Upsala J Med Sci 91: 251-255, 1986 Surface Cryoplaning A Technique f o r Clinical Anatomical Correlations Wolfgang Rauschning Department of Orthopaedic Surgery, University Hospital, Uppsalu, Sweden During t h e p a s t decade, r a p i d advances in t h e c l i n i c a l d i s c i p l i n e s a n d mile- s t o n e achievements i n d i a g n o s t i c imaging such a s x-ray computed tomography, magnetic resonance imaging and u l t r a s o n o g r a p h y , have aroused renewed i n t e r e s t i n g r o s s anatomy a n d d e t a i l e d segmental anatomical c o r r e l a t i o n s . I n o r t h o p a e d i c s u r g e r y , t h e development of new techniques f o r t h e t r e a t m e n t of d i s o r d e r s of t h e j o i n t s and t h e s p i n e such as a l l o p l a s t i c j o i n t replacement, ligament recon- s t r u c t i o n , a r t h r o s c o p i c s u r g e r y , decompressive surgery f o r s p i n a l s t e n o s i s o r improved management of s p i n a l f r a c t u r e s a l l c a l l f o r thorough knowledge of f u n c t i o n a l anatomy a n d biomechanics. In a c l i n i c a l - a n a t o m i c a l i n v e s t i g a t i o n of p o p l i t e a l c y s t s a n d t h e i r r e l - a t i o n s h i p t o t h e gastrocnemio-semimembranosus bursa (4), knee specimens were frozen i n v a r i o u s p o s t u r e s and then s e r i a l l y s e c t i o n e d on a l a r g e , heavy-duty cryomicrotome, devised by Ullberg (7,8) f o r a u t o r a d i o g r a p h i c s t u d i e s i n u n d e c a l c i f i e d experimental animals u p t o t h e s i z e o f monkeys. S p e c i f i c r e q u i r e - ment i n h e r e n t in s t u d i e s of musculoskeletal anatomy prompted m o d i f i c a t i o n s of t h e o r i g i n a l c r y o s e c t i o n i n g t e c h n i q u e . In t h i s a r t i c l e , a d e s c r i p t i o n of t h e cryoplaning technique i s supplemented w i t h i l l u s t r a t i v e examples of s p i n e anatomy. TECHNIQUE The a c q u i s i t i o n of f r e s h c a d a v e r i c specimens i s t h e prime p r e r e q u i s i t e f o r good anatomical s t u d i e s . Since t h e procurement of such m a t e r i a l s from w i l l e d body donor programs is l i m i t e d with r e s p e c t t o number and t h e u s u a l l y high age of t h e deceased, t h e major i t y of specimens i s o b t a i n e d from d e p a r t - ments of pathology and f o r e n s i c medicine d u r i n g r o u t i n e a u t o p s i e s . To prevent t h e deformation of t h e topographic r e l a t i o n s h i p s between t h e bone and t h e s o f t t i s s u e s as well as drainage of blood a n d emptying of f l u i d - f i l l e d c a v i t i e s , f r e e z i n g o f t h e specimen in s i t u i s e s s e n t i a l . A r t e r i e s and v e i n s a r e more r e a d i l y d i s t i n g u i s h e d i f they a r e f i l l e d with c o l o r e d l a t e x o r dye p r i o r t o f r e e z i n g . While a whole c a d a v e r , p r o p e r l y p o s i t i o n e d and s u p p o r t e d , 25 1 may be frozen in t o t o , in s i t u f r e e z i n g of a j o i n t o r a s p i n e segment i s d i f f i - c u l t and time-consuming. The specimen i s prepared by d i v i s i o n of a d j a c e n t s o f t t i s s u e s and i n s u l a t e d a g a i n s t t h e s k i n a n d organs with c e l l u l o s e . Crushed dry i c e i s packed i n t o t h e s e spaces a n d l i q u i d n i t r o g e n i s r e p e a t e d l y poured over t h e i c e t o lower t h e temperatureof t h e c r y o g e n i c . Dependent on t h e s i z e of t h e specimen, i t t a k e s two t o f o u r hours b e f o r e i t i s frozen s o l i d and can be c u t o u t with a n o s c i l l a t i n g e l e c t r i c saw. For d i r e c t c o r r e l a t i o n s w i t h computed tomographic s c a n s , t h e specimen may be em- bedded i n orthogonal Styrofoam boxes f i l l e d with precooled carboxymethyl c e l l u - l o s e gel (CMCIwhich i s f r o z e n t o i c e . Scanning may be performed in o p t i o n a l p l a n e s prior t o s e c t i o n i n g ( 5 ) . Other specimens may be embedded d i r e c t l y on t h e s t a g e by f i l l i n g t h e box a n d surrounding metal frame with CMC gel a n d f r e e z i n g a t -70' C . t a p e , b u t by r e p e a t e d l y photographing t h e specimen s u r f a c e ( 4 , 5 ) . a t m i l l i m e t e r i n t e r v a l s . A f t e r t e s t s w i t h cinematographic equipment had shown t h a t t h e microtome s l e d g e s t o p s e x a c t l y i n t h e same p o s i t i o n a f t e r each s t r o k e , t h e following p r o v i s i o n s were made f o r s e q u e n t i a l image r e g i s t r a t i o n : A s t a b l e camerastand w i t h a f i x t u r e f o r t h e v e r t i c a l l y moving k n i f e h o l d e r a l s o c a r r i e s a h o r i z o n t a l b a r towhich a n a d j u s t a b l e hinged r u l e r w i t h a m i l l i m e t e r s c a l e i s a t t a c h e d . The r u l e r i s a l i g n e d along t h e lower b a s e l i n e of t h e image frame a n d t h e camera then focused on t h e plane specimen s u r f a c e . As t h e k n i f e i s f e d down during s e c t i o n i n g , t h e camera and s c a l e a r e lowered, rendering i d e n t i c a l alignment a n d m a g n i f i c a t i o n of t h e images throughout t h e specimen. Previously embedded specimens a r e t r a n s f e r r e d t o t h e microtome s t a g e . The anatomical images a r e not o b t a i n e d from s e c t i o n s c o l l e c t e d on adhesive Modern scanners "zoom" through a n orthogonal block of t i s s u e i n t h e p a t i e n t In s t i l l cameraspthe image frame i s not a c c u r a t e l y r e l a t e d t o t h e t r a n s - p o r t s p r o c k e t h o l e s of the f i l m . A 35 mm s i n g l e lens r e f l e x camera was t h e r e f o r e r e b u i l t f o r e x a c t p i n - r e g i s t r a t i o n . Photographic images a r e taken on 25 A S A c o l o r r e v e r s a l f i l m w i t h f l a t f i e l d l e n s e s a n d e x t e n s i o n t u b e s allowing f o r v a r i o u s m a g n i f i c a t i o n s . Automatic e l e c t r o n i c f l a s h e s a r e used f o r i l l u m i n a t i o n . In a d d i t i o n t o i n - r e g i s t e r overview images, c l o s e u p s a r e taken with a second, hand-held camera i n a r e a s of p a r t i c u l a r i n t e r e s t . Specimens c o n t a i n i n g l a r g e r p r o p o r t i o n s of hard o r b r i t t l e bone (such a s t h e temporal bone) a r e cryoplaned a t a few microns' t h i c k n e s s whereas l a r g e r , s o f t t i s s u e specimens can be s e c t i o n e d a t 30-40 microns' t h i c k n e s s . Photographs a r e taken a t i n t e r v a l s ranging from . 1 mm t o 1 . 0 m m . The microtome i s programmed t o c u t a c e r t a i n number of s e c t i o n s of t h e s e l e c t e d t h i c k n e s s a n d s t o p s a u t o m a t i c a l l y i n t h e p o s i t i o n f o r photography. Depending o n t h e anatomical r e g i o n , t h e c u t t i n g h e i g h t i n t e r v a l s vary. Fig.1. was f r o z e n i n f l e x i o n . Therefore t h e upper a r t i c u l a r p r o c e s s e s of C7 l a c k c o n t a c t w i t h t h e i n f e r i o r a r t i c u l a r processes of C6. Also t h e e p i d u r a l v e i n s and the venous s i n u s e s i n t h e foramina a r e engorged.The arrow p o i n t s a t a small paramedian d i s c h e r n i a t i o n . G= dorsal r o o t ganglion UP= u n c i n a t e process V A = v e r t e b r a l a r t e r y Axial anatomy i n t h e lower c e r v i c a l s p i n e (C6-C7 d i s c ) . T h e specimen F i g . 2 . Axial anatomy of t h e lumbar s p i n a l canal a t L3-L4. T h e t h e c a l s a c c o n t a i n i n g t h e cauda equina r o o t s i s surrounded by e p i d u r a l f a t and f i r m l y a f f i x e d t o t h e concave p o s t e r i o r d i s c margin. The v e n t r a l i n t e r n a l v e i n s a r e d e s i g n a t e d by arrows. Fig.3. S a g i t t a l s e c t i o n t h r o u g h t h e c e r v i c a l s p i n e o f a 63 y.0. male with a f r a c t u r e d i s l o c a t i o n a t t h e C5-C6 l e v e l . Note t h e complete dis- r u p t i o n of the d i s c , rupture o f the ligamentum flavum and r e t r o p u l s i o n of f r a c t u r e d spondylophytic r i d g e i n t o t h e v e r t e b r a l canal (arrow) 253 Exacting care must be taken in the preparation of the specimen surface for photography. Especially at higher magnification, the ice crystals on the fresh- ly cut specimen surface are disturbing. Compressed air of room temperature is used to remove debries and t o thaw the specimen surface. Fraying of soft tiss- ues bordering pneumatic cavities (e.g. in the skull) is avoided by filling them with water and allowing it to freeze to ice. To prevent recrystallization, the specimen surface is gently wiped with a warm cloth pad slightly soaked in ethy- len glykol. APPLICATIONS IN RESEARCH AND TEACHING The great number of atlases on segmental correlative anatomy published in recent years and the rapid advances in diagnostic imaging underscore the need for improved reference material for the interpretation of computed tomo- grams. These tomographic scans are taken not only in the axial, sagittal and coronal plane, but increasingly in oblique, nonorthogonal planes. Familiarity with the topographic anatomy in these unconventional planes is essential for a correct diagnosis. A systematic assessment of the multiplanar human anatomy in series of closely spaced sections should also encompass the variations of the normal anatomy with respect to shape, dimensions and intrinsic relationships, define borderline cases and abnormalities and should also demonstrate significant pathoanatomical changes typical for each region. macroscopic pathoanatomical changes in experimental animal models ( 3 ) . It a1 so is used to supplement biomechanical studies, when specimens are frozen under a specific load. Subsequent cryoplaning then renders a quantitative assessment of the magnitude of dislocation and deformation and morphometric data ( 1 ) . The technique of surface cryoplaning allows to study the normal and pathol- ogical anatomy in specimens containing large amounts of undecalcified bone. The undistorted and also the functional relationships of the skeleton to contiguous soft tissues is maintained as well as the natural colors. Sequences o f detailed images taken at submillimeter intervals, high magnification and in exact reg- istration allow to "zoom" through a block of tissue in a movie mode. Cryoplaning is a powerful tool in basic research, notably for quantifying The irrefutable need for an improved knowledge of multiplanar anatomy for better diagnosis and treatment may entail the necessity of incorporating it in undergraduate anatomy curricula. Conventional atlases and textbooks can only accommodate a limited number of illustrations (6). New electronic media with the capacity of storing vast numbers of images (2) and facilitating computer aided interactive use would seem to have a greater potential for teaching of complex three dimensional anatomy. 254 CONCLUSION The Uppsala t e c h n i q u e f o r s u r f a c e c r y o p l a n i n g o f f r o z e n specimens c o n t a i n i n g l a r g e p r o p o r t i o n s o f u n d e c a l c i f i e d bone, r e n d e r s s e r i e s o f a c c u r a t e l y r e g i s t e r e d p h o t o g r a p h i c images w h i c h i n h i g h d e t a i l and n a t u r a l c o l o r s d e p i c t t h e u n d i s t o r - t e d r e l a t i o n s h i p s between s k e l e t a l elements and c o n t i g u o u s s o f t t i s s u e s . I t i s used t o c l a r i f y c o m p l i c a t e d t o p o g r a p h i c and f u n c t i o n a l a n a t o m i c a l r e l a t i o n s h i p s i n j o i n t and s p i n e specimens and f o r d i a g n o s t i c c o r r e l a t i o n w i t h computed tomo- g r a p h i c scans. A p a r t f r o m i t s h i g h i m p a c t on t e a c h i n g and e n h a n c i n g d i a g n o s t i c acumen, c r y o p l a n i n g f a c i l i t a t e s m o r p h o m e t r i c s t u d i e s o f p a t h o l o g i c a l c o n d i t i o n s i n d u c e d i n e x p e r i m e n t a l a n i m a l models and a q u a n t i t a t i v e assessment o f p a t h o l - o g i c a l c o n d i t i o n s i n t h e m u s c u l o s k e l e t a l system, w h i c h a l s o i n c l u d e s m u l t i p l a n - a r 1. 2. 3. 4. 5. 6. 7. 8. r e f o r m a t t i n g and t h r e e - d i m e n s i o n a l m o d e l i n g REFERENCES Asplund, S. : B i o m e c h a n i c a l s t u d i e s o f c o n g e n i t a l d i s l o c a t i o n o f t h e h i p . E x p e r i m e n t s i n human a u t o p s y specimens and r a b b i t s . D o c t o r a l T h e s i s a t Uppsala U n i v e r s i t y , 1983. Glenn, W.V. ,Jr. & Rauschning, W . : The knee: m u l t i p l a n a r anatomy, m a g n e t i c resonance i m a g i n g and computed tomography. I n s t r u c t i o n a l L a s e r V i d e o d i s c . American Academy o f O r t h o p a e d i c Surgeons and N a t i o n a l L i b r a r y o f M e d i c i n e , 1986 M i c h e l s s o n , J-E. & Rauschning, W. : P a t h o g e n e s i s o f e x p e r i m e n t a l h e t e r o t o p i c bone f o r m a t i o n f o l l o w i n g t e m p o r a r y f o r c i b l e e x e r c i s i n g o f i m m o b i l i z e d j o i n t s . C l i n O r t h o p Re1 Res 176: 265-272, 1983. Rauschning, W . : P o p l i t e a l c y s t s and t h e i r r e l a t i o n t o t h e g a s t r o c n e m i o - s e m i - membranosus b u r s a . A c l i n i c a l and a n a t o m i c a l s t u d y . D o c t o r a l D i s s e r t a t i o n a t Uppsala U n i v e r s i t y , 1979. Rauschning, W. : Computed tomography and c r y o m i c r o t o m y o f l u m b a r s p i n e s p e c i - mens. A new t e c h n i q u e f o r m u l t i p l a n a r a n a t o m i c c o r r e l a t i o n . S p i n e 8 : 170- 180, 1985. Rauschning, W . : D e t a i l e d s e c t i o n a l anatomy o f t h e s p i n e . I n : M u l t i p l a n a r CT o f t h e s p i n e (ed. S.L.G. Rothman & W.V.Glenn, J r . ) , pp 33-85. W i l l i a m s and W i l k i n s . B a l t i m o r e . 1985. U l l b e r g , S . : S t u d i e s on t h e d i s t r i b u t i o n and f a t e o f S 3 5 - l a b e l l e d b e n z y l - p e n i c i l l i n i n t h e body. A c t a Radio1 ( S t o c k h ) Suppl 118: 1-110, 1954. U l l b e r g , S. : The t e c h n i q u e o f whole body a u t o r a d i o g r a p h y . C r y o s e c t i o n i n g o f l a r g e specimens. S c i e n c e T o o l s , S p e c i a l I s s u e : 2-29, 1977. Adress f o r r e p r i n t s and f u r t h e r b i b l i o g r a p h y : Wolfgang Rauschning, M.D. Department o f O r t h o p a e d i c S u r g e r y U n i v e r s i t y H o s p i t a l Sweden S- 751 85 UPPSALA 255