h’eurosrience Vol. 38, No. I, Printed in Great Britain

pp.205-212.

0306422/W

1990

53.00 + 0.00

Pergamon Press plc 0 1990IBRO

ALTERATION OF SUBSTANCE P AFTER TRAUMA TO THE SPINAL CORD: AN EXPERIMENTAL STUDY IN THE RAT H. S. Sr%W&$*t$ F. NYBERG,~Y. OLsaoNt and P. IL DEYQ TLaboratory of Neuropathology, Institute of Pathology, University Hospital and SDepartment of Pharmacology, Uppsala University, Uppsala, Sweden §Neurophysiology Research Unit, Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221 005, India Abstract-The distribution of substance P was determined in the rat spinal cord and brain after a focal traumatic injury to the thoracic region (T,,,, ) of the spinal cord. There was at 1 and 2 h after the injury a statistically significant increase of the substance P content not only in the injured segment but also in samples removed 5 mm proximal (T9) and distal (T,r) to the lesion. At 5 h the substance P content of the injured segment of the cord was reduced by 30% compared with controls. However, there was a significant increase in the concentration of this peptide in segments located 5 mm cranial and caudal to the injury (65% and 22%, respectively). Interestingly, the whole brain content of substance P showed a statistically significant 22% increase from control values at 5 h after the injury. At 1 and 2 h after the spinal cord injury there was a significant decrease in whole brain substance P concentration by 25% and 65%, respectively. Pretreatment with pthlorophenylalanine (a serotonin synthesis inhibitor) markedly reduced the endogenous content of substance P in whole brain of normal animals. In these animals, the spinal cord content of the peptide was elevated by 83-123% as compared to untreated control animals. Spinal cord trauma inflicted on pchlorophenylalanine-treated animals did not affect the brain peptide level at all. However, a profound decrease was noted in all the spinal cord segments at 5 h as compared to the untreated traumatized group. The decrease in this peptide was more pronounced in the cranial and the injured segments as compared to the caudal one. Our observations show that (1) substance P takes part in the early tissue reactions occurring in spinal cord trauma, (2) endogenous depletion of serotonin causes a redistribution of this peptide in the CNS, and (3) there is a functional interaction between serotonin and substance P in a trauma model, not reported earlier.

Neuropeptides in the spinal cord form a vast, rapidly expanding group of compounds, many of which may qualify as candidates for neurotransmitter or neuromodulator functions 3~4~8~13~16~17~2e31~41~50~52~53~58.63 core than 20 neuroactive peptides have now been localized in the cord. The rapid discovery of new peptides and peptide families challenges physiologists to define their modes of action as those of neurotranmitters, neuromodulators or trophic agents. Their roles in different pathological circumstances also need to be clarified. There are three possible sources of peptidecontaining nerves in the spinal cord.31.57 One is extrinsic, from afferent fibres derived from neurons within the dorsal ganglia, another is intrinsic, from cell bodies and terminals of interneurons, and the third one axons and collaterals from descending systems. 22*‘8 The presence of peptide-containing

*To whom correspondence should be addressed. HPLC, high performance hquid chromatography; p-CPA, p-chlorophenylalanine; RIA, radioimmunoassay; SP, substance P; TRH, thyrotropin releasing hormone.

Abbreuidons:

ascending and descending pathways in the spinal cord has been determined by examining the effect of spinal cord lesions, namely hemisections or transections on peptide levels (build up or depletion) rostra1 and caudal to the lesions.17 The dorsal horn is particularly rich in peptides and, indeed, every peptide so far identified in the spinal cord, with the exception of thyrotropin releasing hormone (TRH), is concentrated in the upper dorsal laminae I-III.*9~21~2s~2”M This region of the spinal cord contains thousands of closely packed neurons and its complexity derives from its role in processing and sorting incoming information. The majority of terminals of afferent fibres ends in laminae I-II and consequently many peptidecontaining fibres and terminals are concentrated here; substance P (SP), cholecystokinin, gastrin-like peptide and calcitonin gene-related peptides are some of the most abundant. Other peptides localized in the upper dorsal horn which are not found in afferents and are thought to be intrinsic to the CNS include the enkephalins, neurotensin and neuropeptide Y3e.59.M.In general, the distribution of any given peptide in the dorsal horn is similar throughout the entire length of the spinal cord, but there is a trend

205

H. S. SHARMAet al

206

for some peptides like SP to be most concentrated in sacral segments.“*‘” The region around the central canal (lamina X) and the dorsally adjacent area are also rich in these peptides. In the ventral spinal cord (laminae VI, VII, Vi11 and IX), the relative abundance of peptide is small compared to the dorsal horn and central canal region. In certain parts of the ventral spinal cord, peptidergic fibres tend to aggregate around specific groups of motoneurons. In lumbal segments of the rat, SP-containing ftbres are concentrated around a mediolateral motor nucleus and appear to form a peptidergic link between the ventral and dorsal spinal cord.20 l2 The origin of many peptides in the ventral cord remains to be established. Hemisection and transection markedly reduce leveis of SP and TRH caudal to a lesion, while no change in enkephalin, neurotensin or neuropeptide Y immunoreactivity has yet been reported.3i.53 Interestingly, by chemical, knife-cut or eiectrocoagulation lesions of brainstem nuclei of serotonergic neurons in the medullary raphe complex, not only serotonin-containing, but also SP-containing and TRH-containing fibres disappear from the ventral spinal cord, This suggests a coexistence and functional interrelationship between the three substances.2b,28.2955 We earlier reported that there is an increased serotonin content after a unilateral, longitudinal 5 mm long lesion in the dorsal horn (about lamina VII), also evident 5 mm rostra1 and caudal to the In addition, these spinal cord segments injury. 48,60.6i exhibited a profound increase in water content6’ Pretreatment with ~~hloroph~nylalanine @-CPA), a specific depletor of serotonin in the central nervous system.2.“,5’ resulted in the trauma-induced increase of the serotonin content being markedly attenuated. The increase in water content after the trauma was thwartedm.“’ (cf. Ref. 69). Since the dorsal horn contains almost all neuropeptides discovered so far, we have in this study investigated the effect of a spinal cord trauma, limited to the dorsal horn (lamina VII). The content of SP, [Metlenkephalin and dynorphin A in the injured as well as 5 mm rostra1 and caudal segments of the cord was determined at 5 h after the trauma. We also examined the concentration of these peptides in whole brain after making a lesion in the spinal cord to find out if there were any alterations in the peptides. Furthermore, to investigate the possible relationship between the peptides and serotonergic systems 1~4~9~15~*3~26~32-33~3g we depleted the endogenous seroionin content of the spinal cord prior to trauma by p-CPA, and measured the peptides in the same model. Comparisons were then made between untreated and drug-treated animals. This paper is concentrated on SP redistribution after spinal cord trauma.

The alteration

of opioid pcptides

in the same model will be reported in a forthcoming

paper.

The experiments were carried out on 30 inbred Sprague.Dawley rats (AIab, Stockholm, Sweden, 300-350 g) housed at controlled ambient temperature (22 f 1°C) with a 12 h light and 12 h dark schedule. Food and tap water were supplied ad libitum. Spinal cord trauma

Under urethane anaesthesia (1.5gfkg i.p.), a dorsal laminectomy was done between the Tie_,, segments. One about 3 mm deep and about 5 mm long unilateral incision into the dorsal horn was made 2mm to the right of the midline using a scalpel blade. The injured spinat cord was covered with cotton soaked in sahne. In a separate group of rats, examination of fo~~n~x~ injured segments shows that the lesion was mainfy Iocahzed to lammae I-VII.“.W p-Chlorophenylalanine

treatment

In a separate group of animals, p-CPA (Sigma Chemical Co., U.S.A.) was administeredin~~~t~~~ in a dose of IO0 mglkg daily for three consecutive days. Oa the fourth day these animals were subjected to the spinai cord trauma.3.X.5i.W Peptides and chemicals

Synthetic peptides used in this study were purchased from Bachem (Bubendorf, Stitzetfand). ?he c&sumskqrqhii material (SP-Seahadex C-2JI was obtained from %armacia

(Uppsala; SW&~). All other chemiib and solvents weic:of analytical reagent grade from commercIa1 sources. Experimental

protocol

The animals were divided into the following groups. (a) Conrrol group. Urethane anaesthetized intact animals

served as control (n = 5). (b) Spinaf cord traumut~~edgrou~. The spinal cord trauma was inflicted in 15 animals. At the end of I (n = 5). 2 (n = 5) and 5 h (n = 5) survival Periods, the spinal cord and brain samples were removed after decapitation. (c) pChlorophenyl&ninepretreatedgroup. In 10 an+Is, p-CPA was given according to the dose and thnc scWutC mentioned above. Five animals served as intact emttih. The remaining five animals survived 5 h after the spinalcord injury. Sampling protocol After 1, 2 and 5 h survival periods, the animals were decapitated. The traumatiaed spinal cord and 3mm rostra1 and caudal segmentsadO were removed quickly and placed in preweighed plastic vials on dry ice. In addition, the ivhofe brain was also included for analysis of the pepridc. The samples were imm~iat~y weighed, frozen and sto+ at minus 90°C until assay.” The site of thG spif&t%Wd varied between I20 and 14Omg. The whob brain W#%s weighed between 1.98 and 2.2Og. Sample preparation

The frozen tissues were thawed and hoe at 90°C as described.‘O The extract was cohected a~~~~~n and separated on minicohtmns for ion e~~hangG&rwartoe_ raphy LSP-Sephadcx).’ Prior to application, the ompk was diluted with 4 ml of 0.01% M pyridine in 0.1 M formic rcid (pH 3.2) before separation on s--x llriaioakrmns The column was wethaI with the above pyddimc Bruit; and thereafter eluta! acquantialIy with 4mI of 0.3J and I.6 M of pyridine-formate buffer (pki 4.4). The f-ions collected at I .6 M py~dine-fo~ate were evaporated and

207

Substance P in spinal cord trauma analysed for SP immunoreactivity. The recovery of SP in the SP-Sephadex procedure was 80%.

higher than that of the T9 values, but the difference was not statistically significant.

Radioimmunoassay

Spinal cord traumatized animals

The radioimmunoassay (RIA) for SP was described in detail in a recent paper. cI The procedure is based on the charcoal adsorption technique. The detection limit of the RIA was about 5 fmol/tube. The cross-reactivity of the antiserum with SP fragments (3-l I), (5-l 1) and (6-l 1) was 100, 60 and 20% respectively, with all SP N-terminal fragments and other SP related peptides less than 0.1%. High performance

liquid chromatography

identification

The immunoreactivity detected in the different samples was identified by high performance liquid chromatography (HPLC). Reversed-phase separation was performed using an LKB instrument (LKB Produkter AB, Broman, Sweden) equipped with an LKB 2134 Super Pat Spherisorb column (5 FM particle size, 4.5 mm x 250mm). The column was eluted with a linear gradient of 15-45% acetonitrile containing 0.04% trifluoroacetic acid. Fractions of 0.5ml were collected at a flow rate of 0.5 ml/min and evaporated before RIA. Statistical

treatment

of the data

data were computed using commercial software (Excel, Microsoft Corp., Statview 512 + SE+, Abacus Concepts Inc., U.S.A.) on a personal computer (Macintosh Plus, Apple Computer Inc., U.S.A.). Dunnett’s test for multiple group comparison was used to evaluate the statistical significance of the data obtained.‘” A P-value less than 0.05 was considered significant. The

RESULTS

Normal anirna/s

The distribution of SP in brain and three spinal cord segments in the intact control group is shown in Table 1. There was a gradual increase in spinal cord content of SP in rostrocaudal direction using Tg values as reference. This increase in peptide was significant at the T,, level. On the other hand, though, the whole brain content of the peptide was apparently

Spinal cord trauma markedly affected the SP values in all the spinal cord segments and in brain tissues examined (Table 1). Thus, there was an initial significant increase in SP content on all the segments of the spinal cord after I h which tended to decline after 2 h injury. At the end of 5 h there was a 30% decrease in the injured segment as compared to the control value. The corresponding rostra1 and caudal segments showed a significant 65% and 22% increase from the control group, respectively. On the other hand, the response of brain peptide to the spinal cord trauma was different. Thus, there was a progressive decrease of SP activity by 25% after 1 h and 65% after 2 h injury but, at the end of 5 h survival period, the brain peptide content showed a significant 22% increase as compared to the intact control group. E#ect of pchlorophenylalanine

pretreatment

Normal animals. p-CPA pretreatment markedly affected the endogenous content of SP in brain and spinal cord of untraumatized animals (Table 2). Thus, there was an 87% decrease in whole brain SP content in p-CPA pretreatment animals as compared to the untreated control group. On the other hand, the spinal cord substance P was remarkably enhanced in all the segments examined. Thus, there were 123%, 117% and 83% increases in the SP activity in T,, TIC,, and T12 segments, respectivety as compared to the untreated corresponding control (Table 2). Spinal cord traumatized animals. As is evident from Table 2, p-CPA pretreatment markedly modified the SP response of spinal cord trauma in both the spinal

Table 1. Brain and spinal cord substance P content in control and spinal cord traumatized rats Substance P [fmol/lOOmg tissue (wet wt)] Spinal cord segments TP rostra1

T l&lI injured

T,Z

Type of experiment

Brain

Intact control n= 5 Spinal cord injury lh

185 k 30

154f26

177 + 33

186f6

138 4 37* (-25)

320 f 17** (+ 107)

279 f 26+* (+57)

249 f 22* (-t33)

63 f 15** (-65)

274 f 26** (+79j

276 + 18*” (+55)

266 f 16* (f43)

227k 17+* (+22)

254+ ll+* (+65)

124&-42* (-30)

227 + 17’ (+22)

caudal

n=5

Spinal cord injury 2h n= 5 Spinal cord injury 5h n-5

The animals were allowed to survive 1, 2 or 5 h after the injury and samples were taken from the entire brain and three spinal cord segments. Values are mean + S.D. Figures in parentheses indicate % change from intact control. + = increase, - = decrease. *P < 0.05, l*P < 0.01 Dunnet’s test for multiple group comparison.

208

H. S.

SHARMA et

al.

Table 2. Effect of p-chlorophenylalanine pretreatment on substance P distribution in brain and spinal cord of normal and spinal cord traumatized rats --

Type of experiment Intact control tl=5 p-CPA + intact control n=S Spinal cord injury 5 h survival n=5 p-CPA + spinal cord injury 5 h survivaf a= 5

Substance P [fmo1/1OOmg tissue (wet wt)] --Spinal cord segments

Brain

T9

T m-11

rostra1

injured

T,,

caudal

185 _+30

154+26

177 * 33

186*6

23 + 2** (-87) 227& 17** (+22)

344 + 28** (+ 123) 254f II** (+65)

385 i 46** (+117) 124f42* (-30)

341 f 34** (+83) 227 f 17” (+22)

32 + 12** (-79)

74 f 19** (-58)

224 f 34** (+20)

24 + 2** (-87)

The animals were allowed to survive 5 h after the injury and samples were taken from the entire brain and three spinal cord segments. p-CPA was administered intraperitoneally (100 mg/kg) daily for 3 days. On the fourth day the animals were subjected to the traumatic injury of the spinal cord. Values are mean & S.D. Figures in parentheses indicate % change from intact control. + = increase. - = decrease. *P < 0.05. **P < 0.01 Dunnet’s test for multiple group comparison.

cord and in the brain as compared to the untreated traumatized group. There was a marked decrease in SP activity after 5 h spinal cord trauma in all the segments (Table 2). On the other hand, there was no change in brain SP activity after trauma in p-CPAtreated animals as compared to the drug-treated control group.

The principal difference between the p-CPAtreated and untreated group 5 h after the trauma was that there was a marked profound decrease in the SP activity not only in the injured spinal cord segments but also in the cranial and caudal segments. In the untreated injured group there was a sig&eant increase in SP activity in rostra1 and caudai~segmcnts at this time period and the decrease of SP in the injured segment was very small. High performance

liquid chromarqraphy cation of substance P immunoreactivity

i&ntip-

By reversed-phase HPLC the SP immunoreactivity present in the spinal cord samples was resolved in two separate components (Fig. 1). The major peak ebrted identically to synthetic SP, whereas a minor peak was observed in a position close to that of SP sutphoxide. DISCUSSION

Ra@fwon

time 6nh)

Fig. 1. Reversed-phase WPLC of SP-like immunoreactivity from the rat spinal cord. The immunoreactivity is rep resented by the hatched area. The retention times for synthetic SP and its sulphoxide were 32 and 25 min. respectively. For other conditions, see text.

The most implant o~~a~o~ in the present study is that there are idled changes in the concentration of SP in the spiaal cord and in brain after a focal traumatic lesion to the dorsal horn of rat spinal cord. Depletion of the endwous serotonin content by Q-CPA marked& a#%etedtlpe dMfibutioi% of SP in the brain and spinai cord of normal a&&s. This treatment also sj~~~~ altered the traumainduced changes in this peptide in bram aad spinal cord. These observations indicate a functional relationship between serotonin and SF which may be rvlati to their coexistence qorted ~arfii.*~~ Furthermore, this study demantita, for thy &st time, a signi~~nt alteration in whok brain content of

209

Substance P in spinal cord trauma

SP after a lesion made in the spinal cord. This opens

tonin

several new questions

on the probable

pathways

peptidergic

influence

on various

information

processing

a new concept, may tional

induce

involvement

of

pathophysiological

in the brain. It also illustrates

namely that a focal injury of the cord biochemical

changes

within

and the

probably brain

as

also well.

func-

Obviously, further efforts should be made to clarify their significance. SP has attracted great interest for its proposed function in neurotransmission or neuromodulation and has been extensively studied in various biologiSeveral cal and clinical aspects. 7.18.37.43.46c7.50.52.56.70.71 studies in neurological diseases have been directed toward SP analysis of cerebrospinal fluid samples in humans.63 Data collected from different studies of SP in human cerebrospinal fluid show higher values in patients with psychiatric disorderqs6 while decrease levels were associated with chronic neurogenic or psychogenic pain67 and in cluster headache.62 The initial decrease in brain SP and concurrent increase in spinal cord suggest a probable release of the peptide or its activation in certain brain regions. SP is located in nerve fibres of small diameter and primary sensory neurons, the central branches of which terminate in the dorsal horn of the spinal cord.2S.49Another anatomical pool of nerve terminals, mainly in the ventral horn of the spinal cord, derives from nerve fibres descending from midbrain areas. In brain large amounts of SP are found in areas like striatum, substantia nigra, globus pallidus and hypothalamus.” Biochemical, psychological and immunological observations support the involvement of SP in spinal function 11.12.15.17.18.34.42.50.53.65 SP is released in the spinal cord by direct stimulation of sciatic nerve, and the release of SP from sensory neurons is decreased by opiates in the intact spinal cord.“.72 Its role in pain transmission is well documented in the literature (cf. Ref. 8). Our results show that this peptide is also involved in traumatic insults to spinal cord. The release of SP from sensory endings in the spinal cord is regulated by descending, intersegmental and segmental intemeurons. This is evident by the fact that this release from dorsal root ganglion cells is not only inhibited by the opiates, but also by serotonin, GABA and norepinephrine.” The sero-

influence

is suggested

in the intact

through

its descending

spinal

cord. Our results further show that p-CPA, a 5-hydroxytryptamine synthesis inhibitor drug, could markedly affect the distribution of SP in brain and spinal cord. A marked decrease of SP in the brain and profound build up in spinal cord segment after treatment with p-CPA would indicate that there is a functional association between S-hydroxytryptamine and the redistribution of SP content.‘.6.9 Previous studies have also shown that there is a marked decrease in SP activity after degeneration of S-hydroxytryptamine neurons with the neuro~~~~ toxin 5,6_dihydroxytryptamine. 1.23.24,32,33.35,39.54~6! ther studies are needed until we can understand the association between SP and serotonin in the spinal cord. The accumulation of SP caudal to the injury point and its decrease in brain suggests that the peptide may be involved as a transmitter in ascending sensory pathways. The segmental extent of these rostrally directed pathways has not been identified. Similarly, the peptides have been observed to accumulate on the sensory ganglion side of ligation of both the peripheral nerves and the dorsal roots,‘* indicating that the peptide is not only transported along the afferent axon into the spinal cord, but also enters the peripheral neurite. The functional significance of the alterations of SP content in brain and spinal cord after trauma is not known. Our earlier studies showed a gradual progressive edema after spinal cord trauma in the same mode1.48.60.6L p-CPA pretreatment remarkedly thwarted this edema development after 5 h injury. 48*60*6’ On the basis of this information it may be suggested that marked build up of SP in the spinal cord segments prior to injury and its profound release over 5 h after trauma may be important in mitigating the edema formation. However, further studies are needed to clarify these points. study was supported by grants from the Swedish Medical Research Council, projects 12X03020, 04X-3766, Wallenius Line, Soderbergs Stiftelser, RTP, Rikesbankens Jubileums Fond and from TryggHansa. Miss Sari Kankaanranta is acknowledged for skilful technical assistance and Mrs Aruna Misra for secretarial assistance. Acknowledgements-This

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-

__

34. Jessels T. M. and Yamamoto M. (1981) Identification and interactions of substance P, enkephalin and serotonin neurons in vivo and in dissociated cell culture. In Neuropeptides: Basic and Clinical Aspecls. Proceedings of the Xlth Pfizer Inrernafional Symposium (eds Fink G. and Whall&L. J.), pp. 73-87. Churchill Livingstone, London. 35. Johansson 0.. HBkfelt T., Pemow B., Jeffcoate S. L., White N., Steinbuseh H. W. M., Verhofstnd A. A. J., Emson P. C. and Spindel E. (1981) Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P and thyrotropin releasing hormone-like immunoreactivity in medulhiry neurons projecting to the spinal cord. Neuroscience 6, 1857-l 88 1.

Substance

211

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