JOURNAL
OF SURGICAL
Standardized
RESEARCH
26,
87-93 (1979)
Observation
Procedures
in Brain Injury Research
B. VALLFORS,' H.-A. HANSSON,AND S. LARSSON Department
of Neurosurgery,
Sahlgrenska sjukhuset, Giiteborg, and the Institute University of Giiteborg, Giiteborg, Sweden
of Neurobiology,
Submitted for publication July 18, 1977 An experimental model has been designed for testing the injurious effects of surgical methods and of physical and chemical agents in vivo on the brain surface of the dog and cat. It permits experimental exposure of the brain for several hours without causing damage to the surface cells of the arachnoid or to the blood-brain barrier. The model is easily set up and may be used for both acute and chronic experiments.
General Preparation
INTRODUCTION
Animals. These procedures have been Progress in neurosurgery has increased the demand for an in viva model for testing developed using dogs and cats as experinew surgical methods and instruments mental models. Anesthesia. The animals were anesthebefore their clinical use, as well as for monitoring the injurious effects of different tized intravenously or intraperitoneally with physical and chemical agents. However, no Nembutal, 30 to 40 mg/kg of body weight. suitable experimental system seems to be They were intubated and ventilated artiavailable. As recently as 1975, Pudenz et al. ficially by respirator with 0, and N,O in the [17], in connection with their experimental proportion 1:2. Measures were taken to constudies of the damage induced by electrical trol paOz and pC0,. A muscle relaxant was stimulation of the brain, described the given2 and blood loss compensated by problems associated with prolonged expo- Ringerdex solution. Body temperature was sure of the brain surface of the cat. In spite kept normal by heating pads. Arterial blood of spraying the surface with Ringer’s solu- pressure was recorded. tion, they had difficulty preventing the Head holder for the animal. The heads of occurrence of edema and diffuse breakdown the dogs were attached to a fixture on a of the blood-brain barrier (BBB). The aim lockable ball-and-socket joint3 (Fig. 1). A of this paper is to present a system which modified Horsley-Clark head holder was allows access to the intact brain surface of used for the cats. experimental animals without injurious efProtective pool. The protective pool is of fects due to exposure. special importance for avoiding injury to the brain. The pool (Figs. l-3) consists of MATERIALS AND METHODS polyvinyl chloride (PVC) film, attached on Details of the routine procedures will only its underside to the skin by staples4 and on be described very briefly. The techniques its upper side to a metal ring (300 mm in for control of the possible occurrence of diameter, 3 mm thick) (Fig. 3). The brain is damage to the brain have been described immersed in Elliott’s solution type B at 34 to in detail elsewhere and will not be reiterated 35°C [7] to avoid damage caused by expohere. ’ To whom correspondence should be sent: Neurokirurgiska kliniken, Sahlgrenska sjukhuset, S-413 45 Goteborg, Sweden. 87
* Pavulon. 3 Miniclamp 1200, Spencer Franklin Ltd., London, England. 4 Bostitch B8 Plier, Bostitch, East Greenwich, R. I. 0022-4804/79/010087-07$01.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
88
JOURNAL OF SURGICAL RESEARCH: VOL. 26, NO. 1, JANUARY
1979
FIG. 1. Head holder, pool, and coordinate manipulator. The brain surface could easily be set in the desired position in relation to the manipulator, thus enabling tests on both hemispheres.
sure to air or blood [6, 9, 11, 16, 211. Observation and testing of instrumental equipment on the brain surface explored in the pool was facilitated by a modified Horsley-Clark coordinate manipulator, which could be moved into any desired position and combined with a sleigh [26] (Figs. 1 and 3).
tion field with Steri-drape,6 a long sagittal skin incision is made. The attachments of the temporal muscles are cut parasagittally by diathermy while being cooled with Elliott’s solution, The muscles are then subperiostally detached over the temporal areas down to the zygomatic arch and sutured to the skin. A hole (diameter: 80 mm for the dog, and 45 mm for the cat) is cut in the PVC film. The edge of the hole is stapled Surgical Preparation to the margin of the skin incision (Fig. 2), The entire procedure of exposing the thus keeping the temporal muscles away brain surface is of importance for avoiding from the brain surface. The junction beinjurious effects and will therefore be de- tween skin and film is sprayed with Nobescribed in some detail. The head skin is cutan from the periphery, and a second line shaved and washed with a commercial of staples is applied outside the first to depilator and with 70% ethanol. It is dried produce a tight junction. The film is then with an electric hair dryer and Nobecutan stapled to the ring (Fig. 3). The pool is spray5 is applied. After covering the opera- checked for water-tightness and lowered. s AB Bofors Nobel-Pharma.
6 3M.
VALLFORS,
HANSSON,
AND LARSSON:PROCEDURES
The bone is trephined with a dental drill cooled with rinsing solution [24] and then carefully removed by rongeur forceps from the frontal sinuses to the posterior rim of the temporal fossa. Bleeding from the bone is controlled by bone wax and that from the dura, by bipolar electrocoagulation. Under magnification the dura is opened with small injection needles and microscissors. The isolated brain surface is rinsed with Elliott’s solution to avoid exposure to air (Fig. 2). In chronic experiments the dura is closed under magnification with Ethiflex 6:0 sutures. The sectioned temporal muscles are sutured toward their origins and the skin is closed after excising those parts which have been traumatized by staples. Znjury Evaluation For demonstration of BBB injury, a freshly made and filtered Evans blue solution (2 ml of a 2% (w/v) solution per kilogram of body weight) was administered intravenously before preparation. For postmortem studies the animal was rinsed transcardially [ 151with Ringerdex solution (with added Xylocaine and heparin) followed by fixation with 4% formaldehyde at pH 7.2 or 3% glutaraldehyde for electron microscopy. The perfusion pressure was controlled. The fluid in the pool could be replaced by fixative solution. Identification of the isolated brain area was achieved by markings on a full scale Polaroid photograph. RELIABILITY
OF THE PROCEDURES
These procedures were tested in 25 animals. Exposure of the brain surface for up to 3 hr did not cause any marked damage to the surface cells of the arachnoid or to the brain tissue, as demonstrated by the following investigative methods: (1) fluorescence microscopy [12]; (2) isotope studies [25]; (3) scanning electron microscopy (SEM); and (4) histopathology. Fluorescence microscopy showed minimal extravasation of Evans blue albumin (EBA) in the lepto-
IN BRAIN
INJURY
RESEARCH
89
meninges (Fig. 4). In control animals there was hardly visible fluorescence or none at all. Isotope studies demonstrated minimal activity in the exposed tissue [25]. SEM showed that the flat mesothelial cells of the arachnoid were morphologically intact (Fig. 5). Histopathology showed a normal appearance . COMMENTS
The procedures described make it possible to isolate a comparatively large cortical area easily and safely. The liquid pool is rapidly set up and, owing to its conical shape, it does not restrict surgical manipulations. The intracranial pressure can be varied by selecting an appropriate level of the liquid filling the pool. By creating physiologically optimal conditions, it is possible to avoid any marked damage to the brain due to the procedures themselves. The cortex of the brain is an excellent surface on which to measure surgical trauma, owing to the architecture of its blood supply [lo]. The numerous surface leptomeningeal arterioles and venules, as well as capillaries, penetrate the cortex closely interrelated to each other perpendicularly and form a dense, homogeneous vascular network below the surface, from which the connections to the white matter are relatively few. Thus, much of the cortical vascular system is supplied from the leptomeninx and trauma to the latter will produce cortical venous congestion with BBB damage, bleeding to the gray matter, insufficient blood supply, and combinations of these changes. The procedures described were designed to study the injurious effects of neurosurgical procedures applied to the brain surface, such as electrocoagulation, irrigation, suction, and the influence of physical and chemical agents. Identification of tested areas was facilitated by photographic methods. To evaluate damage produced by various surgical procedures, the following methods are of special interest: (1) BBB injury
VALLFORS,
HANSSON,
AND LARSSON:
PROCEDURES
FIG. 4. Fluorescence micrograph of exposed brain cortex of the dog. EBA tracer. Section perpendicular to the brain surface. Slight fluorescence of leptomeninx. X 150.
studies with Evans blue albumin [2, 121 and 1311-labeledalbumin [5, 251 to indicate injury to the cortex and the white matter; and (2) SEM, as an extremely sensitive indicator of damage to the surface cells and collagenous network of the arachnoid [23, 251.The tracer protein complexes are sensitive indicators of BBB damage and edema. It should be noted that bound Evans blue need not be used since it binds strongly to plasma proteins in vivo, particularly albumin [l, 19, 221. When extravasation occurs, the tracer is detected macroscopitally and more sensitively by fluorescence microscopy [22]. Uptake of tracer in neurons has been interpreted as an indication of neuronal injury [2] and even irreversible damage [ 181. 1311-labeled albumin is a very stable complex in blood [5] and is suitable for quantitative studies of BBB dysfunction.
IN BRAIN
INJURY
RESEARCH
91
FIG. 5. Scanning electron micrograph of exposed arachnoid surface of the dog. Mesothelial cells morphologically intact. x4800.
When evaluating BBB damage, it is important to control the biological variables. p,O, and pC0, should be adjusted to normal values [8, 141. Slight hypercapnia may aggravate a BBB dysfunction [13], whereas more severe hypercapnia per se may alter the properties of the BBB [3, 41. Since blood pressure influences the degree of extravasation of tracers [ 121,it should be maintained at a normal level of 110 to 130 mm Hg. Since the temperature of the liquid bathing the brain most likely alters the properties of the BBB, it is maintained at a normal level of 34 to 35°C. The effect on the brain of monkeys of cutting the temporal muscles by diathermy has been studied by Hudgins and Garcia [9]. Extensive BBB damage, visible swelling of the brain, and neurological deficits were noted. These changes, as well as those reported by West and Edvinson [24], were probably due to excessive temperatures.
FIG. 2. The exposed left hemisphere of the dog made available for testing by rotating the head to the right. Note the irregular anatomy of the gyri and vessels, making it easy to find an indicated area. Staples are attached in two lines to obtain a liquid-tight seal between the PVC film and the skin. FIG. 3. Setup for testing suction systems. Dog nose fastened by latex tubing in an atraumatic manner. The orifice of the pool can be lowered during surgical manipulations.
92
JOURNAL OF SURGICAL RESEARCH: VOL. 26, NO. 1, JANUARY
However, no such damage was seen with the procedures here described probably due to continuous cooling, the use of the lowest possible power setting, and the fact that the thinnest part of the muscles was cut. Bipolar electrocoagulation is preferable for hemostasis. However, bipolar coagulation may sometimes produce unexpected damage to the brain owing to current leak. This has been described [20], but requires further investigation. Silk sutures proved unsuitable for closure of the dura, since they produced proliferative changes in the dura and subdural adherences in 2 to 4 weeks. This has been described previously by Echlin [6] but was accidently repeated. Ethiflex sutures did not produce such changes during the same period of observation. In conclusion, the procedures described permit exposure of the brain surface of the dog and cat without producing any marked injury, thus satisfying the requirement for a test system for measuring injurious effects of surgical manipulations and of different physical and chemical agents. The use of SEM, the study of BBB damage, and histopathology provide an excellent basis for assessment of the degree of injury produced. ACKNOWLEDGMENTS The study was supported by grants from the Swedish Medical Research Council (12X-2543), SPRI (P-6007), Goteborgs Llikaresallskap, Colliander’s Foundation for Medical Research, and the University of Goteborg, Sweden.
REFERENCES 1. Allen, T. H., and Orahovats, P. D. Spectrophotometric measurement of the dye T-1824 by extraction with cellophane from both serum and urine of normal dogs. Amer. J. Physiol. 154: 27, 1948. 2. Brightman, M. W., Klatzo, I., Olsson, Y., and Reese, T. S. The blood-brain barrier to proteins under normal and pathological conditions. J. Neural. Sci. 10: 215, 1970. 3. Clemedson, C. J., Hartelius, H., and Holmberg, G. The influence of carbon dioxide inhalation on the cerebral vascular permeability to trypan blue
1979
(“the blood-brain barrier”). Acta Pathol. Micro42: 137, 1958. 4. Cutler, R. W. P., and Barlow, C. F. The effect of hypercapnia on brain permeability to protein. Arch. Neurol. 14: 54, 1966. 5. Cutler, R. W., Walters, G. V., and Barlow, C. F. Fz5-Labeled protein in experimental brain edema. Arch. Neurol. 11: 225, 1964. 6. Echlin, F. A. Cerebral Ischemia and Its Relation to Epilepsy. Thesis, Faculty of Graduated Studies and Research, McGill University, 1939. 7. Elliott, K. A. C., and Jaspar, H. Physiological salt solutions for brain surgery. J. Neurosurg. 6: 140, 1949. 8. Herbert, D. A., and Mitchell, R. A. Blood gas tensions and acid-base balance in awake cats. J. Appl. Physiol. 30: 434, 1971. 9. Hudgin, M. D., and Garcia, J. H. The effect of electrocautery, atmospheric exposure, and surgical retraction on the permeability of the blood-brain barrier. Stroke 1: 376, 1970. 10. Iida, T. Elektronenmikroskopische Untersuchungen am oberfllchlichen Anteil des Gehims bei Hund und Katze. Arch. Histol. Japan 27: 267, 1966. 11. Jackson, I. J. Aseptic hemogen meningit. AMA biol. Stand.
Arch. Neural.
Psychiat.
62: 572, 1949. Barrier Dysfunction Hypertension. Thesis, University
12. Johansson, B. Blood-Brain in Acute Arterial
of Goteborg, Sweden, 1974. 13. Johansson, B. Some factors influencing the damaging effect of acute arterial hypertension on cerebral vessels in rats. Clin. Sci. Mol. Med. 51: 41, 1976. 14. Jones, E. W. Ventilation and resuscitation in the dog. Fed. Proc. 28: 1471, 1969. 15. Karlsson, U., and Schultz, R. Fixation of the central nervous system for electron microscopy by aldehyde perfusion. J. Ultrastruct. Res. 12: 160, 1965. 16. Prados, M., Strowger, G., and Feindel, W. H. Studies on cerebral edema. I. Reaction of the brain to air exposure. Pathologic changes. Arch. Neural. Psychiat. 54: 163, 1945. 17. Pudenz, R. H., Bullara, L. A., Dru, D., and Talalla, A. Electrical stimulation of the brain. II. Effects on the blood-brain barrier. Surg. Neural. 4: 265, 1975. 18. Pudenz, R. H., Bullara, L. A., Jacques, S., and Hambrecht, F. T. Electrical stimulation of the brain. III. The neural damage model. Surg. Neurol. 4: 389, 1975. 19. Rawson, R. A. The binding of T-1824 and structurally related diazo dyes by the plasma proteins. Amer. J. Physiol. 138: 708, 1943. 20. Robinson, J. L., and Davies, N. J. Bipolar diathermi. Canad. J. Surg. 17: 287, 1974. 21. Samorajski, T., and Moody, R. A. Changes in the blood-brain barrier after exposure of the brain. Arch. Neural.
Psychiat.
78: 369, 1957.
VALLFORS,
HANSSON, AND LARSSON: PROCEDURES IN BRAIN INJURY RESEARCH
22. Steinwall, O., and Klatzo, I. Selective vulnerability of the blood-brain barrier in chemically induced lesions. J. Neuropathol. Exp. Neural. 25: 542, 1966. 23. Suzuki, S., Ishii, M., Ottomo, M., and Iwabuchi, T. Changes in the subarachnoidal space after experimental subarachnoid hemorrhage in the dog: Scanning electron microscopic observation. Acfa Neurochir.
39: 1, 1977.
24. West, K. A., and Edvinson, L. Experimental
93
cerebral heat lesion produced by trephine craniotomy in rabbits. Acta Pathol. Microbial. &and. Sect. A 80: 134, 1972.
25. Vallfors, B. Neurosurgical
Suction
Systems. An
Thesis, University of Goteborg, Sweden, 1976. 26. Vallfors, B., Hansson, H.-A., Johansson, G., and Larsson, S. Studies on optimal conditions in surgical suction systems. Acta Chir. Stand. Suppl.. 474, 1976. Experimental
Study.
OF SURGICAL
Standardized
RESEARCH
26,
87-93 (1979)
Observation
Procedures
in Brain Injury Research
B. VALLFORS,' H.-A. HANSSON,AND S. LARSSON Department
of Neurosurgery,
Sahlgrenska sjukhuset, Giiteborg, and the Institute University of Giiteborg, Giiteborg, Sweden
of Neurobiology,
Submitted for publication July 18, 1977 An experimental model has been designed for testing the injurious effects of surgical methods and of physical and chemical agents in vivo on the brain surface of the dog and cat. It permits experimental exposure of the brain for several hours without causing damage to the surface cells of the arachnoid or to the blood-brain barrier. The model is easily set up and may be used for both acute and chronic experiments.
General Preparation
INTRODUCTION
Animals. These procedures have been Progress in neurosurgery has increased the demand for an in viva model for testing developed using dogs and cats as experinew surgical methods and instruments mental models. Anesthesia. The animals were anesthebefore their clinical use, as well as for monitoring the injurious effects of different tized intravenously or intraperitoneally with physical and chemical agents. However, no Nembutal, 30 to 40 mg/kg of body weight. suitable experimental system seems to be They were intubated and ventilated artiavailable. As recently as 1975, Pudenz et al. ficially by respirator with 0, and N,O in the [17], in connection with their experimental proportion 1:2. Measures were taken to constudies of the damage induced by electrical trol paOz and pC0,. A muscle relaxant was stimulation of the brain, described the given2 and blood loss compensated by problems associated with prolonged expo- Ringerdex solution. Body temperature was sure of the brain surface of the cat. In spite kept normal by heating pads. Arterial blood of spraying the surface with Ringer’s solu- pressure was recorded. tion, they had difficulty preventing the Head holder for the animal. The heads of occurrence of edema and diffuse breakdown the dogs were attached to a fixture on a of the blood-brain barrier (BBB). The aim lockable ball-and-socket joint3 (Fig. 1). A of this paper is to present a system which modified Horsley-Clark head holder was allows access to the intact brain surface of used for the cats. experimental animals without injurious efProtective pool. The protective pool is of fects due to exposure. special importance for avoiding injury to the brain. The pool (Figs. l-3) consists of MATERIALS AND METHODS polyvinyl chloride (PVC) film, attached on Details of the routine procedures will only its underside to the skin by staples4 and on be described very briefly. The techniques its upper side to a metal ring (300 mm in for control of the possible occurrence of diameter, 3 mm thick) (Fig. 3). The brain is damage to the brain have been described immersed in Elliott’s solution type B at 34 to in detail elsewhere and will not be reiterated 35°C [7] to avoid damage caused by expohere. ’ To whom correspondence should be sent: Neurokirurgiska kliniken, Sahlgrenska sjukhuset, S-413 45 Goteborg, Sweden. 87
* Pavulon. 3 Miniclamp 1200, Spencer Franklin Ltd., London, England. 4 Bostitch B8 Plier, Bostitch, East Greenwich, R. I. 0022-4804/79/010087-07$01.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
88
JOURNAL OF SURGICAL RESEARCH: VOL. 26, NO. 1, JANUARY
1979
FIG. 1. Head holder, pool, and coordinate manipulator. The brain surface could easily be set in the desired position in relation to the manipulator, thus enabling tests on both hemispheres.
sure to air or blood [6, 9, 11, 16, 211. Observation and testing of instrumental equipment on the brain surface explored in the pool was facilitated by a modified Horsley-Clark coordinate manipulator, which could be moved into any desired position and combined with a sleigh [26] (Figs. 1 and 3).
tion field with Steri-drape,6 a long sagittal skin incision is made. The attachments of the temporal muscles are cut parasagittally by diathermy while being cooled with Elliott’s solution, The muscles are then subperiostally detached over the temporal areas down to the zygomatic arch and sutured to the skin. A hole (diameter: 80 mm for the dog, and 45 mm for the cat) is cut in the PVC film. The edge of the hole is stapled Surgical Preparation to the margin of the skin incision (Fig. 2), The entire procedure of exposing the thus keeping the temporal muscles away brain surface is of importance for avoiding from the brain surface. The junction beinjurious effects and will therefore be de- tween skin and film is sprayed with Nobescribed in some detail. The head skin is cutan from the periphery, and a second line shaved and washed with a commercial of staples is applied outside the first to depilator and with 70% ethanol. It is dried produce a tight junction. The film is then with an electric hair dryer and Nobecutan stapled to the ring (Fig. 3). The pool is spray5 is applied. After covering the opera- checked for water-tightness and lowered. s AB Bofors Nobel-Pharma.
6 3M.
VALLFORS,
HANSSON,
AND LARSSON:PROCEDURES
The bone is trephined with a dental drill cooled with rinsing solution [24] and then carefully removed by rongeur forceps from the frontal sinuses to the posterior rim of the temporal fossa. Bleeding from the bone is controlled by bone wax and that from the dura, by bipolar electrocoagulation. Under magnification the dura is opened with small injection needles and microscissors. The isolated brain surface is rinsed with Elliott’s solution to avoid exposure to air (Fig. 2). In chronic experiments the dura is closed under magnification with Ethiflex 6:0 sutures. The sectioned temporal muscles are sutured toward their origins and the skin is closed after excising those parts which have been traumatized by staples. Znjury Evaluation For demonstration of BBB injury, a freshly made and filtered Evans blue solution (2 ml of a 2% (w/v) solution per kilogram of body weight) was administered intravenously before preparation. For postmortem studies the animal was rinsed transcardially [ 151with Ringerdex solution (with added Xylocaine and heparin) followed by fixation with 4% formaldehyde at pH 7.2 or 3% glutaraldehyde for electron microscopy. The perfusion pressure was controlled. The fluid in the pool could be replaced by fixative solution. Identification of the isolated brain area was achieved by markings on a full scale Polaroid photograph. RELIABILITY
OF THE PROCEDURES
These procedures were tested in 25 animals. Exposure of the brain surface for up to 3 hr did not cause any marked damage to the surface cells of the arachnoid or to the brain tissue, as demonstrated by the following investigative methods: (1) fluorescence microscopy [12]; (2) isotope studies [25]; (3) scanning electron microscopy (SEM); and (4) histopathology. Fluorescence microscopy showed minimal extravasation of Evans blue albumin (EBA) in the lepto-
IN BRAIN
INJURY
RESEARCH
89
meninges (Fig. 4). In control animals there was hardly visible fluorescence or none at all. Isotope studies demonstrated minimal activity in the exposed tissue [25]. SEM showed that the flat mesothelial cells of the arachnoid were morphologically intact (Fig. 5). Histopathology showed a normal appearance . COMMENTS
The procedures described make it possible to isolate a comparatively large cortical area easily and safely. The liquid pool is rapidly set up and, owing to its conical shape, it does not restrict surgical manipulations. The intracranial pressure can be varied by selecting an appropriate level of the liquid filling the pool. By creating physiologically optimal conditions, it is possible to avoid any marked damage to the brain due to the procedures themselves. The cortex of the brain is an excellent surface on which to measure surgical trauma, owing to the architecture of its blood supply [lo]. The numerous surface leptomeningeal arterioles and venules, as well as capillaries, penetrate the cortex closely interrelated to each other perpendicularly and form a dense, homogeneous vascular network below the surface, from which the connections to the white matter are relatively few. Thus, much of the cortical vascular system is supplied from the leptomeninx and trauma to the latter will produce cortical venous congestion with BBB damage, bleeding to the gray matter, insufficient blood supply, and combinations of these changes. The procedures described were designed to study the injurious effects of neurosurgical procedures applied to the brain surface, such as electrocoagulation, irrigation, suction, and the influence of physical and chemical agents. Identification of tested areas was facilitated by photographic methods. To evaluate damage produced by various surgical procedures, the following methods are of special interest: (1) BBB injury
VALLFORS,
HANSSON,
AND LARSSON:
PROCEDURES
FIG. 4. Fluorescence micrograph of exposed brain cortex of the dog. EBA tracer. Section perpendicular to the brain surface. Slight fluorescence of leptomeninx. X 150.
studies with Evans blue albumin [2, 121 and 1311-labeledalbumin [5, 251 to indicate injury to the cortex and the white matter; and (2) SEM, as an extremely sensitive indicator of damage to the surface cells and collagenous network of the arachnoid [23, 251.The tracer protein complexes are sensitive indicators of BBB damage and edema. It should be noted that bound Evans blue need not be used since it binds strongly to plasma proteins in vivo, particularly albumin [l, 19, 221. When extravasation occurs, the tracer is detected macroscopitally and more sensitively by fluorescence microscopy [22]. Uptake of tracer in neurons has been interpreted as an indication of neuronal injury [2] and even irreversible damage [ 181. 1311-labeled albumin is a very stable complex in blood [5] and is suitable for quantitative studies of BBB dysfunction.
IN BRAIN
INJURY
RESEARCH
91
FIG. 5. Scanning electron micrograph of exposed arachnoid surface of the dog. Mesothelial cells morphologically intact. x4800.
When evaluating BBB damage, it is important to control the biological variables. p,O, and pC0, should be adjusted to normal values [8, 141. Slight hypercapnia may aggravate a BBB dysfunction [13], whereas more severe hypercapnia per se may alter the properties of the BBB [3, 41. Since blood pressure influences the degree of extravasation of tracers [ 121,it should be maintained at a normal level of 110 to 130 mm Hg. Since the temperature of the liquid bathing the brain most likely alters the properties of the BBB, it is maintained at a normal level of 34 to 35°C. The effect on the brain of monkeys of cutting the temporal muscles by diathermy has been studied by Hudgins and Garcia [9]. Extensive BBB damage, visible swelling of the brain, and neurological deficits were noted. These changes, as well as those reported by West and Edvinson [24], were probably due to excessive temperatures.
FIG. 2. The exposed left hemisphere of the dog made available for testing by rotating the head to the right. Note the irregular anatomy of the gyri and vessels, making it easy to find an indicated area. Staples are attached in two lines to obtain a liquid-tight seal between the PVC film and the skin. FIG. 3. Setup for testing suction systems. Dog nose fastened by latex tubing in an atraumatic manner. The orifice of the pool can be lowered during surgical manipulations.
92
JOURNAL OF SURGICAL RESEARCH: VOL. 26, NO. 1, JANUARY
However, no such damage was seen with the procedures here described probably due to continuous cooling, the use of the lowest possible power setting, and the fact that the thinnest part of the muscles was cut. Bipolar electrocoagulation is preferable for hemostasis. However, bipolar coagulation may sometimes produce unexpected damage to the brain owing to current leak. This has been described [20], but requires further investigation. Silk sutures proved unsuitable for closure of the dura, since they produced proliferative changes in the dura and subdural adherences in 2 to 4 weeks. This has been described previously by Echlin [6] but was accidently repeated. Ethiflex sutures did not produce such changes during the same period of observation. In conclusion, the procedures described permit exposure of the brain surface of the dog and cat without producing any marked injury, thus satisfying the requirement for a test system for measuring injurious effects of surgical manipulations and of different physical and chemical agents. The use of SEM, the study of BBB damage, and histopathology provide an excellent basis for assessment of the degree of injury produced. ACKNOWLEDGMENTS The study was supported by grants from the Swedish Medical Research Council (12X-2543), SPRI (P-6007), Goteborgs Llikaresallskap, Colliander’s Foundation for Medical Research, and the University of Goteborg, Sweden.
REFERENCES 1. Allen, T. H., and Orahovats, P. D. Spectrophotometric measurement of the dye T-1824 by extraction with cellophane from both serum and urine of normal dogs. Amer. J. Physiol. 154: 27, 1948. 2. Brightman, M. W., Klatzo, I., Olsson, Y., and Reese, T. S. The blood-brain barrier to proteins under normal and pathological conditions. J. Neural. Sci. 10: 215, 1970. 3. Clemedson, C. J., Hartelius, H., and Holmberg, G. The influence of carbon dioxide inhalation on the cerebral vascular permeability to trypan blue
1979
(“the blood-brain barrier”). Acta Pathol. Micro42: 137, 1958. 4. Cutler, R. W. P., and Barlow, C. F. The effect of hypercapnia on brain permeability to protein. Arch. Neurol. 14: 54, 1966. 5. Cutler, R. W., Walters, G. V., and Barlow, C. F. Fz5-Labeled protein in experimental brain edema. Arch. Neurol. 11: 225, 1964. 6. Echlin, F. A. Cerebral Ischemia and Its Relation to Epilepsy. Thesis, Faculty of Graduated Studies and Research, McGill University, 1939. 7. Elliott, K. A. C., and Jaspar, H. Physiological salt solutions for brain surgery. J. Neurosurg. 6: 140, 1949. 8. Herbert, D. A., and Mitchell, R. A. Blood gas tensions and acid-base balance in awake cats. J. Appl. Physiol. 30: 434, 1971. 9. Hudgin, M. D., and Garcia, J. H. The effect of electrocautery, atmospheric exposure, and surgical retraction on the permeability of the blood-brain barrier. Stroke 1: 376, 1970. 10. Iida, T. Elektronenmikroskopische Untersuchungen am oberfllchlichen Anteil des Gehims bei Hund und Katze. Arch. Histol. Japan 27: 267, 1966. 11. Jackson, I. J. Aseptic hemogen meningit. AMA biol. Stand.
Arch. Neural.
Psychiat.
62: 572, 1949. Barrier Dysfunction Hypertension. Thesis, University
12. Johansson, B. Blood-Brain in Acute Arterial
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