anaesthetised cats and rabbits when the diaphragm is paralysed. In a previous paper, we suggested that the change in respiration of the bilaterally phrenicotomised rat to a faster, shallower pattern of breathing would significantly reduce alveolar ventilation ('~A) (Maskrey et al., 1990a). The present experiments were designed to test this hypothesis and to assess the effectiveness of the immediate compensation mechanisms in maintaining blood gases and pH within normal limits during quiet wake (QW) and grooming (G) episodes. Some aspects of the study have been briefly reported in abstract form (Maskrey et al., 1990b).
Methods Animals Hooded male Wistar rats (250-340 g) were used in this study. Initially it was found that when the right carotid artery was catheterised to obtain blood samples many of the animals became distressed and had to be sacrificed. It appears that phrenicotomy and the impairment of the cerebral circulation which follows right carotid artery iigation is a lethal combination in many rats. When the femoral artery was catheterised instead of the carotid the animals were not unduly stressed. Surgical procedures The rats were anaesthetised initially with Nembutal (45 mg.kg -1, i.p.). Ketamine (10 mg.kg-l, l.m.) was given subsequently as required to maintain surgical anaesthesia. With minor modifications the procedure for cannulating the right femoral artery was that recommended by Raft and Fagin (1984). PE-10 polyethylene tubing (4 cm) was inserted into the expanded end of PE-50 polyethylene tubing (40 cm) and the junction was sealed on the outside with 'Super Glue gel'. The PE-50 catheter was passed subcutaneously and attached to a subminiature socket which was cemented to the cranium in the midline. Phrenic nerves were identified in the root of the neck as previously described by Maskrey et al. (1990a). Silk threads were loosely tied around both of the nerves and
PHRENICOTOMY: BLOOD GASES
49
passed subcutaneously to the headpiece. When sham operations were performed, the silk threads were placed in the vicinity of the phrenic nerves but not around them. After separating the sterno-hyoid muscles, a thermocouple was placed in the superior mediastinum and the leads passed subcutaneously to the headpiece. Recordings of body temperature were made on a Digi-Sense (Cole Parmer) thermocouple thermometer. To avoid damage, the catheter and the thermocouple leads were taped to a cable, one end of which was plugged into the subminiature headsocket and the other into a slip ring connector attached to the roof of the incubator. When the catheters were inserted at surgery they were filled ~.,h heparinised saline (20 U.ml-l). After surgery, when the rats were awake and moving around, the catheters were flushed with 0.1 ml heparinised saline (5000 U.ml-~). When taking samples, the dead space volume was aspirated and then returned after 0.3 ml of blood had been taken.
Analysis of blood gases and pH These measurements were made on a Radiometer (Copenhagen) BMS3 MK2. The oxygen and carbon dioxide electrodes were calibrated with humidified gases using Coming 192 and WOsthoff 2M301/A-F gas mixers, pH electrodes were calibrated using Radiometer S 1500 and S 1510 precision buffer solutions. The analyser temperature was kept as close as possible to the animal's body temperature.
Experimental protocol The rats were allowed 24 h to recover from surgery. After the operation and throughout the experiment they were housed in a Gallenkamp incubator at a temperature of 29 + 1 ° C, this being the thermoneutral temperature of the adult rat (Bernet et al., 1975). After calibration of the pH, Po, and Pco, electrodes two blood samples, each of 0.3 ml, were taken approximately half an hour apart. One sample was taken during quiet wakefulness and the other during grooming. The ties round the phrenic nerves were then pulled leaving the rat with a paralysed diaphragm. After this procedure the animal was checked to make sure it displayed paradoxical respiration, typified by inward movement of the abdominal wall on inspiration, instead of the normal outward movement. In other studies, the effectiveness of phrenicotomy has been established by the total loss of electromyographic activity in the diaphragm following the pulling of the ties (Sherrey and Megirian, 1990). After I h, two further 0.3-ml samples were taken, again during quiet wakefulness and during grooming. When each blood sample was taken the animal's temperature was recorded. After the last sample had been taken the rat was sacrificed by injecting a lethal dose of Nembutal. The management of the sham operated animals was the same, except that the pulling of the ties did not result in paralysis of the diaphragm.
50
M. MASKREY et al.
Statistical methods The differences in each of the variables for each rat before and after PNX or before and after SHAMX were subjected to statistical analysis using Student's paired t-test. Results
The effects of bilateral phrenicotomy (PNX) on arterial blood gases and pH are shown in Fig. 1. Table 1 provides a statistical summary of these data. It can be seen that whether samples were taken during periods of quiet wakefulness (QW) or during grooming, Pao~ was significantly decreased following PNX. At the same time Paco: tended to increase, though there was a great deal of variability, especially during QW
Quiet Wake
PNX
100
SHAMX
~ 9o o E e0 IZ~k~----. O4 ¢~
~A
Grooming
o-.._..._~ oo===-:o o--
70
SHAMX
e ~
c--
o
'
'
--o
6O
40 E
PNX
A
B
35
°"'''--'°
25
,
7.55
,
C
== ¢~ 7,50
o.......~
7.45
i
8~=--,~
~
e ~
7.40 ,
,
,l
,
,
i
,
,
,
,
r
D
40
o~ 3e ~ 3s 34
32
o_______o
7
Before After
'
""
V
Before After
W
Before After
!
Before After
Fig. 1. Blood gas, pH and body temperature measurements from seven rats before and after phrenicotomy (PNX) and from five rats before and after sham operations (SHAMX) while quietly awake and during grooming. From top to bottom, graphs show effects of PNX and $HAMX on: A, arterial Po: (Pao,); B, arterial Pco: (Paco2); C, arterial pH (pHa); and D, body temperature (Tb).
P H R E N I C O T O M Y : BLOOD G A S E S
51
TABLE 1 Mean values ( _ SD) of changes in Pao,, Paco,, p H a and Tb in phrenicotomised rats and sham-operated rats while quietly awake and during grooming Quiet awake
Grooming
Before
After
Before
After
Phrenicotomy (n ffi 7) Pao2 (mmHg) Paco_, (mmHg) pHa Tb (°C)
85.15 + 2.48 3 !. 19 + 2.88 7.471 + 0.003 37.96 + 1.86
73.98 + 4.26" 33.76 + 2.28 7.435 + 0.016.. 37.31 + 1.56'
83.62 + 4.01 30.45 + 3.49 7.465 + 0.026 38.29 + !.49
73.61 + 7.46" 32.81 + 2.64.. 7.434 + 0.014" 36.83 + 1.93..
Sham-operated control (n = 5) Pao2 (mmHg) Paco2 (mmHg) pHa Tb (°C)
83.51 + 7.09 31.07 + 2.05 7.462 + 0.023 38.06+0.49
82.82 + 5.63 30.83 + 3.57 7.459 + 0.021 38.21 +0.75
84.81 + 12.35 27.71 + !.81 7.471 + 0.024 38.28+0.19
86.65 + 10.49 29.81 + 2.43 7.471 + 0.015 38.14+0.72
" P
47
RESP 01943
Phrenicotomy in the rat: Acute changes in blood gases, pH and body temperature M. Maskrey, S.E. Evans, U. Mesch, N.A. Andersen and J.H. Sherrey Department of Physiology, Tasmanian School of Medicine. Universityof Tasmania at Hobart, Tasmania, Australia (Accepted 4 May 1992) Abstract. Adult male rats were used to compare blood gases, pH and body temperature (Tb) before and after acute bilateral phrenicotomy. Under anaesthesia a femoral artery was catheterised and ties were placed round the phrenic nerves of seven rats (PNX group), while in five rats the ties were placed in the vicinity of the phrenic nerves (SHAM group). Twenty-four hours after surgery arterial blood samples were collected during quiet wakefulness (QW) and grooming (G), before and I h after the ties were pulled, and analysed for Po:, Pco: and pH. No changes were detected in the SHAM samples taken before and after the ties were pulled. In the PNX group a significant decrease in Tb occurred (QW, 0.6 °C; G, 1.5 °C). Following PNX Pao, decreased by 11.2 mmHg (QW) and 10.0 mmHg (G); Paco: increased by 2.6 mmHg (QW) and 2.4 mmHg (G) and pH fell by 0.04 (QW) and 0.03 (G). All changes except in Pa¢o: (QW) were significant. It is concluded that the changes in Tb, blood gases and pH which follow phrenicotomy in the rat are due to an increase in dead space ventilation (~'D) and a small reduction in alveolar ventilation (VA) associated with a faster, shallower pattern of breathing.
Blood gases, phrenicotomy; Body temperature, phrenicotomy: Mammals, rat; Phrenicotomy, blood gases, body temperature
When a rat loses the activity of its diaphragm the frequency of respiration (f) increases and the tidal volume (VT) falls, however the minute ventilation (V) remains almost unchanged (Maskrey et al., 1990a). The maintenance of V is accomplished by reorchestrating the activity of the remaining respiratory muscles and recruiting muscles not normally involved in quiet respiration (Sherrey and Megirian, 1990). The above investigations were carried out during different states of consciousness, the former while the animals were awake and the latter during sleep. Other investigators report that phrenicotomy causes changes in ventilation and breathing pattern in the rat which depend on the preparation used. In anaesthetised, tracheostomised rats tested in the supine position the phrenicotomised rat suffers from hypoventilation due to reductions in both f and VT: Pao2 is decreased and Paco, is increased (Nachazel and Palecek, 1990). Although no blood gas data are provided, Correspondence to: M. Maskrey, Department of Physiology, University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001, Australia.
48
M. MASKREY et ai.
experiments carried out by the same workers using awake rats confirm the breathing pattern changes observed in this laboratory, i.e. a decrease in VT but an increase in f (Nachazel and Palecek, 1991). Following hemidiaphragm paralysis the awake rat's ventilatory drive is capable of overcompensating for any deficit in diaphragm movement with a consequent rise in Pao,_ (Goshgarian et al., 1986). Blood gas estimations have been carded out on both humans and other animals with a paralysed diaphragm. Newsom-Davis et al. (1976) studied seven patients with neuromuscular disease involving a paralysed diaphragm and found that three had a lowered Pao2 and a raised Paco2 while standing. Five patients showed similar changes while lying down, awake or asleep. Stradling et al. (1987) found that in the dog, the Paco., increased during rapid eye movement sleep (REMS). Sant'Ambrogio et al. (1970) have shown that Paco2 is increased in both anaesthetised cats and rabbits when the diaphragm is paralysed. In a previous paper, we suggested that the change in respiration of the bilaterally phrenicotomised rat to a faster, shallower pattern of breathing would significantly reduce alveolar ventilation ('~A) (Maskrey et al., 1990a). The present experiments were designed to test this hypothesis and to assess the effectiveness of the immediate compensation mechanisms in maintaining blood gases and pH within normal limits during quiet wake (QW) and grooming (G) episodes. Some aspects of the study have been briefly reported in abstract form (Maskrey et al., 1990b).
Methods Animals Hooded male Wistar rats (250-340 g) were used in this study. Initially it was found that when the right carotid artery was catheterised to obtain blood samples many of the animals became distressed and had to be sacrificed. It appears that phrenicotomy and the impairment of the cerebral circulation which follows right carotid artery iigation is a lethal combination in many rats. When the femoral artery was catheterised instead of the carotid the animals were not unduly stressed. Surgical procedures The rats were anaesthetised initially with Nembutal (45 mg.kg -1, i.p.). Ketamine (10 mg.kg-l, l.m.) was given subsequently as required to maintain surgical anaesthesia. With minor modifications the procedure for cannulating the right femoral artery was that recommended by Raft and Fagin (1984). PE-10 polyethylene tubing (4 cm) was inserted into the expanded end of PE-50 polyethylene tubing (40 cm) and the junction was sealed on the outside with 'Super Glue gel'. The PE-50 catheter was passed subcutaneously and attached to a subminiature socket which was cemented to the cranium in the midline. Phrenic nerves were identified in the root of the neck as previously described by Maskrey et al. (1990a). Silk threads were loosely tied around both of the nerves and
PHRENICOTOMY: BLOOD GASES
49
passed subcutaneously to the headpiece. When sham operations were performed, the silk threads were placed in the vicinity of the phrenic nerves but not around them. After separating the sterno-hyoid muscles, a thermocouple was placed in the superior mediastinum and the leads passed subcutaneously to the headpiece. Recordings of body temperature were made on a Digi-Sense (Cole Parmer) thermocouple thermometer. To avoid damage, the catheter and the thermocouple leads were taped to a cable, one end of which was plugged into the subminiature headsocket and the other into a slip ring connector attached to the roof of the incubator. When the catheters were inserted at surgery they were filled ~.,h heparinised saline (20 U.ml-l). After surgery, when the rats were awake and moving around, the catheters were flushed with 0.1 ml heparinised saline (5000 U.ml-~). When taking samples, the dead space volume was aspirated and then returned after 0.3 ml of blood had been taken.
Analysis of blood gases and pH These measurements were made on a Radiometer (Copenhagen) BMS3 MK2. The oxygen and carbon dioxide electrodes were calibrated with humidified gases using Coming 192 and WOsthoff 2M301/A-F gas mixers, pH electrodes were calibrated using Radiometer S 1500 and S 1510 precision buffer solutions. The analyser temperature was kept as close as possible to the animal's body temperature.
Experimental protocol The rats were allowed 24 h to recover from surgery. After the operation and throughout the experiment they were housed in a Gallenkamp incubator at a temperature of 29 + 1 ° C, this being the thermoneutral temperature of the adult rat (Bernet et al., 1975). After calibration of the pH, Po, and Pco, electrodes two blood samples, each of 0.3 ml, were taken approximately half an hour apart. One sample was taken during quiet wakefulness and the other during grooming. The ties round the phrenic nerves were then pulled leaving the rat with a paralysed diaphragm. After this procedure the animal was checked to make sure it displayed paradoxical respiration, typified by inward movement of the abdominal wall on inspiration, instead of the normal outward movement. In other studies, the effectiveness of phrenicotomy has been established by the total loss of electromyographic activity in the diaphragm following the pulling of the ties (Sherrey and Megirian, 1990). After I h, two further 0.3-ml samples were taken, again during quiet wakefulness and during grooming. When each blood sample was taken the animal's temperature was recorded. After the last sample had been taken the rat was sacrificed by injecting a lethal dose of Nembutal. The management of the sham operated animals was the same, except that the pulling of the ties did not result in paralysis of the diaphragm.
50
M. MASKREY et al.
Statistical methods The differences in each of the variables for each rat before and after PNX or before and after SHAMX were subjected to statistical analysis using Student's paired t-test. Results
The effects of bilateral phrenicotomy (PNX) on arterial blood gases and pH are shown in Fig. 1. Table 1 provides a statistical summary of these data. It can be seen that whether samples were taken during periods of quiet wakefulness (QW) or during grooming, Pao~ was significantly decreased following PNX. At the same time Paco: tended to increase, though there was a great deal of variability, especially during QW
Quiet Wake
PNX
100
SHAMX
~ 9o o E e0 IZ~k~----. O4 ¢~
~A
Grooming
o-.._..._~ oo===-:o o--
70
SHAMX
e ~
c--
o
'
'
--o
6O
40 E
PNX
A
B
35
°"'''--'°
25
,
7.55
,
C
== ¢~ 7,50
o.......~
7.45
i
8~=--,~
~
e ~
7.40 ,
,
,l
,
,
i
,
,
,
,
r
D
40
o~ 3e ~ 3s 34
32
o_______o
7
Before After
'
""
V
Before After
W
Before After
!
Before After
Fig. 1. Blood gas, pH and body temperature measurements from seven rats before and after phrenicotomy (PNX) and from five rats before and after sham operations (SHAMX) while quietly awake and during grooming. From top to bottom, graphs show effects of PNX and $HAMX on: A, arterial Po: (Pao,); B, arterial Pco: (Paco2); C, arterial pH (pHa); and D, body temperature (Tb).
P H R E N I C O T O M Y : BLOOD G A S E S
51
TABLE 1 Mean values ( _ SD) of changes in Pao,, Paco,, p H a and Tb in phrenicotomised rats and sham-operated rats while quietly awake and during grooming Quiet awake
Grooming
Before
After
Before
After
Phrenicotomy (n ffi 7) Pao2 (mmHg) Paco_, (mmHg) pHa Tb (°C)
85.15 + 2.48 3 !. 19 + 2.88 7.471 + 0.003 37.96 + 1.86
73.98 + 4.26" 33.76 + 2.28 7.435 + 0.016.. 37.31 + 1.56'
83.62 + 4.01 30.45 + 3.49 7.465 + 0.026 38.29 + !.49
73.61 + 7.46" 32.81 + 2.64.. 7.434 + 0.014" 36.83 + 1.93..
Sham-operated control (n = 5) Pao2 (mmHg) Paco2 (mmHg) pHa Tb (°C)
83.51 + 7.09 31.07 + 2.05 7.462 + 0.023 38.06+0.49
82.82 + 5.63 30.83 + 3.57 7.459 + 0.021 38.21 +0.75
84.81 + 12.35 27.71 + !.81 7.471 + 0.024 38.28+0.19
86.65 + 10.49 29.81 + 2.43 7.471 + 0.015 38.14+0.72
" P
