A simple method for high resolution light microscopy of nervous tissue

Journal o] Neurov¢teme Methods 15 (1985) 213-218

213

Flsevler

NSM 00540

A simple method for high resolution light microscopy of nervous tissue M P Pender Department of Erpertmental Pathology, John Curtm School of Medwal Research Austrahan National Umt,er~ttv Canberra Cttl A C T 2601 (Austraha) (Received August l l t h , 1985) (Accepted September 29th, 1985)

hey word~ histology - nervous system - light microscop~ - demyehnatlon - HlstoResm - experimental allergic encephalomyelitis A simple method for high resolution hght microscopy of nervous tissue is described Tissue perfused with glutaraldehyde and formaldehyde and postflxed with osmium tetroxlde is embedded In HlstoResln (LKB Bromma) a new glycol methacrylate based embedding medium and sectioned (2 t~m) on a standard mlcrotorne The method gives excellent preservation of tissue structure, which is far superior to that in paraffin sections and in fact resembles that m epoxy resin-embedded material It does not however require the time expemse and ultramicrotome needed to cut epoxy resin sections This technique is partlcularl) useful for assessing demyehnation

Introduction

Many published papers concerning the hlstopathology of the nervous system rely solely upon paraffin-embedded material to examine t~ssue structure This method has severe hmitat~ons, especially when used to assess demyelinatlon, as for example m experimental allergic encephalomyelitis (EAE), a possible ammal model of multiple sclerosis Semlthm (0 5-1 #m) sections of epoxy resin-embedded material stained with tolmdme blue provide extremely good light microscopic detad (Pender and Sears, 1984), but the time, expertise and ultramlcrotome required for this procedure are not available in many histological laboratories The present report describes a simple procedure giving excellent preservation of nervous tissue structure for light microscopy It employs a new glycol methacrylate based embedding medmm, HistoResm (LKB BrommaL which can be sectioned on a standard

microtome

Correspondence M P Pender, Department of Experimental Pathology, John Curtm School of Medical Research Australian National University P O Box 334 Canberra City A C T 2601, Australia 0165-0 270/85/$03 30 ': 1985 Elsevier ScLence Pubhshers B V (Blomedl~.al DlvlsLon)

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Fig 1 Transverse sections through sacral dorsal root gangha of a normal rat A HlstoResln section stained with toluldlne blue B paraffin section ~talned with chromoxane cyanln-R and neutral red Cahbratlon bars 25 p.m

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Materials and Methods The studtes were p e r f o r m e d on tissues from a n o r m a l adult Lewts rat a n d on 3 a d u l t Lewis rats with E A E i n d u c e d by i n o c u l a t i o n into the f o o t p a d s with a h o m o g e n a t e of g u i n e a - p i g spinal cord a n d i n c o m p l e t e F r e u n d ' s a d j u v a n t c o n t a l m n g ktlled Mvcobactertum butyrwum (Dlfco) The rats with E A E were killed 2 - 3 d a y s after the onset of neurological signs, which c o m m e n c e d 1 1 - 1 2 d a y s after inoculation U n d e r ether anaesthesia the rats were perfused t h r o u g h the left ventricle with 0 9% sahne until the effluent was clear and then with 120 ml of 2% g l u t a r a l d e h y d e , 2% f o r m a l d e h y d e in 0 1 M s o d i u m c a c o d y l a t e buffer ( p H 7 3 - 7 4) T h e brain, spinal cord, dorsal and ventral roots, dorsal root ganglia, spinal nerves a n d the sciatic a n d tatl nerves were r e m o v e d a n d i m m e r s e d in fixative The fixed tissues were cut into 1 m m thick shces which were postfixed with 2% o s m i u m tetroxlde, d e h y d r a t e d in a s c e n d i n g ethanols, and, after 1 h in a 1 1 mixture of ethanol a n d H l s t o R e s l n , infiltrated with full strength resm overnight a n d then e m b e d d e d Sections were cut on a Sorvall JB4 m l c r o t o m e (2 /~m) a n d stained wtth toluldlne blue m p h o s p h a t e

F~g 2 Trans'~erse section through a ventral horn of the sacral spinal cord of a normal rat An arrow indicates the soma of a motor neurone HlstoResm section stained with cresvl violet (_allbrat~on bar 25 p~m

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F~g 3 Transverse sections through sacral dorsal root gangha of a rat with EAE 2 days after the onset of neurological signs A HistoResm section stained with tolmdme blue Demyehnated axons (arrows) can be seen w~thm a region of mononuclear lrffIltrat*on B paraffin sect,on stained v,lth chromoxane c,~anln-R and neutral red Cal*bration bars 25 # m

217 buffer (pH 7 6) or with cresyl violet Specimens exceeding 2 m m m diameter had to be lnfdtrated with H l s t o R e s i n for longer periods a n d u n d e r v a c u u m in order to o b t a i n even i n f i l t r a t i o n of the osm~cated tissue For c o m p a r i s o n , some of the tissues fixed with glutaraldehyde a n d formaldehyde were prepared routmely--'ln paraffin, sectioned (4 # m ) a n d stained with c h r o m o x a n e c y a n l n - R a n d neutral red

Results H l s t o R e s l n sections stained with t o l m d m e blue or cresyl violet give very good preservation of tissue structure of the peripheral nervous system (PNS) a n d the central nervous system (CNS), i n c l u d i n g neurones, axons a n d m y e h n sheaths (Figs 1A a n d 2) The hght microscopic detad is m u c h greater than that m paraffin sections stained with c h r o m o x a n e c y a n l n - R a n d n e u t r a l red (Fig 1B) F u r t h e r m o r e , de-

F~g 4 Transverse section through the dorsal columns of the mld-thoraoc spinal cord of a rat w~th EAE 2 days after the onset of neurological signs Demyehnated axons (arrows) can be seen within a region of penvascular mononuclear cellular mfdtratlon (asterisk m one vessel) HlstoResm section stained with tolmdme blue Cahbratlon bar 25 #m

218 myehnatlon and inflammation in the PNS and CNS of rats with EAE can be clearly recognized in HistoResln sections (Figs 3A and 4) Of particular importance was the clear demonstration of naked axons, which is essential in distinguishing primary demyehnatlon (in which axons are spared) from demyellnat~on secondary to axonal degeneration Intracellular myelin debris was also revealed In the HlstoResln sec/ions In contrast, in paraffin sections stained w~th chromoxane cyanln-R and neutral red the naked axons and other details were only poorly defined (Fig 3B)

Discussion HlstoResln sections of normal and diseased nervous tissues provide excellent histological detail approaching that in semlthln epoxy resin sections and yet their preparation requires no more time, expertise or equipment than paraffin sections For best results, particularly when examining myelin, it is necessary to fix the tissues with glutaraldehyde and formaldehyde and to post-fix them with osmium tetroxlde Thin (2 /tm) serial sections can be cut from HlstoResin blocks by the ribboning technique Furthermore, HlstoResin enables the use of all the commonly employed stains and histochemlcal techniques This is an advantage over epoxy resin sections which are more difficult to stain A disadvantage of HlstoResln embedding is that it is not satisfactory for electron microscopy In conclusion, HlstoResln embedding of tissue post-fixed with osmium tetroxlde is a simple method which allows high resolution light microscopy of the nervous system It is particularly useful for demonstrating demyellnation

Acknowledgements I thank Dr Gutta Schoefl for helpful discussions and Ms Wendy Hughes and Ms Ailsa Rohnson for excellent technical assistance This work was supported by the National Multiple Sclerosis Society of Australia

Reference Pender, M P and Sears, T A (1984)The pathophyslologyof acute experimental allergic encephalomyehtl~ m the rabbit, Brain, 107 699-726