BRES 23905

The effect of peripheral newe section on iectJn binding in the superficial dorsal horn of the rat spinal cord M B Plenderlelth and P.J Snow Mammahan Neurobtology Laboratory, DepartmentoJ Anatomy, Umverstty of Queensland, St Lucia, Queensland(Austraha) (Accepted 26 September 1989)

Key words Soybean agglutmm, Substance P, Fluoride resistant aod phosphatase Peripheral nerve section

The effect of peripheral nerve section on bmdmg of the plant lectm soybean agglutlnm (SBA) to the dorsal horn of the spinal cord has been investigated in the rat Four, 15 and 43 days after unilateral sclauc nerve section no effect on SBA binding m the 1_,4segment of the spinal cord was apparent However 90 days after nerve section a marked dechne m SBA binding was observed It is suggested that the long latency m effect on SBA binding may be due to the slow rate of utd~satlon of the SBA bmdmg site

It ts now well estabhshed that in most m a m m a h a n species, the m a j o r i t y of small d m m e t e r dorsal root ganghon ( D R G ) cells support u n m y e h n a t e d or small d m m e t e r m y e l m a t e d axons 3 s,15 16 These cells have peripheral processes which m n e r v a t e a n u m b e r of organs (including the skin) and they are acttvated chiefly by stlmuh which t h r e a t e n to d a m a g e the integrity of such organs These cells also send an axon into the spinal cord which t e r m m a t e wtthm the superficml laminae of the dorsal horn 25 Hlstochemlcal analysis has revealed that a n u m b e r of p u t a t w e neurotransm~tters, enzymes and o t h e r neurochemlcal m a r k e r s are assocmted exclusively with this population of small d m m e t e r p r i m a r y sensory neurones These include the n e u r o a c t w e peptlde, substance P (SP) and the e n z y m e , fluoride resistant a o d phosphatase ( F R A P ) 4 9 13,15 21 22 B o t h SP and F R A P have been found m small d i a m e t e r D R G cells, their p e r i p h e r a l processes and central terminals tn the spinal cord Recently we have shown that the plant lectin soybean agglutmm ( S B A ) brads to small d m m e t e r D R G cells, u n m y e h n a t e d axons and t e r m m a l s in the superficial dorsal horn m both the rat and the cat 17 is We have therefore p r o p o s e d that hke SP and F R A P , S B A m a y be used as a hlstochemlcal m a r k e r for a class of small d m m e t e r p r i m a r y sensory neurones In recent years a n u m b e r of studies have shown that d a m a g e to the p e r i p h e r a l processes of small d i a m e t e r p r i m a r y sensory n e u r o n e s has dramatic effects on their neurochemxstry F o r e x a m p l e both SP and F R A P are d e p l e t e d from the central t e r m m a l s of primary sensory

neurones following section of their p e r i p h e r a l processes l' 11 14 20 In the present study we have investigated the effect of p e r i p h e r a l nerve section on the binding of the plant lectln S B A in the dorsal horn of the rat spinal cord In the course of this investigation, 12 adult mate, Wtstar strain rats u n d e r w e n t unilateral section of the left sciatic nerve U n d e r d e e p h a l o t h a n e anaesthesia ( 3 - 4 % halothane m air) and using aseptic procedures, the sciatic nerve was e x p o s e d at the level of the p o p h t e a l fossa The nerve was tightly hgated using a silk suture and a 6 - 8 mm portion of the nerve trunk r e m o v e d d~stal to th~s ligature The lnOslon was then closed in layers and anaesthesia discontinued A f t e r p o s t o p e r a t i v e survival times of 4, 15, 43 and 90 days, rats were d e e p l y anaesthettsed with sodium pentob a r b l t o n e ( N e m b u t a l , 60 mg/kg l p ) and perfused through the a o r t a with 4 % p a r a f o r m a l d e h y d e and 0 05% glutaraldehyde using the p H change p r o t o c o l of B e r o d et al z The fourth l u m b a r (L4) segment of the spinal cord was then r e m o v e d and postfLxed in the same fixatwe for 4 h at 4 °C Following an overnight wash m 30% sucrose in 0 1 M p h o s p h a t e - b u f f e r e d saline (PBS), the entire L4 segment was serially sectioned (50-/~m transverse sections) wnh a freezmg m l c r o t o m e Sections were then screened alternately for either, SP-llke ~mmunoreactivtty (-LI), F R A P activity or S B A binding The pattern of SP-LI was studied usmg a polyclonal SP antiserum ( A m e r s h a m ) raised in rabb~ts SecUons were incubated m a 1 500 dilution of this p r i m a r y antlsera (m PBS containing 0 05% Triton X-100) for 48 h at 4 °C

Correspondence M B Plenderleith, Mammahan Neurob~ology Laboratory, Department of Anatomy, Umverslty of Queensland St Luoa 4067, Qld, Austraha 0006-8993/90/$03 50 © 1990 Elsevier Soence Pubhshers B V (Biomedical Division)

147 After washing in PBS, sections were incubated for 4 h in a biotinylated secondary antiserum (dtlutton 1 200 m PBS) raised against rabbit IgG and again washed in PBS Finally sections were Incubated m a streptawdm-horseradish peroxldase complex (ddutlon 1 200 in PBS), washed m PBS and developed in a solution of 0 5 mg/ml dlammobenztdine containing 0 015% H2Oe in phosphate buffer Fluoride-resistant acid phosphatase activity was v~suahzed by incubating sections in Gomorl medmm ~°,

containmg 0 15 mM sodmm fluoride, tor 3 h at 50 °C After washmg in PBS the sections were developed w~th 1% ammonium sulphide in distilled water The distribution of SBA-bindmg was carried out using a s~mtlar protocol to that described previously TM Briefly, sections were incubated in 12 5 /~g/ml of an S B A horseradish peroxidase complex m PBS for 24 h at 4 °C They were then washed to remove unbound conjugate and developed in a solution of PBS contaming 0 5 mg/ml diaminobenzidine and 0 015% H~O~

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Fig 1 Three adjacent sections through the L4 segment of spinal cord m a rat 15 days following scmtic nerve section The sections had been processed for F R A P activity (top), SP-LI (middle) and S B A binding (bottom) The right-hand panels (control side) exhibit a normal pattern of F R A P activity SP-LI and S B A binding The left panels show the ~.ltect of sciatic nerve section on the contralateral s~de ol thc same sections Note the virtual absence of F R A P actwltV m the medial portion of the dorsal horn The same region s h o w s a marked depiction of SP-LI but no effect on S B A binding Bar = 500/ira

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Fig 2 The distribution of FRAP activity (top), SP-LI (m~ddle) and SBA bmdlng (bottom) m the dorsal horn of the L4 segment of the spinal cord m a rat subjected to sctat~c nerve secUon 90 days prior to sacrifice The right-hand panels show the control (right) s~de and the panels on the left, the side subjected to nerve section Note the virtual absence of FRAP actlwty m the medial 2/3 of the dorsal horn on the left The same me&olateral region shows a marked depletion of SP-LI and SBA bmdmg when compared to the control side Bar = 500 um Following the htstochemlcal procedures outlined above all sections were m o u n t e d on glass slides m serial order, dehydrated, cleared and m o u n t e d m P e r m o u n t Results were obtained from 3 rats at each of 4 postoperative survtval periods The effect of sciatic nerve section on SP-LI, F R A P actlvtty and SBA binding were stmllar for each rat m a particular group Four days after sclattc nerve section both SP-LI and F R A P activity were reduced m the medial portion of the dorsal horn m the L4 segment of the spinal cord A l t h o u g h the medlolaterai extent of this depletion was similar for SP-LI and FRAP, there was a marked

difference in the a m o u n t of depleUon between the two W h e n compared to the me&al portion of the contralateral (control) side F R A P actwtty was found to be virtually abolished followmg peripheral nerve section while SP-LI m the same region was stgnlficantly reduced but sttll present Thts pattern of depletton of SP-LI and F R A P activity was also found in rats 15 43 and 90 days after SClatlc nerve section (Figs 1, 2) These results provided us with two important pieces of mformatlon Firstly they told us that our peripheral nerve section had been effective and secondly revealed exactly which portion of the spinal cord had been denervated

149 These controls were essential because 4, 15 and 43 days following p e r i p h e r a l nerve sectton we were unable to detect any effect on S B A binding m the dorsal horn In all rats there was no detectable difference m S B A binding m the medial portion of the superficial laminae of the dorsal horn, despite depletion of SP-LI and F R A P activity in a d j a c e n t sections (Fig 1) H o w e v e r by 90 days after sclattc nerve section there was a m a r k e d dechne in S B A binding In the dorsal horn (Fig 2) This depletion was similar in m e d l o l a t e r a l extent to that of SP-LI and F R A P (Fig 2) H o w e v e r at this postoperative time p e r i o d S B A binding was not completely a b o h s h e d m the m e d t o l a t e r a l portion of the dorsal horn These results, m agreement with previous reports, show that 4 days after sciatic nerve section both SP-LI and F R A P acttvtty are lost from the medial portion of the dorsal horn In the L4 segment of the spinal cord I s 11 14 20 No stgmficant recovery of either SP-LI or F R A P activity was found at survival times of up to 90 days s 2o Surpnsmgly however, 4, 15 and 43 days alter nerve section we observed no effect on S B A bmdmg in the same region of the spinal cord By 90 days however, depletton of S B A binding was found and this had a similar topographical distribution as that found for SP-LI and F R A P activity Clearly then, like SP-L1 and F R A P activity, S B A binding is lost from the central terminals of small diameter primary sensory neurones following section of their peripheral processes However, whilst the effect of the peripheral nerve lesion on SP-LI and F R A P activity ts seen in days, the effect on S B A binding is delayed by months The simplest explanation for the loss of SP-LI, F R A P aettvlty and S B A binding from the central terminals ot neurones following section of their p e r i p h e r a l processes would be that the entire neurone degenerates following such a lesion H o w e v e r m adult animals, p e r i p h e r a l nerve section appears to result in a loss of only a small p r o p o r t i o n of lesioned dorsal root ganglion ceils 6 1,~ F u r t h e r m o r e , although d e g e n e r a t i o n of axonal profiles has been r e p o r t e d m the superficial dorsal horn following sclattc nerve section 14, D e v o r et al 6 were unable to find m o r e than an occasional trace of d e g e n e r a t i o n and many a x o t o m l z e d terminals have been shown to exhibit essentially n o r m a l ultrastructure 24 Clearl~ then, d e g e n e r a t i o n of prtmary sensory neurones is unhkely to account for the dramatic loss of SP-LI, F R A P activity and S B A binding following a p e r i p h e r a l nerve lesion T h e r e f o r e an alternative explanation tor the loss of these 3 substances must be sought O n e posslbillt3 is that following section of their peripheral process, cells close down protein synthesis 27 W h e t h e r this is caused by the absence of a peripheral n e u r o t r o p h l c factor (such as nerve growth factor) or simply by the t r a u m a ot such an insult, the end result

would be mhlbltlOn of the production of SP, F R A P and the S B A hgand and consequently the loss of these from the central terminals of the neurones This m itself is not sufficient to explain the different latencles we observed for the depletion of S P / F R A P and S B A b m d m g However it is well established that SP and F R A P are syntheslsed in the cell b o d y of small d i a m e t e r primary sensory neurones and t r a n s p o r t e d in the axoplasm to their central terminals 1° 12 Based on electron microscoplc studies it has been p o s t u l a t e d that the S B A hgand is a m e m b r a n e associated glycoconlugate which is syntheslsed in the cell's Golgl a p p a r a t u s and then inserted into the plasma m e m b r a n e and t r a n s p o r t e d to the central terminals in the m e m b r a n e ls2~ These two differing mechanisms of transport b e t w e e n the soma and central terminals may be a consequence of the different rates of utdlsatton of different neurochemlcals In the intact neurone, p e p t l d e and enzyme t u r n o v e r m a y both be high which is reflected in the rapid t r a n s p o r t of SP and F R A P from the soma to the terminals along the mlcrotubule system On the o t h e r hand, the rate of turnover of the S B A binding molecule may be slower and thus is renewed by the p r e s u m a b l y slower m e m b r a n e transport mechanism If this really is the case and p e r i p h e r a l nerve section does cause cessation ot synthesis of SP, F R A P and the S B A binding then SP and F R A P would be expected to d i s a p p e a r from the central terminals faster than S B A b m d m g Two o t h e r studies have dealt with eftect ot peripheral nerve section on lectln binding on primary sensory neurones Fischer and Csflhk 7 have shown that the lectm Ule:t europaeus I ( U E A - I ) binds to both small and large D R G cells and lamina III and the ventral portion of lamina 1I in spinal cord from human cadavers Interestlngly in subjects who had u n d e r g o n e lower limb amputation between 2 and 36 months before death, U A E - I binding was lost from the lpsdateral lumbosacral spinal cord M o r e recently Tajtl et al 26 have r e p o r t e d that the two lectlns Rtcmus communts-I and Gr~fJonta stmphctfoha lsolectm B4 (both of which bind to small d i a m e t e r p r i m a r y sensory neurones and terminals within the superficial dorsal horn) are also d e p l e t e d trom the spinal cord 2 - 3 months after sciatic nerve section in he rat Despite differences in the sugar speclhclty of these different lectlns and the laminar termination oI the populations of p r i m a r y sensory n e u r o n e s that they label, the effects ot p e r i p h e r a l nerve section on the binding of these lectins are r e m a r k a b l y similar to those described for S B A in this study

Supported by t h e N H & M R C We wlsh to thank Dr P Wilson for crlncallv reading the manuscript and Ms D K Crook and Mrs A Duns for techmcal assistance

150

1 Barbut, D , Polak, J M and Wall, P D , Substance P m spinal dorsal horn decreases following peripheral nerve injury, Bram Research, 205 (19811 289-298 2 Berod A , Hartman, B K and Pujol, J F , Importance of fixation m lmmunoh~stochem~stry use of formaldehyde solutaons at variable pH for the locallsatlon of tyrosme hydroxylase, J Hlstochem Cvtochem, 29 (1981) 844-850 3 Cameron, A A , Leah, J D and Snow P J The electrophysiolog~cal and morphological characteristics of fehne dorsal root ganghon cells, Bram Research. 362 (19861 1-6 4 Cameron, A A , Leah, J D and Snow, P J The coexistence of neuropept~des in feline sensor) neurons, Neurosctence, 27 (1988) 969-979 5 Devor, M and Claman, D , Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn, Brain Research, 19(1 (1980) 17-28 6 Devor, M , Govnn-Llppmann, R Frank, I and Raber P Prohferat~on of primary sensory neurones in adult rat dorsal root ganglion and the kinetics of retrograde cell loss after scmtIC nerve section, Somatosens Res 3 (1985) 139-167 7 Fischer, J and Csllhk, B , Lectm binding a genuine marker for transganghomc regulation of human primary sensory neurones, Neurosct Lett, 54 (1985) 263-267 8 Harper, A A and Lawson, S N , Conduction velocity is related to cell type m rat dorsal root ganghon neurones J Phvszol, 359 (1985) 31-46 9 Hokfelt, T Kellerth, J - 0 , Nilsson, G and Pernow, B Substance P localization m the central nervous system and m some primary sensory neurones, Science, 190 (1975) 889-890 10 Hunt, S P and Ross1, J , PepUde- and non-peptlde-contaming primary afferents the parallel processing of noclceptlve information, Phil Trans R Soc Lond B, 308 (1985)283-290 11 Jessell T M , Tsunoo, A , Kamazawa, I and Otsuka M Substance P, depletion m the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurones, Bram Research, 168 (1979) 247-259 12 Keen P , Harmar, A J , Spears, F and Winter, E , Biosynthesis, axonal transport and turnover of neuronal substance P In Substance P m the nervous system C1BA symposium 91, Pitman London, 1982, pp 145-164 13 Knylhar, E , Laszlo, I and Tornyos, S , Fine structure and fluoride resistant acid phosphatase actlwty of electron dense slnuso~d terminals in the substantta gelatmosa Rolandl of the rat after dorsal root transection, Exp Brain Research, 19 (1974) 529-544 14 Knylhar, E and Csllhk, B , Effect of peripheral axotomy on the fine structure and hlstochemistry of the Rolando substance degenerative atrophy of central processes of pseudoumpolar ceils Exp Bram Res, 26 (1976) 73-87

15 Leah, J D Cameron A A , Kell), ~ L and Snov, PJ Coexistence of peptlde lmmunoreactlVlt) in sensory neurons of the cat Neurowlence, 16 (1985) 683-690 16 Lee, K H Chung, K , Chung, J M and Coggeshall, R E Correlation of cell body size, axon size and signal conduction velocity for individually labelled dorsal root ganghon cells m the cat J Comp Neurol 24~ (19861 335-346 17 Plenderlelth, M B , Cameron, A A , Key B and Snow, PJ Soybean agglutmin binds to a subpopulatlon ot primary sensory neurones in the cat Neuro~t Lett. 86 (1988) 252-262 18 Plenderleith, M B , Cameron A A Key B and Snow PJ The plant lectin soybean agglutlnln brads to a subpopulahon of small diameter primary sensory neurones m the rat and eat Neurosctence, 31 (1989) 683-695 19 Rlshng M Aldskoglus, H Hildebrand, ( and Remahl, S , Effects of sciatic nerve resection on L7 spinal dorsal roots and dorsal root gangha m adult cats Erp Neurol. 82 (1983) 568-580 20 Shehab S A S and Atkinson M E , Vasoactlve intestinal polypeptlde increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral nerve axotomy, Exp Brain Re~ , 62 (1986) 422-430 21 Sdva-Pinto, M and Colmbra, A , Comparative studies of the central nervous system phosphatases employing the Gomon and azo-dye methods Acta Anat 52 (1963) 157-173 22 Sllverman, J D and Kruger, L , Acid phosphatase as d selectwe marker for a class of small sensory ganglion cells in several mammals spinal cord d~stributlon, hlstochem~cal properties and relation to fluoride resistant acid phosphatase (FRAP) of rodents Somato~ens Res, 5 (1988) 219-246 23 Strelt, W J , Schulte 1 Balentlne, D and Spicer S S Evidence for glycoconlugates in nociceptlve primary sensory neurons and its origin from the Golgi complex, Brain Research, 377 (1986) 1-17 24 Sugimoto T and Gobel S , Primary neurones retain their central axonal arbors m the spinal dorsal horn following peripheral nerve injury an anatomical analysis using transganghontc transport of horseradish peroxtdase, Bram Research, 248 (19821 377-381 25 Suglura, Y , Lee C R and Perl, E R , Central projections of ~dentlfied unmyehnated (C) afferent fibres innervating mammahan skin, Science, 34 (19861 358-361 26 Tajtl, J , Fischer, J , Knylhar-Csdhk, E and CsIlhk, B , Transganghomc regulation and fine structural locahzat~on of lectm-reactwe carbohydrate epitopes m primary sensory neurons of the rat, Hlstochemtstry, 88 (1988) 213-218 27 Wells M R and Vaidya, U . Morphological alterations m dorsal root ganglion neurons after peripheral axon injury assocmtlon with changes m metabolism, Exp Neurol, 104 (19891 32-38