77
Journal of Physiology (1990), 430, pp. 77-86 With 5 figures Printed in Great Britain
ALTERATIONS IN CAT KNEE JOINT BLOOD FLOW INDUCED BY ELECTRICAL STIMULATION OF ARTICULAR AFFERENTS AND EFFERENTS BY A. KHOSHBATEN AND W. R. FERRELL From the Institute of Physiology, University of Glasgow, G12 8QQ
(Received 10 January 1990) SUMMARY
1. Experiments were performed in cats anaesthetized with pentobarbitone. Laser Doppler flowmetry was used to assess the responses of knee joint blood vessels to nerve stimulation under control conditions and in the presence of different adrenoceptor antagonists in order to establish the nature of neurotransmitters released from articular nerve fibres. 2. The posterior articular nerve (PAN) supplying the knee was stimulated at different intensities, and frequency-response curves were obtained. In fourteen animals electrical stimulation of PAN produced an initial vasoconstriction during stimulation which in eight of these was followed by a prolonged dilatation on cessation of stimulation. The constrictor response was increased as a function of frequency but was little altered with increasing intensity beyond a threshold level. 3. The constrictor response to electrical stimulation of PAN was markedly reduced by the a-adrenergic antagonist phentolamine (10-5 M), the al-blocker prazosin (10-5 M), and guanethidine (10-5 M) which inhibits the release of noradrenaline, ATP, and neuropeptide Y from sympathetic nerve endings. 4. The constrictor response to PAN stimulation was unaffected by the ot2-blocker rauwolscine and the P2-purinoceptor desensitizer a,,/-methylene ATP. 5. The dilator response was due to activation of afferent fibres as it could also be produced by direct electrical stimulation of the L7 dorsal roots. 6. The dilator response to stimulation of PAN or the L7 dorsal root was reduced by prior intra-articular injection of 100 jug of the substance P antagonist D-Pro4-D-
Trp7' 9 lo-Sp 7. These results suggest that the vasoconstrictor response to electrical stimulation of PAN is most likely to be mediated via noradrenaline acting mainly upon ocladrenoceptors. As the dilator response to articular nerve stimulation is reduced by a substance P antagonist, the mediator inducing this response may be substance P or a related neurokinin. INTRODUCTION
Although there have been some studies concerning the innervation of the knee joint in the cat (Gardner, 1944; Freeman & Wyke, 1967), less is known about the action of joint nerves in the regulation of articular blood flow. Cobbold & Lewis MS 8191
78
A. KHOSHBA TEN AND W. R. FERRELL
(1956) observed that electrical stimulation of the medial articular nerve (MAN) resulted in reduction of blood flow to the dog knee joint. However, the electrical stimulus parameters used were not stated. In a more recent investigation, electrical stimulation of the posterior articular nerve (PAN) was found to produce an initial vasoconstriction of blood vessels in the cat knee (Ferrell & Cant, 1987). However, in neither of these investigations was the nature of the neurotransmitters mediating these responses examined. In the study of Ferrell & Cant (1987) it was also observed that neurogenically mediated vasodilatation occurred after PAN stimulation. These authors concluded that stimulation of unmyelinated efferent nerve fibres, which are known to be present in PAN (Langford & Schmidt, 1983), produced the initial vasoconstriction whilst the vasodilatation resulted from activation of unmyelinated afferents. These findings were confirmed in a more recent study in the rabbit by Khoshbaten & Ferrell (1990). It was shown that stimulation of nerves supplying knee joint blood vessels in the rabbit resulted in vasoconstriction during stimulation followed by a prolonged vasodilatation on cessation of stimulation. In other sites such as skin, it has been observed that a neurogenically mediated increase in blood vessel permeability is accompanied by dilatation of these blood vessels (Couture & Cuello, 1984). For many years, studies of the neurohumoral control of the vasculature have been dominated by the role of catecholamines released from sympathetic perivascular nerves and from the adrenal medulla into the blood stream (Burnstock, 1975; Burnstock, Chanley & Campbell, 1986). Attention has also been paid to the cholinergic innervation of some blood vessels (Burnstock, 1980). In the last decade, however, non-adrenergic, non-cholinergic components of the autonomic nervous system have become established. Many neurotransmitters such as ATP, vasoactive intestinal polypeptide, substance P, dopamine and neuropeptide Y are now considered to play a role in the regulation of blood vessel calibre in many vascular beds (Burnstock, 1985). However, whether any of these substances are released from nerves innervating articular blood vessels has not been established. There is evidence for the involvement of noradrenaline as a neurotransmitter in nerves supplying rabbit knee joint blood vessels. In an in vitro study carried out by Ferrell & Khoshbaten (1990) it was found that the vasoconstrictor response to electrical stimulation of the rabbit knee joint capsule was mediated via noradrenaline acting upon ax-adrenoceptors. The dilator response observed by Ferrell & Cant (1987) was abolished by intra-articular injection of a substance P antagonist, suggesting that this was mediated by substance P or a related neuropeptide. The present study was performed to further examine the response of cat knee joint blood vessels, as measured by laser Doppler flowmetry, to electrical stimulation of the nerve supply to the posterior aspect of the knee, and determine the nature of neurotransmitters released from nerve endings on these vessels. METHODS
Experiments were performed on fourteen adult cats, their weights varying between 1-8 and
4.5 kg. Anaesthesia was by intraperitoneal injection of pentobarbitone (Sagatal, May & Baker Ltd)
in a dose of 45 mg kg-'. Maintenance of anaesthesia was by means of further intra-arterial injections of pentobarbitone in doses of 03-1 ml (30 mg ml-'), depending on the weight of cat. The
NEURAL REGULATION OF KNEE JOINT BLOOD FLOW
79
right common carotid artery was exposed and cannulated for monitoring arterial blood pressure. A laminectomy was performed to expose the spinal cord, and isolation of the dorsal roots L7 and S1. After laminectomy PAN was also prepared for electrical stimulation. Relative changes in knee joint blood flow were assessed by laser Doppler flowmetry (Moor Instruments MBF 3). The fibre-
Capsule
A T'ibia
emur
Nee
Pate
r.7-077.0
a
Probe
C L5
Ei 3 :
o1 en)
21
1 min
Fig. 1. Diagrammatic representation of (A) the lateral aspect of the cat knee joint and (B) the posterior aspect showing the intra-articular location of the laser probe which was inserted from the antero-lateral side through the infra-patellar region. C, responses to stimulation of PAN (10 V, 10 Hz, 1 ms) with the laser probe sited externally on the surface of the postero-medial capsule. The arrow indicates that a black polythene film (opaque to laser radiation) located between the internal surface of the posterior capsule and the medial femoral condyle was removed. Bars indicate the period of stimulation. Little change occurred in either the basal flowmeter signal or the response to nerve stimulation. optic probe (2 mm in diameter) was either located on the external surface of the posterior capsule or a smaller probe (0 9 mm in diameter) was inserted (via a 18 gauge needle) from the antero-lateral side of knee into the joint cavity and advanced dorsally to contact the internal surface of the posterior capsule (Fig. 1A and B). These probes contained two fibres, one of which conducted laser radiation (780 nm) to the tissue with the other fibre conducting the back-scattered light to
80
A. KHOSHBA TEN AND W. R. FERRELL
photodetectors. The rectal temperature was maintained at 37+1 °C by a heating lamp. The temperatures of the spinal and popliteal paraffin pools were also maintained at the same level by means of additional heating lamps. Stimulation of L7 and Si In experiments involving electrical stimulation of the L7 and SI dorsal roots, these were sectioned close to the spinal cord and placed on a pair of silver electrodes with an inter-electrode distance of 2-5 mm. These electrodes were connected to a Harvard advanced stimulator which produced squarewave pulses whose duration, intensity, and frequency could be varied. The parameters used for both PAN and dorsal root stimulation were chosen to be suprathreshold for group IV afferent fibres, using the criteria of Sato, Schaible & Schmidt (1983), and are included in the figure legends. Stimulation of PAN In experiments where PAN was to be electrically stimulated, it was necessary to transect this nerve to avoid reflex alterations in systemic blood pressure. Prior to transection, PAN was located on a pair of silver electrodes and then cut proximally whilst knee joint blood flow, arterial blood pressure, and heart rate were monitored continuously on a pen recorder. Although only shown once in subsequent figures, blood pressure and heart rate were monitored throughout the experiments and recordings only obtained whilst these remained constant. Intra-articular placement of the flowmeter probe resulted in only the capsular tissue being sampled, although this did not distinguish between synovial and peri-articular blood flow. Placing the probe on the external aspect of the posterior capsule could potentially sample structures other than the capsule (e.g. ligaments, bone). To test this, control experiments were performed which indicated that the flowmeter signal only related to changes in blood flow in the posterior region of the capsule (Fig. 1C).
Drug administration Adrenoceptor antagonists were administred by bolus injection (0-2 ml) of these agents via a polythene cannula inserted into an artery distal to the popliteal artery. The cannula was then advanced proximally until the tip was placed just distal to the knee joint supply and tied into position. The substance P antagonist D-Pro4-D-Trp7 9 ,'-SP4.11 was administered by intra-articular injection in a dose of 100 jug and a volume of 0-5 ml. Control experiments indicated that a similar volume of saline had little effect on nerve-mediated responses. Statistical data analysis was carried out by either paired or unpaired t test. An F test was also used to test the assumption of homogeneity of variances. Where this exceeded tabled F values, modified t values were generated using the formula described by Phillips (1978). All data expressed on graphs are means + S.E.M. Differences between means were considered significant if the P values were 5 % or less. RESULTS
Transection of PAN As shown in Fig. 2, in some experiments it was observed that transection of PAN resulted in an obvious rise in blood flow to the joint which was not accompanied by any reflex rise in arterial blood pressure, suggesting loss of pre-existing sympathetically mediated vasoconstrictor 'tone'.
Electrical stimulation of PAN In all fourteen cats a constrictor response was observed on electrical stimulation of PAN (Fig. 3A). Responses to PAN stimulation were obtained both by increasing the number of pulses per second and also by increasing the stimulus voltage (Fig. 4). These showed considerable enhancement of constrictor response to increasing the frequency (with a maximum at 30 Hz) but little significant change to increasing the voltage. As illustrated in Fig. 3A there was a consistent response to PAN stimulation
r-*r _
NEURAL REGULATION OF KNEE JOINT BLOOD FLOW
81
at intervals of 5 min. In eight out of fourteen cats, stimulation of articular nerve fibres resulted in a characteristic pattern of a rapid initial fall in the blood flow of the knee joint during stimulation followed by a prolonged vasodilatation on cessation of stimulation (Fig. 5A). It was noticeable that in these animals the 1 min
>4-
, 3
Journal of Physiology (1990), 430, pp. 77-86 With 5 figures Printed in Great Britain
ALTERATIONS IN CAT KNEE JOINT BLOOD FLOW INDUCED BY ELECTRICAL STIMULATION OF ARTICULAR AFFERENTS AND EFFERENTS BY A. KHOSHBATEN AND W. R. FERRELL From the Institute of Physiology, University of Glasgow, G12 8QQ
(Received 10 January 1990) SUMMARY
1. Experiments were performed in cats anaesthetized with pentobarbitone. Laser Doppler flowmetry was used to assess the responses of knee joint blood vessels to nerve stimulation under control conditions and in the presence of different adrenoceptor antagonists in order to establish the nature of neurotransmitters released from articular nerve fibres. 2. The posterior articular nerve (PAN) supplying the knee was stimulated at different intensities, and frequency-response curves were obtained. In fourteen animals electrical stimulation of PAN produced an initial vasoconstriction during stimulation which in eight of these was followed by a prolonged dilatation on cessation of stimulation. The constrictor response was increased as a function of frequency but was little altered with increasing intensity beyond a threshold level. 3. The constrictor response to electrical stimulation of PAN was markedly reduced by the a-adrenergic antagonist phentolamine (10-5 M), the al-blocker prazosin (10-5 M), and guanethidine (10-5 M) which inhibits the release of noradrenaline, ATP, and neuropeptide Y from sympathetic nerve endings. 4. The constrictor response to PAN stimulation was unaffected by the ot2-blocker rauwolscine and the P2-purinoceptor desensitizer a,,/-methylene ATP. 5. The dilator response was due to activation of afferent fibres as it could also be produced by direct electrical stimulation of the L7 dorsal roots. 6. The dilator response to stimulation of PAN or the L7 dorsal root was reduced by prior intra-articular injection of 100 jug of the substance P antagonist D-Pro4-D-
Trp7' 9 lo-Sp 7. These results suggest that the vasoconstrictor response to electrical stimulation of PAN is most likely to be mediated via noradrenaline acting mainly upon ocladrenoceptors. As the dilator response to articular nerve stimulation is reduced by a substance P antagonist, the mediator inducing this response may be substance P or a related neurokinin. INTRODUCTION
Although there have been some studies concerning the innervation of the knee joint in the cat (Gardner, 1944; Freeman & Wyke, 1967), less is known about the action of joint nerves in the regulation of articular blood flow. Cobbold & Lewis MS 8191
78
A. KHOSHBA TEN AND W. R. FERRELL
(1956) observed that electrical stimulation of the medial articular nerve (MAN) resulted in reduction of blood flow to the dog knee joint. However, the electrical stimulus parameters used were not stated. In a more recent investigation, electrical stimulation of the posterior articular nerve (PAN) was found to produce an initial vasoconstriction of blood vessels in the cat knee (Ferrell & Cant, 1987). However, in neither of these investigations was the nature of the neurotransmitters mediating these responses examined. In the study of Ferrell & Cant (1987) it was also observed that neurogenically mediated vasodilatation occurred after PAN stimulation. These authors concluded that stimulation of unmyelinated efferent nerve fibres, which are known to be present in PAN (Langford & Schmidt, 1983), produced the initial vasoconstriction whilst the vasodilatation resulted from activation of unmyelinated afferents. These findings were confirmed in a more recent study in the rabbit by Khoshbaten & Ferrell (1990). It was shown that stimulation of nerves supplying knee joint blood vessels in the rabbit resulted in vasoconstriction during stimulation followed by a prolonged vasodilatation on cessation of stimulation. In other sites such as skin, it has been observed that a neurogenically mediated increase in blood vessel permeability is accompanied by dilatation of these blood vessels (Couture & Cuello, 1984). For many years, studies of the neurohumoral control of the vasculature have been dominated by the role of catecholamines released from sympathetic perivascular nerves and from the adrenal medulla into the blood stream (Burnstock, 1975; Burnstock, Chanley & Campbell, 1986). Attention has also been paid to the cholinergic innervation of some blood vessels (Burnstock, 1980). In the last decade, however, non-adrenergic, non-cholinergic components of the autonomic nervous system have become established. Many neurotransmitters such as ATP, vasoactive intestinal polypeptide, substance P, dopamine and neuropeptide Y are now considered to play a role in the regulation of blood vessel calibre in many vascular beds (Burnstock, 1985). However, whether any of these substances are released from nerves innervating articular blood vessels has not been established. There is evidence for the involvement of noradrenaline as a neurotransmitter in nerves supplying rabbit knee joint blood vessels. In an in vitro study carried out by Ferrell & Khoshbaten (1990) it was found that the vasoconstrictor response to electrical stimulation of the rabbit knee joint capsule was mediated via noradrenaline acting upon ax-adrenoceptors. The dilator response observed by Ferrell & Cant (1987) was abolished by intra-articular injection of a substance P antagonist, suggesting that this was mediated by substance P or a related neuropeptide. The present study was performed to further examine the response of cat knee joint blood vessels, as measured by laser Doppler flowmetry, to electrical stimulation of the nerve supply to the posterior aspect of the knee, and determine the nature of neurotransmitters released from nerve endings on these vessels. METHODS
Experiments were performed on fourteen adult cats, their weights varying between 1-8 and
4.5 kg. Anaesthesia was by intraperitoneal injection of pentobarbitone (Sagatal, May & Baker Ltd)
in a dose of 45 mg kg-'. Maintenance of anaesthesia was by means of further intra-arterial injections of pentobarbitone in doses of 03-1 ml (30 mg ml-'), depending on the weight of cat. The
NEURAL REGULATION OF KNEE JOINT BLOOD FLOW
79
right common carotid artery was exposed and cannulated for monitoring arterial blood pressure. A laminectomy was performed to expose the spinal cord, and isolation of the dorsal roots L7 and S1. After laminectomy PAN was also prepared for electrical stimulation. Relative changes in knee joint blood flow were assessed by laser Doppler flowmetry (Moor Instruments MBF 3). The fibre-
Capsule
A T'ibia
emur
Nee
Pate
r.7-077.0
a
Probe
C L5
Ei 3 :
o1 en)
21
1 min
Fig. 1. Diagrammatic representation of (A) the lateral aspect of the cat knee joint and (B) the posterior aspect showing the intra-articular location of the laser probe which was inserted from the antero-lateral side through the infra-patellar region. C, responses to stimulation of PAN (10 V, 10 Hz, 1 ms) with the laser probe sited externally on the surface of the postero-medial capsule. The arrow indicates that a black polythene film (opaque to laser radiation) located between the internal surface of the posterior capsule and the medial femoral condyle was removed. Bars indicate the period of stimulation. Little change occurred in either the basal flowmeter signal or the response to nerve stimulation. optic probe (2 mm in diameter) was either located on the external surface of the posterior capsule or a smaller probe (0 9 mm in diameter) was inserted (via a 18 gauge needle) from the antero-lateral side of knee into the joint cavity and advanced dorsally to contact the internal surface of the posterior capsule (Fig. 1A and B). These probes contained two fibres, one of which conducted laser radiation (780 nm) to the tissue with the other fibre conducting the back-scattered light to
80
A. KHOSHBA TEN AND W. R. FERRELL
photodetectors. The rectal temperature was maintained at 37+1 °C by a heating lamp. The temperatures of the spinal and popliteal paraffin pools were also maintained at the same level by means of additional heating lamps. Stimulation of L7 and Si In experiments involving electrical stimulation of the L7 and SI dorsal roots, these were sectioned close to the spinal cord and placed on a pair of silver electrodes with an inter-electrode distance of 2-5 mm. These electrodes were connected to a Harvard advanced stimulator which produced squarewave pulses whose duration, intensity, and frequency could be varied. The parameters used for both PAN and dorsal root stimulation were chosen to be suprathreshold for group IV afferent fibres, using the criteria of Sato, Schaible & Schmidt (1983), and are included in the figure legends. Stimulation of PAN In experiments where PAN was to be electrically stimulated, it was necessary to transect this nerve to avoid reflex alterations in systemic blood pressure. Prior to transection, PAN was located on a pair of silver electrodes and then cut proximally whilst knee joint blood flow, arterial blood pressure, and heart rate were monitored continuously on a pen recorder. Although only shown once in subsequent figures, blood pressure and heart rate were monitored throughout the experiments and recordings only obtained whilst these remained constant. Intra-articular placement of the flowmeter probe resulted in only the capsular tissue being sampled, although this did not distinguish between synovial and peri-articular blood flow. Placing the probe on the external aspect of the posterior capsule could potentially sample structures other than the capsule (e.g. ligaments, bone). To test this, control experiments were performed which indicated that the flowmeter signal only related to changes in blood flow in the posterior region of the capsule (Fig. 1C).
Drug administration Adrenoceptor antagonists were administred by bolus injection (0-2 ml) of these agents via a polythene cannula inserted into an artery distal to the popliteal artery. The cannula was then advanced proximally until the tip was placed just distal to the knee joint supply and tied into position. The substance P antagonist D-Pro4-D-Trp7 9 ,'-SP4.11 was administered by intra-articular injection in a dose of 100 jug and a volume of 0-5 ml. Control experiments indicated that a similar volume of saline had little effect on nerve-mediated responses. Statistical data analysis was carried out by either paired or unpaired t test. An F test was also used to test the assumption of homogeneity of variances. Where this exceeded tabled F values, modified t values were generated using the formula described by Phillips (1978). All data expressed on graphs are means + S.E.M. Differences between means were considered significant if the P values were 5 % or less. RESULTS
Transection of PAN As shown in Fig. 2, in some experiments it was observed that transection of PAN resulted in an obvious rise in blood flow to the joint which was not accompanied by any reflex rise in arterial blood pressure, suggesting loss of pre-existing sympathetically mediated vasoconstrictor 'tone'.
Electrical stimulation of PAN In all fourteen cats a constrictor response was observed on electrical stimulation of PAN (Fig. 3A). Responses to PAN stimulation were obtained both by increasing the number of pulses per second and also by increasing the stimulus voltage (Fig. 4). These showed considerable enhancement of constrictor response to increasing the frequency (with a maximum at 30 Hz) but little significant change to increasing the voltage. As illustrated in Fig. 3A there was a consistent response to PAN stimulation
r-*r _
NEURAL REGULATION OF KNEE JOINT BLOOD FLOW
81
at intervals of 5 min. In eight out of fourteen cats, stimulation of articular nerve fibres resulted in a characteristic pattern of a rapid initial fall in the blood flow of the knee joint during stimulation followed by a prolonged vasodilatation on cessation of stimulation (Fig. 5A). It was noticeable that in these animals the 1 min
>4-
, 3
