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Focal lesions in the brain of growing rabbits produced by focused ultrasound
EXPERIMENTAL
NEUROLOGY
Focal
9,
502-511
Lesions in the Produced by G. F.
Medical
Acoustics
YOUNG
(1964)
Brain of Growing Focused Ultrasound AND
P. P.
LELE~
Research Group, Massachusetts Boston, Massachusetts Received
February
Rabbits
General
Hospital,
24, 1964
Trackless focal lesions could be p!aced at preselected sites within the brain of rabbits at various stages of development by irradiation with a single beam of focused ultrasound. Similar lesions could also be made in the fetal rabbits in situ at gestational age of 24 days. The histological characteristics and evolution of ultrasonic lesions, studied during 2 months after irradiation, did not differ significantly regardless of the age at operation (1 day to adult), and they were similar in all respects to lesions similarly produced in the brain of the adult cat. Introduction
Transdural irradiation with a single beam of focused ultrasound produced trackless focal lesions of desired size at predetermined sites within the nervous system of adult animals (2). The lesions were found to have unvarying neuropathological features and freedom from any delayed local or remote spread ( 1). The present study was undertaken to ascertain whether similar results were obtained in the brain of growing animals or if the physical or biological factors governing the production of ultrasound lesions differ at different stages of maturation of the nervous system, and if the nature and the evolution of the resultant lesions, neuropathologically, differs with the age of the animal. Materials
and
Methods
New Zealand rabbits 1, 14 and 28 days old and young adults weighing about 2 kg were used. Since stereotaxic atlases for young animals at dif1 This investigation was supported by the U.S. Public Health Service Grants NB-00816-09 and NB-0322-02. The authors are thankful to Dr. H. Thomas Ballantine, Jr. for much helpful advice and encouragement. Dr. Young is a NINDB Fellow in Pediatric Neurology, Departments of Neurology, Neuropathology and the Joseph P. Kennedy, Jr. Memorial Laboratories, Massachusetts General Hospital and the Department of Neurology-Neuropathology, Harvard Medical School. Dr. Lele’s address is Department of Physiology, Harvard Medical School. 502
ULTRASONIC
LESIONS
IN
BRAIN
503
ferent ages are not available, animals of different ages were killed to determine the suitable site for placement of lesions, the desired depth from the dural surface and the landmarks on the overlying skull. The posterior portion of the diencephalon and the subjacent mesencephalon were selected as irradiation sites (Fig. 1). The animals were anesthetized with pento-
FIG. 1. Diagram showing the planes of cutting brain (broken lines). For histological studies a block of brain obtained by cutting along the vertical dotted lines was divided into halves by a horizontal knife cut. AC-anterior commissure, AQaqueduct, CC-corpus callosum, MI-massa intermedia, SC-superior colliculi, SPseptum pellucidurn, OC-optic chiasma, PC-posterior commissure. Shaded region represents the target site.
barbital sodium (30 mg/kg) parenterally, the level of anesthesia being kept low to prevent excessive mortality. A left parietal craniectomy adequate in extent to permit unimpeded passage of the beam of ultrasound was performed. Since the solid angle of irradiation was 42”, the minimum diameter of the craniectomy required was approximately equal to the depth of the target site from the dural surface (4). For irradiation, the
504
YOUNG
AND
LELE
I- and 14-day-old animals were positioned by manually holding the head firmly against a plasticine cushioned window (1 cm in diameter) which had been cut in a plexiglass platform fixed perpendicular to the main axis of irradiation. The 28-day-old and young adult animals were kept in the desired position by an improvised head holder. The ultrasound equipment and the irradiation techniques as described previously (2, 4) were used. Since the level of anesthesia of the animals was not deep enough, nor their heads held rigidly enough to preclude movement during irradiation, requisite ultrasonic dosage was administered as a single pulse (its duration was varied between 0.5 and 5.0 set) at an average focal intensity of 42 X 15 w/cm’. As a control, a stab wound was made with a ZO-gauge hypodermic needle into the corresponding region of the right cerebral hemisphere. Animals used for the relation between ultrasonic dosage and lesion size were given a 0.5-Z per cent solution of Trypan Blue intravenously after irradiation and were killed 30 min later. The brain was removed and after fixation was cut in a plane parallel to the axis of irradiation, just anterior and posterior to the target site. This block of tissue was then cut on a freezing microtome until the maximum dimensions of the lesion were reached (2 ) . Acute lesions were made in several fetuses at 24 days gestational age. The mother rabbit was kept anesthetized with pentobarbital sodium and a uterotomy was performed in such a way as to expose a fetus and also maintain its blood supply intact. A left parietal craniectomy was performed on some fetuses and in others ultrasonic irradiation was delivered through scalp and skull. Trypan Blue was injected into the umbilical vein immediately postoperatively. The brain was removed 20-30 min later and was cut after fixation for 24 hours in Bouin’s fixative. Animals used for neuropathological studies were irradiated with a dose of ultrasound adequate to produce a lesion approximately 4 mm in length. Of each age group, one or more animals each were killed 1, 3 and 7 days and 2, 4 and 8 weeks after irradiation by decapitation or a lethal dose of pentobarbital sodium. The brain was removed immediately, placed in a 10 per cent isotonic solution of neutralized formalin for 24 hours, after which the irradiated part was cut into two blocks for subsequent preparation of stained sections (Fig. 1). The basal block was used for frozen sectioning and staining for microglia (Hortega silver carbonate method), astrocytes (Cajal gold sublimate method) and lipids (scarlet red). The
ULTRASONIC
LESIONS
IN
BRAIN
505
other was embedded in paraffin and the sections stained with hematoxylin and eosin, cresyl violet and mordinated hematoxylin (Heidenhane’s method for myelin sheaths). Some sections were also stained with periodic acid Schiff (PAS) and phosphotungstic acid-hematoxylin (PTAH) . No attempt was made to accomplish survival in irradiated fetuses. Results
Immediately after irradiation, approximately 25 per cent of the younger animals (newborn and 2 weeks old) assumed a posture with the head and body bent toward the operated side. This torsion of the body gradually disappeared during the first two postoperative days. No other neurological defect was apparent in any of the surviving animals. Some 26 per cent of the newborn and 2-week-old operated rabbits died during the first 48 hours, probably due to the anesthesia and surgery; an additional 29 per cent of those surviving died because of maternal neglect or an illness which usually caused diarrhea in the infected animals. The mortality dropped to 13-14 per cent at the age of 4 weeks. All operated adults survived. Ultrasonic Dosage-Lesion Size Relationship. Although no precise placement of lesions was attempted, their location (Fig. 2) was entirely satisfactory for the purposes of this study. The relation of the lesion size to ultrasonic dosage at two pulse durations for different ages of the animal is given in Fig. 3. The larger dose produced the larger lesions, but there was considerable variability in lesion size at each dosage level and particularly so in the younger animals. However, for equivalent dosages of ultrasound, there was a general trend for the younger animals to have smaller lesions than the older animals. Lesions were also successfully placed in the brain of two fetuses by irradiation through a craniectomy and two additional fetuses by irradiation through intact scalp and skull (Fig. 4). Pulse durations of 2 and 5 set, respectively, were used. The size of the lesions was comparable to that found in newborn animals irradiated for 2 set (Fig. 3). Histological Observations. Discrete foci of coagulation necrosis were found in the target zone of twenty-nine of the thirty animals thus studied. The only rabbit which did not show a discrete lesion (but a diffuse astrocytic reaction instead) had been operated upon at the age of 1 day and killed 4 weeks later. The characteristics of the earliest lesion, the progress of its breakdown
506
YOUNG
AND
LELE
and the tissue reaction to the lesion were all the same for each age group of rabbits. The lesion had the characteristic moat-island form (1). The center (island), in cross section, was almost circular and was composed of a plug of coagulated, necrosed brain tissue (Fig. 2C & 5). Surrounding this coagulum there was a necrotic and damaged zone which had a loose, reticulated appearance in which extravasate of serum and a few fibrin strands were seen. Vessels at the periphery of the lesion were dilated and
FIG. 2. A & B. Photographs of two supravitally stained lesions produced in brain of 4-week-old rabbits by irradiation with different doses of ultrasound. Each brain of the cut was cut in a plane parallel to the vertical lines in Fig. 1. C. Photograph horizontal surface of a block of brain showing an ultrasonic lesion on the left and an India-ink needle track on the right. Note the characteristic moat-island form of the ultrasonic lesion. Magnification: A & B, 2.5 X ; C, 3 X.
congested. Reaction to this focus of necrosis was prompt and vigorous. A few polymorphonuclear leucocytes were seen during the first 1 to 3 days but were very sparse by 1 week. Microglia cells and macrophages were found to have accumulated about the periphery of the lesion at 24 hours and they increased greatly in numbers up to 1 or 2 weeks. At the rim of the lesion, neuroglia cells appeared to have survived in some areas where all nerve cells were necrotic. Plump astrocytes were evident in undamaged tissue near the periphery of the lesion. The astrocytes showed
ULTRASONIC
LESIONS
IN
507
BRAIN
evidence of hypertrophy and proliferation and by 7 days there was a definite zone of astrocytic gliosis about the moat (Fig. 6B). During the first 1 or 2 weeks, swollen axons could be identified within and near the periphery of the lesion. Phagocytosis of necrotic debris from the moat appeared to progress rapidly; but not until several weeks later was the
6
c
-d--J2 AGE
7
14
OF RABB/T
26
ADULT
fDays1
FIG. 3. Relation of lesion size to ultrasonic dosage at two at different ages of the rabbit. Depth of the target site from also indicated as a measure of the size of the brain.
pulse durations and the dural surface is
coagulated island lysed and phagocytized. During the first 2 months the density of the astrocytic glial scar was found to increase. New vessels with a stroma of fibrous tissue were seen to grow into the lesion; but there were few fibroblasts present except in close proximity to vessel walls. In one of the rabbits which was operated upon at age 2 weeks, there were fossilized fragments (concretions of “pseudocalcium”) of cell processes at the periphery of the lesion 2 months later.
508
YOUNG
AND
LELE
Four of the thirty lesions contained hemorrhage, confined to the necrotic focus and the immediately surrounding tissue. There was a large abscess in the left cerebral hemisphere of one animal which was operated upon at age 1 day and killed 2 weeks later. A perivascular accumulation of plasma cells and lymphocytes, histological evidence of mild encephalitis,
FIG. 4. Photograph of a coronal section of brain 24 days) showing a lesion resultin, v from irradiation H. & E. stain. Calibration mark = 1 mm.
FIG.
rabbit. rabbit.
5. A. Photograph of a lesion 20 min. after H. &3 E. stain. B. A similar lesion 7 days H. & E. stain. Calibration marks = 500 u.
of fetal through
rabbit intact
irradiation of after irradiation
(gestational scalp and
age skull.
a Z-week-old of an adult
ULTRASONIC
LESIONS
IN
509
BRAIN
was seen in the brain of two of the adult animals. None of these three infected animals showed overt signs of illness.
FIG.
Red
6. Photographs and B, with Cajal
of lesions 7 days after irradiation, stained gold stain. Calibration marks = 100 CL.
A, with
Scarlet
510
YOUNG
AND
LELE
Discussion
The ability to produce trackless foci of necrosis within specific regions of brain of fetuses and growing animals makes focused ultrasound a valuable tool in experimental neuroembryology. Production of such lesions in fetuses in situ by irradiation through intact scalp and skull, in particular, opens up many new possibilities for such investigations. The major deterrent to such use, at this time, is the lack of an effective head-holding device which will provide the requisite stability and yet will not damage the uterus, placenta and fetus. The methods used for positioning and holding of the head in this investigation were only just adequate: but no great precision in the placement of the lesions was necessary. In a previous study (2) with adequate instrumentation and technique, the probability of accurate lesion placement was restricted only by the variability in the stereotaxic location of the structure itself. Irradiation with an adequate dosage of focused ultrasound almost invariably produced a discrete lesion in the brain of the rabbit, as was found to be the case in that of the cat (2). The average size of the lesion is again related directly to the duration of irradiation. But, for the same dosage of ultrasound, average lesion size was smallest in the newborn and increased with increasing age and size of the brain (as indicated approsimately by the depth of the lesion from the dural surface; Fig, 3). At any dosage level there was considerable variability in the lesion size but particularly marked in the younger animals. This would indicate the existence, in the case of the smaller brains, of some varying factor, such as interference patterns resulting from the brain-bone-air interface at the base of the skull. Existence of such interference patterns was found to cause much variability in the effects of ultrasonic radiation on the peripheral nerve (5). Other factors, such as increasing attenuation of ultrasound by brain with increasing age of the animal, possibly due in part to decreasing water content (3), increased myelination of fiber tracts, and growth of dendrites, may play some role which, however, could not be expected to contribute to variability. The influence of some of these factors is currently under investigation. The incidence of hemorrhage in the necrotic focus (13% ) is higher than that found in the adult cat ( 1, 2). This may be related to the animal’s movements during irradiation, and to inadequacy of the technique of holding the head without squeezing it. Furthermore, to minimize the risk of the head moving during irradiation, the duration of irradiation was
ULTRASONIC
LESIONS
IN
BRAIN
511
kept short by resorting to higher intensity of ultrasound. Basauri and Lele (2) found that the incidence of hemorrhage increases directly with the intensity of radiation. The histological characteristics of the lesion and the reparative processes which were consequent upon it were the same for all of the animals regardless of the age when the lesion was made and were similar to those described by iistrijm and his co-workers (1) in the adult cat. This is congruent with the results of the study of Young and Dodge (6) in which the histopathology of stab wounds in fetal rabbits was compared with the stab wounds produced in the animals of the present study. In each study the propensity for producing healing through phagocytosis of necrotic tissue by microglia and macrophages and the concurrent production of an astrocytic gliosis is equally strong in newly born and adult rabbits. This concept holds both for small lesions (produced by a to-gauge hypodermic needle puncture) and for the relatively large volume of brain tissue necrosis which was produced by ultrasound. References 1.
2.
3. 4. 5.
6.
K. E., E. BELL, H. T. BALLANTINE, JR., and E. HEIDENSLEBEN. 1961. An experimental neuropathological study of the effects of high-frequency focused ultrasound on the brain of the cat. J. Neuropath. Exptk Neurol. 20: 484-520. 1962. A simple method for production of trackBASAURI, L., and P. P. LELE. less focal lesions with focused ultrasound: Statistical evaluation of the effects of irradiation on the central nervous system of the cat. J. Physiol. London 160: 513-534. 1955. Age and water content of rabbit brain GRAVES, J., and H. E. HIMWICH. parts. Am. J. Physiol. 180: 205-208. LELE, P. P. 1962. A simple method for production of trackless focal lesions with focused ultrasound: Physical factors. I. Physiol. London 160: 494-512. LELE, P. P. 1963. Effects of focused ultrasonic radiation on peripheral nerve with observations on local heating. Exptl. Neural. 8: 47-83. YOUNG, G. F., and P. R. DODGE. Experiments in developmental neuropathology: Stab wounds into the brain of rabbits at various ages. In preparation.
ASTR~~M,
NEUROLOGY
Focal
9,
502-511
Lesions in the Produced by G. F.
Medical
Acoustics
YOUNG
(1964)
Brain of Growing Focused Ultrasound AND
P. P.
LELE~
Research Group, Massachusetts Boston, Massachusetts Received
February
Rabbits
General
Hospital,
24, 1964
Trackless focal lesions could be p!aced at preselected sites within the brain of rabbits at various stages of development by irradiation with a single beam of focused ultrasound. Similar lesions could also be made in the fetal rabbits in situ at gestational age of 24 days. The histological characteristics and evolution of ultrasonic lesions, studied during 2 months after irradiation, did not differ significantly regardless of the age at operation (1 day to adult), and they were similar in all respects to lesions similarly produced in the brain of the adult cat. Introduction
Transdural irradiation with a single beam of focused ultrasound produced trackless focal lesions of desired size at predetermined sites within the nervous system of adult animals (2). The lesions were found to have unvarying neuropathological features and freedom from any delayed local or remote spread ( 1). The present study was undertaken to ascertain whether similar results were obtained in the brain of growing animals or if the physical or biological factors governing the production of ultrasound lesions differ at different stages of maturation of the nervous system, and if the nature and the evolution of the resultant lesions, neuropathologically, differs with the age of the animal. Materials
and
Methods
New Zealand rabbits 1, 14 and 28 days old and young adults weighing about 2 kg were used. Since stereotaxic atlases for young animals at dif1 This investigation was supported by the U.S. Public Health Service Grants NB-00816-09 and NB-0322-02. The authors are thankful to Dr. H. Thomas Ballantine, Jr. for much helpful advice and encouragement. Dr. Young is a NINDB Fellow in Pediatric Neurology, Departments of Neurology, Neuropathology and the Joseph P. Kennedy, Jr. Memorial Laboratories, Massachusetts General Hospital and the Department of Neurology-Neuropathology, Harvard Medical School. Dr. Lele’s address is Department of Physiology, Harvard Medical School. 502
ULTRASONIC
LESIONS
IN
BRAIN
503
ferent ages are not available, animals of different ages were killed to determine the suitable site for placement of lesions, the desired depth from the dural surface and the landmarks on the overlying skull. The posterior portion of the diencephalon and the subjacent mesencephalon were selected as irradiation sites (Fig. 1). The animals were anesthetized with pento-
FIG. 1. Diagram showing the planes of cutting brain (broken lines). For histological studies a block of brain obtained by cutting along the vertical dotted lines was divided into halves by a horizontal knife cut. AC-anterior commissure, AQaqueduct, CC-corpus callosum, MI-massa intermedia, SC-superior colliculi, SPseptum pellucidurn, OC-optic chiasma, PC-posterior commissure. Shaded region represents the target site.
barbital sodium (30 mg/kg) parenterally, the level of anesthesia being kept low to prevent excessive mortality. A left parietal craniectomy adequate in extent to permit unimpeded passage of the beam of ultrasound was performed. Since the solid angle of irradiation was 42”, the minimum diameter of the craniectomy required was approximately equal to the depth of the target site from the dural surface (4). For irradiation, the
504
YOUNG
AND
LELE
I- and 14-day-old animals were positioned by manually holding the head firmly against a plasticine cushioned window (1 cm in diameter) which had been cut in a plexiglass platform fixed perpendicular to the main axis of irradiation. The 28-day-old and young adult animals were kept in the desired position by an improvised head holder. The ultrasound equipment and the irradiation techniques as described previously (2, 4) were used. Since the level of anesthesia of the animals was not deep enough, nor their heads held rigidly enough to preclude movement during irradiation, requisite ultrasonic dosage was administered as a single pulse (its duration was varied between 0.5 and 5.0 set) at an average focal intensity of 42 X 15 w/cm’. As a control, a stab wound was made with a ZO-gauge hypodermic needle into the corresponding region of the right cerebral hemisphere. Animals used for the relation between ultrasonic dosage and lesion size were given a 0.5-Z per cent solution of Trypan Blue intravenously after irradiation and were killed 30 min later. The brain was removed and after fixation was cut in a plane parallel to the axis of irradiation, just anterior and posterior to the target site. This block of tissue was then cut on a freezing microtome until the maximum dimensions of the lesion were reached (2 ) . Acute lesions were made in several fetuses at 24 days gestational age. The mother rabbit was kept anesthetized with pentobarbital sodium and a uterotomy was performed in such a way as to expose a fetus and also maintain its blood supply intact. A left parietal craniectomy was performed on some fetuses and in others ultrasonic irradiation was delivered through scalp and skull. Trypan Blue was injected into the umbilical vein immediately postoperatively. The brain was removed 20-30 min later and was cut after fixation for 24 hours in Bouin’s fixative. Animals used for neuropathological studies were irradiated with a dose of ultrasound adequate to produce a lesion approximately 4 mm in length. Of each age group, one or more animals each were killed 1, 3 and 7 days and 2, 4 and 8 weeks after irradiation by decapitation or a lethal dose of pentobarbital sodium. The brain was removed immediately, placed in a 10 per cent isotonic solution of neutralized formalin for 24 hours, after which the irradiated part was cut into two blocks for subsequent preparation of stained sections (Fig. 1). The basal block was used for frozen sectioning and staining for microglia (Hortega silver carbonate method), astrocytes (Cajal gold sublimate method) and lipids (scarlet red). The
ULTRASONIC
LESIONS
IN
BRAIN
505
other was embedded in paraffin and the sections stained with hematoxylin and eosin, cresyl violet and mordinated hematoxylin (Heidenhane’s method for myelin sheaths). Some sections were also stained with periodic acid Schiff (PAS) and phosphotungstic acid-hematoxylin (PTAH) . No attempt was made to accomplish survival in irradiated fetuses. Results
Immediately after irradiation, approximately 25 per cent of the younger animals (newborn and 2 weeks old) assumed a posture with the head and body bent toward the operated side. This torsion of the body gradually disappeared during the first two postoperative days. No other neurological defect was apparent in any of the surviving animals. Some 26 per cent of the newborn and 2-week-old operated rabbits died during the first 48 hours, probably due to the anesthesia and surgery; an additional 29 per cent of those surviving died because of maternal neglect or an illness which usually caused diarrhea in the infected animals. The mortality dropped to 13-14 per cent at the age of 4 weeks. All operated adults survived. Ultrasonic Dosage-Lesion Size Relationship. Although no precise placement of lesions was attempted, their location (Fig. 2) was entirely satisfactory for the purposes of this study. The relation of the lesion size to ultrasonic dosage at two pulse durations for different ages of the animal is given in Fig. 3. The larger dose produced the larger lesions, but there was considerable variability in lesion size at each dosage level and particularly so in the younger animals. However, for equivalent dosages of ultrasound, there was a general trend for the younger animals to have smaller lesions than the older animals. Lesions were also successfully placed in the brain of two fetuses by irradiation through a craniectomy and two additional fetuses by irradiation through intact scalp and skull (Fig. 4). Pulse durations of 2 and 5 set, respectively, were used. The size of the lesions was comparable to that found in newborn animals irradiated for 2 set (Fig. 3). Histological Observations. Discrete foci of coagulation necrosis were found in the target zone of twenty-nine of the thirty animals thus studied. The only rabbit which did not show a discrete lesion (but a diffuse astrocytic reaction instead) had been operated upon at the age of 1 day and killed 4 weeks later. The characteristics of the earliest lesion, the progress of its breakdown
506
YOUNG
AND
LELE
and the tissue reaction to the lesion were all the same for each age group of rabbits. The lesion had the characteristic moat-island form (1). The center (island), in cross section, was almost circular and was composed of a plug of coagulated, necrosed brain tissue (Fig. 2C & 5). Surrounding this coagulum there was a necrotic and damaged zone which had a loose, reticulated appearance in which extravasate of serum and a few fibrin strands were seen. Vessels at the periphery of the lesion were dilated and
FIG. 2. A & B. Photographs of two supravitally stained lesions produced in brain of 4-week-old rabbits by irradiation with different doses of ultrasound. Each brain of the cut was cut in a plane parallel to the vertical lines in Fig. 1. C. Photograph horizontal surface of a block of brain showing an ultrasonic lesion on the left and an India-ink needle track on the right. Note the characteristic moat-island form of the ultrasonic lesion. Magnification: A & B, 2.5 X ; C, 3 X.
congested. Reaction to this focus of necrosis was prompt and vigorous. A few polymorphonuclear leucocytes were seen during the first 1 to 3 days but were very sparse by 1 week. Microglia cells and macrophages were found to have accumulated about the periphery of the lesion at 24 hours and they increased greatly in numbers up to 1 or 2 weeks. At the rim of the lesion, neuroglia cells appeared to have survived in some areas where all nerve cells were necrotic. Plump astrocytes were evident in undamaged tissue near the periphery of the lesion. The astrocytes showed
ULTRASONIC
LESIONS
IN
507
BRAIN
evidence of hypertrophy and proliferation and by 7 days there was a definite zone of astrocytic gliosis about the moat (Fig. 6B). During the first 1 or 2 weeks, swollen axons could be identified within and near the periphery of the lesion. Phagocytosis of necrotic debris from the moat appeared to progress rapidly; but not until several weeks later was the
6
c
-d--J2 AGE
7
14
OF RABB/T
26
ADULT
fDays1
FIG. 3. Relation of lesion size to ultrasonic dosage at two at different ages of the rabbit. Depth of the target site from also indicated as a measure of the size of the brain.
pulse durations and the dural surface is
coagulated island lysed and phagocytized. During the first 2 months the density of the astrocytic glial scar was found to increase. New vessels with a stroma of fibrous tissue were seen to grow into the lesion; but there were few fibroblasts present except in close proximity to vessel walls. In one of the rabbits which was operated upon at age 2 weeks, there were fossilized fragments (concretions of “pseudocalcium”) of cell processes at the periphery of the lesion 2 months later.
508
YOUNG
AND
LELE
Four of the thirty lesions contained hemorrhage, confined to the necrotic focus and the immediately surrounding tissue. There was a large abscess in the left cerebral hemisphere of one animal which was operated upon at age 1 day and killed 2 weeks later. A perivascular accumulation of plasma cells and lymphocytes, histological evidence of mild encephalitis,
FIG. 4. Photograph of a coronal section of brain 24 days) showing a lesion resultin, v from irradiation H. & E. stain. Calibration mark = 1 mm.
FIG.
rabbit. rabbit.
5. A. Photograph of a lesion 20 min. after H. &3 E. stain. B. A similar lesion 7 days H. & E. stain. Calibration marks = 500 u.
of fetal through
rabbit intact
irradiation of after irradiation
(gestational scalp and
age skull.
a Z-week-old of an adult
ULTRASONIC
LESIONS
IN
509
BRAIN
was seen in the brain of two of the adult animals. None of these three infected animals showed overt signs of illness.
FIG.
Red
6. Photographs and B, with Cajal
of lesions 7 days after irradiation, stained gold stain. Calibration marks = 100 CL.
A, with
Scarlet
510
YOUNG
AND
LELE
Discussion
The ability to produce trackless foci of necrosis within specific regions of brain of fetuses and growing animals makes focused ultrasound a valuable tool in experimental neuroembryology. Production of such lesions in fetuses in situ by irradiation through intact scalp and skull, in particular, opens up many new possibilities for such investigations. The major deterrent to such use, at this time, is the lack of an effective head-holding device which will provide the requisite stability and yet will not damage the uterus, placenta and fetus. The methods used for positioning and holding of the head in this investigation were only just adequate: but no great precision in the placement of the lesions was necessary. In a previous study (2) with adequate instrumentation and technique, the probability of accurate lesion placement was restricted only by the variability in the stereotaxic location of the structure itself. Irradiation with an adequate dosage of focused ultrasound almost invariably produced a discrete lesion in the brain of the rabbit, as was found to be the case in that of the cat (2). The average size of the lesion is again related directly to the duration of irradiation. But, for the same dosage of ultrasound, average lesion size was smallest in the newborn and increased with increasing age and size of the brain (as indicated approsimately by the depth of the lesion from the dural surface; Fig, 3). At any dosage level there was considerable variability in the lesion size but particularly marked in the younger animals. This would indicate the existence, in the case of the smaller brains, of some varying factor, such as interference patterns resulting from the brain-bone-air interface at the base of the skull. Existence of such interference patterns was found to cause much variability in the effects of ultrasonic radiation on the peripheral nerve (5). Other factors, such as increasing attenuation of ultrasound by brain with increasing age of the animal, possibly due in part to decreasing water content (3), increased myelination of fiber tracts, and growth of dendrites, may play some role which, however, could not be expected to contribute to variability. The influence of some of these factors is currently under investigation. The incidence of hemorrhage in the necrotic focus (13% ) is higher than that found in the adult cat ( 1, 2). This may be related to the animal’s movements during irradiation, and to inadequacy of the technique of holding the head without squeezing it. Furthermore, to minimize the risk of the head moving during irradiation, the duration of irradiation was
ULTRASONIC
LESIONS
IN
BRAIN
511
kept short by resorting to higher intensity of ultrasound. Basauri and Lele (2) found that the incidence of hemorrhage increases directly with the intensity of radiation. The histological characteristics of the lesion and the reparative processes which were consequent upon it were the same for all of the animals regardless of the age when the lesion was made and were similar to those described by iistrijm and his co-workers (1) in the adult cat. This is congruent with the results of the study of Young and Dodge (6) in which the histopathology of stab wounds in fetal rabbits was compared with the stab wounds produced in the animals of the present study. In each study the propensity for producing healing through phagocytosis of necrotic tissue by microglia and macrophages and the concurrent production of an astrocytic gliosis is equally strong in newly born and adult rabbits. This concept holds both for small lesions (produced by a to-gauge hypodermic needle puncture) and for the relatively large volume of brain tissue necrosis which was produced by ultrasound. References 1.
2.
3. 4. 5.
6.
K. E., E. BELL, H. T. BALLANTINE, JR., and E. HEIDENSLEBEN. 1961. An experimental neuropathological study of the effects of high-frequency focused ultrasound on the brain of the cat. J. Neuropath. Exptk Neurol. 20: 484-520. 1962. A simple method for production of trackBASAURI, L., and P. P. LELE. less focal lesions with focused ultrasound: Statistical evaluation of the effects of irradiation on the central nervous system of the cat. J. Physiol. London 160: 513-534. 1955. Age and water content of rabbit brain GRAVES, J., and H. E. HIMWICH. parts. Am. J. Physiol. 180: 205-208. LELE, P. P. 1962. A simple method for production of trackless focal lesions with focused ultrasound: Physical factors. I. Physiol. London 160: 494-512. LELE, P. P. 1963. Effects of focused ultrasonic radiation on peripheral nerve with observations on local heating. Exptl. Neural. 8: 47-83. YOUNG, G. F., and P. R. DODGE. Experiments in developmental neuropathology: Stab wounds into the brain of rabbits at various ages. In preparation.
ASTR~~M,