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Electrical activity of the isolated macaque brain
EXPERIMENTAL
NEUROLOGY
Electrical
22,
Activity
L. C. MASSOPUST,
303-325
(1968)
of
the
Isolated
Macaque
JR., R. J. WHITE, L. R. WOLIN, D. YASHON, AND N. TASLITZ~
M.
Brain S.
ALBIN,
Laboratory of Nellroph~lsiology, Cleveland Psychiatric Institute; Divisiokk of Neurosurgery and the Brain Rescarclk Laboratory, Case Western Ressme University School of ilIcdicirke at Clezfela?kd Metropolitakk Gcfkeral Hospital, Clcz,elarld. Ohio 44109 Recehvd
Jzkrze 28 > 1968
The completely isolated brain (vascular as well as neurologic isolation) on donor perfusion showed excellent electrical activity of the cortex and reticular formation during all phases of its preparation including 14 hours of donor perfusion. As the various cranial nerves and spinal cord connections were severed from the brain stem, faster moderate voltage electrical activity appeared in cortex and reticular formation. These results were found in isolated preparations where no difficulties occurred in maintaining proper blood pressure, with minimal blood loss, and normothermic brain temperature. The neurogenically isolated brain (all cranial nerves severed) was maintained with little difficulty and appeared to show electrocortical activity similar to that in the completely isolated brain. Fast electrical activity appeared after the removal of the cranial nerves and spinal cord. Control of blood pressure, blood loss, and brain temperature was a prerequisite for optimum cortical activity. Severance of the brain stem between the mesencephalon and diencephalon (cerzvan isole’) created first: a short period of synchronized slowing of electrical activity, followed by a longer period of extremely fast, low-voltage, desynchronized activity, then by phasic slow and fast activity. The intervals of slow activity increased in number and duration as the blood pressure became difficult to maintain, finally becoming very slow with highvoltage activity in the failing preparation. It appears that the isolated primate brain shows a range of electrophysiological activity including states characteristic of the aroused brain of an intact monkey. Introduction
The feasibility of isolating the mammalian brain and its extended survival was indicated by the work of Heymans and his colleagues (6) and by Chute and Smyth (4) as early as 1937. However, the Heymans esperiments involved more of a perfused severed head preparation than a truly isolated brain. The excellent preparation by Chute and Smyth was incom1 This work was supported by NIH Grants NB03859 and NB06552. The authors acknowledge the engineering and fabrication talents of Mr. Ronald Meder, E.E., in the construction of the highly specialized supportive and recording devices for the various isolated brain preparations, the surgical assistance of Satoru Kadoya, M.D., and the general technical assistance of Mr. James Austin and Mr. Paul Austin. 303
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pletely isolated since the contents of the orbit and nasal cavities remained intact and failure to remove some of the soft tissue rendered vascular isolation incomplete as well. Another so-called isolated brain was prepared with the cat by Geiger and Magnes (5) in 1947. These experiments were criticized because of incomplete vascular isolation and the use of heterologous perfusion media in place of blood. Complete neural and vascular isolation and the subsequent maintenance techniques for the rhesus monkey brain, using compatible donor monkey blood perfusion, were developed by White and his associates (21, 24, 25). Their isolated preparation, which is employed in the present study, provides not only a biological model for the study of neurological dysfunction, but also a model for examining electrical phenomena inherent within the brain without impinging neural and vascular connections to and from other regions of the body (24). During total isolation of the brain most of the cranial nerves (except VIII), extracranial vasculature, muscle and soft tissue, and spinal cord connections are severed or removed. Sympathetic innervation of the brain is eliminated by the stripping and surgical division of the internal carotid arteries. As the longevity of the isolated monkey brain was increased by improvements in support systems and protective techniques (11, 22, 23), a standard stereotaxic localization of cortical and brain stem recording electrodes was established, providing intersubject comparability of the spontaneous electrocortical activity and reticular formation activity. Evoked responses from the visual pathway were also recorded in several incompletely isolated preparations. Two other brain preparations were examined for their electrophysiological activity for the purpose of comparison with the completely isolated brain. We refer to the first of these as the “neurogenically isolated brain” because all the cranial nerves were divided from the brain stem, or in the case of the optic, oculomotor, trochlear and abducens nerves, the receptor or effector structures were removed. Finally, the spinal cord was severed from the medulla at the C-l or C-2 level. The second preparation was a “cerveau isoli,” in which the brain stem was transected between the diencephalon and mesencephalon. Bremer (2) described a “deafferent” cat preparation which today would be called a cerveau isolk, since deafferentation was accomplished by brain stem transection which interrupted most of the cranial nerve pathways to the cerebral cortex. However, he did not attempt to interrupt the olfactory or optic nerves and therefore, his preparation was not completely deafferented. The major purpose of the present study was to examine the spontaneous and evoked electrical activity of the isolated brain which was recorded: (a) throughout the dissection of soft tissue from the skull ; (b) during and after cannulation of the carotid arteries; (c) after ligation of the
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305
vertebral arteries; (d) before, during, and after ligation of the spinal cord at the second cervical level (C-Z) ; (e) after severing the spinal cord and removal of the body; and (f) finally, during several hours of donor monkey perfusion of the completely isolated brain. Methods
Twenty-two rhesus monkeys (Macaca wulafta) were used in this investigation. They were divided into four experimental groups: seven monkeys for the completely isolated brain preparation, five for neurogenically isolated brain preparations, four cerveau isolhs. and one control with chronically implanted electrodes. For surgical isolation of the brain small animals (2.6-3.5 kg) were selected in order to reduce the overall operating time. Five large hematologically compatible donor monkeys (9-18 kg) were employed to provide continuous circulatory support for the completely isolated brain preparation. The surgical technique for total isolation of the primate brain and the method of preparation of the donor monkey have been detailed elsewhere and will not he elaborated upon here (21. 23, 25). Completely Isolated Brain. Two weeks before subjecting the animals to the isolation procedure, silver-ball recording electrodes 3.5 mm in diameter were chronically implanted through small trephine holes and fixed to the skull with dental cement. Two pairs of deep recording electrodes (0.3mm diameter stainless-steel needle electrode with 0.5~mm exposed tip’) were placed in the diencephalic reticular formation and posterior reticular formation near the posterior border of the pons. Each electrode had a 3%mm lead wire, which was coiled under the scalp when the wound was closed. Using the frontal coordinates of the stereotaxic system (19) superimposed on the cortex, pairs of the electrodes were implanted epidurally on the medial portion of the precentral gyrus, the superior part of the postcentral gyrus, and 10 mm IateraI on each side of the occipital lobe. The two occipital electrodes were eliminated from the neurogenically isolated and isol& preparations because they interfered with the surgical approach to the brain. The posterior pair of deep electrodes was also eliminated in these preparations and in some isolated preparations. All standard recordings from both cortical surface and brain stem were bipolar, The four main channels consisted of the paired left and right precentral gyri (motor areas), the paired left and right postcentral gyri (sensory areas), the paired occipital leads, and the two pairs of deep electrodes. Evoked potentials from the macular area of the occipital visual cortex were also recorded using bipolar connections. Each animal was initially anesthetized with intravenous sodium pentobarbital (Nembutal) 20 mg/kg or 2.576 thiamylal sodium (Sm-ital).
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MASSOPUST
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Anesthesia was maintained during the surgical preparation and supplemented with topical anesthetics when indicated. Upon completion of surgery, when all sensory inputs had been eliminated, anesthesia was discontinued. Following a tracheotomy, a wire spiral endotracheal tube was inserted into the trachea and a Bird respirator with a pediatric “Q” circle was used to ventilate the animal. To facilitate mechanical respiration, gallamine triethiodide (Flaxedil) was administered. The large donor monkeys were anesthetized with Nembutal (25 mg/kg) or 2.5% Surital. Cannulation of the femoral artery and vein was then performed. In order to limit the length of the cannulation system, the donor was placed in a special restraining chair and brought up to the isolated brain preparation with the lower extremities lying just lateral to, and on a level with the suspended isolated brain. The incisions in the donor monkey were kept heavily infiltrated with local anesthetic. After anesthetization, the scalp and soft tissue were stripped from the head of the monkeys on which the isolation procedure was being performed. Following this, the short wires attached to the recording electrodes were dissected free and longer wire leads were soldered to the short leads. These leads were inserted into the junction box of an eight-channel Grass electroencephalograph, which was coupled with a Tektronix four-channel oscilloscope. Th us, bipolar recordings of the spontaneous electrocortical activity could be taken from any pair of leads and, simultaneously, evoked responses could be recorded from the occipital pair of cortical electrodes. In specific experiments where stimulation of the visual pathway was contemplated, the operative procedure was altered by preserving the contents of one of the orbits. An incandescent light stimulator with a camera shutter was used under light-adapted conditions to evoke visual responses in the occipital visual cortex. Continuous monitoring of blood pressure, pulse, electrocardiogram rectal temperature, and periodic measurements of hematocrit, (EKG), pH, PaC02, PaO,, and oxygen saturations were performed during the length of the experiment to observe and, when necessary, to adjust homeostatic balance. At the time the spinal cord was severed and the body was removed, a subcortical thermistor was inserted through the dura mater into the cortex for temperature recordings. Prior to that the body temperature was monitored rectally. The temperature of the donor monkey was monitored rectally throughout the perfusion of the isolated brain. Unoperated Control. In one monkey, recording electrodes were chronically implanted in the samemanner as described for the completely isolated monkey brain. An amphenol connector was attached to the skull after the leads from each recording electrode were attached to it. The animal was allowed to recover partially from the anesthetic and then was placed in
ISOLATED
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a Foringer monkey chair. For 6 days after the operation, standard previously described electrocorticogram (ECoG) and deep recordings were made from this animal in the awake state and in various sleep states. Neurogenically Isolated Brain. The purpose of this preparation was to examine the effect that neurogenic isolation-as distinguished from vascular and tissue isolation-has on the electrical activity of the monkey brain. The same anesthetic procedures mentioned previously were used. The head was fixed in the special orbital-oral fixation unit and bilateral exenteration of the orbits was carried out with careful dissection of all structures; cautery was used to reduce bleeding. In this way, any input to the central nervous system via cranial nerves II, III, IV, and VI was eliminated. After orbital exenteration, the animal’s head was flexed in such a manner that an extensive bilateral occipital craniectomy, as well as a laminectomy of the first three cervical vertebrae, could easily be carried out. The dura mater was reflected widely, exposing both cerebellar hemispheres. Each cerebellar hemisphere was carefully retracted medially and away from the clivus in such a way that cranial nerves XII, XI, X. and IX were exposed. In turn, each of these cranial nerves was divided first on one side and then the other. Care was taken to free each nerve of its blood supply (if possible) and its arachnoid covering. It has been our practice to divide a set of nerves on one side, to control any bleeding, and then to proceed to the other side. This has assisted in anatomical definition and also has reduced the duration of retraction of the cerebellar hemspheres. Kext, both cranial nerves VIII and VII were exposed, and here great care was necessary in separating the auditory artery from the nervous elements. These nerves were individually teased free and partially divided with sharp scissors. After this procedure, a blunt hook was placed behind the nerve and the nerve was drawn tense toward the operator in such a way that the nerve was fragmented and completely transected. Throughout the entire procedure, an attempt was made to minimize the effect of surgery and retraction on the brain stem. Bleeding was controlled by Gelfoam and cotton pledgets and on the rare occasion when the bleeding was heavy, the vessel was cauterized. Considerable care was taken to elevate the cerebellar hemisphere and to displace slightly the brain stem in order to visualize the trigeminal nerve. Once this nerve (motor and sensory) was brought into view, the same procedure followed, whereby the nerve was partially divided with sharp scissorsand then teased apart with a blunt hook. Again, this was done on one side and then the other. With completion of the trigeminal section, all cranial nerves with the exception of the olfactory had been eliminated. The olfactory nerve was sectioned by performing a small craniectomy
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just above the orbital roof, opening the dura mater, and elevating the frontal lobe. Both olfactory nerves were located and sectioned through this small opening. Using the Ferguson ligature carrier, two 000 silk threads were placed under the cervical spinal cord at C-l and C-3 and, after completion of an ECoG recording period, the cord was tied and divided. Bleeding was controlled with cautery and blood loss was minimal, usually S-10 ml. If a larger volume of blood was lost, Dextran replacement was routinely initiated. Since these animals were instrumented for arterial pressures via femoral arterial catheters the delivery of fluids, drugs, and blood replacement was easily accomplished. “Ceuveazz Is012 Prefaration. In this preparation the brain stem of the rhesus monkey was sectioned just rostra1 to the collicular structures, basically through the dorsal and ventral midbrain. This was accomplished easily by performing a small occipitoparietal craniectomy, followed by resection of one of the occipital lobes. This permitted easy access to the collicular area with displacement of the medial portion of the parietal lobe away from the falx. Within an anatomical triangle formed by the tentorium and the falx, the collicular structures were easily seen when the parietal lobe was properly displaced. When the region of the brain stem just proximal to the colliculi and in direct relationship to the posterior portion of the corpus callosum was located, the small vascular structures (usually veins) were carefully dissected away from the brain stem with an appropriately sized blunt hook. If the veins were inadvertently opened they could be controlled with temporary Gelfoam packing or judicious use of cautery. Once the proper plane was decided upon by studying the configuration of the skull and the position and alignment of the skull in the fixation unit, a surgical transection was made in the brain stem by using blunt instruments (after using a sharp scalpel for opening of the arachnoid membrane). The transection was worked completely through the brain stem in steps, first severing the dorsal medial connections and then the lateral portions on the side from which the brain stem was approached. Finally, the most ventral portions were sectioned by reaching the base of the skull with the blunt instrument. Here great care was taken SOIthat the basilar artery was not severed. Some experience was required to limit properly the dissection to the lateral boundary of the brain stem. The raw surfaces of the cut brain were packed with Gelfoam or wet Cottonoid to reduce bleeding to a minimum. Since some blood loss was inevitable during this procedure, volume replacement was accomplishment with Dextran. When necessary, normal blood pressure was maintained by administration of vasopressors. After completion of each experiment, the brain was removed and examined. The neurogenically isolated preparations were checked to
ISOLATED
BRAIN
assure severance of all cranial nerves. The cerveau isolB preparations examined to determine the plane and extent of the lesion.
309 were
Results
COMPLETELY
ISOLATED BRAIN
ON DONOR PERFUSION
Prior to dissection of the head and neck, the predominant frequency found in the motor and sensory areas of the cerebral cortex was 20-25 Hz of approximately SO-150 pv. In those isolated preparations on donor perfusion which were anesthetized with Nembutal, the common frequencies were between 10 and 16 Hz with voltages of about 100 ,uv. Occasional barbiturate spikes and spindling were observed during early phases of the isolation procedure. Sparse intervals of 3-8 Hz sometimes appeared in the sensory cortex recordings. The ECoG activity appearing in the occipital cortex was generally slower (5-15 Hz) and of higher voltages (ZOO-250 pv). Occasional periods of 3-5 Hz were also noted in this area. In preparations given Surital and Flaxedil, the predominant frequencies were generally faster (25-30 Hz) and of higher voltage (300 pv). No intervals of slow frequencies were noted, not even in the occipital leads. Electrical activity f ram the anterior reticular formation (thalamus) was unaffected by either Nembutal or Surital, since the predissection records showed little or no differences. This activity consisted of moderately fast potentials ( 1 S-20 Hz) with voltages around 30-50 pv. The posterior reticular formation (pans) exhibited typical fast activity with voltages around 20~~. This latter set of eelctrodes was included in only two preparations, since manipulation of the lower brain stem during ligation of the vertebral arteries, and ligation and transection of the spinal cord eventually caused these electrodes to short. The electrocortical and reticular (thalamic) activity reported above suf fered little or no change during dissection of the soft tissue from the head, removal of the necessary bony parts, cannulation of the carotid arteries, or ligation of the vertebral arteries. However, after the spinal cord was ligated and severed at the C-2 level, the electrical activity from all cortical areas became extremely fast with high voltages in isolated brain preparations previously anesthetized with Surital and Flaxedil. The predominant frequencies were found to be on the order of 35-40 Hz with voltages of 100-150 pv. After approximately 10 min spindle-burst activity appeared, sometimes lasting for 2-3 set and recurred periodically for the ensuing l-l.5 hours. This burst activity gradually subsided in preparations that remained in good condition over the next 2 hours. No slow potentials or slow-wave intervals were noted in these monkeys up to the termination of the experiment, which lasted as long as 4 hours post-donor perfusion (Fig. 1).
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FIG. 1. Electrical activity in the completely isolated brain prepared with Surital and Flaxedil: (1) predissection activity ; (2) during dissection of the soft tissue and vascular supply from the head and neck; (3) after cannulation of the carotid arteries ; (4) after ligation of the vertebral arteries ; (5) immediately after transection of the spinal cord at C-2; (6) 1 hour after perfusion transfer to the donor monkey’s circulatory system; (7) after 4 hours on donor perfusion. See Table 1.
On the other hand, in isolated brain experiments in which Nembutal was used during the early stages of the surgery, little or no change was noted in EC& activity throughout the rest of the experiment. We emphasize that the cortical records were generally much slower before dissection of the
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ISOLATED
FIG. 2. Electrical activity in the completely (l-6) same as listed in Fig. 1 ; (7) 2 hours monkey. See Table 1 for key and calibration.
BRAIN
isolated brain after perfusion
313
prepared with Nembutal: of isolated brain by donor
soft tissue and never showed any increase in frequencies throughout the various isolation phases, even after transection of the spinal cord (Fig. 2). C7isually-evoked Cortical Responses. In those experiments (three), in which the right eye and its central vascular and neural connections were left intact, the evoked responses recorded were similar to the responses obtained from the optic cortex of an intact monkey. The “on” potential followed the stimulus onset by about 10-15 msec and had a voltage averag-
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MASSOPUST TABLE
ET AL. 1
KEY FOR ALL FIGURES
LH LHo oc
PCG PO PoCG RH RHO TH
= Bipolar trace from precentral and postcentral electrodes in the left hemisphere. = Bipolar electrical activity from the parietal-occipital electrodes in the left cerebral hemisphere. = l%polar spontaneous electrical activity from the macular representation in the occipital cortices. = Bipolar spontaneous electrical activity from the precentral gyri (motor). = Pontine reticular formation electrical activity recorded near the anterior border of the pons. = Bipolar activity from the postcentral gyri (sensory). = Precentral and postcentral electrode cross in the right hemisphere. ; = Bipolar electrical activity from the parietal-occipital electrodes in the right cerebral hemisphere. = Bipolar thalamic activity.
Calibration:
Vertical mark = 50 gv ; horizontal
mark = 1 sec.
ing about 150 pv. The “off” potential followed the shutter closure by about 7-10 msec. During the first hour of donor perfusion, four or five afterpotentials followed the initial “on-off” responseas long as the intracerebral temperatures remained about 34 C. The afterpotentials usually disappeared at 32 C, but could be recovered if the intracerebral temperature was brought back to 34 C within a reasonableperiod of time. UNOPERATEDCONTROL
A monkey with an electrode array chronically implanted (as described for the completely isolated monkey brain), showed the following characteristic electrocortical activity and reticular formation electrical activity (thalamus) (Fig. 3). Awake State. The precentral gyrus (sensory) tracings had predominant frequencies of 20-25 Hz and voltages of not more than 20 pv. No slower frequencies were observed while the animal occupied the comfortable restraint of the Foringer monkey chair. The postcentral gyrus (motor) tracings also contained the above low voltage fast activity, but had short intervals of 5-15 Hz activity of 30 pv. The occipital lobe tracings were generally slower, 15-20 Hz activity predominating with voltages ranging between 30 and 60 pv. The thalamic electrical activity was found to be of extremely low voltage (less than 20 pv) with frequencies matching those in the precentral and postcentral gyri. When the animal was alerted from a light sleep state by a loud noise the cortical and thalamic activity became extremely fast (35-45 HZ) for
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315
FIG. 3. Represensentative tracings of various awake and sleep states in a macaque under conditions of comfortable restraint of the Foringer monkey chair: (1) resting awake state ; (2) light sleep (paradoxical sleep?) characterized by partially closed eyelids with rapid eye movements ; (3) deep sleep characterized by closed eyelids with no eye movements. See Table 1 for key and calibration.
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ET AL.
about 4-6 set after the noise stimulus, then reverted to the usual awake frequencies. Activated Sleep. This state is characterized by a partial closure of the eyelids with rapid eye movements and probably corresponds to, what is currently termed “paradoxical sleep” (7). The tracings from the precentral and postcentral gyri remained generally fast and of low voltage. Since the standard electrode pattern was used in the unoperated control monkey, it was not possible to observe all the criteria which determine the paradoxical sleep phenomenon. However, based on eyelid closure and rapid eye movements, the ECoG corresponded well with what is usually found in paradoxical sleep. The most dramatic changeswere observed in the anterior reticular formation tracings, Here the S-10 Hz activity completely dominated. Only sparse very short intervals of the low voltage, fast activity were found (intervals of 0.2-0.6 set). The voltages of the S-10 Hz potentials measured 70-150 pv. The occipital lobe tracings also showed 5-10 Hz activity, but only about 50% of the time; the remainder of the tracings continued as relatively fast moderate voltage activity. Sloz~ Wazfe State. This state is characterized by 5-10 Hz potentials (30-60 pv) with momentary periods of faster activity interspersed. When complete relaxation was attained, high-voltage (300 pv peak to peak) multiphasic waves appeared in precentral and postcentral gyri. In the occipital cortex these multiphasic waves were of moderate voltage (lOO150 pv). This state was characterized by complete closure of the eyelids without eye movements. The thalamic tracing was somewhat different from the cortical traces in that these high-voltage waves were monophasic, always positive in deflection, and seemedto trigger the multiphasic waves of the cortex. NEUROGENICALLY
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The basic spontaneous electrical activity from the sensory and motor cortices and from the reticular formation in the neurogenically isolated brain was similar to that observed in those completely isolated brains prepared under Surital and Flaxedil (compare Figs. 1 and 4). The presence of predominant intervals of 20-25 Hz activity of moderately high voltages (100-150 pv) is comparable to an intact monkey’s electrocorticograms. After the removal of cranial nerves V and VII through XII. phasic intervals of 25-35 Hz activity appeared. Generally, however, aft& removal of all the cranial nerves, the intervals of 25-35 Hz activity became longer and more synchronous throughout all leads except the anterior reticular formation. These fast moderate-voltage potentials reached a steady state before the spinal cord was severed from the brain stem. High-voltage slow-wave activity was seldom observed at this point. After transection
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of the spinal cord at the C-2 level, intervals of 40 to 45-Hz activity and occasional high-frequency spindle-burst activity was also noted for as long as 20 min after severance of the spinal cord. These spindle bursts gradually disappeared and none was observed l-1.5 hours after spinal cord transection. Loss of the intervals of 25-35 Hz and the 40 to 45-Hz activity accompanied a drop in the animal’s blood pressure or heart rate or both. If the blood pressure was allowed to fall below 100 mm Hg (systolic) the 25 to 35Hz activity disappeared. When blood pressure was increased by the use of vasopressors it was accompanied by the return of this highfrequency electrocortical activity (Fig. 4 ) . Manipulation of the medullary region or the mesencephalic-diencephalic regions of the brain stem tended to cause an instability in the cardiovascular system, as shown by changes in EKG and blood pressure recordings. Even though blood loss was kept to a minimum, it was difficult to maintain the monkey at its preisolation pressure levels so that the use of vasopressor agents was often necessary. Crrvrnlr
Isoli
In the CUVEULC isolt preparation in good homeostatic balance. the spontaneous electrical activity was generally 20-25 Hz with some short intervals of 25-33 Hz, This activity was characterized by moderate voltages (100 pv). Immediately after section of the brain stem the activity in the motor and sensory cortices and the reticular formation changed to 3-7 Hz with voltages measuring about SO pv in the sensory cortex and reticular formation, while voltages of 75-100 pv were fomid in the motor cortex. This slow electrical activity lasted 2-10 min, then changed rapidly to 16-25 Hz of low voltage (50 pv) and became clesynchronous. At about 30 min after transection, phasic slowing of the electrocorticogram and reticular formation cativity occurred. This was concurrent with difficulties in maintaining the systolic blood pressure above 120 mm Hg. Finally, in the failing preparation the activity slowed to 2-3 Hz with voltages measuring aroutld 1.~0~~ (Fig. 5). No evoked potentials were elicited in either the neurogenicaily isolated or the CCYVCUUisolh preparations because of the interruption of the visual pathway by removal of the contents of the orbits, or removal of the occipital lobe. Discussion
Reports by Kennard and Nims (9), Robert de Ramirez de Arellano ( 16)) and Jurko and Andy (8) show that the highest frequency of electrocortical activity observed in their 1 to 2-year-old awake and alert macaques was on the order of 12-20 Hz. Ours, of approximately the same age, were found to have a 20 to 25-Hz predominant frequency before surgery and
ISOLATED
FIG.
Surital nerves VIII; nerve tion of
319
BRAIN
4. Electrical activity of the neurogenically isolated brain prepared with and Flaxedil: (I) presurgical; (2) exenteration of orbits destroying cranial II, III, IV, and VI; (3) after section of cranial nerves XII, XI, X, IX, and (4) after section of cranial nerves VII and V; (5) after severance of cranial I ; (6) after transection of the spinal cord at C-Z ; (7) 2.5 hours after transecthe spinal cord at C-Z. See Table 1 for key and calibration.
under Surital and Flaxedil. Except under light Nembutal anesthesia, 10 to 12-Hz activity was never seen. The unoperated control monkey with epidural recording electrodes in the awake and alert state further confirmed
the above-mentioned higher frequencies. However, the frequencies reported above
were
taken
from
recordings
with
scalp
electrodes,
while
the fre-
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I
.,I
321
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‘.
I
i
.I.
FIG. 5. Electrical activity during various preparation phases of a cerveatc isolh in which the brain stem was transected between the mesencephalon and diencephalon: (1) presurgical; (2) after partial section of the brain stem on the right side approximately to the midline; (3) immediately after total transection of the brain stem between the mesencephalon and diencephalon ; (4) 10 min after brain stem transection ; (5) 30 min after transection; (6) 1 hour after transection; (i) 2 hours after transection. See Table 1 for key and calibration.
quencies observed in this study were taken from recordings with dural electrodes. Immediately upon completion of the surgery in the completely isolated
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preparation and in the neurogenically isolated brain, frequencies of 25 to 35-Hz activity appeared in both the sensory and motor cortices (Figs. 1 and 4). Several possible factors can be cited which may explain this unusual phenomena. It is possible that the removal of the afferent inputs from the cranial nerves and spinal cord eliminates inhibitory mechanisms which are localized in the mesencephalic reticular formation or other centers within the brain stem (7, 10, 12, 13, 20). Thus, the two isolated brain preparations are similar in the sense that the complex electrical activity generated in the reticular formation and cortex is no longer influenced by the afferent inputs. Changes in frequencies and amplitudes occur after changes in the metabolic conditions, or the internal brain temperature, or blood pressure, or all three. Spindle-burst activity appeared in our preparations approximately 10 min after isolation. Andersson and Wolpow (1) reported that transection of the spinal cord, including the specific short latency cortical projection pathways at the C-3 or C-4 level, increased the spindle-burst activity in localized areas of the somatosensory cortex in cats. This spindle activity also appeared after section of the dorsal funiculi of the spinal cord. It is possible that total severance of the spinal cord in our monkeys also removes these short-latency projection pathways and thus could account for the appearance of similar spindle-burst activity. However, after 3 hours of postoperative time, these spindle bursts subsided and at 4 hours could hardly be detected. In those isolated brain preparations which were given Nembutal, no changes in cortical or reticular formation activity were noted. Since electrocortical activity was slow and of high voltage after anesthesia and usually remained at this level throughout the procedure, the true release phenomena (appearance of the 25 to 35-Hz activity and of the spindle-burst activity) was completely masked by the long term anesthetic effect (15) (Fig. 2). Because of this, Nembutal and other moderately long-acting barbiturates were discarded and Surital and Flaxedil with controlled ventilation were used to obtain optimum electrophysiological activity. The cerveau is016 preparation (severance of brain stem between the mesencephalon and diencephalon) showed some of the classical signs of electrocortical activity. However, after an initial appearance of slow, moderate-voltage activity (for 2 min after brain-stem section), phasic slow and fast intervals of electrocortical activity appeared. Short intervals (l-2 set) of fast, low-voltage activity were observed (Fig. 5). These intervals became increasingly longer and eventually became the predominant pattern as the cerveau isolk preparation was carried out in time. Finally, when the brain began to fail, high-voltage slow activity became more prominent
ISOLATED
BRAIN
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until the low-voltage fast activity was no longer observed (Fig. 5). It is possible that the slow, high-voltage, spontaneous electrical activity reported in cerveau isol6 preparations in other species (7, 10, 12, 13, 20) may have been due to failure in the ability to prevent a detrimental change in cardiovascular homeostasis (i.e., blood loss and maintenance of proper levels of blood pressure, or perfusion of the brain, or both). Another factor which could account for the activity reported in these cervealc isolt preparations could be failure to maintain intracerebral temperature, which reduces the metabolic activity and thus energy output. The ultimate failure of any of our three preparations paralleled the gradual development of brain edema which is due to increases in the circulatory resistance of the brain, and which finally causes general ischemia. Ischemic cortical damage is first indicated by the dropout of the faster frequencies in the ECoG, then by the prominent appearance of slow-wave activity (3-5 Hz), and finally by the appearance of damage spindles. In well-developed cerebral edema the ECoG is completely isoelectric. Reticular modulation of cortical activity is manifold. Both activating and inhibiting influences have been described (3, 7, 10, 12, 13, 20). Afferent inputs to the reticular system are likewise manifold and, undoubtedly, also exert a modulating influence on reticular activity. When all afferent inputs to the brain are eliminated, something like a release phenomenon or “disinhibition” occurs, as manifested by exceptional high-frequency cortical activity. This was observed in both the isolated and deafferent brain preparations. The lack of accord between our results and the vast reticular formation literature, based primarily on the cat, was previously explained by Routtenberg (17). More recently he has suggested (18) the existence of a dual arousal system, one being the classical reticular activating system of Moruzzi and Magoun (12), the other a limbic-midbrain system described anatomically by Nauta (14). Routtenberg has attributed the recovery of low-voltage, fast activity following precollicular brain-stem transection- to “denervation supersensitivity” and has suggested that the medial forebrain bundle running through the hypothalamus and telencephalon mediates the low-voltage fast activity because Villablanca (20) showed desynchronization during stimulation of the olfactory nerve. In our preparations (except the cerveau is&), the central connections of the olfactory nerve were severed or the olfactory receptor areas destroyed and we still obtained the moderate voltage fast activity in the cortex of our monkeys. This indicates that the olfactory nerve input per se is not necessary to produce the low-voltage fast activity. It is possible that some other more central
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ET
AL.
limbic structure (i.e., septum, hippocampus, amygdala) may generate this fast cortical electrical activity after elimination of the classical reticular activating system. In addition to a second arousal system, this also suggests a possible inhibiting mechanism in the posterior reticular formation. In this case the posterior reticular formation was severed from the diencephalon and telencephalon, but afferent inputs via the first three cranial nerves remained. Under these conditions fast electrocortical activity was also obtained. While differing somewhat in character from the fast cortical activity noted in the completely isolated and neurogenically isolated preparations, this activity was still unusual and suggestive of a similar “release” phenomenon. We conclude that there exists a delicate balance between the various portions of the reticular system, and that disruption of this balance may produce several possible effects, one of them being the unusual sustained high-frequency arousal observed in the preparations reported herein. Thus, it appears that the cerebral cortex, at least in primates, can recover its aroused electrical state after external and internal influences (i.e., reticular activating system, cranial nerve inputs) have been removed. It is true that interruption of these influences causes temporary slowing and deactivation of the cortex; however, it seems that when homeostasis has been reestablished the electrocorticogram returns to its activated condition (20). Removal of reticular inhibitory mechanisms may cause this activated or aroused state to be maintained in the cerebral cortex indefinitely. It is also possible that diencephalic structures (i.e., thalamus) control the aroused state, particularly when the mesencephalic-medullary reticular influences have been removed. Under these conditions, inhibitory influences appear to be permanently eliminated, while activation is sustained either by diencephalic mechanisms or directly by the telencephalon. References 1. ANDERSSEN, S. A., and samatosensory cortex 2. BREMER, F. 1935. Cerveau Biol. 118 : 1235-1241.
E. R. WOLPOW. 1964. Localized slow wave activity of the cat. Actn Physiol. Stand. 61: 130-140. (isole)
et physiologie
du sommeil.
Compt.
Rend.
in the Sot.
3. BREMER, F., and C. TERZUOLO. 1954. Contribution a l’ktude des mkanismes physiologiques du maintien de l’activitk vigile du cerveau. Interaction de la formation r&iculCe et de l’korce dr&brale dans le processus du r&eil. Arc/z. Intern. Physiol. 62 : 157-178. 4. CHUTE, A. L., and D. H. SMYTH. 1939. Metabolism of isolated perfused cat’s brain. Quart. J. Exptl. Physiol. 29 : 379-394 5. GEIGER, A., and J. MAGNES. 1947. The isolation of the cerebral circulation and the perfusion of the brain in the living cat. Am. J. Physiol. 149: 517-537.
ISOLATED
BRAIN
325
6. HEYMANS, C., J. J. BAUKAERT, F. JOURDAN, S. J. G. NOWAK, and S. FARBER. 1937. Survival and revival of nerve centers following acute anemia. .4.M.*4. rlrch. Neurol. Psychiat. 38 : 304-307. 7. JOUVET, M. 1967. Neurophysiology of the states of sleep. Physiol. Rezl. 47: 117-177. 8. JURKO, M. F., and 0. J. ANDY. 1967. Comparative EEG frequencies in rhesus, stumptail, and cynomolgus monkeys. Elcctvocnccphaloy. Clin. Naurophgsiol. 23 : 270-272. 9. KENNARD, M. A., and L. F. NIMS. 1942. Changes in normal electroencephalogram of Macaca mulatta with growth. 1. Nczcrophgsiol. 5 : 325-333. 10. MAGOUN, H. W. 1952. An ascending reticular activating system in the brain stem. A.M.A. Arch. Nrurol. Psychitrt. 67 : 145-154. 11. MEDER, R., L. C. MASSOP~ST, JH., R. J. WHITE, J. VERDURA, and M. S. ALBIN. 1963. Isolated brain perfusion : electro-mechanical requirements. 16th ilnn. Conf. Eny. Med. Riol. 5 : 28-29. 12. MORUZZI, G., and H. W. MACOCN. 1949. Brain stem reticular formation and activation of the EEG. Elcctrocwephaloy. Clin. Nenropltgsiol. 1: 455-473. 13. MORUZZI, G. 1964. Reticular influences 011 the EEG. Electrocncrphalog. Clip Neurophysiol. 16 : 2-17. 14. NAUTA, W. J. H. 1958. Hypocampal projections and related neural pathways to the midbrain in the cat. Brain 81: 319-340. 15. PRINCE, D. A., and S. SIIANZER. 1966. Effects of anesthetics upon the EEG response to reticular stimulation. Elrctroerzcepkalog. Clin. Ncurophysiol. 21: 578-588. 16. ROBERT DE RAMIREZ DE ARELLANO, M. I. 1961. Maturational changes in the electroencephalogram of normal monkeys. Exptl. Ncuvol 3 : 209-224. 17. ROUTTENBERG, A. 1966. Neural mechanisms of sleep: changing view of reticular formation function. Psychol. Rerj. 73 : 481-499. 18. ROUTTENBERG, A. 1968. The two-arousal hypothesis: reticular formation and limbic system. Psycho/. Rezj. 75 : 51-80. 19. SNIDER, R. S., and J. C. LEE. 1961. “A Stereotaxic Atlas of the Monkey Brain.” Univ. of Chicago Press, Chicago, Illinois. 20. VILLABLANCA, J. 1965. The electrocorticogram in the chronic cewears isol6 cat Electrocncephalog. Clin. Nrnroplk)qsiol. 19 : 576-586. 21. WHITE, R. J., M. S. ALBIN, and J. VERDURA. 1963. Isolation of the monkey brain: in vitro preparation and maintenance. Sciotce 141: 106&1061. 22. WHITE, R. J., M. S. ALBIN, L. C. MASSOPUST, JR., and R. MEDER. 1964. Design of a simplified perfusion system for differential cooling of brain. 17th Am Cowf. Eng. Med. Biol. 6: 85. 23. WHITE, R. J., M. S. AI.BIN, and J. VERDURA. 1965. Maintenance of the isolated brain by mechanical perfusion. Tram. Am. Sot. drtificial Intrvnal Organs 11: 201-204. 24. WHITE, R. J. 1965. Isolating the brain. L)iscoevery 26 : 34-37. 25. WHITE, R. J., M. S. ALBIN, J. VERDURA, and G. E. LOCKE. 1967. The isolated brain : Operative preparation and design of support systems. J. Nc~r~osu~g. 27 : 216-225.
NEUROLOGY
Electrical
22,
Activity
L. C. MASSOPUST,
303-325
(1968)
of
the
Isolated
Macaque
JR., R. J. WHITE, L. R. WOLIN, D. YASHON, AND N. TASLITZ~
M.
Brain S.
ALBIN,
Laboratory of Nellroph~lsiology, Cleveland Psychiatric Institute; Divisiokk of Neurosurgery and the Brain Rescarclk Laboratory, Case Western Ressme University School of ilIcdicirke at Clezfela?kd Metropolitakk Gcfkeral Hospital, Clcz,elarld. Ohio 44109 Recehvd
Jzkrze 28 > 1968
The completely isolated brain (vascular as well as neurologic isolation) on donor perfusion showed excellent electrical activity of the cortex and reticular formation during all phases of its preparation including 14 hours of donor perfusion. As the various cranial nerves and spinal cord connections were severed from the brain stem, faster moderate voltage electrical activity appeared in cortex and reticular formation. These results were found in isolated preparations where no difficulties occurred in maintaining proper blood pressure, with minimal blood loss, and normothermic brain temperature. The neurogenically isolated brain (all cranial nerves severed) was maintained with little difficulty and appeared to show electrocortical activity similar to that in the completely isolated brain. Fast electrical activity appeared after the removal of the cranial nerves and spinal cord. Control of blood pressure, blood loss, and brain temperature was a prerequisite for optimum cortical activity. Severance of the brain stem between the mesencephalon and diencephalon (cerzvan isole’) created first: a short period of synchronized slowing of electrical activity, followed by a longer period of extremely fast, low-voltage, desynchronized activity, then by phasic slow and fast activity. The intervals of slow activity increased in number and duration as the blood pressure became difficult to maintain, finally becoming very slow with highvoltage activity in the failing preparation. It appears that the isolated primate brain shows a range of electrophysiological activity including states characteristic of the aroused brain of an intact monkey. Introduction
The feasibility of isolating the mammalian brain and its extended survival was indicated by the work of Heymans and his colleagues (6) and by Chute and Smyth (4) as early as 1937. However, the Heymans esperiments involved more of a perfused severed head preparation than a truly isolated brain. The excellent preparation by Chute and Smyth was incom1 This work was supported by NIH Grants NB03859 and NB06552. The authors acknowledge the engineering and fabrication talents of Mr. Ronald Meder, E.E., in the construction of the highly specialized supportive and recording devices for the various isolated brain preparations, the surgical assistance of Satoru Kadoya, M.D., and the general technical assistance of Mr. James Austin and Mr. Paul Austin. 303
304
MASSOPUST
ET
AL.
pletely isolated since the contents of the orbit and nasal cavities remained intact and failure to remove some of the soft tissue rendered vascular isolation incomplete as well. Another so-called isolated brain was prepared with the cat by Geiger and Magnes (5) in 1947. These experiments were criticized because of incomplete vascular isolation and the use of heterologous perfusion media in place of blood. Complete neural and vascular isolation and the subsequent maintenance techniques for the rhesus monkey brain, using compatible donor monkey blood perfusion, were developed by White and his associates (21, 24, 25). Their isolated preparation, which is employed in the present study, provides not only a biological model for the study of neurological dysfunction, but also a model for examining electrical phenomena inherent within the brain without impinging neural and vascular connections to and from other regions of the body (24). During total isolation of the brain most of the cranial nerves (except VIII), extracranial vasculature, muscle and soft tissue, and spinal cord connections are severed or removed. Sympathetic innervation of the brain is eliminated by the stripping and surgical division of the internal carotid arteries. As the longevity of the isolated monkey brain was increased by improvements in support systems and protective techniques (11, 22, 23), a standard stereotaxic localization of cortical and brain stem recording electrodes was established, providing intersubject comparability of the spontaneous electrocortical activity and reticular formation activity. Evoked responses from the visual pathway were also recorded in several incompletely isolated preparations. Two other brain preparations were examined for their electrophysiological activity for the purpose of comparison with the completely isolated brain. We refer to the first of these as the “neurogenically isolated brain” because all the cranial nerves were divided from the brain stem, or in the case of the optic, oculomotor, trochlear and abducens nerves, the receptor or effector structures were removed. Finally, the spinal cord was severed from the medulla at the C-l or C-2 level. The second preparation was a “cerveau isoli,” in which the brain stem was transected between the diencephalon and mesencephalon. Bremer (2) described a “deafferent” cat preparation which today would be called a cerveau isolk, since deafferentation was accomplished by brain stem transection which interrupted most of the cranial nerve pathways to the cerebral cortex. However, he did not attempt to interrupt the olfactory or optic nerves and therefore, his preparation was not completely deafferented. The major purpose of the present study was to examine the spontaneous and evoked electrical activity of the isolated brain which was recorded: (a) throughout the dissection of soft tissue from the skull ; (b) during and after cannulation of the carotid arteries; (c) after ligation of the
lSO$ATED
BRAIN
305
vertebral arteries; (d) before, during, and after ligation of the spinal cord at the second cervical level (C-Z) ; (e) after severing the spinal cord and removal of the body; and (f) finally, during several hours of donor monkey perfusion of the completely isolated brain. Methods
Twenty-two rhesus monkeys (Macaca wulafta) were used in this investigation. They were divided into four experimental groups: seven monkeys for the completely isolated brain preparation, five for neurogenically isolated brain preparations, four cerveau isolhs. and one control with chronically implanted electrodes. For surgical isolation of the brain small animals (2.6-3.5 kg) were selected in order to reduce the overall operating time. Five large hematologically compatible donor monkeys (9-18 kg) were employed to provide continuous circulatory support for the completely isolated brain preparation. The surgical technique for total isolation of the primate brain and the method of preparation of the donor monkey have been detailed elsewhere and will not he elaborated upon here (21. 23, 25). Completely Isolated Brain. Two weeks before subjecting the animals to the isolation procedure, silver-ball recording electrodes 3.5 mm in diameter were chronically implanted through small trephine holes and fixed to the skull with dental cement. Two pairs of deep recording electrodes (0.3mm diameter stainless-steel needle electrode with 0.5~mm exposed tip’) were placed in the diencephalic reticular formation and posterior reticular formation near the posterior border of the pons. Each electrode had a 3%mm lead wire, which was coiled under the scalp when the wound was closed. Using the frontal coordinates of the stereotaxic system (19) superimposed on the cortex, pairs of the electrodes were implanted epidurally on the medial portion of the precentral gyrus, the superior part of the postcentral gyrus, and 10 mm IateraI on each side of the occipital lobe. The two occipital electrodes were eliminated from the neurogenically isolated and isol& preparations because they interfered with the surgical approach to the brain. The posterior pair of deep electrodes was also eliminated in these preparations and in some isolated preparations. All standard recordings from both cortical surface and brain stem were bipolar, The four main channels consisted of the paired left and right precentral gyri (motor areas), the paired left and right postcentral gyri (sensory areas), the paired occipital leads, and the two pairs of deep electrodes. Evoked potentials from the macular area of the occipital visual cortex were also recorded using bipolar connections. Each animal was initially anesthetized with intravenous sodium pentobarbital (Nembutal) 20 mg/kg or 2.576 thiamylal sodium (Sm-ital).
306
MASSOPUST
E,T
AL.
Anesthesia was maintained during the surgical preparation and supplemented with topical anesthetics when indicated. Upon completion of surgery, when all sensory inputs had been eliminated, anesthesia was discontinued. Following a tracheotomy, a wire spiral endotracheal tube was inserted into the trachea and a Bird respirator with a pediatric “Q” circle was used to ventilate the animal. To facilitate mechanical respiration, gallamine triethiodide (Flaxedil) was administered. The large donor monkeys were anesthetized with Nembutal (25 mg/kg) or 2.5% Surital. Cannulation of the femoral artery and vein was then performed. In order to limit the length of the cannulation system, the donor was placed in a special restraining chair and brought up to the isolated brain preparation with the lower extremities lying just lateral to, and on a level with the suspended isolated brain. The incisions in the donor monkey were kept heavily infiltrated with local anesthetic. After anesthetization, the scalp and soft tissue were stripped from the head of the monkeys on which the isolation procedure was being performed. Following this, the short wires attached to the recording electrodes were dissected free and longer wire leads were soldered to the short leads. These leads were inserted into the junction box of an eight-channel Grass electroencephalograph, which was coupled with a Tektronix four-channel oscilloscope. Th us, bipolar recordings of the spontaneous electrocortical activity could be taken from any pair of leads and, simultaneously, evoked responses could be recorded from the occipital pair of cortical electrodes. In specific experiments where stimulation of the visual pathway was contemplated, the operative procedure was altered by preserving the contents of one of the orbits. An incandescent light stimulator with a camera shutter was used under light-adapted conditions to evoke visual responses in the occipital visual cortex. Continuous monitoring of blood pressure, pulse, electrocardiogram rectal temperature, and periodic measurements of hematocrit, (EKG), pH, PaC02, PaO,, and oxygen saturations were performed during the length of the experiment to observe and, when necessary, to adjust homeostatic balance. At the time the spinal cord was severed and the body was removed, a subcortical thermistor was inserted through the dura mater into the cortex for temperature recordings. Prior to that the body temperature was monitored rectally. The temperature of the donor monkey was monitored rectally throughout the perfusion of the isolated brain. Unoperated Control. In one monkey, recording electrodes were chronically implanted in the samemanner as described for the completely isolated monkey brain. An amphenol connector was attached to the skull after the leads from each recording electrode were attached to it. The animal was allowed to recover partially from the anesthetic and then was placed in
ISOLATED
BRAIN
307
a Foringer monkey chair. For 6 days after the operation, standard previously described electrocorticogram (ECoG) and deep recordings were made from this animal in the awake state and in various sleep states. Neurogenically Isolated Brain. The purpose of this preparation was to examine the effect that neurogenic isolation-as distinguished from vascular and tissue isolation-has on the electrical activity of the monkey brain. The same anesthetic procedures mentioned previously were used. The head was fixed in the special orbital-oral fixation unit and bilateral exenteration of the orbits was carried out with careful dissection of all structures; cautery was used to reduce bleeding. In this way, any input to the central nervous system via cranial nerves II, III, IV, and VI was eliminated. After orbital exenteration, the animal’s head was flexed in such a manner that an extensive bilateral occipital craniectomy, as well as a laminectomy of the first three cervical vertebrae, could easily be carried out. The dura mater was reflected widely, exposing both cerebellar hemispheres. Each cerebellar hemisphere was carefully retracted medially and away from the clivus in such a way that cranial nerves XII, XI, X. and IX were exposed. In turn, each of these cranial nerves was divided first on one side and then the other. Care was taken to free each nerve of its blood supply (if possible) and its arachnoid covering. It has been our practice to divide a set of nerves on one side, to control any bleeding, and then to proceed to the other side. This has assisted in anatomical definition and also has reduced the duration of retraction of the cerebellar hemspheres. Kext, both cranial nerves VIII and VII were exposed, and here great care was necessary in separating the auditory artery from the nervous elements. These nerves were individually teased free and partially divided with sharp scissors. After this procedure, a blunt hook was placed behind the nerve and the nerve was drawn tense toward the operator in such a way that the nerve was fragmented and completely transected. Throughout the entire procedure, an attempt was made to minimize the effect of surgery and retraction on the brain stem. Bleeding was controlled by Gelfoam and cotton pledgets and on the rare occasion when the bleeding was heavy, the vessel was cauterized. Considerable care was taken to elevate the cerebellar hemisphere and to displace slightly the brain stem in order to visualize the trigeminal nerve. Once this nerve (motor and sensory) was brought into view, the same procedure followed, whereby the nerve was partially divided with sharp scissorsand then teased apart with a blunt hook. Again, this was done on one side and then the other. With completion of the trigeminal section, all cranial nerves with the exception of the olfactory had been eliminated. The olfactory nerve was sectioned by performing a small craniectomy
308
MASSOPUST
ET
AL.
just above the orbital roof, opening the dura mater, and elevating the frontal lobe. Both olfactory nerves were located and sectioned through this small opening. Using the Ferguson ligature carrier, two 000 silk threads were placed under the cervical spinal cord at C-l and C-3 and, after completion of an ECoG recording period, the cord was tied and divided. Bleeding was controlled with cautery and blood loss was minimal, usually S-10 ml. If a larger volume of blood was lost, Dextran replacement was routinely initiated. Since these animals were instrumented for arterial pressures via femoral arterial catheters the delivery of fluids, drugs, and blood replacement was easily accomplished. “Ceuveazz Is012 Prefaration. In this preparation the brain stem of the rhesus monkey was sectioned just rostra1 to the collicular structures, basically through the dorsal and ventral midbrain. This was accomplished easily by performing a small occipitoparietal craniectomy, followed by resection of one of the occipital lobes. This permitted easy access to the collicular area with displacement of the medial portion of the parietal lobe away from the falx. Within an anatomical triangle formed by the tentorium and the falx, the collicular structures were easily seen when the parietal lobe was properly displaced. When the region of the brain stem just proximal to the colliculi and in direct relationship to the posterior portion of the corpus callosum was located, the small vascular structures (usually veins) were carefully dissected away from the brain stem with an appropriately sized blunt hook. If the veins were inadvertently opened they could be controlled with temporary Gelfoam packing or judicious use of cautery. Once the proper plane was decided upon by studying the configuration of the skull and the position and alignment of the skull in the fixation unit, a surgical transection was made in the brain stem by using blunt instruments (after using a sharp scalpel for opening of the arachnoid membrane). The transection was worked completely through the brain stem in steps, first severing the dorsal medial connections and then the lateral portions on the side from which the brain stem was approached. Finally, the most ventral portions were sectioned by reaching the base of the skull with the blunt instrument. Here great care was taken SOIthat the basilar artery was not severed. Some experience was required to limit properly the dissection to the lateral boundary of the brain stem. The raw surfaces of the cut brain were packed with Gelfoam or wet Cottonoid to reduce bleeding to a minimum. Since some blood loss was inevitable during this procedure, volume replacement was accomplishment with Dextran. When necessary, normal blood pressure was maintained by administration of vasopressors. After completion of each experiment, the brain was removed and examined. The neurogenically isolated preparations were checked to
ISOLATED
BRAIN
assure severance of all cranial nerves. The cerveau isolB preparations examined to determine the plane and extent of the lesion.
309 were
Results
COMPLETELY
ISOLATED BRAIN
ON DONOR PERFUSION
Prior to dissection of the head and neck, the predominant frequency found in the motor and sensory areas of the cerebral cortex was 20-25 Hz of approximately SO-150 pv. In those isolated preparations on donor perfusion which were anesthetized with Nembutal, the common frequencies were between 10 and 16 Hz with voltages of about 100 ,uv. Occasional barbiturate spikes and spindling were observed during early phases of the isolation procedure. Sparse intervals of 3-8 Hz sometimes appeared in the sensory cortex recordings. The ECoG activity appearing in the occipital cortex was generally slower (5-15 Hz) and of higher voltages (ZOO-250 pv). Occasional periods of 3-5 Hz were also noted in this area. In preparations given Surital and Flaxedil, the predominant frequencies were generally faster (25-30 Hz) and of higher voltage (300 pv). No intervals of slow frequencies were noted, not even in the occipital leads. Electrical activity f ram the anterior reticular formation (thalamus) was unaffected by either Nembutal or Surital, since the predissection records showed little or no differences. This activity consisted of moderately fast potentials ( 1 S-20 Hz) with voltages around 30-50 pv. The posterior reticular formation (pans) exhibited typical fast activity with voltages around 20~~. This latter set of eelctrodes was included in only two preparations, since manipulation of the lower brain stem during ligation of the vertebral arteries, and ligation and transection of the spinal cord eventually caused these electrodes to short. The electrocortical and reticular (thalamic) activity reported above suf fered little or no change during dissection of the soft tissue from the head, removal of the necessary bony parts, cannulation of the carotid arteries, or ligation of the vertebral arteries. However, after the spinal cord was ligated and severed at the C-2 level, the electrical activity from all cortical areas became extremely fast with high voltages in isolated brain preparations previously anesthetized with Surital and Flaxedil. The predominant frequencies were found to be on the order of 35-40 Hz with voltages of 100-150 pv. After approximately 10 min spindle-burst activity appeared, sometimes lasting for 2-3 set and recurred periodically for the ensuing l-l.5 hours. This burst activity gradually subsided in preparations that remained in good condition over the next 2 hours. No slow potentials or slow-wave intervals were noted in these monkeys up to the termination of the experiment, which lasted as long as 4 hours post-donor perfusion (Fig. 1).
310
MASSOPUST
ET
AL.
ISOLATED
BRAIN
311
FIG. 1. Electrical activity in the completely isolated brain prepared with Surital and Flaxedil: (1) predissection activity ; (2) during dissection of the soft tissue and vascular supply from the head and neck; (3) after cannulation of the carotid arteries ; (4) after ligation of the vertebral arteries ; (5) immediately after transection of the spinal cord at C-2; (6) 1 hour after perfusion transfer to the donor monkey’s circulatory system; (7) after 4 hours on donor perfusion. See Table 1.
On the other hand, in isolated brain experiments in which Nembutal was used during the early stages of the surgery, little or no change was noted in EC& activity throughout the rest of the experiment. We emphasize that the cortical records were generally much slower before dissection of the
312
MASSOPUST
ET
AL.
ISOLATED
FIG. 2. Electrical activity in the completely (l-6) same as listed in Fig. 1 ; (7) 2 hours monkey. See Table 1 for key and calibration.
BRAIN
isolated brain after perfusion
313
prepared with Nembutal: of isolated brain by donor
soft tissue and never showed any increase in frequencies throughout the various isolation phases, even after transection of the spinal cord (Fig. 2). C7isually-evoked Cortical Responses. In those experiments (three), in which the right eye and its central vascular and neural connections were left intact, the evoked responses recorded were similar to the responses obtained from the optic cortex of an intact monkey. The “on” potential followed the stimulus onset by about 10-15 msec and had a voltage averag-
314
MASSOPUST TABLE
ET AL. 1
KEY FOR ALL FIGURES
LH LHo oc
PCG PO PoCG RH RHO TH
= Bipolar trace from precentral and postcentral electrodes in the left hemisphere. = Bipolar electrical activity from the parietal-occipital electrodes in the left cerebral hemisphere. = l%polar spontaneous electrical activity from the macular representation in the occipital cortices. = Bipolar spontaneous electrical activity from the precentral gyri (motor). = Pontine reticular formation electrical activity recorded near the anterior border of the pons. = Bipolar activity from the postcentral gyri (sensory). = Precentral and postcentral electrode cross in the right hemisphere. ; = Bipolar electrical activity from the parietal-occipital electrodes in the right cerebral hemisphere. = Bipolar thalamic activity.
Calibration:
Vertical mark = 50 gv ; horizontal
mark = 1 sec.
ing about 150 pv. The “off” potential followed the shutter closure by about 7-10 msec. During the first hour of donor perfusion, four or five afterpotentials followed the initial “on-off” responseas long as the intracerebral temperatures remained about 34 C. The afterpotentials usually disappeared at 32 C, but could be recovered if the intracerebral temperature was brought back to 34 C within a reasonableperiod of time. UNOPERATEDCONTROL
A monkey with an electrode array chronically implanted (as described for the completely isolated monkey brain), showed the following characteristic electrocortical activity and reticular formation electrical activity (thalamus) (Fig. 3). Awake State. The precentral gyrus (sensory) tracings had predominant frequencies of 20-25 Hz and voltages of not more than 20 pv. No slower frequencies were observed while the animal occupied the comfortable restraint of the Foringer monkey chair. The postcentral gyrus (motor) tracings also contained the above low voltage fast activity, but had short intervals of 5-15 Hz activity of 30 pv. The occipital lobe tracings were generally slower, 15-20 Hz activity predominating with voltages ranging between 30 and 60 pv. The thalamic electrical activity was found to be of extremely low voltage (less than 20 pv) with frequencies matching those in the precentral and postcentral gyri. When the animal was alerted from a light sleep state by a loud noise the cortical and thalamic activity became extremely fast (35-45 HZ) for
ISOLATED
BRAIN
315
FIG. 3. Represensentative tracings of various awake and sleep states in a macaque under conditions of comfortable restraint of the Foringer monkey chair: (1) resting awake state ; (2) light sleep (paradoxical sleep?) characterized by partially closed eyelids with rapid eye movements ; (3) deep sleep characterized by closed eyelids with no eye movements. See Table 1 for key and calibration.
316
MASSOPUST
ET AL.
about 4-6 set after the noise stimulus, then reverted to the usual awake frequencies. Activated Sleep. This state is characterized by a partial closure of the eyelids with rapid eye movements and probably corresponds to, what is currently termed “paradoxical sleep” (7). The tracings from the precentral and postcentral gyri remained generally fast and of low voltage. Since the standard electrode pattern was used in the unoperated control monkey, it was not possible to observe all the criteria which determine the paradoxical sleep phenomenon. However, based on eyelid closure and rapid eye movements, the ECoG corresponded well with what is usually found in paradoxical sleep. The most dramatic changeswere observed in the anterior reticular formation tracings, Here the S-10 Hz activity completely dominated. Only sparse very short intervals of the low voltage, fast activity were found (intervals of 0.2-0.6 set). The voltages of the S-10 Hz potentials measured 70-150 pv. The occipital lobe tracings also showed 5-10 Hz activity, but only about 50% of the time; the remainder of the tracings continued as relatively fast moderate voltage activity. Sloz~ Wazfe State. This state is characterized by 5-10 Hz potentials (30-60 pv) with momentary periods of faster activity interspersed. When complete relaxation was attained, high-voltage (300 pv peak to peak) multiphasic waves appeared in precentral and postcentral gyri. In the occipital cortex these multiphasic waves were of moderate voltage (lOO150 pv). This state was characterized by complete closure of the eyelids without eye movements. The thalamic tracing was somewhat different from the cortical traces in that these high-voltage waves were monophasic, always positive in deflection, and seemedto trigger the multiphasic waves of the cortex. NEUROGENICALLY
ISOLATED
BRAIN
The basic spontaneous electrical activity from the sensory and motor cortices and from the reticular formation in the neurogenically isolated brain was similar to that observed in those completely isolated brains prepared under Surital and Flaxedil (compare Figs. 1 and 4). The presence of predominant intervals of 20-25 Hz activity of moderately high voltages (100-150 pv) is comparable to an intact monkey’s electrocorticograms. After the removal of cranial nerves V and VII through XII. phasic intervals of 25-35 Hz activity appeared. Generally, however, aft& removal of all the cranial nerves, the intervals of 25-35 Hz activity became longer and more synchronous throughout all leads except the anterior reticular formation. These fast moderate-voltage potentials reached a steady state before the spinal cord was severed from the brain stem. High-voltage slow-wave activity was seldom observed at this point. After transection
ISOLATED
RHAIN
317
of the spinal cord at the C-2 level, intervals of 40 to 45-Hz activity and occasional high-frequency spindle-burst activity was also noted for as long as 20 min after severance of the spinal cord. These spindle bursts gradually disappeared and none was observed l-1.5 hours after spinal cord transection. Loss of the intervals of 25-35 Hz and the 40 to 45-Hz activity accompanied a drop in the animal’s blood pressure or heart rate or both. If the blood pressure was allowed to fall below 100 mm Hg (systolic) the 25 to 35Hz activity disappeared. When blood pressure was increased by the use of vasopressors it was accompanied by the return of this highfrequency electrocortical activity (Fig. 4 ) . Manipulation of the medullary region or the mesencephalic-diencephalic regions of the brain stem tended to cause an instability in the cardiovascular system, as shown by changes in EKG and blood pressure recordings. Even though blood loss was kept to a minimum, it was difficult to maintain the monkey at its preisolation pressure levels so that the use of vasopressor agents was often necessary. Crrvrnlr
Isoli
In the CUVEULC isolt preparation in good homeostatic balance. the spontaneous electrical activity was generally 20-25 Hz with some short intervals of 25-33 Hz, This activity was characterized by moderate voltages (100 pv). Immediately after section of the brain stem the activity in the motor and sensory cortices and the reticular formation changed to 3-7 Hz with voltages measuring about SO pv in the sensory cortex and reticular formation, while voltages of 75-100 pv were fomid in the motor cortex. This slow electrical activity lasted 2-10 min, then changed rapidly to 16-25 Hz of low voltage (50 pv) and became clesynchronous. At about 30 min after transection, phasic slowing of the electrocorticogram and reticular formation cativity occurred. This was concurrent with difficulties in maintaining the systolic blood pressure above 120 mm Hg. Finally, in the failing preparation the activity slowed to 2-3 Hz with voltages measuring aroutld 1.~0~~ (Fig. 5). No evoked potentials were elicited in either the neurogenicaily isolated or the CCYVCUUisolh preparations because of the interruption of the visual pathway by removal of the contents of the orbits, or removal of the occipital lobe. Discussion
Reports by Kennard and Nims (9), Robert de Ramirez de Arellano ( 16)) and Jurko and Andy (8) show that the highest frequency of electrocortical activity observed in their 1 to 2-year-old awake and alert macaques was on the order of 12-20 Hz. Ours, of approximately the same age, were found to have a 20 to 25-Hz predominant frequency before surgery and
ISOLATED
FIG.
Surital nerves VIII; nerve tion of
319
BRAIN
4. Electrical activity of the neurogenically isolated brain prepared with and Flaxedil: (I) presurgical; (2) exenteration of orbits destroying cranial II, III, IV, and VI; (3) after section of cranial nerves XII, XI, X, IX, and (4) after section of cranial nerves VII and V; (5) after severance of cranial I ; (6) after transection of the spinal cord at C-Z ; (7) 2.5 hours after transecthe spinal cord at C-Z. See Table 1 for key and calibration.
under Surital and Flaxedil. Except under light Nembutal anesthesia, 10 to 12-Hz activity was never seen. The unoperated control monkey with epidural recording electrodes in the awake and alert state further confirmed
the above-mentioned higher frequencies. However, the frequencies reported above
were
taken
from
recordings
with
scalp
electrodes,
while
the fre-
320
MASSOPUST
ET
AL.
ISOLATED
I
.,I
321
BRAIN
‘.
I
i
.I.
FIG. 5. Electrical activity during various preparation phases of a cerveatc isolh in which the brain stem was transected between the mesencephalon and diencephalon: (1) presurgical; (2) after partial section of the brain stem on the right side approximately to the midline; (3) immediately after total transection of the brain stem between the mesencephalon and diencephalon ; (4) 10 min after brain stem transection ; (5) 30 min after transection; (6) 1 hour after transection; (i) 2 hours after transection. See Table 1 for key and calibration.
quencies observed in this study were taken from recordings with dural electrodes. Immediately upon completion of the surgery in the completely isolated
322
MASSOPUST
ET
AL.
preparation and in the neurogenically isolated brain, frequencies of 25 to 35-Hz activity appeared in both the sensory and motor cortices (Figs. 1 and 4). Several possible factors can be cited which may explain this unusual phenomena. It is possible that the removal of the afferent inputs from the cranial nerves and spinal cord eliminates inhibitory mechanisms which are localized in the mesencephalic reticular formation or other centers within the brain stem (7, 10, 12, 13, 20). Thus, the two isolated brain preparations are similar in the sense that the complex electrical activity generated in the reticular formation and cortex is no longer influenced by the afferent inputs. Changes in frequencies and amplitudes occur after changes in the metabolic conditions, or the internal brain temperature, or blood pressure, or all three. Spindle-burst activity appeared in our preparations approximately 10 min after isolation. Andersson and Wolpow (1) reported that transection of the spinal cord, including the specific short latency cortical projection pathways at the C-3 or C-4 level, increased the spindle-burst activity in localized areas of the somatosensory cortex in cats. This spindle activity also appeared after section of the dorsal funiculi of the spinal cord. It is possible that total severance of the spinal cord in our monkeys also removes these short-latency projection pathways and thus could account for the appearance of similar spindle-burst activity. However, after 3 hours of postoperative time, these spindle bursts subsided and at 4 hours could hardly be detected. In those isolated brain preparations which were given Nembutal, no changes in cortical or reticular formation activity were noted. Since electrocortical activity was slow and of high voltage after anesthesia and usually remained at this level throughout the procedure, the true release phenomena (appearance of the 25 to 35-Hz activity and of the spindle-burst activity) was completely masked by the long term anesthetic effect (15) (Fig. 2). Because of this, Nembutal and other moderately long-acting barbiturates were discarded and Surital and Flaxedil with controlled ventilation were used to obtain optimum electrophysiological activity. The cerveau is016 preparation (severance of brain stem between the mesencephalon and diencephalon) showed some of the classical signs of electrocortical activity. However, after an initial appearance of slow, moderate-voltage activity (for 2 min after brain-stem section), phasic slow and fast intervals of electrocortical activity appeared. Short intervals (l-2 set) of fast, low-voltage activity were observed (Fig. 5). These intervals became increasingly longer and eventually became the predominant pattern as the cerveau isolk preparation was carried out in time. Finally, when the brain began to fail, high-voltage slow activity became more prominent
ISOLATED
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323
until the low-voltage fast activity was no longer observed (Fig. 5). It is possible that the slow, high-voltage, spontaneous electrical activity reported in cerveau isol6 preparations in other species (7, 10, 12, 13, 20) may have been due to failure in the ability to prevent a detrimental change in cardiovascular homeostasis (i.e., blood loss and maintenance of proper levels of blood pressure, or perfusion of the brain, or both). Another factor which could account for the activity reported in these cervealc isolt preparations could be failure to maintain intracerebral temperature, which reduces the metabolic activity and thus energy output. The ultimate failure of any of our three preparations paralleled the gradual development of brain edema which is due to increases in the circulatory resistance of the brain, and which finally causes general ischemia. Ischemic cortical damage is first indicated by the dropout of the faster frequencies in the ECoG, then by the prominent appearance of slow-wave activity (3-5 Hz), and finally by the appearance of damage spindles. In well-developed cerebral edema the ECoG is completely isoelectric. Reticular modulation of cortical activity is manifold. Both activating and inhibiting influences have been described (3, 7, 10, 12, 13, 20). Afferent inputs to the reticular system are likewise manifold and, undoubtedly, also exert a modulating influence on reticular activity. When all afferent inputs to the brain are eliminated, something like a release phenomenon or “disinhibition” occurs, as manifested by exceptional high-frequency cortical activity. This was observed in both the isolated and deafferent brain preparations. The lack of accord between our results and the vast reticular formation literature, based primarily on the cat, was previously explained by Routtenberg (17). More recently he has suggested (18) the existence of a dual arousal system, one being the classical reticular activating system of Moruzzi and Magoun (12), the other a limbic-midbrain system described anatomically by Nauta (14). Routtenberg has attributed the recovery of low-voltage, fast activity following precollicular brain-stem transection- to “denervation supersensitivity” and has suggested that the medial forebrain bundle running through the hypothalamus and telencephalon mediates the low-voltage fast activity because Villablanca (20) showed desynchronization during stimulation of the olfactory nerve. In our preparations (except the cerveau is&), the central connections of the olfactory nerve were severed or the olfactory receptor areas destroyed and we still obtained the moderate voltage fast activity in the cortex of our monkeys. This indicates that the olfactory nerve input per se is not necessary to produce the low-voltage fast activity. It is possible that some other more central
324
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ET
AL.
limbic structure (i.e., septum, hippocampus, amygdala) may generate this fast cortical electrical activity after elimination of the classical reticular activating system. In addition to a second arousal system, this also suggests a possible inhibiting mechanism in the posterior reticular formation. In this case the posterior reticular formation was severed from the diencephalon and telencephalon, but afferent inputs via the first three cranial nerves remained. Under these conditions fast electrocortical activity was also obtained. While differing somewhat in character from the fast cortical activity noted in the completely isolated and neurogenically isolated preparations, this activity was still unusual and suggestive of a similar “release” phenomenon. We conclude that there exists a delicate balance between the various portions of the reticular system, and that disruption of this balance may produce several possible effects, one of them being the unusual sustained high-frequency arousal observed in the preparations reported herein. Thus, it appears that the cerebral cortex, at least in primates, can recover its aroused electrical state after external and internal influences (i.e., reticular activating system, cranial nerve inputs) have been removed. It is true that interruption of these influences causes temporary slowing and deactivation of the cortex; however, it seems that when homeostasis has been reestablished the electrocorticogram returns to its activated condition (20). Removal of reticular inhibitory mechanisms may cause this activated or aroused state to be maintained in the cerebral cortex indefinitely. It is also possible that diencephalic structures (i.e., thalamus) control the aroused state, particularly when the mesencephalic-medullary reticular influences have been removed. Under these conditions, inhibitory influences appear to be permanently eliminated, while activation is sustained either by diencephalic mechanisms or directly by the telencephalon. References 1. ANDERSSEN, S. A., and samatosensory cortex 2. BREMER, F. 1935. Cerveau Biol. 118 : 1235-1241.
E. R. WOLPOW. 1964. Localized slow wave activity of the cat. Actn Physiol. Stand. 61: 130-140. (isole)
et physiologie
du sommeil.
Compt.
Rend.
in the Sot.
3. BREMER, F., and C. TERZUOLO. 1954. Contribution a l’ktude des mkanismes physiologiques du maintien de l’activitk vigile du cerveau. Interaction de la formation r&iculCe et de l’korce dr&brale dans le processus du r&eil. Arc/z. Intern. Physiol. 62 : 157-178. 4. CHUTE, A. L., and D. H. SMYTH. 1939. Metabolism of isolated perfused cat’s brain. Quart. J. Exptl. Physiol. 29 : 379-394 5. GEIGER, A., and J. MAGNES. 1947. The isolation of the cerebral circulation and the perfusion of the brain in the living cat. Am. J. Physiol. 149: 517-537.
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6. HEYMANS, C., J. J. BAUKAERT, F. JOURDAN, S. J. G. NOWAK, and S. FARBER. 1937. Survival and revival of nerve centers following acute anemia. .4.M.*4. rlrch. Neurol. Psychiat. 38 : 304-307. 7. JOUVET, M. 1967. Neurophysiology of the states of sleep. Physiol. Rezl. 47: 117-177. 8. JURKO, M. F., and 0. J. ANDY. 1967. Comparative EEG frequencies in rhesus, stumptail, and cynomolgus monkeys. Elcctvocnccphaloy. Clin. Naurophgsiol. 23 : 270-272. 9. KENNARD, M. A., and L. F. NIMS. 1942. Changes in normal electroencephalogram of Macaca mulatta with growth. 1. Nczcrophgsiol. 5 : 325-333. 10. MAGOUN, H. W. 1952. An ascending reticular activating system in the brain stem. A.M.A. Arch. Nrurol. Psychitrt. 67 : 145-154. 11. MEDER, R., L. C. MASSOP~ST, JH., R. J. WHITE, J. VERDURA, and M. S. ALBIN. 1963. Isolated brain perfusion : electro-mechanical requirements. 16th ilnn. Conf. Eny. Med. Riol. 5 : 28-29. 12. MORUZZI, G., and H. W. MACOCN. 1949. Brain stem reticular formation and activation of the EEG. Elcctrocwephaloy. Clin. Nenropltgsiol. 1: 455-473. 13. MORUZZI, G. 1964. Reticular influences 011 the EEG. Electrocncrphalog. Clip Neurophysiol. 16 : 2-17. 14. NAUTA, W. J. H. 1958. Hypocampal projections and related neural pathways to the midbrain in the cat. Brain 81: 319-340. 15. PRINCE, D. A., and S. SIIANZER. 1966. Effects of anesthetics upon the EEG response to reticular stimulation. Elrctroerzcepkalog. Clin. Ncurophysiol. 21: 578-588. 16. ROBERT DE RAMIREZ DE ARELLANO, M. I. 1961. Maturational changes in the electroencephalogram of normal monkeys. Exptl. Ncuvol 3 : 209-224. 17. ROUTTENBERG, A. 1966. Neural mechanisms of sleep: changing view of reticular formation function. Psychol. Rerj. 73 : 481-499. 18. ROUTTENBERG, A. 1968. The two-arousal hypothesis: reticular formation and limbic system. Psycho/. Rezj. 75 : 51-80. 19. SNIDER, R. S., and J. C. LEE. 1961. “A Stereotaxic Atlas of the Monkey Brain.” Univ. of Chicago Press, Chicago, Illinois. 20. VILLABLANCA, J. 1965. The electrocorticogram in the chronic cewears isol6 cat Electrocncephalog. Clin. Nrnroplk)qsiol. 19 : 576-586. 21. WHITE, R. J., M. S. ALBIN, and J. VERDURA. 1963. Isolation of the monkey brain: in vitro preparation and maintenance. Sciotce 141: 106&1061. 22. WHITE, R. J., M. S. ALBIN, L. C. MASSOPUST, JR., and R. MEDER. 1964. Design of a simplified perfusion system for differential cooling of brain. 17th Am Cowf. Eng. Med. Biol. 6: 85. 23. WHITE, R. J., M. S. AI.BIN, and J. VERDURA. 1965. Maintenance of the isolated brain by mechanical perfusion. Tram. Am. Sot. drtificial Intrvnal Organs 11: 201-204. 24. WHITE, R. J. 1965. Isolating the brain. L)iscoevery 26 : 34-37. 25. WHITE, R. J., M. S. ALBIN, J. VERDURA, and G. E. LOCKE. 1967. The isolated brain : Operative preparation and design of support systems. J. Nc~r~osu~g. 27 : 216-225.