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Split-brain preparation by ultrasonic lesions in the rat
Physiology & Behavior, Vol. 24, pp. 123-129. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.
Sprit-Brain Preparation by Ultrasonic Lesions in the R a t I ANDREW
J. L E E A N D P E T E R V. T A B E R N E R
Department of Pharmacology, University of Bristol, Bristol BS8 ITD, England AND MICHAEL
HALLIWELL
Department of Medical Physics, Bristol General Hospital, Bristol BS1 6SY, England R e c e i v e d 5 F e b r u a r y 1979 LEE, A. J., P. V. TABERNER AND M. HALLIWELL. Split-brain preparation by ultrasonic lesions in the rat. PHYSIOL. BEHAV. 24(1) 123-129, 1980.--Ultrasound has been used to selectively lesion the corpus callosum in 18 rats. A focused beam, at 3.3 MHz of 3.5 W total acoustic output with 2 see continuous wave exposure, was used to create approximately spherical lesions of 1 mm diameter. A series of overlapping lesions enabled severance of the entire corpus callosum at the midline. To make the technique more reliable an ultrasonic method was developed to accurately locate the corpus callosum. Verification of the lesions was made histologically and also electrophysiologically by observing the disappearance of trans-callosal evoked potentials. Damage to fibres other than the corpus callosum was minimal. Possible applications of this lesioning technique are mentioned.
Split brain rat
Corpus callosum
Ultrasound
Lesioning
IN most mammals the cerebral hemispheres are connected by a band of fibres, the corpus caliosum, which connects regions of one hemisphere to homotopic regions in the opposite hemisphere [13]. F o r various studies of perceptual processes and clinical treatments it is necessary to sever these intercortical fibres so that the two hemispheres can function independently under specific conditions. In large animals such as man [1], monkeys [9,21] and cats [24] this operation is not too difficult as the cerebral hemispheres are large and, after cutting through the dura the presence of a falx dividing the two hemispheres above the dorsal callosum aids access to the corpus callosum with little cortical damage. With smaller animals, such as the rat, this approach is more difficult as there is no falx and it is easy to cause damage not only to the hemispheres, but also the superior sagittal and transverse sinuses and the inferior sagittal sinus almost directly overlying the corpus callosum. In behavioural studies where large numbers of animals are often needed, rats have several advantages as the experimental animal of choice. Previous methods for obtaining "split brain" rats have included cutting with a small scalpel under hypothermia [8], manipulation of a thread in a saw-like motion [6,20] and, more recently, a shielded-knife technique which approaches the corpus callosum through the cerebellum [14]. F o r behavioural experiments such methods are not optimal as they cause unwanted tissue damage which in some cases is considerable. In any subsequent behavioural tasks where the effect of lesioning particular neuronal path-
Trans-callosal evoked potentials
ways is being studied, damage other than at the target site is undesirable. Ultrasound offers an alternative means of creating lesions. When an alternating voltage is applied across a piezoelectric crystal, the crystal changes in thickness, vibrates and produces a beam of sound at the frequency of the applied voltage [25]. With a concave piezoelectric ceramic, ultrasound acts in a similar way to light passing through an optical lens and is focused at a given distance from the ceramic. The minimum focal " s p o t " which can be produced is proportional to the wavelength and frequencies in the range of 1-10 MHz can provide focal " s p o t s " of 1.5-0.15 mm dia. (measured in water) [11,15]. Under appropriate conditions sufficient energy can be dissipated at this focal region to cause irreversible tissue damage, possibly as a consequence of mechanical stress factors with a thermal component (although the thermal effect is not thought to be the major component at doses used in this study) [3, 10, 11, 12, 18]. A focused ultrasound beam can create small lesions specifically at its focus without causing tissue damage elsewhere along the ultrasound pathway. In order to specifically lesion the corpus callosum it was necessary to locate the structure accurately. Two location methods have been used: one, using stereotaxic coordinates, and a second, more accurate, method employing an ultrasound technique. To ascertain the validity of the technique for severing the corpus callosum, the disappearance of transcallosal evoked
1Reprint requests should be sent to: A. J. Lee, Department of Pharmacology, University of Bristol, Bristol BS8 1TD, England.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/010123-07502.00/0
124
LEE, T A B E R N E R AND H A L L I W E L L
potentials was studied on rats in acute experiments. A preliminary account of ultrasonic lesioning has already been reported [16].
•To Micromanipulator Saline
To Ultrasound Generator
1
METHOD
Animals and Materials
,i
Twenty-three albino rats, Porton strain from our inbred colony, 200-250 g, at the start of each experiment [23]. Chloral hydrate was obtained from May and Baker Ltd., Dagenham, and chloramphenicol sodium succinate (chloromycetin succinate) from Parke Davis and Co., Pontypool, Gwent.
Brass Body
Histology
PZT-4 Ceramic
Sectioning was carried out in a Bright BMCS cryostat. Photomicrographs were taken with a Leitz Ortholux II.
Truncated Hollow Cone Preparatory Surgery
II
00 V
Removal of the overlying area of the skull prior to ultrasound exposure is necessary as bone will absorb approximately 50% of the incident energy dissipating it largely in the form of heat which may damage the underlying cortex [4]. Furthermore, the uneven thickness of the skull and the different acoustic properties of bone and soft tissue will scatter the ultrasound at their boundary to give a poorly defined focal region which would make it difficult to obtain the small lesions required. Under chloral hydrate anaesthesia (500 mg/kg, IP, with incremental doses as necessary to keep a constant depth of surgical anaesthesia) the scalp was shaved and the rats placed in a head holder such that the corpus callosum was approximately on a horizontal plane--this was necessary for cleavage of the corpus callosum using stereotaxic methods. Animals were injected with chloramphenicol (80 mg/kg, IM) to help counter infection. A midline incision was made through the scalp and underlying membranes scraped away from the skull. A 4 mm hole was trephined in the skull, centered at the skull suture landmark lambda. Using a modified Daniels miniature rongeur and Holth corneal scleral punch, a rectangle of bone approximately 10 mm long and 6 mm wide, centered on the midline suture, was then removed, with the dura left intact.
Location of the Corpus Callosum by Stereotaxic Coordinates Manipulation of the ultrasound transducer, shown schematically in Fig. 1, was carried out with a Prior micromanipulator. The focal region of the ultrasound was indicated by a detachable cone. With the corpus callosum aligned horizontally (the angle of the head having been previously established using freshly decapitated heads with the corpus callosum exposed at the midline), the parameters required for locating the lesioning sites are the stereotaxic coordinates for the caudal margin of the corpus callosum, on the midline, and the length of the corpus callosum. A preliminary study indicated that the position of the intersection of the superior sagittal and transverse sinuses proved to be more consistent than skull landmarks such as lambda as a surface zero-reference point (x,y,z), as illustrated in Fig. 2, to determine the caudal margin of the corpus callosum. The mean coordinates of the caudal margin were x+2.0 mm
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1 cm FIG. 1. Ultrasound transducer used to lesion the corpus callosum.
TopViewof Skull .6,'i
*Yx~
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-Y
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Sagittal Section
FIG. 2. Schematic diagrams to illustrate removal of the bone overlying the cerebral hemispheres and axes used to locate the corpus callosum (shown stippled in the transverse and sagittal sections).
( _+ 0.5 mm), y ( - 0.25 mm), z - 0 . 2 mm ( -+ 0.3 mm), and the length of the corpus callosum was 7-8 mm at the midline.
Location of the Corpus Callosum by Ultrasound Using a technique similar to sonar, a 10 MHz ultrasound A-mode scanner of very low intensity (5 mW pulsed square wave 0.2/xsec on, 1 msec off), with a slightly focused beam, was used to detect the corpus callosum. Physiological saline (0.9% w/v) acoustically coupled the ultrasound between the
U L T R A S O N I C L E S I O N I N G O F T H E CORPUS C A L L O S U M
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A. SURFACE OF BRAIN B. CORPUS CALLOSUM FIG. 3. Oscilloscope trace from A-scan used to locate the corpus callosum. The first large peak is at the A-scan transducer surface. Calibration mark 1 mm.
transducer (air effectively blocks ultrasound transmission). To prevent the formation of air bubbles in the transmission path and to reduce the possibility of cavitation effects, the saline was previously degassed by boiling for 1 min, then bottled, sealed and allowed to cool. A silicon rubber well was placed over the skull and sealed, by pulling the skin edges from the longitudinal cut over a flange at the bottom of the well. This enabled a pool of saline to be retained over the brain. A constant flow of saline at 37°C was maintained over the open skull area. Suitable precautions were taken to keep the rat warm and dry. The transducer used for the A-scan was mounted similarly to the lesioning transducer using an alignment device so that the focus of the lesioning transducer was in a fixed relationship with the crystal surface of the locating transducer. At tissue interfaces ultrasound is reflected, the echoes detected by the A-scan transducer itself and the resulting voltages displayed on an oscilloscope. The oscilloscope was calibrated using the proximal surface of a block of perspex under saline, so that depth readings could be obtained directly from the time base (Fig. 3).
Ultrasonic Lesioning of the Corpus Callosum The lesioning transducer was fitted with a hollow cone truncated 7 mm from the focus. This cone was used to contain the coupling medium (0.9% saline) which conducted the ultrasound from the face of the transducer to the surface of the brain. A total acoustic output of approximately 3.5 W at
125
3.3 MHz was used to create lesions within the corpus callosum from 2 sec continuous wave exposures. This dose was selected from trial experiments as being suitable to create lesions that (when the focus is accurately placed in the centre of the callosal fibre tract) would be bounded by the limits of the upper and lower fibres--rather than a dose that produced a set lesion size in the corpus callosum. The power output was set, using a radiation pressure balance, immediately before lesioning and the monitored transducer driving voltage held at this value for each lesion. After lesioning the acoustic output was checked on the radiation pressure balance to confirm the settings used. A series of lesions at 0.3 mm intervals was made in a caudal-rostral direction along the length of the corpus callosum. Where the stereotaxic locating method was used (in 10 rats) the y and z coordinates for the transducer were held constant. F o r the A-scan technique (in 8 rats) the y-coordinate was adjusted as appropriate.
Verification of Lesions Electrophysiology. Electrical stimulation of a small area of cerebral cortex results in an evoked potential at the homotopic point on the opposite hemisphere [7]. Severance of the corpus callosum may be expected to abolish this evoked potential if the callosal fibres provide the only means of transmission between homotopic areas of each hemisphere under these conditions. Electrophysiological studies were therefore useful for providing a functional test for severance of the corpus callosum. For the acute electrophysiological experiments carried out during and immediately after the ultrasonic lesioning, a larger portion of the skull had to be removed than that required to carry out the lesioning alone, so that the lesioning transducer could pass unobstructed down the midline with the electrodes peripherally placed. Pairs of insulated silver electrodes rested lightly on the pia of each hemisphere. Each pair was used to record bipolar E E G when not used for recording transcallosal evoked potentials. An interpolar distance of less than 1 mm helped to localize the stimulating current (0.25 mA measured across a 10 I~ resistor in series with one electrode monitored periodically throughout each experiment). Chloriding of the electrodes was not a problem as the stimulating current remained at 0.25 mA at the end of each experiment. Square wave pulses, 0.4 msec duration at 30 V, were used to stimulate the surface of the cortex. The electrodes, placed under visual control on homotopic areas of each hemisphere, were adjusted until a maximal evoked potential was recorded. Relative positon of the electrodes are illustrated in Fig. 4. Initial placement of the electrodes was 4.5 mm from the intersection of the sagittal and transverse sinuses and 2 mm either side of the midline (3.5 mm when evoked potentials were monitored during ultrasound lesioning). Having finalized the position of the electrodes the stimulating pulses were changed from monophasic to biphasic to help reduce chloriding of the electrodes and to decrease the stimulus artifact. Evoked potentials and E E G were monitored before, during and after lesioning. Fifteen animals were used in this study: five controls (no ultrasonic lesioning), five rats lesioned after prior location of the corpus callosum by stereotaxic means, and five rats lesioned after location of the corpus callosum by ultrasonic means. Two animals from each category were used for the electrophysiological experiments during and immediately after ultrasonic lesioning and from each category for acute exper-
126
LEE, TABERNER AND HALLIWELL
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FIG. 4. Electrophysiologicalverification of severance of the corpus callosum. (A). Position of stimulating (a) and recording (b) electrodes; (c) surface of cerebral hemisphere, (d) skull. (B) Trans-callosal potentials before ultrasound lesioning. (C) and (D) Evoked potentials 2 hr and l 1 hr, respectively, after ultrasound lesioning. (E) Evoked potentials with incomplete severance of the corpus callosum. Calibration marks for the abscissa and ordinate, respectively, are 10 msec and 20/~V.
iments at 2, 10 and 30 days after lesioning (one control and two rats, one from each corpus callosum location method, at each of the indicated days). Histology. Rats were killed 1-30 days after lesioning, their brains removed, rapidly frozen in a stream of CO2powder and then sectioned, in coronal or sagittal planes, at 20/~ intervals in a cryostat. Luxol fast blue and cresyl violet were used to stain myelin sheaths and Nissl granules respectively. RESULTS
Histology Lesions were seen within the corpus callosum as regions of pallor in which fibre disruption had occurred. Examples are shown in Fig. 5. In coronal section these were of approximately 1 mm diameter. Examination of serial 20 t~ coronal and sagittal sections indicated that the entire length of the corpus callosum was severed on the midline in six of the eight cases with the ultrasound location method, and only in three of the ten cases with the stereotaxic location method. A successfully lesioned animal was taken as one having lesions similar to those illustrated in Fig. 5 over at least 75% of the length of the corpus callosum; and, with extracallosal damage immediately above and below a lesion not extending for more than 0.3 mm in the remaining 25% of the corpus callosum. In the unsuccessfully lesioned animals m o r e damage was observed either immediately above the corpus callosum (to the cingulum), and below the corpus callosum (to the fornix commissures and into the hippocampus), and in places there was incomplete severance of the corpus callosum. The size of the lesions varied only slightly from 1-30 days after sonication.
Electrophysiology Before lesioning, biphasic transcallosal evoked potentials could be recorded, with the first phase approximately 8 msec after stimulation of 30/xV peak intensity and lasting approximately 15 msec. The second phase followed as a phase reversal from the first and was of 50/zV peak intensity with a 30 msec decay to the baseline, as shown in Fig. 4. During lesioning no change was seen in the evoked potentials except when the transducer was lesioning directly between the stimulating and recording electrodes: towards the end of this sonication period the evoked potentials totally disappeared but started to reappear within 1 min and fully returned within 2 min. This effect was observed in all the animals lesioned, but not in the two control animals where the ultrasound was not switched on. In all cases where subsequent histological results showed that the corpus callosum had been thoroughly severed at the level where the cortex was electrically stimulated, the evoked potentials remained for 30-90 min after lesioning, after which both phases slowly disappeared until no evoked potentials could be detected (Fig. 4). Up to 11 hr later, with continuous monitoring, no evoked potentials were observed. Evoked potentials could not be recorded up to 30 days after lesioning. Of the five rats with the corpus callosum located by ultrasound four were successfully lesioned, while of the five rats operated after the stereotaxic location method only two were successfully lesioned. In the cases where subsequent histological examination showed incomplete severance of the callosal fibres in the region of transcallosal stimulation (some of the upper regions of the fibre tract remaining intact), the magnitude of the first peak of the evoked potential slightly decreased, while the
ULTRASONIC LESIONING OF THE CORPUS CALLOSUM
127
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FIG. 5. Ultrasound lesions in the corpus callosum of the rat, 7 days after irradiation at 250 W/cm 2 for 2 sec at 3.3 MHz. Twenty/.~ sections stained with Luxol blue and cresyl violet. (a)-(d) Nearly spherical lesions in the corpus callosum on the midline, × 5. (e) Detail of a single lesion, x 80. (f) Detail of (e), x 150.
128
LEE, T A B E R N E R AND H A L L I W E L L
second peak was substantially reduced in height (see Fig. 4(e)). DISCUSSION The electrophysiological and histological data indicated that complete severance of the corpus callosum could be achieved by ultrasound. The disappearance of the evoked potentials 30-90 min after sonication was not due to the brain under the electrodes becoming electrically inactive as the EEG could still be recorded. The temporary loss of the evoked potentials when lesioning directly between the stimulating and recording electrodes would appear to be due to a short-lasting primary blocking action by the ultrasound of neurotransmission of electrical events. This may be a consequence of intracellular "stirring" brought about by sonication [17]. Reversible suppression of evoked cortical potentials, in response to a flash of light, has been reported [12], although this study used doses which did not produce histologically observable lesions. The evoked potentials did not reappear even up to 11 hr after the operation. If a bypass of the corpus callosum were to be used, it may be expected that a longer latency evoked potential would appear within this period providing the ultrasound had not affected subcortical structures. The absence of evoked potentials in successfully lesioned animals up to 30 days after lesioning provides evidence for the electrophysiological permanency of the lesion over this time period. The presence of evoked potentials up to 90 min after lesioning would not be expected if the effects were due to thermocoagulation means, and this lends support to the mechanical stress properties of ultrasound at the dose used [15]. Such temporary recovery of nerve axons after sonication, only to cease function 90 min later, has not previously been reported in the literature, and the mechanism by which this is achieved is as yet unclear. As the size of the lesion varied only slightly from 1-30 days after sonication, this suggests that gross retrograde degeneration did not occur. In 15 of the animals examined, when lesions in the corpus callosum were viewed in coronal section the central band of fibres within the corpus callosum often distended the "sphericity" of the lesion. This occurred
particularly in the splenium of the corpus callosum. This "central spreading" of the lesion may imply that certain fibre tracts within the corpus callosum were differentially susceptible to the action of ultrasound. Accurate location of the corpus callosum was found to be essential for complete severance of the callosal fibres. The A-scan location method indicated that in many cases the actual position of the corpus callosum could vary by - 0 . 5 mm from the reference level obtained by the intersection of the sinuses. Severing the corpus callosum using the A-scan location technique was more successful than using the stereotaxic location method, and for this reason it was preferred. By closely controlling the ultrasound dose and, if necessary, making overlapping lesions, it is feasible that practically any prescribed area of brain tissue can be destroyed. The use of the A-scan technique for accurate location of many regions of the brain may not be feasible, but for some uses stereotaxic coordinate location may be adequate [5]. The effectiveness of ultrasound in creating lesions within the lateral geniculate body [3], and the mamillo-thalamic tract [12] has been demonstrated in cats. With smaller animals, such as the rat, location of structures such as the nigrostriatal pathway may be feasible using stereotaxic coordinates, although the method would not be expected to be highly accurate. The potential use of ultrasound to create lesions in any part of the brain makes it a valuable tool for studies where tissue damage other than at the target side could be disadvantageous, particularly in human neurosurgery [2, 19, 22], or in experimental studies where non-specific damage may make interpretation of results difficult. ACKNOWLEDGEMENTS Grateful acknowledgement is given for the advice from Dr. P. N. T. Wells (Bristol General Hospital, Bristol), Mr. H. Griffith and Dr. E. Brownwell (Frenchay Hospital, Bristol), Dr. P. Keen, Dr. R. G. Hill and Mr. R. Morris (Department of Pharmacology, University of Bristol). The ultrasonic lesioning apparatus and A-scan were made at, and were on loan from, Bristol General Hospital. The Leitz Ortholux II was obtained on a Royal Society Grant to Dr. P. Keen. A.J.L. is a Bristol University Scholarship holder.
REFERENCES 1. Akelaitis, A. J. Studies on the corpus callosum II. The higher visual functions in each homonymous field following complete section of the corpus callosum. Archs Neurol. Psychiat., Lond. 45: 788-796, 1941. 2. Akelaitis, A. J. Psychobiological studies following section of the corpus callosum. A preliminary report. Am. J. Psychiat. 97: 1147-1157, 1941. 3. Ballantine, H. T., Jr., T. F. Hueter, W. J. H. Nauter and D. M. Sosa. Focal destruction of nervous tissue by focused ultrasound: Biophysical factors influencing its application. J. exp. Med. 104: 337-360, 1956. 4. Barnard, J. W., W. J. Fry, F. J. Fry and R. F. Krumins. Effects of high intensity ultrasound on the central nervous system of the cat. J. comp. Neurol. 103: 459-484, 1955. 5. Basauri, L. and P. P. Lele. A simple method for production of trackless focal lesions with focused ultrasound: Statistical evaluation of the effects of irradiation on the central nervous system of the cat. J. Physiol., Lond. 160: 513-534, 1962. 6. Bures, J., O. Buresova and E. Fifkova. Interhemispheric transfer of a passive avoidance response. J. comp. physiol. Psychol. 57: 326-330, 1964.
7. Curtis, H. J. Intercortical connections of corpus callosum as indicated by evoked potentials. J. Neurophysiol. 3: 407--413, 1940. 8. CydeU, A. The use of hypothermia in split-brain surgery on the rat. Behav. Res. Meth. lnstrum. 2: 65-66, 1970. 9. Downer, J. L. De C. Changes in visual guided behaviour following mid-sagittal division of optic chiasma and corpus callosum in monkey (Macaca mulatta). Brain 82: 251-259, 1959. 10. Fry, W. J., D. Tucker, F. J. Fry and V. J. Wulff. Physical factors involved in ultrasonically induced changes in living systems. II. Amplitude duration relations and the effect of hydrostatic pressure for nerve tissue. J. acoust. Soc. Am. 23: 364368, 1951. 11. Fry, W. J. Ultrasound in neurology. Neurology 6: 693-704, 1956. 12. Fry, W. J. Intense ultrasound in investigations of the central nervous system. Adv. biol. reed. Phys. 6: 281-348, 1958. 13. Gazzaniga, M. S. Partial commissurotomy and cerebral localization of function. In: Cerebral Localization, edited by K. J. Zulch, O. Creutzfeldt and G. C. Galbraith. Berlin: SpringerVerlag, 1975, pp. 133-143.
ULTRASONIC LESIONING OF THE CORPUS CALLOSUM 14. Goodman, E. D. and I. S. Russell. Split brain rat: a new surgical approach. Physiol. Behav. 13: 327-330, 1974. 15. Hueter, T. F., H. T. Ballantine, Jr. and W. C. Cotter. Production of lesions in the central nervous system with focused ultrasound. J. acoust. Soc. Am. 28: 192-201, 1956. 16. Lee, A. J. and P. V. Taberner. A non-invasive method for the preparation of split brain rats using ultrasonics. Br. J. Pharmac. 59: 531P, 1977. 17. Lehmann, J. F. The biophysical mode of action of biologic and therapeutic ultrasonic reactions. J. acoust. Soc. Am. 25" 17-25, 1953. 18. Lerner, R., E. L. Carstensen and F. Dunn. Frequency dependence of the acoustic intensity threshold for ultrasonic production of thermal lesions. J. acoust. Soc. Am. 54: 504-506, 1973. 19. Lindstrom, P. A. Prefrontal ultrasonic irradiation--A substitute for prefrontal lobotomy. Archs. Neurol. Psychiat., Lond. 72: 399--425, 1954.
129 20. Mayes, A. R. and A. Cowey. The interhemispheric transfer of avoidance learning: an examination of the stimulus control hypothesis. Behav. Biol. 8: 193-205, 1973. 21. Myers, R. E. Function of corpus callosum in interoccular transfer. Brain 79: 358-363, 1956. 22. Oka, K., T. Okumura, H. Yokoi, T. Murao, Y. Miyashita, K. Ota, S. Yoshitatsu, K. Yoshioka, H. Hirano and Y. Kawashima. Surgical application of the high intensity focused ultrasound. Med. J. Osaka Univ. 10: 427--442, 1960. 23. Sharpe, R. M., A. Morris, A. C. Wyatt and P. S. Brown. Variability of response in cross-fostered rats. Lab. Anita. 6: 225234, 1972. 24. Sperry, R. W. Cerebral organization and behaviour. Science 133: 1749-1757, 1961. 25. Wells, P. N. T. Ultrasonics in Clinical Diagnosis. London: Churchill Livingstone, 1977.
Sprit-Brain Preparation by Ultrasonic Lesions in the R a t I ANDREW
J. L E E A N D P E T E R V. T A B E R N E R
Department of Pharmacology, University of Bristol, Bristol BS8 ITD, England AND MICHAEL
HALLIWELL
Department of Medical Physics, Bristol General Hospital, Bristol BS1 6SY, England R e c e i v e d 5 F e b r u a r y 1979 LEE, A. J., P. V. TABERNER AND M. HALLIWELL. Split-brain preparation by ultrasonic lesions in the rat. PHYSIOL. BEHAV. 24(1) 123-129, 1980.--Ultrasound has been used to selectively lesion the corpus callosum in 18 rats. A focused beam, at 3.3 MHz of 3.5 W total acoustic output with 2 see continuous wave exposure, was used to create approximately spherical lesions of 1 mm diameter. A series of overlapping lesions enabled severance of the entire corpus callosum at the midline. To make the technique more reliable an ultrasonic method was developed to accurately locate the corpus callosum. Verification of the lesions was made histologically and also electrophysiologically by observing the disappearance of trans-callosal evoked potentials. Damage to fibres other than the corpus callosum was minimal. Possible applications of this lesioning technique are mentioned.
Split brain rat
Corpus callosum
Ultrasound
Lesioning
IN most mammals the cerebral hemispheres are connected by a band of fibres, the corpus caliosum, which connects regions of one hemisphere to homotopic regions in the opposite hemisphere [13]. F o r various studies of perceptual processes and clinical treatments it is necessary to sever these intercortical fibres so that the two hemispheres can function independently under specific conditions. In large animals such as man [1], monkeys [9,21] and cats [24] this operation is not too difficult as the cerebral hemispheres are large and, after cutting through the dura the presence of a falx dividing the two hemispheres above the dorsal callosum aids access to the corpus callosum with little cortical damage. With smaller animals, such as the rat, this approach is more difficult as there is no falx and it is easy to cause damage not only to the hemispheres, but also the superior sagittal and transverse sinuses and the inferior sagittal sinus almost directly overlying the corpus callosum. In behavioural studies where large numbers of animals are often needed, rats have several advantages as the experimental animal of choice. Previous methods for obtaining "split brain" rats have included cutting with a small scalpel under hypothermia [8], manipulation of a thread in a saw-like motion [6,20] and, more recently, a shielded-knife technique which approaches the corpus callosum through the cerebellum [14]. F o r behavioural experiments such methods are not optimal as they cause unwanted tissue damage which in some cases is considerable. In any subsequent behavioural tasks where the effect of lesioning particular neuronal path-
Trans-callosal evoked potentials
ways is being studied, damage other than at the target site is undesirable. Ultrasound offers an alternative means of creating lesions. When an alternating voltage is applied across a piezoelectric crystal, the crystal changes in thickness, vibrates and produces a beam of sound at the frequency of the applied voltage [25]. With a concave piezoelectric ceramic, ultrasound acts in a similar way to light passing through an optical lens and is focused at a given distance from the ceramic. The minimum focal " s p o t " which can be produced is proportional to the wavelength and frequencies in the range of 1-10 MHz can provide focal " s p o t s " of 1.5-0.15 mm dia. (measured in water) [11,15]. Under appropriate conditions sufficient energy can be dissipated at this focal region to cause irreversible tissue damage, possibly as a consequence of mechanical stress factors with a thermal component (although the thermal effect is not thought to be the major component at doses used in this study) [3, 10, 11, 12, 18]. A focused ultrasound beam can create small lesions specifically at its focus without causing tissue damage elsewhere along the ultrasound pathway. In order to specifically lesion the corpus callosum it was necessary to locate the structure accurately. Two location methods have been used: one, using stereotaxic coordinates, and a second, more accurate, method employing an ultrasound technique. To ascertain the validity of the technique for severing the corpus callosum, the disappearance of transcallosal evoked
1Reprint requests should be sent to: A. J. Lee, Department of Pharmacology, University of Bristol, Bristol BS8 1TD, England.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/010123-07502.00/0
124
LEE, T A B E R N E R AND H A L L I W E L L
potentials was studied on rats in acute experiments. A preliminary account of ultrasonic lesioning has already been reported [16].
•To Micromanipulator Saline
To Ultrasound Generator
1
METHOD
Animals and Materials
,i
Twenty-three albino rats, Porton strain from our inbred colony, 200-250 g, at the start of each experiment [23]. Chloral hydrate was obtained from May and Baker Ltd., Dagenham, and chloramphenicol sodium succinate (chloromycetin succinate) from Parke Davis and Co., Pontypool, Gwent.
Brass Body
Histology
PZT-4 Ceramic
Sectioning was carried out in a Bright BMCS cryostat. Photomicrographs were taken with a Leitz Ortholux II.
Truncated Hollow Cone Preparatory Surgery
II
00 V
Removal of the overlying area of the skull prior to ultrasound exposure is necessary as bone will absorb approximately 50% of the incident energy dissipating it largely in the form of heat which may damage the underlying cortex [4]. Furthermore, the uneven thickness of the skull and the different acoustic properties of bone and soft tissue will scatter the ultrasound at their boundary to give a poorly defined focal region which would make it difficult to obtain the small lesions required. Under chloral hydrate anaesthesia (500 mg/kg, IP, with incremental doses as necessary to keep a constant depth of surgical anaesthesia) the scalp was shaved and the rats placed in a head holder such that the corpus callosum was approximately on a horizontal plane--this was necessary for cleavage of the corpus callosum using stereotaxic methods. Animals were injected with chloramphenicol (80 mg/kg, IM) to help counter infection. A midline incision was made through the scalp and underlying membranes scraped away from the skull. A 4 mm hole was trephined in the skull, centered at the skull suture landmark lambda. Using a modified Daniels miniature rongeur and Holth corneal scleral punch, a rectangle of bone approximately 10 mm long and 6 mm wide, centered on the midline suture, was then removed, with the dura left intact.
Location of the Corpus Callosum by Stereotaxic Coordinates Manipulation of the ultrasound transducer, shown schematically in Fig. 1, was carried out with a Prior micromanipulator. The focal region of the ultrasound was indicated by a detachable cone. With the corpus callosum aligned horizontally (the angle of the head having been previously established using freshly decapitated heads with the corpus callosum exposed at the midline), the parameters required for locating the lesioning sites are the stereotaxic coordinates for the caudal margin of the corpus callosum, on the midline, and the length of the corpus callosum. A preliminary study indicated that the position of the intersection of the superior sagittal and transverse sinuses proved to be more consistent than skull landmarks such as lambda as a surface zero-reference point (x,y,z), as illustrated in Fig. 2, to determine the caudal margin of the corpus callosum. The mean coordinates of the caudal margin were x+2.0 mm
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( _+ 0.5 mm), y ( - 0.25 mm), z - 0 . 2 mm ( -+ 0.3 mm), and the length of the corpus callosum was 7-8 mm at the midline.
Location of the Corpus Callosum by Ultrasound Using a technique similar to sonar, a 10 MHz ultrasound A-mode scanner of very low intensity (5 mW pulsed square wave 0.2/xsec on, 1 msec off), with a slightly focused beam, was used to detect the corpus callosum. Physiological saline (0.9% w/v) acoustically coupled the ultrasound between the
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transducer (air effectively blocks ultrasound transmission). To prevent the formation of air bubbles in the transmission path and to reduce the possibility of cavitation effects, the saline was previously degassed by boiling for 1 min, then bottled, sealed and allowed to cool. A silicon rubber well was placed over the skull and sealed, by pulling the skin edges from the longitudinal cut over a flange at the bottom of the well. This enabled a pool of saline to be retained over the brain. A constant flow of saline at 37°C was maintained over the open skull area. Suitable precautions were taken to keep the rat warm and dry. The transducer used for the A-scan was mounted similarly to the lesioning transducer using an alignment device so that the focus of the lesioning transducer was in a fixed relationship with the crystal surface of the locating transducer. At tissue interfaces ultrasound is reflected, the echoes detected by the A-scan transducer itself and the resulting voltages displayed on an oscilloscope. The oscilloscope was calibrated using the proximal surface of a block of perspex under saline, so that depth readings could be obtained directly from the time base (Fig. 3).
Ultrasonic Lesioning of the Corpus Callosum The lesioning transducer was fitted with a hollow cone truncated 7 mm from the focus. This cone was used to contain the coupling medium (0.9% saline) which conducted the ultrasound from the face of the transducer to the surface of the brain. A total acoustic output of approximately 3.5 W at
125
3.3 MHz was used to create lesions within the corpus callosum from 2 sec continuous wave exposures. This dose was selected from trial experiments as being suitable to create lesions that (when the focus is accurately placed in the centre of the callosal fibre tract) would be bounded by the limits of the upper and lower fibres--rather than a dose that produced a set lesion size in the corpus callosum. The power output was set, using a radiation pressure balance, immediately before lesioning and the monitored transducer driving voltage held at this value for each lesion. After lesioning the acoustic output was checked on the radiation pressure balance to confirm the settings used. A series of lesions at 0.3 mm intervals was made in a caudal-rostral direction along the length of the corpus callosum. Where the stereotaxic locating method was used (in 10 rats) the y and z coordinates for the transducer were held constant. F o r the A-scan technique (in 8 rats) the y-coordinate was adjusted as appropriate.
Verification of Lesions Electrophysiology. Electrical stimulation of a small area of cerebral cortex results in an evoked potential at the homotopic point on the opposite hemisphere [7]. Severance of the corpus callosum may be expected to abolish this evoked potential if the callosal fibres provide the only means of transmission between homotopic areas of each hemisphere under these conditions. Electrophysiological studies were therefore useful for providing a functional test for severance of the corpus callosum. For the acute electrophysiological experiments carried out during and immediately after the ultrasonic lesioning, a larger portion of the skull had to be removed than that required to carry out the lesioning alone, so that the lesioning transducer could pass unobstructed down the midline with the electrodes peripherally placed. Pairs of insulated silver electrodes rested lightly on the pia of each hemisphere. Each pair was used to record bipolar E E G when not used for recording transcallosal evoked potentials. An interpolar distance of less than 1 mm helped to localize the stimulating current (0.25 mA measured across a 10 I~ resistor in series with one electrode monitored periodically throughout each experiment). Chloriding of the electrodes was not a problem as the stimulating current remained at 0.25 mA at the end of each experiment. Square wave pulses, 0.4 msec duration at 30 V, were used to stimulate the surface of the cortex. The electrodes, placed under visual control on homotopic areas of each hemisphere, were adjusted until a maximal evoked potential was recorded. Relative positon of the electrodes are illustrated in Fig. 4. Initial placement of the electrodes was 4.5 mm from the intersection of the sagittal and transverse sinuses and 2 mm either side of the midline (3.5 mm when evoked potentials were monitored during ultrasound lesioning). Having finalized the position of the electrodes the stimulating pulses were changed from monophasic to biphasic to help reduce chloriding of the electrodes and to decrease the stimulus artifact. Evoked potentials and E E G were monitored before, during and after lesioning. Fifteen animals were used in this study: five controls (no ultrasonic lesioning), five rats lesioned after prior location of the corpus callosum by stereotaxic means, and five rats lesioned after location of the corpus callosum by ultrasonic means. Two animals from each category were used for the electrophysiological experiments during and immediately after ultrasonic lesioning and from each category for acute exper-
126
LEE, TABERNER AND HALLIWELL
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iments at 2, 10 and 30 days after lesioning (one control and two rats, one from each corpus callosum location method, at each of the indicated days). Histology. Rats were killed 1-30 days after lesioning, their brains removed, rapidly frozen in a stream of CO2powder and then sectioned, in coronal or sagittal planes, at 20/~ intervals in a cryostat. Luxol fast blue and cresyl violet were used to stain myelin sheaths and Nissl granules respectively. RESULTS
Histology Lesions were seen within the corpus callosum as regions of pallor in which fibre disruption had occurred. Examples are shown in Fig. 5. In coronal section these were of approximately 1 mm diameter. Examination of serial 20 t~ coronal and sagittal sections indicated that the entire length of the corpus callosum was severed on the midline in six of the eight cases with the ultrasound location method, and only in three of the ten cases with the stereotaxic location method. A successfully lesioned animal was taken as one having lesions similar to those illustrated in Fig. 5 over at least 75% of the length of the corpus callosum; and, with extracallosal damage immediately above and below a lesion not extending for more than 0.3 mm in the remaining 25% of the corpus callosum. In the unsuccessfully lesioned animals m o r e damage was observed either immediately above the corpus callosum (to the cingulum), and below the corpus callosum (to the fornix commissures and into the hippocampus), and in places there was incomplete severance of the corpus callosum. The size of the lesions varied only slightly from 1-30 days after sonication.
Electrophysiology Before lesioning, biphasic transcallosal evoked potentials could be recorded, with the first phase approximately 8 msec after stimulation of 30/xV peak intensity and lasting approximately 15 msec. The second phase followed as a phase reversal from the first and was of 50/zV peak intensity with a 30 msec decay to the baseline, as shown in Fig. 4. During lesioning no change was seen in the evoked potentials except when the transducer was lesioning directly between the stimulating and recording electrodes: towards the end of this sonication period the evoked potentials totally disappeared but started to reappear within 1 min and fully returned within 2 min. This effect was observed in all the animals lesioned, but not in the two control animals where the ultrasound was not switched on. In all cases where subsequent histological results showed that the corpus callosum had been thoroughly severed at the level where the cortex was electrically stimulated, the evoked potentials remained for 30-90 min after lesioning, after which both phases slowly disappeared until no evoked potentials could be detected (Fig. 4). Up to 11 hr later, with continuous monitoring, no evoked potentials were observed. Evoked potentials could not be recorded up to 30 days after lesioning. Of the five rats with the corpus callosum located by ultrasound four were successfully lesioned, while of the five rats operated after the stereotaxic location method only two were successfully lesioned. In the cases where subsequent histological examination showed incomplete severance of the callosal fibres in the region of transcallosal stimulation (some of the upper regions of the fibre tract remaining intact), the magnitude of the first peak of the evoked potential slightly decreased, while the
ULTRASONIC LESIONING OF THE CORPUS CALLOSUM
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128
LEE, T A B E R N E R AND H A L L I W E L L
second peak was substantially reduced in height (see Fig. 4(e)). DISCUSSION The electrophysiological and histological data indicated that complete severance of the corpus callosum could be achieved by ultrasound. The disappearance of the evoked potentials 30-90 min after sonication was not due to the brain under the electrodes becoming electrically inactive as the EEG could still be recorded. The temporary loss of the evoked potentials when lesioning directly between the stimulating and recording electrodes would appear to be due to a short-lasting primary blocking action by the ultrasound of neurotransmission of electrical events. This may be a consequence of intracellular "stirring" brought about by sonication [17]. Reversible suppression of evoked cortical potentials, in response to a flash of light, has been reported [12], although this study used doses which did not produce histologically observable lesions. The evoked potentials did not reappear even up to 11 hr after the operation. If a bypass of the corpus callosum were to be used, it may be expected that a longer latency evoked potential would appear within this period providing the ultrasound had not affected subcortical structures. The absence of evoked potentials in successfully lesioned animals up to 30 days after lesioning provides evidence for the electrophysiological permanency of the lesion over this time period. The presence of evoked potentials up to 90 min after lesioning would not be expected if the effects were due to thermocoagulation means, and this lends support to the mechanical stress properties of ultrasound at the dose used [15]. Such temporary recovery of nerve axons after sonication, only to cease function 90 min later, has not previously been reported in the literature, and the mechanism by which this is achieved is as yet unclear. As the size of the lesion varied only slightly from 1-30 days after sonication, this suggests that gross retrograde degeneration did not occur. In 15 of the animals examined, when lesions in the corpus callosum were viewed in coronal section the central band of fibres within the corpus callosum often distended the "sphericity" of the lesion. This occurred
particularly in the splenium of the corpus callosum. This "central spreading" of the lesion may imply that certain fibre tracts within the corpus callosum were differentially susceptible to the action of ultrasound. Accurate location of the corpus callosum was found to be essential for complete severance of the callosal fibres. The A-scan location method indicated that in many cases the actual position of the corpus callosum could vary by - 0 . 5 mm from the reference level obtained by the intersection of the sinuses. Severing the corpus callosum using the A-scan location technique was more successful than using the stereotaxic location method, and for this reason it was preferred. By closely controlling the ultrasound dose and, if necessary, making overlapping lesions, it is feasible that practically any prescribed area of brain tissue can be destroyed. The use of the A-scan technique for accurate location of many regions of the brain may not be feasible, but for some uses stereotaxic coordinate location may be adequate [5]. The effectiveness of ultrasound in creating lesions within the lateral geniculate body [3], and the mamillo-thalamic tract [12] has been demonstrated in cats. With smaller animals, such as the rat, location of structures such as the nigrostriatal pathway may be feasible using stereotaxic coordinates, although the method would not be expected to be highly accurate. The potential use of ultrasound to create lesions in any part of the brain makes it a valuable tool for studies where tissue damage other than at the target side could be disadvantageous, particularly in human neurosurgery [2, 19, 22], or in experimental studies where non-specific damage may make interpretation of results difficult. ACKNOWLEDGEMENTS Grateful acknowledgement is given for the advice from Dr. P. N. T. Wells (Bristol General Hospital, Bristol), Mr. H. Griffith and Dr. E. Brownwell (Frenchay Hospital, Bristol), Dr. P. Keen, Dr. R. G. Hill and Mr. R. Morris (Department of Pharmacology, University of Bristol). The ultrasonic lesioning apparatus and A-scan were made at, and were on loan from, Bristol General Hospital. The Leitz Ortholux II was obtained on a Royal Society Grant to Dr. P. Keen. A.J.L. is a Bristol University Scholarship holder.
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129 20. Mayes, A. R. and A. Cowey. The interhemispheric transfer of avoidance learning: an examination of the stimulus control hypothesis. Behav. Biol. 8: 193-205, 1973. 21. Myers, R. E. Function of corpus callosum in interoccular transfer. Brain 79: 358-363, 1956. 22. Oka, K., T. Okumura, H. Yokoi, T. Murao, Y. Miyashita, K. Ota, S. Yoshitatsu, K. Yoshioka, H. Hirano and Y. Kawashima. Surgical application of the high intensity focused ultrasound. Med. J. Osaka Univ. 10: 427--442, 1960. 23. Sharpe, R. M., A. Morris, A. C. Wyatt and P. S. Brown. Variability of response in cross-fostered rats. Lab. Anita. 6: 225234, 1972. 24. Sperry, R. W. Cerebral organization and behaviour. Science 133: 1749-1757, 1961. 25. Wells, P. N. T. Ultrasonics in Clinical Diagnosis. London: Churchill Livingstone, 1977.