Chronic Cervical Compressive Myelopathy

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Hughes TT (1966) Pathology of the Spinal Cord, Year Book MedicalPublishers,Chicago

Hukuda S. Mochizuki T, O gata M, Shichikawa K and Shimomura Y (1985)

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Wright F and Palmer AC (1969 ) Pathol Vet 6:355

Yovich JV, tashak TS and Powers BE (1987) Proc 33rd Annu Meet Am

(Acceptedfor publication 1August 1991)

(1979 ) Vet Surg 8 8 4

Assoc Equine Pract, p 595

Chronic cervical compressive myelopathy in horses:

patterns of astrocytosis in the spinal cord

JV YOVICH*, DH GOULD', and RA LeCOUTEURt

SUMMARY: The distribution and morphology of fibrous astrocytes in the cervical spinal

cord of normal horses and horses with chronic compressive myelopathy weredemonstrated uslng lmmunohlstochemical staining for glial flbrlllary acidic protein.

In the spinal cord from normal horses, astrocytes with stellate cell bodles and short

processes were Irregularly dlstributed Ingrey matter. In he white matter, their cell bodleswere small and angular In areas adjacent to grey matter and larger and more stellate-shaped in the subplal area. Astrocyte processes were fine, and evenly distributed In apredominantly radial pattern In transverse sections of cord.Glloslswas marked n he spinal cords of horses with cervical compressive myelopathy.

In he grey matter at the level of compression astrocyteswere often enlarged and rounded,

with short, blunt processes, but the gllosis was generally mild. in he white matter, gllosiswas obvious inareas of nerve ibre swelling and degeneration at the level of compression

and in areasof ascending and descending Wallerian degeneration. The fine radial pattern

of astrocyte fibres was replaced by a dense, Irregular arrangement. Giiosis persisted Inthe cords of chronlcally affected horses after active nerve flbre degeneration had sub-sided. The areas of gllosis coincided with the areas of March1 staining for degeneratingmyelln and with areas of myelln loss in osmium tetroxlde post-fixed tissue. Histologicalobservations were consistent with astrocytes replacing areas of extracellular space thatremained after nerve fibre degeneration. It is concluded that astrocytic gllosis is aprominent and perslstent alteration of the spinal cord of horses with chronic cervicalCompressive myelopathy.

Introduction

Astrocytes undergo extensive proliferation and hypertrophy, aprocess called fibrous gliosis, in response to trauma and certaindiseases of the central nervous system (Sternaas e t a1 1987).Reactive astrocytes are thought to perform several functions intraumatised neural tissue including removal of myelin andneuronal debris (Nathaniel and Nathaniel 1977), filling in ofenlarged extracellular spaces, maintenance of the contents of theextracellular space including ions and neurotransm itters (Barrettet al 1981; Eng et a1 1987) and re-establishment of the glialimitans (Reier et a1 1983). Despite these seemingly beneficialeffects, reactive astrocytes may block axonal regeneration,prevent remyelination and perhaps disturb the function ofremaining nerve fibres (Reier e t a1 1983; Eng e t a1 1987; Stensaase t a1 1987).

School of Veterinary Studies, Murdoch University. Mu rdoch, Western

Australia6150

t Departmentsof Clinical Sciences and Veterinary Pathology, CollegeofVeterinary Medicine and Biomedical Sciences, Colorado StateUniversity, Fort Collins, Colorado 80523

Aust Vet J 68: 3 3 4 4 3 7

Investigations inlo astrocyte response in spinal cord injury arecomplicated when damage to adjacent tissueresults inconnectivetissue ingrowth (Reier et a1 1983; Stensaas e t a1 1987). Accord-ingly, investigators have examined the response in areas of

Wallerian degeneration away from the traumatised site (Barrette t a1 1981; Stensaas et af 1987). Chronic cervical compressivemyelopathy in horses provides an opportunity to examine themorphology of astrocyte responses in circumstances where theglia limitans remains intact an d the spinal cord is not penetratedby meningeal connective tissue.

Reactive aseocytes accumulate ilaments, he major componentof

which is glial fibrillary acidic protein (GFAP) (DeArmond et a1

1980). In the mature central nervous system, GFAP is limited toastrocytes (Eng et al 1987). In this study an immunohistochemicaltechnique was used to identlfy GFA P in order to examine astrocyteresponses in the spinal cord of horses with chroniccervical compres-sive myelopathy of varying du ration.

*Dako Corporation, Santa Barbara, CA (primary antibody)5 Vectastain ABC Kit, Vector Laboratories. Burlingham, CA

g Sigm a Chemical Co, St Louis, MO

334 Australian VeterinaryJournal, Vo l68 , N o 10, October 1991



Figure 1 . Grey matter from a transverse section of the cervical spinalcord of a normal horse (No 14) (A), and from horse No 7 (B) at thelevel of injury from cervical compressive myelopathy of 14 monthsduration. Stained or glial fibri llary acidic protein. Astrocyte cell bodiesand processes (dark reaction product) are more numerous in thecompressed spinal cord. Bar = 100 pm.

Materials and Methods

Thirteen horses with stable clinical signs of cervical compres-

sive m yelopathy (generalised ataxia and tetraparesis) of 4 to 29months duration and 2 normal horses (No 14 an d 15)were used,and spinal cord sections from these were prepared as previouslydescribed (Yovich et a1 1991).

Spinal cord for immunohistochemistry was dehydrated ingraded ethanol solutions and chloroform and em bedded in paraf-fin Sections cut at 5 pm were deparaff i ised in xylene and

ethanol,rinsedindistilledwaterfor5min,thenincubatedin0.3%

hydrogen peroxide in Tris buffer (0.5 M, pH 7.6) to quenchendogenous peroxidase activity, and rinsed in Tris buffer. Afterincubation with diluted normal godt serum (blocking serum) andblotting to remove excess serum, the sections were sequentiallyincubated with rabbit anti-GFAP S. biotinylated goat antirabbitIgC and avidin-biotin-horseradish peroxidase complex'.

A primary antibody dilution of 1:400 was used and sectionswere w ashed in Tri s buffer between incubations. Rabbit wholenonimmune serumn(1:400 dilution) or buffer (negative control)

Figure 2. White matter of the ventral funiculus from a transversesection of spinal cord from a normal horse (No 15) (A), and from thelevel of spinal cord injury in horse No 2 with cervical compressivemyelopathy of 4 months duration (B), and 14 months duration (C) (Noa) , stained or glial fibrillary acidic protein. Compared with A, astrocytecell bodies and processes (dark reaction product) are more numerous

in the compressed spinal cords, either with (B), or without (C), exten-sive nerve fibre swelling. Bar = 200 pm.

were substituted for the primary antibody on consecutive spinalcord sections. Diaminobenzidine tetrahydrochloride, 0.1% in

Tris buffer with an equal volume of 0.02% hydrogen peroxide,was the peroxidase substrate solution. The sections were cover-slipped and examined by light microscopy. Sections were com-pared with H an d E, Marchi and osm ium stained sections of the

same spinal cord segm ents as previously described (Yovich et al

19 91).

Results

Reaction product staining was not present in sections wherebuffer or nonimmune rabbit serum was substituted for primary

Australian VeterinaryJownal,Vol68, No 10,October 1991 335



antibody. In spinal cord sections from all horses, staining ofGFA P was restricted to astrocyte bodies and processes.

Astrocytcs in the grey matter of the spinal cord from normalhorses were irregularly distributed and had stellate bodies withshort, tapered processes (Figure 1A ). In the white matter adjacentto grey matter, their bodies were small and angular, but in theouter third of the white matter, adjacent to the pia m ater, the cellswere larger, with stellate bodies (Figure 2A). Throughout thewhite matter, astrocyte processes were long and fine, with aslightly granular staining pattern. They surrounded nerve fibres

and vessels, giving an ovcrall radial pattern to the staining andwere concentrated also in the subependym al area, with an overallorientation perpendicular to ependym al cells. The glia limitansadjacent to the pia mater was formed by a continuous layer of

Figure 3. White matter of the lateral funiculus of a transverse section

of the spinal cord, cranial to the injured segment inNo 3with cervical

compressive myelopathy. Areas of nerve fibre degeneration, darkly

stained by the Marchi technique (A), correspond with pale areas of

nerve fibre loss in osmium tetroxide post-fixed tissue (B), and areas

of gliosis indicated by staining of glial fibrillary acidic protein(C). Bar

= 200pm .

astrocyte processes that w ere thicker w here vessels entered thespinal cord parenchyma. A dense, parallel accum ulation of theprocesses accompanied dorsal nerve root fibres and fibres cours-ing through the grey matter.

In cord from horses with cervical compressive myelopathy, atthe level of compression there w as m ild gliosis characterised bymore numerous astrocyte bodies and processes in the grey matter(Figure 1B ). Where gliosis in the grey matter was most obvious,many of the astrocyte bodies were enlarged and rounded, withintense GFA P staining of the cytoplasm. The processes of these

enlarged cells were blunt and short.In the white m atter of all cords there w as extens ive gliosis at the

level of compression. Astrocyte bodies were numerous, enlarged,angular and stellate. Cell processes were thickened and had more

.granular staining than in normal spinal cord. Gliosis was mostmarked in areas of nerve fibre swelling and degeneration,where thetine radial pattern of GFAP staining in normal spinal cords wasreplaced by a dense, irregular mosaic of staining of cell bodies andprocess es. The mos t severely aCfected horses w ere No 1 to5 and 11.which, except for No 11, were those with the shortest duration ofclinical signs (4 o 6 months). How ever, som e gliosis persisted inh o r n with a longer duration of clinical signs (14 to29 months), andwhich had less nerve fibre swelling (Figures 2B, C).

Gliosis also was obvious in areas of W allerian degenerationaway from the level of compression, with astrocytes similar tothose in the com pressed segments, except that their bodies wereno t as enlarged. Although this gliosis was m ore extensive in cordswith the most nerve fibre swelling, some gliosis was present inall horses in areas where nerve fibre degeneration had occurredin ascending and descending tracts. Nevertheless, the gliosisassociated with degeneration in descending tracts adjacent to h eventral median fissure was more dcnse than that in ascendingtracts. Areas of gliosis consistently matched the areas of Marchistaining for degenerating myelin and w ere most prominent where

osmiu m tetroxide post-fixation illustrated myelin loss (Figurc 3).

The extent of gliosis did not correlate with the age of affectedhorses or the level of spinal cord compression.

Discussion

Gliosis was sev ere and persisted in the spinal cord of horseswith chronic cervical compressive myelopathy. It was associatedwith nerve fibre degeneration at the level of compress ion and inwell delineated areas of ascending and descending nerve fibretracts. In most cases gliosis of the grey matter was mild at thelcvel of compression, although extensive gliosis has been

described in areas of modcrate grey matter degeneration fromother causes (Barrett el a1 1981; Reier and Houle 1988).

Substances liberated from degenerating nerve fibres have beenproposed as gliosis stimulation factors, based on observationsthat myelin bas ic protein caus es proliferation of culturedastrocytes (Shcffield and K im 1977), whileneuronal sonicate canstimulate astrocyte proliferation in virro and can also stimulatecell process extension in a dose dependent fashion (Hansen and

Yartlow 1978,1980). Both myelin and neurone derived substan-ces may have stimulated gliosis in our horses since it waslocalised to areas of nerve fibre degeneration and was greatestwhere this wa s most extcnsivc.

The reaction of astrocytes in the spinal cord in areas of Wal-

lenan degeneration distant from the primary injury has beenstudied in other species (Lampert and C ressman 1966; Bignamiand Ralston 1969; Barrett et a1 1981). For example, in catssubjected to dorsal nerve root transection (Rignami and Ralston1969), by 60 days, in areas of degeneration in the white matterthere was enlargement of the extracellular space by amorphousmaterial; by 100 days this space had been filled by astrocyte'processes. A similar process of astrocyte filling of extracellularspac e appears to have o ccurred in the spinal cord of horses in thisstudy, both at, and distant to, the compressive lesion. Gliosispersisted in the chronically affccted horses, after the amount of

336 Australian VererinaryJournaI,Vol68, No 10,October 1991



nerve fibre swelling and degeneration had greatly decreased. Th epresence of g liosis in the cords of horses w ith clinical signs of 14to29months duration, when active nerve fibre degeneration wasminimal, indicates that there may be some permanent residualdamage in these chronic cases.

Because haem orrhagic necrosis, spinal cord cavitation o r dis-ruption of the pia and glia limitans, which a re mo re characteristicof acute impact trauma (Wagner et a1 1978), were not a feature

of the spinal cord injury in horses in this study, con nective tissueformation by mesenchymal cells did not interfere with the reac-

tion of astrocytes in response to compressive spinal cord injury.Th e full influence of gliosis on axonal regeneration in chronic

compressive myelopathy is unknown, although it is generallyconsidere d that this regeneration is blocked by astrocyte proces-ses (Stensaas et a1 1987). Astrocytes can ha lt regenerating axonsby activating a "physiological stop pathway" inherent in axonmetabolism, which normally operates when axons form ter-minals on target cells (Liuzzi and Lasek 1987). Hence, a densescar producing physical obstruction is not the only means ofastrocyte impediment of axon regeneration. By whatever means,the persistent astrocytic gliosis present in chronic cervical com-

pressive myelopathy of horses may prevent, orslow, recovery ofneurologic function in affected horses.

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Liuzzi FJ and Lasek RJ (1987) Science 237642

Nathaniel EJH and Nathaniel DR (1977) ExpN e w o l 5 4 6 0

Reier PJ and HouleJD (1988) In Advances inNeurology, edited by Waxman

Reier PJ, Stensaas LT and Guth L (1983) In Spinal Cord Reconstruction,

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Stensaas LT, a d o w LM, urgess PR and Horch KW (1987)ProgBrainRes

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(Accepted or publicatio n: August I 1991)

S G . Raven Press, New York, p 87

edited by Kao CC et al, Raven Press.New York, 163

71:457

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In the authors' words "the purpose of this text is to provideveterinary practitioners, residents, interns and students withinformation ab out the basic process of wound hcaling and severa laspects of wound management and repair". The text "is notmeant to provide an exhaustive coverage of pathophysiology,diagnostic alternatives, or perioperative care options" but

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Pfizer Annual Res earch Conference, 39th, 1991,PfizerAgricare Pty Ltd, Wharf Road, W est Ryde, NSW 2114

The feeling of being overwhelmed by the vast literature of

science is somewhat mitigated when an organisation manages tocondense, correlate and coordinate at least som e of the m aterial.Pfizer, though the Annual Research Conferences d o just this, ina very useful way. The Proceed ings of the 1991Conference deal

with the literature for 1990: Poultry (RD Miles, U niversity of

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The presentations at the Conference are comprehensive and areexcellent leads into the current literature - but not much fromAustralia or New Zealand. Perhaps we are not doing sufficient

research!Hugh MeL Gordon

Australian VeterinaryJournaI.Vol68, No 10,October 1991 337

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Chronic Cervical Compressive Myelopathy