Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes

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~e~~~c~e~c~ Vol. 35, No. 1, pp. 195-202, 1990 Printed &IGreat Britain

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RELEASE, SPREAD AND PERSISTENCE OF IMMUNOREACTIVE NEUROKININ A IN THE DORSAL HORN OF THE CAT FOLLOWING NOXIOUS CUTANEOUS STIMULATION. STUDIES WITH ANTIBODY MICROPROBES A. W. DUXAN,*~

P. J. HOPE,?

B. JARROTT,$H.-G. SCHAIBL@and S. M. FLEETWOOD-WALKER?

tDepartment

of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, Edinburgh EH9 IQH, U.K. fClinica1 Pharmacology and Therapeutics Unit, Austin Hospital, Melbourne, Victoria 3084, Australia §Physiologi~hes Institut der Universitlt Wiirzburg, D8700 Wiirzburg, F.R.G. Abstract-In barbiturate anaesthetized spinal cats antibody microprobes were used to examine release of immunoreactive neurokinin A following cutaneous thermal and mechanical stimulation. In the absence of peripheral stimuli, microprobes detected a diffuse basal presence of immunoreactive neurokinin A. Noxious mechanical and to a lesser extent noxious thermal stimuli increased the levels of immunoreactive neurokinin A diffusely throughout the dorsal horn which, in many cases, spread into the adjacent white matter. These diffuse stimulus-evoked increases contrast with previous experiments where the same stimuli produced discrete focal increases in levels of immunor~ctive substance P. Evidence was obtained that released immunoreactive neurokinin A persisted in the spinal cord for at least 30 min beyond the period of stimulation. Neurokinin A needs consideration as the agent responsible for the long-lasting increases in excitability of some spinal neurons found by several laboratories to follow a brief input from unmyelinated primary afferents.

In the dorsal roots, dorsal root ganglia and dorsal horn of the spinal cord the distributions of the two tachykinins substance P (SP) and neurokinin A (NKA) are similar. 23 In contrast, the third mammalian tachykinin, ne~okinin B, is practically absent from dorsal roots and dorsal root ganglia and the amounts present in the spinal cord appear to be derived from intrinsic neuronsz3*** In the rat, immunohistochemical studies have shown that all dorsal root ganglion cells which contain immunoreactive NKA (IR NKA) also contain IR SP. In addition, however, there exists a population of ganglion cells which contain IR SP but not IR NKA.’ This is not surprising since studies of translation products of mRNA encoding tachykinin precursors have found three molecules, one containing the sequence of SP and NKA, one containing the sequence of SP and neuropeptide K an N-terminal extended form of NKA and the other possessing that of SP alone.15 Both in the periphery2J and centra11y2~“~r9 there is evidence for at least three binding sites for tachykinins with the implication that SP acts mainly at the NKI receptor with NKA and NKB preferring the NK2 and NK3 receptors, respectively. *To whom correspondence should be addressed. IR, immunoreactive; NKA, neurokinin A; PBS, phosphate-buffered saline; SP, substance P.

Abbreviations:

The functional implications of release of more than one tachykinin from primary afferent fibres are not well understood. Administered intrathecally to rats, analogues active at one or more tachykinin receptor have facilitated nociceptive responses.4*24,26*29 When a number of tachykinin analogues were administered iontophoretically in the substantia gelatinosa of the cat while recording from deeper spinocervical tract neurons, it was found that NKA enhanced excitation by thermal but not mechanical noxious stimuli, whereas SP reduced responses to inn~uous brushing of the skin without affecting firing by noxious stimuli.“’ These studies highlight the importance of determining which peripheral stimuli produce release of which tachykinins in the spinal cord. Synaptic release of tachykinins in the spinal cord in viuo has been studied by means of su~~usion,*‘,‘8,~ push-pull cannulae,r3 microdialysis’ and antibody microprobes. The latter technique has been used in the present experiments. There are two reports where spinal release of NKA and SP have been concurrently examined. In that of Hua et al.” both NKA and SP were released in a calcium-de~ndent manner from a spinal cord slice following application of capsaicin (IOpM) or potassium (60mM). Linderoth and Brodin” superfused the rat spinal cord in vitro and in uivo and were able to detect equimolar release of both tachykinins

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following peripheral nerve stimulation or superfusion of the spinal cord with capsaicin (IOllm) or potassium (40 mM). No release was detected by noxious thermal, mechanical or chemical stimuli applied to the skin. There is clearly a need to examine whether peripheral stimuli do release NKA in the spinal cord and, if such release occurs, whether it differs from that of SP in terms of the stimuli needed, the sites of release and inactivation within the spinal cord. The antibody microprobe has the best spatial resolution of present techniques for measuring tachykinin release’ and hence it has been used to examine these questions in the anaesthetized spinal cat. EXPERIMENTAL PROCEDURES

Microprobe preparation

Antibody microprobes were prepared as previously described.’ Briefly, fine glass micropipettes heat sealed at both ends were incubated in a 10% solution of aminopropyltriethoxysilane in toluene. This produced a siloxane polymer layer on the outer surface of microprobes and glutaraldehyde was then used to immobilize protein A (Porton) to this polymer. Protein A then bound immunoglobulins present in an antiserum to NKA. This antiserum was purchased from Peninsula Laboratories and data from the manufacturer indicates 100% cross-reactivity with kassinin but only 3% cross-reactivity with neurokinin B and substance P. The antiserum lyophylate as purchased included salts from the suspending buffer which imposed constraints on varying antibody dilution. The reconstituted serum was concentrated IO-fold by dialysis and the final dilution in which microprobes were incubated was approximately one in 3000. All protein binding was performed by inserting microprobes into 5 ~1 capillary tubes containing the relevant solutions and incubating for 24-48 h in a cold room. These antibody coated microprobes readily bound [“‘I]NKA and preincubation with 10es mol/l NKA for 30 min at 37°C suppressed this binding by 50% or more. Animal preparation Experiments were performed on 10 cats anaesthetized with sodium pentobarbitone (35 mg/kg, i.p.). Anaesthesia was maintained by continuous infusion of pentobarbitone, 3 mg/kg per h. All animals were artificially ventilated following neuromuscular paralysis with gallamine 4 mg/kg per h. Blood pressure was monitored via a cannula in a carotid artery and end tidal CO, levels were continually measured. The lumbar spinal cord was exposed by removal of the laminae and the spinal cord was transected at the thoracolumbar junction following injection of 0.1 ml of 2% lignocaine. The lumbar dura mater was cut longitudinally and retracted laterally. A thin layer of Ringer/agar was then placed over the dorsal surface of the exposed spinal cord. At

sites of proposed microprobe insertion an area of agar was removed and a small part of the pia-arachnoid was removed with fine forceps. The exposed dorsal area of the spinal cord was continuously irrigated with warm Ringer solution. Microprobes were inserted into the spinal cord with stepping motor micromanipulators. With the first probe introduced into a particular area of the spinal cord, it was usual to obtain extracellular recordings during introduction to determine the areas of the skin of the hindlimb which activated adjacent neurons during brushing. Nearly all microprobes were inserted to a depth of 3 mm which, in the lumbar dorsal horn of the adult cat, places the tips in the upper ventral horn. Peripheral thermal stimulation was provided by immersing a hindpaw in a water bath of a conlrolled temperature.

The hindpaw was immersed in the bath fbr 3 mm and ~hcn removed for 2min and this cycle repeated for the total duration of the stimulus. Noxious mechanical stimulation was provided by alligator clips applied to the glabrous skin of the hindlimb digits, on for 3 min and removed for 2 min. Stimulus durations varied from 5 to 60 min. The antibody microprobe technique detects bound endogenous ligand by the failure of binding of exogenous radiolabelled ligand. Thus, following removal from the spinal cord, microprobes were washed for I5 min in coid phosphate-buffered saline (PBS) containing Tween (0. I %) and then incubated for 24 h at 6 C in a PBS-azide solution of “51-radiolabelled NKA (Amersham) containing 0.5% bovine serum albumin. The final dilution resulted in approximately 2000 cpm/pl. After this incubation, microprobes were washed for I5 min in PBS-Tween while continually drawing the solution through the patent tips to remove any radiolabelled NKA from within. The tips were then broken off and glued to a sheet of paper, which was placed in an X-ray film cassette with a sheet of monoemulsion film (Kodak, NMC). Examples of the images of microprobes obtained in this way are shown in Fig. 1. Since the concentration of antibody used in these experiments was relatively high, exposure times of 21.-30 days were commonly used. Images of microprobes were analysed with an image analysis system employing an Image Technology PC Vision frame grabber board operating in a DCS 286e (AT-based) computer. A CCD camera scanned each image and, as described previously,” after background subtraction, a transverse integration of the optical density of the image of each microprobe was executed at defined intervals. With the magnification of the system used and the resolution of the image analysis system (5 12 x 512 locations/frame) this corresponds to a lo-pm interval for transverse integrations. The resultant integrals were stored on a hard disk record which included 32 coded values which described the experimental conditions relevant to that particular image. An analysis program subsequently obtained groups of microprobes which met stated criteria and obtained the mean image analysis of each group, which was plotted with respect to the distance within the spinal cord. In addition, differences and the significance of differences between controls and experimental groups were obtained and assigned statistical significance. It is important to emphasize the spatial resolution of the method. Thus. for microprobes inserted into the spinal cord events are examined at 100 sites/mm. Events at each site in the spinal cord are treated as independent of events at other sites

RESULTS

A total of 161 microprobes coated with antibodies to NKA were inserted into the spinal cord and 66 microprobes were used concurrently for in vitro sensitivity tests. In vitro microprobes The mean image density analysis of microprobes not exposed to NKA prior to incubation in [‘*SI]NKA is shown in Fig. 2A. In vitro tests showed that the procedure followed consistently detected NKA when exposed to lO-8 mol/l for 30 min at 37°C (22 microprobes). Control

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A total of 50 microprobes were inserted 3 mm into the spinal cord for periods of 15-60 min and in the absence of any applied peripheral stimulation. With

Antibody microprobes and neurokinin release in the dorsal horn

Fig. 1. Detection of IR NKA and IR SP in the spinal cord of the cat. Photographic enlargements of X-ray film images of microprobes have been superimposed on a section of spinal cord. The microprobe on the right shows uniform binding of [‘251]NKA. No peripheral stimulation was applied before or whilst this probe was in the cord. The two microprobes on the left were in the spinal cord while the digital pads of the ipsilateral hindpaw were pinched. The microprobe on the far left bore antibodies to SP, the middle microprobe bore antibodies to NKA. Note the discrete band of inhibition of binding of [‘251]SPon the former microprobe which contrasts with the diffuse inhibition of binding of [12?]NKA on the latter. Microprobe diameters are considerably smaller than these scattered X-ray film images suggest. Scale: the middle microprobe was inserted 3 mm into the spinal cord. 22 of these, no previous noxious stimulation had been applied to the hind limbs. An example is shown in

Fig. 1. With 23, however, the control (no stimulus) microprobes were inserted within 15 min of the termination of a period of noxious stimulation, Of these 23, the previous noxious stimulation was applied to the hindlimb ipsilateral to the area of the spinal cord examined by 12 microprobes, whereas 11 microprobes were inserted into an area of the spinal cord contralateral to the side of the animal just previously subjected to noxious stimulation. In the absence of previous noxious stimulation to the hindlimbs, control microprobes showed signifi-

cant differences from in vitro microprobes not exposed to unlabelled NKA. The mean image analysis of microprobes of the former group which were in the spinal cord for 30 min are shown in Fig. 2B. The differences between these groups are shown in Fig. 2C. This shows a significant basal presence of IR NKA at many sites in the spinal cord but in particular the dorsal surface and in the superficial dorsal horn. There was, however, a significant basal presence of IR NKA over virtually the whole of the dorsal horn. Similar patterns of decreased binding were shown on comparable control microprobes inserted into the spinal cord for 15 min.

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into the dorsal horn. (A) The mean image scan of in vifro microprobes not exposed to NKA (exogenous or endogenous) prior to incubation in {“‘IJNKA. (B) The mean image density scan of microprobes inserted for 30min into the dorsal horn prior to any noxious stimulation. (C) Upper record: the differences between the mean scans A and B and the standard errors of the differences of means are plotted. Lower record: the calculated t-values for differences of the means are shown, with T= 2 indicating significance at P = 0.05.

surface of the spinal cord and extending for approximately 1 mm ventrally. The mean image analysis of 10 control microprobes inserted for 30min into a side of the spinal cord contralateral to the side of the animal subjected to noxious stimuli showed no significant differences from the mean image analysis of controls in the absence of any prior noxious stimulation.

No evidence was obtained that innocuous thermal stimuli (36-38”C, eight microprobes) or innocuous flexing of the ~ndlimb (11 microprobes) produced images differing from those of control (with no previous noxious stimulus) microprobes. Noxious

The mean image analysis of control microprobes inserted for 30min, after prior ipsilateral noxious stimuli is shown in Fig. 3A. As shown in Fig. 3B, these differ significantly from controls without prior noxious stimuli, in showing a zone of decreased binding of [‘251]NKA starting 0.5 mm from the dorsal

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The analysis of control microprobes suggests the continued presence of IR NKA in the spinal cord beyond the duration of an applied stimulus. Therefore, when considering the effects of applied noxious stimuli, it has been found necessary to examine each type of noxious stimulus in isolation. In previous

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experiments investigating the release of IR SP following peripheral noxious stimulation,* it was sufficient to average the images of microprobes in the spinal cord for the duration of a defined stimulus. This has also been done in the present study, but in addition the analysis has (a) examined microprobe images in which the applied noxious stimulus had not been preceded by a noxious stimulus of another type and (b) examined the effects of sequential noxious stimuli of a given type. Noxious heat

The effects of successive noxious thermal stimuli using a water bath temperature of 48°C is illustrated in Fig. 4. The illustrated control microprobe

Fig. 5. Detection of IR NIL4 in the dorsal horn during successive noxious thermal stimuli. The mean image scan of microprobes inserted into the dorsal horn for 30min. (A) During the first noxious thermal stimulation (4648°C) of the hindpaw. (B) During subsequent noxious thermal stimuli (4648°C). (C) Upper record: the differences between the mean scans of A and control probes (with no prior noxious stimulation) and the standard errors of the differences of the means are plotted. Lower record: the calculated r-values for differences of the means are shown, with T = 2 indicating significance at P = 0.05. (Fig. 4A), inserted into the spinal cord for 20 min, has uniform binding of [‘251]NIL4. Immersing the hindlimb in water at 48°C for 20min (Fig. 4B) and

subsequently for 25 min (Fig. 4C) gave progressive inhibition of binding of [12’I]NKA along virtually the whole length of microprobes within the spinal cord. The pattern persisted in the absence of thermal stimulation (Fig. 4D). This microprobe was 40 min in the spinal cord and it was inserted 15 min after the withdrawal of that shown in Fig. 4C. This figure highlights the difficulty in relating a particular stimulus to the presence of IR NKA in the spinal cord since not only the intensity and duration of the stimulus need to be considered, but also what stimuli had preceded it. The effects of the first noxious

thermal stimulus (48 C for 30 min) with later stimuli are compared in Fig. 5. It is apparent that the later stimuli were associated with greater levels of IR NKA in the dorsal horn. This analysis was not possible when using stimulus temperatures of 52’C since in nearly all cases this stimulus was used after a series of stimuli at lower temperatures. It is important to emphasize that there was considerable variation in the time from the induction of anaesthesia to the completion of surgery and to the introduction of microprobes into the spinal cord. Despite this, the first (control) microprobes never showed the pattern of binding produced by subsequent stimuli. Thus there is little doubt that the progressive accumulation of IR NKA within the spinal cord was stimulus dependent and not the result of a progressive change in an undefined parameter.

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This stimulus consistently resulted in the detection of increased levels of IR NKA in the dorsal horn and, unlike thermal stimuli, this was apparent after the first stimulus of this type. The mean image analysis of all microprobes where the skin was pinched for 30min and had not been previously subjected to thermal noxious stimuli is illustrated in Fig. 6A. The differences from controls (Fig. 6B) are most significant over a zone starting just below the dorsal surface of the spinal cord and extending for 1.3 mm ventrally. There is a second zone of noxious pinch induced decreased binding of [‘*‘I]NKA from 1.8 to 2.3 mm from the spinal cord surface. Figure 6C shows that diffuse inhibition of binding of [“‘I]NKA was produced even by the first pinch stimuli used. Noxious pinch applied to the hindlimb for 15 min was sufficient to produce reduced binding of [‘251]NKA to microprobes. The effects of pinching the skin are also shown in Fig. 1 where photographic enlargements and not computer densitometric scans are illustrated. This figure contrasts the very different patterns produced on microprobes bearing immobilized antibodies to SP and to NKA when the skin of the hindlimb is pinched.

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DISCUSSION

Fig. 6. Detection of IR neurokinin A in the dorsal horn during noxious mechanical stimuli. (A) The mean image density scan of microprobes inserted into the dorsal horn for 30 min during noxious mechanical stimulation of the ipsilatera1 hindpaw. (B) Upper record: differences between the mean density scans of controls with no prior noxious stimulation (Fig. lB), and during noxious mechanical stimulation (Fig. 6A), and the standard errors of the differences of the means are plotted. Lower record: the calculated f-values for differences of the means are shown, with T = 2 indicating significance at P = 0.05. (C) The mean image analysis of microprobes inserted during the first periods of noxious pinching of the skin.

The results of the present experiments on release of IR NKA in the dorsal horn of the cat have remarkable differences from comparable previous experiments on the release of IR SP.* There differences can be listed as follows. (1) Significant IR NKA was detected diffusely in the dorsal horn under control (no added stimulus) conditions. Minimal basal SP was detected. (2) IR SP was not detected above control levels when using noxious thermal stimuli with temperatures below 50°C whereas water bath temperatures of 48°C were effective in the present experiments, at least beyond the first stimulus.

(3) The release of IR SP was relatively focal and approximated to the sites of termination of unmyelinated primary afferents. The present experiments observed diffuse presence of IR NKA in the dorsal horn. (4) Increased levels of IR NKA persisted beyond the duration of an effective stimulus. Moreover, the changes induced by a stimulus were dependent on which stimuli had been previously applied. This was not observed with release of IR SP. This was a surprising result since, in common with other methods used to measure neuropeptide release, the temporal resolution of the antibody microprobe is

Antibody microprobes and neurokinin release in the dorsal horn not good. Times in the spinal cord needed for detection vary from 5 to 30 min.6~8*2’,20 Since the latter time was most commonly used in the present experiments, they indicate a remarkable persistence of a ligand capable of binding to the immobilized antibodies of microprobes. Common to both neuropeptides was the relative ease with which pinching the skin produced release within the dorsal horn. Thus although these peptides have been reported to co-exist extensively within primary afferent fibres of the rat, they behave very differently when studied under in vivo experiments. Consideration of persistent detection of released compounds requires information on the ability of degradation products to bind to microprobe-immobilized antibodies. Neurokinin A appears to be remarkably resistant to the enzymes believed to be important in the degradation of SP. When equimolar amounts of SP, NKA and neuropeptide K, were incubated with plasma in vitro, SP was undetectable after 30 s whereas levels of NKA and neuropeptide K were little changed after 20 min.*’ The enzyme isolated by Nyberg et a1.22which cleaves SP at the 7-8 and 8-9 positions did not degrade NKA and NKB. These considerations make it unlikely that the compound binding to microprobes in the present experiments for periods beyond the application of noxious mechanical stimuli was a degradation product of NKA. An equally important question when considering persistant detection of a stimulus-evoked release of a compound is whether this persistence results from a continued release of the compound beyond the stimulus duration, or whether it results from a slow inactivation process. The above considerations of enzymes suggest that NKA is slowly degraded and hence a persistence of molecules released only during a peripheral stimulus is likely. Also favouring this explanation are comparisons with results from microprobe experiments examining release of IR SP.* With these, stimulus-evoked release of IR SP occurred in discrete bands and such bands approximated to the sites of SP-containing terminals of the upper dorsal horn. In the rat, NKA also has a restricted distribution in the upper dorsal horn’ but in the present experiments detection occurred broadly over the whole of the dorsal horn, a result very different from that with IR SP.6.8 Such a wide distribution could result from diffusion of neurokinin A not rapidly inactivated adjacent to sites of release. Another possibility which cannot be fully excluded is continued firing of nociceptors beyond the stimulus duration as a result of tissue damage. However, no obvious signs of damage were observed when using thermal stimuli of 46-48”C which contrasts with the extensive swelling produced by temperatures of 5&52”C which

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were needed to produce release in the spinal cord of IR SP.* Collectively, these considerations favour the conclusion that NKA acts diffusely and for long periods beyond the stimuli producing its release in the spinal cord. Microprobe experiments cannot establish sources of release with any certainty. The published data on the distribution of NKA5,23 would suggest, however, that release from primary afferent fibres is a significant component. It has heen suggested that the central release of IR SP is better correlated with peripheral inflammation.9 Recent studies of SP release measured with push-pull cannulae have also found spinal cord release with damaging cutaneous stimuli.t6 This association with inflammation rather than simply nociception appears unnecessary with NKA, however, since it was released by thermal temperatures in the range 4648°C.

CONCLUSIONS

The present results are consistent with the presence of NKA within polymodal nociceptors, but a discrepancy with immunocytochemical observations in the rat’ needs to be pointed out. It was found that all fibres containing IR NKA also contained IR SP5 yet in the present experiments thermal stimuli releasing IR NKA were not previously found to release IR SP under the same conditions. This discrepancy may result from the non-quantitative nature of immunocytochemistry. Thus, a neuron mainly synthesizing NKA might produce small amounts of SP given that both are contained in the precursor molecules of NKA. Both peptides might be detected by immunocytochemistry but only one may be present in significant amounts to be physiologically important. Persistence and wide diffusion of a released compound is not what would be expected of a neurotransmitter mediating fast transmission of information between neurons. Long-term changes in the spinal cord, however, have resulted from relatively brief activity in unmyelinated primary afferents’ and persistence of compounds probably better described as neuromodulators could underlie such changes. The present results indicate that NKA needs consideration in the context of long-term alterations in spinal cord excitability. Thus the present experiments may represent the first demonstration of the spread of a neuromodulator in the mammalian central nervous system. Acknowledgements--Supported by the MRC, Wellcome Trust, Heisenburg Foundation and the University of Edinburgh (Principal’s Fund). We wish to thank K. Main, H. Anderson, J. Brown for technical assistance and A. Stirling-Whyte for typing the manuscript.

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(Accepted 27 November 1989)