key: cord-0005302-o3roa7br
authors: Sasaki, Makoto; Ide, Chizuka
title: Demyelination and remyelination in the dorsal funiculus of the rat spinal cord after heat injury
date: 1989
journal: J Neurocytol
DOI: 10.1007/bf01206664
sha: 2de8a8e4d7692a61cd0389c5488b8c301ccbf7a5
doc_id: 5302
cord_uid: o3roa7br

Part of the dorsal funiculus of the adult male rat (Wistar) spinal cord was treated for l h at the thoracolumbar level by running hot water, at approximately 48–50 ° C, through a polyethylene tube 2 mm in diameter in contact with the dura. Animals were fixed 1 day to 4 weeks later and the spinal cords were examined by light and electron microscopy. The affected area in the dorsal funiculus was approximately 1 mm long and less than 1 mm wide at the dorsal surface, and varied from 0.4 to 0.7 mm in depth. Within 3 days after treatment, almost all the myelin sheaths in the affected area were degraded, leaving the axons denuded, and at the same time astrocyte endfeet at the glial limiting membrane were swollen and partly destroyed. Almost all the denuded axons remained intact, exhibiting no noticeable morphological changes. There was evidence of a moderate vasogenic oedema, but minimal signs of haemorrhage in the lesion. Seven days after treatment, many immature Schwann cells but no oligodendrocytes were found between the denuded axons. By 2 weeks many of the denuded axons were remyelinated, and by 4 weeks almost all of those axons located near the pial and perivascular surfaces had been remyelinated by Schwann cells, while most of those located in the deep and marginal zones bordering the adjoining intact areas were remyelinated by oligodendrocytes. Longitudinal sections revealed that at nodes of Ranvier PNS-type myelin sheaths were apposed by either intact or newly formed CNS-type myelin sheaths. A typical glial limiting membrane was not reformed beneath the pial surface, but an inconspicuous one was found between the PNS- and CNS-type fibre areas.

Recently, therapeutic hyperthermia in localized or whole-body applications has been used as a new method of cancer therapy. In focal hyperthermia, the tissue is heated with microwave or ultrasound transducers to about 40 ~ C, a temperature which damages tumour cells but does not affect the surrounding normal tissue (Hahn, 1982) . Changes in the normal brain and spinal cord tissues resulting as an adverse effect of clinical or experimental hyperthermia have been studied only sporadically in the past three decades (Barnard et al., 1956; Fajardo, 1984; Lyons et al., 1986) . In a clinical study (Douglas et al., 1981) on the myelopathy induced by whole-body hyperthermia at 41.8~ for 6h, severe demyelination, axonal and gliaI cell degeneration, and vascular congestion were noted to occur in the white matter; and swelling and chromatolysis of the neurons were seen in the gray matter.

Several experimental studies of hyperthermia (Barnard et al., 1956; Britt et al., 1983; Lyons et aI., 1984) have also demonstrated that acute changes, including myelin sheath breakdown, vasogenic oedema and * To whom correspondence should be addressed.

haemorrhage, were found mainly in the white matter after heat treatment at a temperature of over 42 ~ C for about 60 min. These studies were done by light microscopy only, and no ultrastructural study has so far been reported with regard to the effects of hyperthermia on normal CNS tissues.

To investigate more precisely the histological changes induced in CNS tissues after high temperature treatment, heat injury was induced in the rat spinal cord by transdural heat application, and early degradation and subsequent repair of the affected tissues were examined by light and electron microscopy.

Thirty male rats (Wistar strain), weighing 200-300 g, were used. The animals were anesthetized by intraperitoneal injection of Nembutal (sodium pentobarbital, 100 mg/kg body weight), and the thoracolumbar region of the spinal cord was exposed through a rectangular window made by partial laminectomy. A polyethylene tube, 2 mm in diameter, was bent and the tip of the bent end was placed on the 0300M864/89 $03.00 +. 12 9 Chapman and Hall Ltd. Water was circulated by a flow roller pump through a 2 ram-diameter polyethylene tube. The water was warmed to approximately 60 ~ C in the water bath so that the temperature of the thermocouple at the bent tube could be kept at 48-50 ~ C. The tube was wrapped with adiabatic material, and the tip of the bent tube was in contact with the dural surface of the spinal cord.

dura mater using a stereotaxic device. Hot water, heated in a water bath (Yamato BK33), was circulated through this tube. tube. The temperature was maintained at 48-50 ~ C for 1 h, as monitored with a thermocouple (1.0 mm in diameter) inserted between the proximal and distal limbs of the bent tube, 10 mm away from its tip (Fig. 1 ). Room temperature was kept at 28-30 ~ C, and the tube, except the bent tip, was shielded by adiabatic material to prevent cooling by air. The pressure on the dura exerted by the tube was not so great as to interrupt the blood flow in the dorsal vein of the spinal cord, as checked under a dissecting microscope. Control animals were operated on and treated in the same manner, except that hot water was not circulated through the tuba in two animals, and hot water at 40M5 ~ C was circulated in five animals. The animals were killed 1 (n-4), 3 (n=4), 5 (n~3) and 7 (n-4) days, and 2 (n=4) and 4 weeks (n=4) after the operation by perfusion through the heart with a fixative containing 2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer. The treated parts of the cord were excised, together with the adjacent cranial and caudal segments and stored overnight in the same fixative. Specimens were cut into small blocks, and were postfixed in 1.0% osmium tetroxide solution in 0.1 M cacodylate buffer for 2 h, dehydrated through a graded series of ethanol, and embedded in Epon 812.

Ultrathin sections were cut on a LKB Ultrotome and observed in a JEOL-100B electron microscope after double staining with uranyl acetate and lead citrate. Semithin sections I p~m thick were stained with 1% toluidine blue and observed by light microscopy.

The rats did not show any gait or other behaviour disturbance after the operation. The lesion resulting from the heat treatment was confined within the dorsal funiculus of the spinal cord. The affected area was approximately i mm long, and less than 1ram wide at the dorsal surface of the spinal cord. The depth of the lesion varied from 0.4 to 0.7 mm.

At every post-treatment stage only a few degenerated axons were observed in the dorsal funiculus at levels cranial to the lesion, indicating that the axons themselves were not damaged by the heat treatment.

In the control cases the dorsal surface of the spinal cord was deformed to some extent, presumably due to compression by the tube'. However, no focus of demyelination was found in the dorsal funiculus.

A few fibres whose myelin sheaths were degraded in the treated part of the dorsal funiculus were noted by light microscopy. Myelinated fibres located near the pial surface appeared loosely packed owing to the increase of extracellular spaces ( Fig. 2A, B) .

Electron microscopy showed that myelin sheath lamellae of some myelinated fibres were loosened or even broken up into irregular structures (Fig. 3) . In some fibres the internal loops of myelin sheaths were markedly swollen. These were features of the early stages of myelin sheath degradation which finally led to the complete demyelination of the affected fibres. The extracellular spaces between the axons were enlarged and contained proteinaceous oedema fluid and electron-dense deposits of fibrin. Astrocyte processes, including endfeet of the subpial glial limiting membrane (GLM), were partly swollen and disrupted, but the basal lamina was preserved as a continuous sheet covering the entire surface of the spinal cord. Oligodendrocyte processes forming the myelin sheath outer loop disappeared from the degenerating myelin sheaths. In the affected area astrocytes and oligodendrocytes contained many vacuoles in their cytoplasm suggesting early degenerative changes. The vessel walls appeared undamaged and no haemorrhage occurred in the lesion.

Light microscopy showed that most fibres located near to the pial surface had myelin sheaths with much less opacity, reflecting advanced myelin lamella disintegration and breakdown. Extracellular spaces between the axons were further enlarged, and some macrophages were found in such interaxonal spaces. No haemorrhage was noted (Fig. 2C) .

Electron microscopy showed that many myelin sheath lamellae were split extensively into irregularly arranged sheets and had partially disintegrated into vesicular structures, losing their original electron Fig. 4 . Three days after treatment. This electron micrograph shows a later stage in myelin sheath degradation with interlamellar splitting of all the fibres. Some axons (asterisk) appear to have been damaged by heat injury, but most seem to be intact (Ax). Astrocytes at the GLM beneath the pia are totally destroyed, but the basal laminae are largely preserved (arrowheads). Extracellular spaces of the affected area have become much more conspicuous than in Fig. 3 . x 8600. opacity (Fig. 4) . Other myelin sheaths, while retaining densely packed lamellae, exhibited a greatly decreased electron opacity. Axons whose myelin sheaths had become degraded appeared normal. Astrocyte processes including endfeet of the GLM were entirely disrupted and were not visible. However, the basal laminae of the GLM remained intact separating the CNS from the s u r r o u n d i n g c o n n e c t i v e tissue. No oligodendrocyte cell bodies or processes were found in the areas near the surface or in the centre of the lesion. Fragments of degraded myelin sheaths, as well as fibrin, were scattered in wide interaxonal spaces, and macrophages were phagocytosing cellular debris.

N u m e r o u s d e n u d e d axons were identified by light microscopy as various-sized bundles within the heat-injured areas. Macrophages containing myelin debris were scattered between such d e n u d e d axons. Spindle-shaped cells proliferated and made a dense cell layer above the affected area (Fig. 2D) .

By electron microscopy almost all axons were completely demyelinated and closely packed (Fig. 5A) . A notable finding was the presence, between the demyelinated axons, of cells with round nuclei which contained no myelin debris. These cells often extended cytoplasmic processes around axons in the m a n n e r of developing Schwann and glial cells, suggesting that they were immature Schwann or glial cells migrating through the interaxonal spaces (Fig. 5B ). Cells identifiable as astrocytes and oligodendrocytes were not observed within the affected area at this time. The GLM basal lamina at the pial surface was largely destroyed. Thus naked axons and associated imma-ture Schwann cells were in direct contact with the pial connective tissues at the pial surface. Spindle-shaped non-neural cells were numerous throughout the pial layer.

Most axons near the pial surface appeared thinly myelinated by light microscopy, while most axons located toward the deep or marginal zones of the lesion remained denuded. Also noted were many round cells with relatively dark cytoplasm, intimately associated with the thinly myelinated axons. Spindleshaped non-neuronal cells and bundles of collagen fibres formed a thick pial layer on the surface of the injured cord. Macrophages containing myelin debris were still present in the lesion (Fig. 2E) .

By electron microscopy, many axons were noted near the pial surface covered mostly by very thin, but occasionally by relatively thick myelin sheaths (Fig. 6) . It was evident that the round cells were associated 1 : 1 with the axons, and that they were covered by basal lamina, features typical of myelinating Schwann cells. In the marginal zone bordering the intact area, some axons were thinly myelinated by oligodendrocyte processes (Fig. 7) : the myelin sheath had an interlamellar periodicity of about 130A. At this stage oligodendrocytes tended to be in contact with or near these CNS-type fibres. Astrocytes also appeared in the marginal zone bordering the intact area.

By light microscopy it was determined that almost all the axons located near the pial and perivascular surfaces were covered by thick myelin sheaths. Schwann cells were associated with axons in a 1:1 relationship. In the deep or marginal zones bordering the intact tissue, most axons had thin myelin, which, unlike Schwann cell myelin, had no cytoplasmic layer or basal lamina on the myelin sheath surface, indicating that oligodendrocytes had made the myelin sheaths around them. Blood vessels were abundant throughout the lesion but macrophages were no longer found in the injured area (Fig. 2F) .

By electron microscopy it was determined that almost all the axons near the pia! and perivascular surfaces were myelinated by Schwann cells (Fig. 8) . The myelin sheaths were 0.5-1 #,m thick and had an interlamellar periodicity of about 150A. Unmye-231 linated axons were wrapped in a bundle of 2-3 fibres by a common Schwann cell, as in unmyelinated fibres of the PNS. Schwann cells were invested with basal laminae associated with a few collagen fibrils on their interstitial surfaces. On the other hand, in the deep and marginal zones bordering the intact area almost all the axons were myelinated by oligodendrocytes (Fig.  9) . Such CNS-type myelin sheaths were extremely thin (about 0.1 ~m thick), and had an interlamellar periodicity of about 130 A.

At the oligodendrocyte-and Schwann cellmyelination transition areas, PNS-and CNS-type fibres intermingled with one another with no clear line of demarcation (Fig. 10) . In such transitional zones, the oligodendrocyte-myelinated fibres were incompletely covered by the astrocyte processes; therefore CNS-type myelin sheaths and oligodendrocyte processes were directly contiguous to Schwann cell basal laminae or collagen fibrils.

Peculiar dark cells, which had previously appeared as spindle-shaped cells, proliferated to form a thick layer on the pial surface of the lesion. No astrocytic layer had developed between this 'pial' cell layer and the underlying neural tissue. Blood vessels within the Schwann cell myelinated areas lacked an astrocytic covering; these vessels were thus of similar appearance to those found in the PNS. A few macrophages containing myelin debris were still observed in the affected area.

In longitudinal sections, internodes of CNS-type remyelinated axons were so short that the entire internode could be followed in one section, whereas those of PNS-type internodes were longer and only part of an internode was traceable in one section. Nodes of Ranvier of the PNS-type had a normal structure, while those of a CNS-type occasionally exhibited abnormally wide gaps. Oligodendrocytes sometimes formed a thin myelin sheath on their own cell body. Newly formed PNS-or CNS-type myelin sheaths were apposed to the adjacent intact CNS myelin sheaths at nodes of Ranvier. Sometimes a single axon was remyelinated by oligodendrocytes and by Schwann cells forming alternate PNS-and CNS-type myelin sheaths (Fig. 11 ). Usually only a few astrocyte processes covered these newly formed nodes of Ranvier. Unlike the normal PNS-CNS transition in the nerve root, lateral loops of oligodendrocytes were directly contiguous with Schwann cell processes at the node (Fig. 11, inset. ). 

The present study has documented the morphological changes in the spinal cord injured by heat treatment, as observed by light and electron microscopy. Using hot water circulating in a tube, we made a small (less than I mm~), well-confined lesion in the dorsal funiculus of the rat spinal cord. According to previous reports (Britt et al., 1983; Lyons et al., 1984) , the temperature threshold for cell damage in the CNS is 42-43~ C for a duration of 50-60 min. In the present study the tube was kept at 48-50 ~ C on the surface of the dura: this elevated the temperature in the tissue high enough to cause damage to glial cells but not axons.

The spinal cord is susceptible to compression: demyelination as well as axonal degeneration can occur following mild compression (Gledhill et al., 1973; Harrison et al., 1975) . In the present study, demyelination was not found in the dorsal funiculus of any control animals, including those exposed to a temperature o f 40-45 ~ C. Even in cases in which spinal cords were accidentally deformed by compression, no demyelination nor axonal degeneration was found in the spinal cord indicating that demyelination was not the result of compression but the direct result of heating.

In the present study the widespread demyelination occurred without any noticeable damage to the axons. Most myelin sheaths were degraded by splitting of the myelin lamellae as was reported by Hirano (1972) ,  Fig. 7 . Two weeks after treatment. This electron micrograph was taken from the deep zone bordering the intact area of the affected dorsal funiculus. Almost all the axons (Ax) in the area are thinly myelinated by oligodendrocytes (O1), while a few axons remain unmyelinated. Proliferation of astrocyte processes (As) has occurred in the spaces between these axons, x 7200.

while others initially only lost their electron opacity while retaining their original compact lamellae. Myelin sheath changes of the latter type have also been found in the cryo-treated spinal cord (unpublished observations). Vulnerability of the myelin sheath to heating can probably be related to the high lipid content and structural characteristics of the membrane. Raison et al. (1971) utilizing electron spin resonance demonstrated that heating gives rise to changes in the lipid components of the plasma membrane. Similarly, the organizations of membraneincorporated proteins are considered to be easily altered by heating (Hahn, 1982) .

The finding that the glial cells and associated myelin sheaths were more vulnerable to heat treatment than axons is in contrast to previous studies using ultrasound for heating (Fry et al., 1955; Barnard et al., 1956) which claimed that axons were more vulnerable to heat injury than glial ceils. The discrepancy in the vulnerability between axons and glial cells between their studies and our own may be due to the different methods employed. The conductivity and absorption rate of the heat in the CNS tissues would be different in the two methods which could produce different effects on axons and on glial cells.

Primary demyelination in the CNS occurs in multiple sclerosis and experimental infection of Theiler's murine encephalomyelitis (TME) virus (Dal Canto & Lipton, 1980; Dal Canto & Barbano, 1984) as well as in other experimental injuries using lysophosphatidylcholine (LCP) (Blakemore, 1976) , 6-aminonicotinamide (6-AN) (Blakemore, 1975) , Cuprizone (Ludwin, 1978) and CSF barbotage (Bunge et aI., 1960) , whereas in other experimental injuries such as allergic ence- Fig. 8 . One month after treatment. This electron micrograph shows the subpial area of the lesion. All the axons (Ax)are thickly myelinated by Schwann cells (S). There is no typical GLM at the surface which is covered by a thick cell layer (P). x 7400. phalomyelitis (EAE) (Lampert, 1967) , diphtheria toxin injection (Harrison et aI., 1972) , and impact injury (Griffith & McCulloch, 1983) , axons also undergo degenerative changes. In these latter disorders severe vasogenic oedema and local haemorrhage occur, which are considered to be harmful to axons, and thus may be partly responsible for axonal degeneration. It seems that heat treatment does not produce damage of sufficient severity to the blood-brain barrier (BBB) so as to fatally affect the axons, although permeability of damaged blood vessels seemed to increase to a certain degree (Muel!er, 1979) .

It was shown in the present study that demyelinated axons are effectively remyelinated by either oligo-dendrocytes or Schwann cells. The same pattern of remyelination by oligodendrocytes and Schwann cells has also been noted in CNS lesions induced by the murine corona (JHM) virus (Nagashima et al., 1979) , the TME virus (Dal Canto & Barbano, 1984) , and cryo-treatment (Collins et al., 1986 ; our unpublished observations) as well as by X-ray irradiation of the infant spinal cord (Gilmore & Duncan, 1968; Blakemore & Patterson, 1975; Gilmore & Sims, 1986) . Remyelination by Schwann cells is predominant in 6-AN (Blakemore, 1975) and ethidium bromide injected CNS (Blakemore, 1982; Gra#a & Blakemore, 1986) , whereas remyelination by oligodendrocytes is prominent in ordinary EAE (Lampert, 1965 (Lampert, , 1967 and Cuprizone treatment (Ludwin, 1978) .

It should be noted that Schwann cell-remyelination was dominant in the subpial and perivascular areas, Fig. 9 . One month after treatment. This electron micrograph shows the deep zone bordering the intact areas. Most axons (Ax) have CNS-type myelin sheaths, that is, they have been myelinated by oligodendrocytes, but their myelin sheaths are still very thin. The axons with thick myelin sheaths (C) are those that were unaffected by heat injury. As: astrocyte, x 7800.

whereas oligodendrocyte-remyelination was conspicuous in the deep and border areas of a lesion. The appearance o f Schwann cells in the CNS has been controversial and various prerequisites for ectopic Schwann cells in the CNS have been proposed which include (1) breakdown of the GLM, (2) absence of astrocytes, and (3) presence of denuded axons (Blakemore, 1983; Sims & Gilmore, 1983; Harrison, 1985; Gilmore & Sims, 1986) .

As described by previous authors (Blakemore, 1983; Sims & Gilmore 1983; Harrison, 1985) , the breakdown of the subpial and perivascular GLM might be the most critical factor for the appearance of Schwann cells. Thus in Cuprizone (Ludwin, 1978) and ordinary EAE (Lampert, 1965) injuries in which the GLM is retained intact, few Schwann cells appear in the lesion. Harrison (1985) thinks that Schwann cells invade the CNS tissue through damaged GLM at the perivascular and subpial surfaces. Astrocytes therefore seem to block the invasion of Schwann cells. No astrocytes were found in CNS lesions where axons were exclusively myelinated by Schwann cells (Hammang et al., 1986) . In contrast, Schwann cells are rarely seen at the sites where astrocyte processes have proliferated throughout the CNS lesions as a result of LPC and 6-AN treatment, and JHM virus and TME virus infection (Blakemore, 1975 (Blakemore, , 1976 Nagashima et al., 1979; Dal Canto & Lipton, 1980) , as well as Schwann cell implantation into normal nervous tissue (Blakemore et al., 1986) .

Denuded axons seem necessary for Schwann cells to invade the CNS and proliferate. It was reported that naked axons have a mitogenic influence on Schwann cells in vitro (Wood & Bunge, 1975) . Harrison (1987) Fig. 10. One month after treatment. This micrograph was taken at the transitional zone between the Schwann cell-myelinated area and the oligodendrocyte-myelinated area. Oligodendrocyte-myelinated axons (Ax) and oligodendrocyte cytoplasm (O1) both directly abut (arrows) on the basal laminae of the Schwann cells (S) without any intervening astrocyte processes. x 23 000. demonstrated by autoradiography that Schwann cell division is only initiated and promoted by direct contact with denuded axons. It has also been suggested that ensheathment of denuded axons by oligodendrocytes or astrocytes eliminated mitogenic influences of naked axons on Schwann cells (Sims & Gilmore, 1983) .

Where are the sources of ectopic Schwann cells? Since many ectopic Schwann cells are found near the nerve root entry, it is supposed that Schwann cells in the root could move along the denuded axons into the injured CNS (Gilmore & Duncan, 1968) . However, even when the roots and their entry zones are preserved undamaged, many Schwann cells have been seen in the midline of the dorsal funiculus, far from the root entry (Gilmore & Sims, 1986 ). On the other hand, Schwann cells of the perivascular autonomic nerves (Ghatak et aI., 1973) and of small nerves located in a pial layer (Blakemore, 1983 ) could proliferate and migrate into the lesion. Though the sources of ectopic Schwann cells could not be clearly identified, the present study indicates that damage to glial cells as well as the GLM at the pial and perivascular surfaces might be the most important prerequisite (Blakemore et aI., 1986) .

Oligodendrocytes remyelinate denuded axons after Cuprizone intoxication (Ludwin, 1987 (Ludwin, , 1988 and other CNS lesions including chronic relapsing EAE (Raine & Traugott, 1985) and TME virus infection (Dal Canto & Barbano, 1984) . In these studies, as in the present study, remyelination by oligodendrocytes occurred in deep areas of the lesion, where there might be no influence of the damaged GLM. On the other hand, there is evidence that astrocytes can co-operate to some extent with oligodendrocytes in the facilitation of myelination (Blakemore, 1975 (Blakemore, , 1984 Fig. 11 . One month after treatment. This electron micrograph shows a longitudinal section from the caudal part of the lesion. A myelin sheath newly formed by Schwann cells (S) makes a node of Ranvier (arrow) at the junction with a thick CNS-type myelin sheath (C) which presumably survived the heat treatment. There are two fibres that are partly myelinated by Schwann cells (S) and partly by otigodendrocytes (O1), forming new nodes of Ranvier between them (arrowheads). x 7600. Inset: a high-powered micrograpl~ showing a similar node of Ranvier taken from other section. A lateral loop (asterisk) of the oligodendrocyte is contiguous with a Schwann cell process (arrowhead). No astrocyte process is interposed between them. x 28 000. Harrison, 1985) . In the present study, however, there were relatively greater numbers of oligodendrocytes in the CNS-PNS border zone in which astrocytes and their processes were sparse. This finding indicates that oligodendrocytes can myelinate d e n u d e d axons in the presence of even only a few astrocytes. 

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P~ (1975) Evidence that sensory axons are mitogenic for Schwann cells

The authors wish to thank Dr T. Ushiki and Dr K. Tohyama for their kind suggestions in th e preparation of manuscript. The authors also thank Professor Paul Langman for his suggestions concerning English usage.