Brain Research, 96 (1975) 323-329 6) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
323
The perineurial window m a new model of focal demyelination and remyelination
P E T E R S. S P E N C E R , H A R O L D J. W E I N B E R G , C E D R I C S. R A I N E AND J O H N W. PRINEAS
Department of Pathology (Neuropathology) and Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461, and (J. W.P.) Department of Neurosciences, New Jersey Medical School and V. A. Hospital, East Orange, N.J. 07019 (U.S.A.) (Accepted June 18th, 1975)
In extensive studies on peripheral nerves, Sunderland demonstrated 11 that breaching the perineurium 4 produced an immediate herniation of underlying nerve fibers through the opening. This and a related observation 13 led to the suggestion that the fibrous outer components of the elastic perineurium resist and maintain a positive pressure normally present within the fascicle. Intrafascicular pressure was believed ~e to originate within axons where it served to maintain the shape of nerve fibers ~5 and their elastic endoneurial tubes 5. This interesting concept has received sparse attention in recent years and neither the nature of intrafascicular pressure nor its possible role in the functional integrity of nerve fibers have been ascertained. We have approached these questions by examining the effects on nerve fibers of a focal release of intrafascicular pressure by cutting a small window in the perineurium. It was found that the regions of those nerve fibers contained within the resulting herniated bleb of endoneurium first formed focal axonal swellings, later underwent demyelination and subsequently became remyelinated. The purpose of this communication is to report these unexpected findings and to describe the major events which occur in this lesion as visualized by light microscopy. The peroneal nerve in the mid-thigh of adult Sprague-Dawley rats (250 g) was selected for this study since this nerve is composed of a single, rounded fascicle in which myelinated fibers of different sizes are evenly distributed (Fig. 1). Before producing a perineurial window, the fascia overlying the nerve was focally interrupted and a portion of the epineurium excised. A pair of fine forceps was used to grip and raise the cellular perineurial sheath 9 which was then cut horizontally with springscissors parallel to the nerve. This technique produced an oval opening 1-2 mm long in the perineurium. The incision was associated with a period of rapid movement of the ipsilateral digits but no clinical signs were present after the animal had recovered from anesthesia. Single perineurial windows were studied in 100 peroneal nerves of 62 animals at 25 timepoints between 15 sec and 50 days post-surgery. Three nerves were examined
324
Fig. I. Normal peroneal nerve at the level chosen lbr study. Note the thick collagenous epineurium (e) and the thin perineurial sheath (ps). This figure and Figs. 3 and 5-7 are 1-!~m Epon crosssections, stained with Totuidine blue. :. 110. Fig. 2. Retouched bright-field micrograph of a herniated bleb (b) protruding through the perineurial window of an excised segment of an operated peroneal nerve. Arrow points to exposed endoneurial capillary. Whole mount, fixed with glutaraldehyde and osmium tetroxide. × 35. Fig. 3. Area of the bleb in an operated peroneal nerve 3 days after surgery. Note the widely spaced, abnormally large myelinated fibers in the bleb (top) and the cut edges (arrows) of the perineurium. "~ 110.
in vivo by incident light 14 i m m e d i a t e l y after o p e n i n g the p e r i n e u r i u m . The o t h e r o p e r a t e d nerves, t o g e t h e r with n o r m a l peroneal nerves f r o m the o p p o s i t e sides o f 10 animals, were fixed either by systemic perfusion or by in situ i m m e r s i o n in p h o s p h a t e buffered 2.5Yoo g l u t a r a l d e h y d e followed by i m m e r s i o n in cold, p h o s p h a t e - b u f f e r e d 1 ~ o s m i u m tetroxide, d e h y d r a t e d a n d infiltrated with E p o x y resin. Single teased myelinated fibers with preserved u l t r a s t r u c t u r e were p r e p a r e d 1° f r o m a total o f 25 windows. I s o l a t e d fibers were studied with bright-field a n d N o m a r s k i differential
325
Fig. 4. A series of photographs, taken with Nomarski optics, showing consecutive segments of a single demyelinated fiber from a 7-day perineurial window. The upper 3 photographs show the fiber entering the window and the lower 3, the swollen, twisted and beaded region. Arrows indicate the interfaces between the myelinated and the demyelinated segments. Note the prominent paranuclear Schwann cell cytoplasm (S), a Schwann cell occupying the demyelinated region (arrowhead), phagocytic cells (p) and nodes of Ranvier (n). x 1500.
326
Fig. 5. Demyelinated axons (a) within a perineurial window, 7 days after surgery. Several cells of uncertain identity are seen within the endoneurium (e). ; 1600. Fig. 6. Many remyelinating nerve fibers, each associated with supernumerary cellular processes, m a perineurial window 12 days after surgery. >; 1260. Fig. 7. Opposite side of the fascicle illustrated in Fig. 6 showing undisturbed organization. 1260.
327 interference contrast microscopy either unstained or after a light staining with Toluidine blue. In addition, transverse and longitudinal l /~m sections of embedded tissue were stained with Toluidine blue and examined by bright-field microscopy. It was observed that immediately after the perineurium had been opened, the underlying nerve fibers and associated endoneurial tissue bulged through the window to form an oval bleb (Fig. 2). In vivo inspection revealed that this procedure did not disturb the local capillary circulation. Examination of living and fixed preparations showed that usually only a few of the most superficial fibers were severed during surgery. The vast majority of myelinated fibers within the window displayed an irregular fiber contour and an internodal dilatation of the axon which was most prominent in the center of the lesion. Paranodal displacement of the myelin sheath, a feature of localized, mild compression injury v, did not accompany this immediate focal fiber enlargement and was rarely encountered at later timepoints. The nerve fiber changes remained restricted to the area of the window leaving proximal and distal regions intact. Within a few hours of creating the window, periodic constrictions appeared along the swollen regions of fibers and the paranuclear Schwann cell cytoplasm was enlarged. These two features became progressively more pronounced with time so that the affected regions of fibers appeared swollen (Fig. 3), beaded and twisted after 5 days. The beading of fibers was readily distinguishable from the chains of myelin ovoids seen during Wallerian degeneration 1 and usually was distinct from the changes associated with nerve stretch 8. The first sign of myelin loss in the affected fibers was found after 3 days and, by 7 days, the majority of fibers within the window displayed advanced demyelination (Figs. 4 and 5). This process began both internodally and paranodally and was associated with phagocytic cells laden with myelin debris. After a length of denuded fiber was visible, the phagocytic cells frequently clustered at the interfaces between the myelinated and demyelinated regions. The removal of the myelin sheath appeared to be directly related to the activity of these phagocytic cells. On the basis of smaller and more densely staining nuclei, these phagocytes usually were distinguishable from nearby Schwann cells. Numerous Schwann cells, each with a large, pale-staining nucleus, invested the demyelinated stretches of axons. By 8 days, these cells had begun to produce new myelin sheaths. Remyelination was prominent by 12 days (Fig. 6) and, by 30 days, up to 8 consecutive short, remyelinated internodes occupied the affected regions of fibers. These lengths of remyelinated fiber commonly extended across the entire length of the window. In many fibers, the central portion of the axon of the remyelinated regions retained some of the swollen and twisted appearance seen prior to demyelination. This central portion was also associated with supernumerary cellular processes which encircled the fiber. Less frequently, fibers contained two unequal lengths of consecutive remyelinated internodes separated by a short, central stretch of the original beaded and twisted myelinated fiber. Sometimes, phagocytes were seen at the interfaces between this region and the adjacent remyelinated region, a finding which sug-
328 gested ongoing demyelination of the central stretch of the fiber. By 50 days, however. these interfaces were devoid of phagocytes. The changes which were anticipated in the area of the perineurial window were partial or complete interruption of fibers caused by trauma during surgical manipulation2, 3 or by pinching under the cut edges of the perineurium. Although both of these phenomena were recognized in the fibers sited superficially, the vast majority of fibers within the window remained intact but immediately underwent focal axonal and fiber swelling. The reason for the axonal swelling is unknown. One possibility currently under study is that a shift of water or of axoplasm occurred 6 as a consequence of the rapid reduction in external pressure on this region of nerve fibers associated with opening the perineurium. The subsequent demyelination might be related to the focal axonal swelling or to the new and abnormal external environment of the fiber. Whether there is any correlation between the observed remyelination and the repair of the window is presently uncertain. in conclusion, the perineurial window provides a new experimental tool for the study of phenomena associated with demyelination and remyelination. The lesion is simple to create, highly reproducible and exquisitely focal. Since only the fibers within the window undergo demyelination, the model is provided with a built-in control (Fig. 7) within the same nerve fascicle. Although the model is suited to morphologic study of the cellular events accompanying demyelination and remyelination, it should be noted that since the site of the lesion is grossly visible, the perineurial window might also prove suitable for physiologic and biochemical studies. The authors thank Drs. R. D. Terry and R. Katzman for discussion. This study was supported in part by Research Grants NS 08952, NS 03356 and Training Grant 5T5 GM1674 from the NIH; by grants from the Alfred P; Sloan Foundation, the National Multiple Sclerosis Society and the Veterans Administration. Dr. Spencer is a Joseph P. Kennedy, Jr. Fellow in the Neurosciences and Dr. Raine is the recipient of a Research Career Development Award from the NIH, NS 70265. This paper received the Weil Award at the 51 st Annual Meeting of the American Association of Neuropathologists, New York, May 1975.
1 CAJAL, S. RAM6N Y, Degeneration and Regeneration of the Nervous System, Vol. 1, (English edition), Oxford University Press, London, 1928. 2 DENNY-BROWN, D., AND BRENNER, C., The effect of percussion on nerve, J. Neurol. Neurosurg. Psychiat., 7 (1944) 76-95. 3 HAFTEK,J., AND THOMAS,P. K., Electron-microscope observations on the effects of localized crush injuries on the connective tissues of peripheral nerves, J. Anat. (Lond.), 103 (1968) 233-243. 4 KEY, A., AND RETZlUS, G., Studien in der Anatomie des Nervensystems und des Bindgewebes, VoL 2, Samson and Wallin, Stockholm, 1876. 5 LUBt~SKA, L., Elasticity and distensibility of nerve tubes, Aeta Biol. exp. (Warszawa), 16 (1952) 73-90. 6 MACGREGOR, R. J., SHARPLESS, S. K., AND LUTTGES, M. W., A pressure vessel model for nerve compression, J. neurol. Sci., 24 (1975) 299-304.
329 7 0 c H o a , J., FOWLER, T. J., AND GILLIATT, R. W., Anatomical changes in peripheral nerves compressed by a pneumatic tourniquet, J. Anat. (Land.), 113 (1972) 433-455. 80CHS, S., Beading of myelinated nerve fibers, Exp. Neurol., 12 (1965) 84 95. 9 SHANTHAVEERAPPA,T. R., AND BOURNE, G. H., The perineural epithelium, a metabolically active, continuous, protoplasmic cell barrier surrounding peripheral nerve fascicles, J. Anat. (Lond.), 96 (1962) 527-537. l 0 SPENCER, P. S., AND THOMAS, P. K., The examination of isolated nerve fibres by light and electron microscopy, with observations on demyelination proximal to neuromas, Acta neuropath. (Berl.), 16(1970) 177-186. 11 SUNDERLAND,S., The effect of rupture of the perineurium on the contained nerve fibres, BraN1, 69(1946) 149 152. 12 SUNDERLAND, S., Nerves and Nerve Injuries, Livingstone, Edinburgh, 1968. 13 SUNDERLAND, S., AND BRADLEY, K. C., The perineurium of peripheral nerves, Anat. Ree., 113 (1952) 125 141. 14 WILLIAMS,P. L., AND HALL, S. M., 111 vivo observations on mature myelinated nerve fibres of the mouse, J. Anat. (Lond.), 107 (1970) 31 38. 15 YOUNG, J. Z., The history of the shape of a nerve-fibre. In W. E. L z G r c s CLARK AND P. B. MEDAWAR (Eds.), Essays on Growth attd Form, Presented to D'Aro' Wentworth Thonipsou, Clarendon Press, Oxford, 1945, pp. 41-94.
323
The perineurial window m a new model of focal demyelination and remyelination
P E T E R S. S P E N C E R , H A R O L D J. W E I N B E R G , C E D R I C S. R A I N E AND J O H N W. PRINEAS
Department of Pathology (Neuropathology) and Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461, and (J. W.P.) Department of Neurosciences, New Jersey Medical School and V. A. Hospital, East Orange, N.J. 07019 (U.S.A.) (Accepted June 18th, 1975)
In extensive studies on peripheral nerves, Sunderland demonstrated 11 that breaching the perineurium 4 produced an immediate herniation of underlying nerve fibers through the opening. This and a related observation 13 led to the suggestion that the fibrous outer components of the elastic perineurium resist and maintain a positive pressure normally present within the fascicle. Intrafascicular pressure was believed ~e to originate within axons where it served to maintain the shape of nerve fibers ~5 and their elastic endoneurial tubes 5. This interesting concept has received sparse attention in recent years and neither the nature of intrafascicular pressure nor its possible role in the functional integrity of nerve fibers have been ascertained. We have approached these questions by examining the effects on nerve fibers of a focal release of intrafascicular pressure by cutting a small window in the perineurium. It was found that the regions of those nerve fibers contained within the resulting herniated bleb of endoneurium first formed focal axonal swellings, later underwent demyelination and subsequently became remyelinated. The purpose of this communication is to report these unexpected findings and to describe the major events which occur in this lesion as visualized by light microscopy. The peroneal nerve in the mid-thigh of adult Sprague-Dawley rats (250 g) was selected for this study since this nerve is composed of a single, rounded fascicle in which myelinated fibers of different sizes are evenly distributed (Fig. 1). Before producing a perineurial window, the fascia overlying the nerve was focally interrupted and a portion of the epineurium excised. A pair of fine forceps was used to grip and raise the cellular perineurial sheath 9 which was then cut horizontally with springscissors parallel to the nerve. This technique produced an oval opening 1-2 mm long in the perineurium. The incision was associated with a period of rapid movement of the ipsilateral digits but no clinical signs were present after the animal had recovered from anesthesia. Single perineurial windows were studied in 100 peroneal nerves of 62 animals at 25 timepoints between 15 sec and 50 days post-surgery. Three nerves were examined
324
Fig. I. Normal peroneal nerve at the level chosen lbr study. Note the thick collagenous epineurium (e) and the thin perineurial sheath (ps). This figure and Figs. 3 and 5-7 are 1-!~m Epon crosssections, stained with Totuidine blue. :. 110. Fig. 2. Retouched bright-field micrograph of a herniated bleb (b) protruding through the perineurial window of an excised segment of an operated peroneal nerve. Arrow points to exposed endoneurial capillary. Whole mount, fixed with glutaraldehyde and osmium tetroxide. × 35. Fig. 3. Area of the bleb in an operated peroneal nerve 3 days after surgery. Note the widely spaced, abnormally large myelinated fibers in the bleb (top) and the cut edges (arrows) of the perineurium. "~ 110.
in vivo by incident light 14 i m m e d i a t e l y after o p e n i n g the p e r i n e u r i u m . The o t h e r o p e r a t e d nerves, t o g e t h e r with n o r m a l peroneal nerves f r o m the o p p o s i t e sides o f 10 animals, were fixed either by systemic perfusion or by in situ i m m e r s i o n in p h o s p h a t e buffered 2.5Yoo g l u t a r a l d e h y d e followed by i m m e r s i o n in cold, p h o s p h a t e - b u f f e r e d 1 ~ o s m i u m tetroxide, d e h y d r a t e d a n d infiltrated with E p o x y resin. Single teased myelinated fibers with preserved u l t r a s t r u c t u r e were p r e p a r e d 1° f r o m a total o f 25 windows. I s o l a t e d fibers were studied with bright-field a n d N o m a r s k i differential
325
Fig. 4. A series of photographs, taken with Nomarski optics, showing consecutive segments of a single demyelinated fiber from a 7-day perineurial window. The upper 3 photographs show the fiber entering the window and the lower 3, the swollen, twisted and beaded region. Arrows indicate the interfaces between the myelinated and the demyelinated segments. Note the prominent paranuclear Schwann cell cytoplasm (S), a Schwann cell occupying the demyelinated region (arrowhead), phagocytic cells (p) and nodes of Ranvier (n). x 1500.
326
Fig. 5. Demyelinated axons (a) within a perineurial window, 7 days after surgery. Several cells of uncertain identity are seen within the endoneurium (e). ; 1600. Fig. 6. Many remyelinating nerve fibers, each associated with supernumerary cellular processes, m a perineurial window 12 days after surgery. >; 1260. Fig. 7. Opposite side of the fascicle illustrated in Fig. 6 showing undisturbed organization. 1260.
327 interference contrast microscopy either unstained or after a light staining with Toluidine blue. In addition, transverse and longitudinal l /~m sections of embedded tissue were stained with Toluidine blue and examined by bright-field microscopy. It was observed that immediately after the perineurium had been opened, the underlying nerve fibers and associated endoneurial tissue bulged through the window to form an oval bleb (Fig. 2). In vivo inspection revealed that this procedure did not disturb the local capillary circulation. Examination of living and fixed preparations showed that usually only a few of the most superficial fibers were severed during surgery. The vast majority of myelinated fibers within the window displayed an irregular fiber contour and an internodal dilatation of the axon which was most prominent in the center of the lesion. Paranodal displacement of the myelin sheath, a feature of localized, mild compression injury v, did not accompany this immediate focal fiber enlargement and was rarely encountered at later timepoints. The nerve fiber changes remained restricted to the area of the window leaving proximal and distal regions intact. Within a few hours of creating the window, periodic constrictions appeared along the swollen regions of fibers and the paranuclear Schwann cell cytoplasm was enlarged. These two features became progressively more pronounced with time so that the affected regions of fibers appeared swollen (Fig. 3), beaded and twisted after 5 days. The beading of fibers was readily distinguishable from the chains of myelin ovoids seen during Wallerian degeneration 1 and usually was distinct from the changes associated with nerve stretch 8. The first sign of myelin loss in the affected fibers was found after 3 days and, by 7 days, the majority of fibers within the window displayed advanced demyelination (Figs. 4 and 5). This process began both internodally and paranodally and was associated with phagocytic cells laden with myelin debris. After a length of denuded fiber was visible, the phagocytic cells frequently clustered at the interfaces between the myelinated and demyelinated regions. The removal of the myelin sheath appeared to be directly related to the activity of these phagocytic cells. On the basis of smaller and more densely staining nuclei, these phagocytes usually were distinguishable from nearby Schwann cells. Numerous Schwann cells, each with a large, pale-staining nucleus, invested the demyelinated stretches of axons. By 8 days, these cells had begun to produce new myelin sheaths. Remyelination was prominent by 12 days (Fig. 6) and, by 30 days, up to 8 consecutive short, remyelinated internodes occupied the affected regions of fibers. These lengths of remyelinated fiber commonly extended across the entire length of the window. In many fibers, the central portion of the axon of the remyelinated regions retained some of the swollen and twisted appearance seen prior to demyelination. This central portion was also associated with supernumerary cellular processes which encircled the fiber. Less frequently, fibers contained two unequal lengths of consecutive remyelinated internodes separated by a short, central stretch of the original beaded and twisted myelinated fiber. Sometimes, phagocytes were seen at the interfaces between this region and the adjacent remyelinated region, a finding which sug-
328 gested ongoing demyelination of the central stretch of the fiber. By 50 days, however. these interfaces were devoid of phagocytes. The changes which were anticipated in the area of the perineurial window were partial or complete interruption of fibers caused by trauma during surgical manipulation2, 3 or by pinching under the cut edges of the perineurium. Although both of these phenomena were recognized in the fibers sited superficially, the vast majority of fibers within the window remained intact but immediately underwent focal axonal and fiber swelling. The reason for the axonal swelling is unknown. One possibility currently under study is that a shift of water or of axoplasm occurred 6 as a consequence of the rapid reduction in external pressure on this region of nerve fibers associated with opening the perineurium. The subsequent demyelination might be related to the focal axonal swelling or to the new and abnormal external environment of the fiber. Whether there is any correlation between the observed remyelination and the repair of the window is presently uncertain. in conclusion, the perineurial window provides a new experimental tool for the study of phenomena associated with demyelination and remyelination. The lesion is simple to create, highly reproducible and exquisitely focal. Since only the fibers within the window undergo demyelination, the model is provided with a built-in control (Fig. 7) within the same nerve fascicle. Although the model is suited to morphologic study of the cellular events accompanying demyelination and remyelination, it should be noted that since the site of the lesion is grossly visible, the perineurial window might also prove suitable for physiologic and biochemical studies. The authors thank Drs. R. D. Terry and R. Katzman for discussion. This study was supported in part by Research Grants NS 08952, NS 03356 and Training Grant 5T5 GM1674 from the NIH; by grants from the Alfred P; Sloan Foundation, the National Multiple Sclerosis Society and the Veterans Administration. Dr. Spencer is a Joseph P. Kennedy, Jr. Fellow in the Neurosciences and Dr. Raine is the recipient of a Research Career Development Award from the NIH, NS 70265. This paper received the Weil Award at the 51 st Annual Meeting of the American Association of Neuropathologists, New York, May 1975.
1 CAJAL, S. RAM6N Y, Degeneration and Regeneration of the Nervous System, Vol. 1, (English edition), Oxford University Press, London, 1928. 2 DENNY-BROWN, D., AND BRENNER, C., The effect of percussion on nerve, J. Neurol. Neurosurg. Psychiat., 7 (1944) 76-95. 3 HAFTEK,J., AND THOMAS,P. K., Electron-microscope observations on the effects of localized crush injuries on the connective tissues of peripheral nerves, J. Anat. (Lond.), 103 (1968) 233-243. 4 KEY, A., AND RETZlUS, G., Studien in der Anatomie des Nervensystems und des Bindgewebes, VoL 2, Samson and Wallin, Stockholm, 1876. 5 LUBt~SKA, L., Elasticity and distensibility of nerve tubes, Aeta Biol. exp. (Warszawa), 16 (1952) 73-90. 6 MACGREGOR, R. J., SHARPLESS, S. K., AND LUTTGES, M. W., A pressure vessel model for nerve compression, J. neurol. Sci., 24 (1975) 299-304.
329 7 0 c H o a , J., FOWLER, T. J., AND GILLIATT, R. W., Anatomical changes in peripheral nerves compressed by a pneumatic tourniquet, J. Anat. (Land.), 113 (1972) 433-455. 80CHS, S., Beading of myelinated nerve fibers, Exp. Neurol., 12 (1965) 84 95. 9 SHANTHAVEERAPPA,T. R., AND BOURNE, G. H., The perineural epithelium, a metabolically active, continuous, protoplasmic cell barrier surrounding peripheral nerve fascicles, J. Anat. (Lond.), 96 (1962) 527-537. l 0 SPENCER, P. S., AND THOMAS, P. K., The examination of isolated nerve fibres by light and electron microscopy, with observations on demyelination proximal to neuromas, Acta neuropath. (Berl.), 16(1970) 177-186. 11 SUNDERLAND,S., The effect of rupture of the perineurium on the contained nerve fibres, BraN1, 69(1946) 149 152. 12 SUNDERLAND, S., Nerves and Nerve Injuries, Livingstone, Edinburgh, 1968. 13 SUNDERLAND, S., AND BRADLEY, K. C., The perineurium of peripheral nerves, Anat. Ree., 113 (1952) 125 141. 14 WILLIAMS,P. L., AND HALL, S. M., 111 vivo observations on mature myelinated nerve fibres of the mouse, J. Anat. (Lond.), 107 (1970) 31 38. 15 YOUNG, J. Z., The history of the shape of a nerve-fibre. In W. E. L z G r c s CLARK AND P. B. MEDAWAR (Eds.), Essays on Growth attd Form, Presented to D'Aro' Wentworth Thonipsou, Clarendon Press, Oxford, 1945, pp. 41-94.
