JOURNAL OF NEUROTRAUMA Volume 9, Number 2, 1992 Mary Ann Liebert, Inc., Publishers
Fetal Cortical Cells Survive in Focal Cerebral Infarct After Permanent Occlusion of the Middle Cerebral Artery in Adult Rats MOSHE
HADANI,1
THOMAS
FREEMAN,2 AMAR MUNSIFF,2 WISE YOUNG,2 and EUGENE
FLAMM3
ABSTRACT Fetal rat cortical cells have been shown previously to survive at the periphery of cerebral infarction. The present study was designed to examine the ability of fetal cells to survive at the edge of the central core of ischemia. In three groups of 8 adult Sprague-Dawley rats, fetal cortical cells from ED 16 were stereotactically transplanted at 3 h, 24 h, and 7 days after unilateral middle cerebral artery occlusion. In 6 rats, fetal cells were transplanted by using the same coordinates, without arterial occlusion, for control. In the ischémie groups, overall graft survival was 85%, and in the control group, all grafts survived. Graft survival was determined by light microscopy. No significant difference was found in the survival of grafts transplanted at different intervals after middle cerebral artery occlusion. It is concluded that fetal cortical cells can survive in cerebral tissue undergoing severe ischémie change.
INTRODUCTION iscHEMic insult to cerebral tissue is considered an end die, and recovery. The cells at the center of the ischémie
Focal
point in regard to structural and functional
cerebral tissue is lost. However, cells at region These territory only partially damaged. injured neurons may still have the periphery conditions the and are to signals present. potential regenerate, provided appropriate In a previous study (Hadani et al., 1987), we demonstrated that embryonic cortical grafts survived when transplanted at the periphery of a focus of cerebral infarction. Grafts, however, did not survive in any animal when transplanted into the core of the infarct, which was subsequently transformed into a necrotic cyst. In the present study, we extend these studies in the same model by now transplanting fetal cells into the penumbra just at the edge of the central core of infarction, where damaged but yet potentially viable neurons
the
of the ischémie
are
'Department of Neurosurgery, Sheba Medical Center, Tel Hashomer, Israel.
2The Neurosurgical Research Laboratories, Department of Neurosurgery, New York University Medical Center, New York.
^Division of Neurosurgery Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania. 107
HADANI ET AL. are
present. Further to complement these studies,
tissue
we
also examined the
subjected to acute ischémie insult.
potential of fetal cells to survive in
MATERIALS AND METHODS Middle Cerebral Artery Occlusion (MCAo)
Sprague-Dawley rats weighing 290-300 g were anesthetized with intramuscular injections of ketamine HC1 mg/kg and xylazine (Rumpon) 6 mg/kg. The left middle cerebral artery (MCA) was occluded as previously described by Tamura et al. (1981a). In brief, the left temporal muscle was excised, and the left mandible was retracted to expose the temporal bone. A subtemporal craniectomy (2 mm x 3 mm) was made with a microdrill anterolateral to the foramen ovale and posteromedial to the optic foramen. The dura was opened with a hooked 25-gauge needle, and the main trunk of the MCA was exposed. The arachnoid membrane above the artery was opened. The MCA was coagulated by a bipolar current and divided distal to the lateral striate branch at its junction with the olfactory stria. The wound was irrigated and closed with metal clips. The animals were housed in separate cages and were fed postoperatively with high-calorie liquid food (Ensure, vanilla flavor) 300 cal/kg. Tetracycline (1.5 g/1) was added to the drinking water for 7 days. 30
Graft Preparation harvested from the anterolateral aspect of the telencephalic vesicle of outbred 16. The developing basal ganglia were not included in the graft. Tissue from 12-14 fetuses was collected in plastic dishes containing PBS/glucose and washed at room temperature. The tissue was dissociated mechanically by repeated gentle pipetting in trypsin and subsequently washed in PBS/glucose with fetal calf serum. The suspension was centrifuged at 500g for 5 min for debris removal and then diluted in PBS/glucose to 40,000 cells per microliter. Fetal cortical cells
were
Sprague-Dawley embryos at embryonic day (ED)
FIG. 1.
H&E-stained coronal section of a rat brain 24 h after MCAo. The region of ischemia is pale. The coordinates for mm, lateral 4.0 mm, vertical, 2.0 mm, and (B) anterior 2.1 mm, lateral, 4.1 mm, vertical, 4.0 mm.
transplantation are (A) anterior 2.1
108
FETAL CORTICAL CELLS IN FOCAL CEREBRAL INFARCT
FIG. 2. H&E-stained section of the graft 28 show a normal pattern of arrangement.
at
A trypan blue exclusion test (Paul, the time of transplantation.
days after transplantation.
Note that the cells
are
mature, yet
they do not
1975) was used to ensure that donor tissue showed at least 75% viability
Transplantation Technique The host rat was anesthetized with ketamine and Rumpon. A single injection of cefamezine 500 mg/kg was The rat was placed in a stereotactic frame (Kopf), and the head was fixed. The bregma was exposed, and a craniectomy was drilled anterior to the coronal suture at the left frontal bone. The caudal-rostral zero point was bregma. The tooth bar was 5.0 mm above the interaural line. The coordinates for transplantation were decided according to the location of the infarcted cyst and the penumbra demonstrated in our previous experiments (Young et al., 1986; Hadani et al., 1987). The coordinates for points A and B were as follows (Fig. 1).
given.
A. Anterior 2.1 mm Lateral 4.0 mm Vertical 2.0 mm
B. Anterior 2.1 mm Lateral 4.1 mm Vertical 4.0 mm
Three microliters of the cell suspension (120,000 cells) were injected into each target through a blunt needle connected to a Hamilton syringe.
Histologie Preparation Rats
were
perfused transcardially under nembutal
anesthesia with 50 ml of 10%
formaldehyde
and 30%
removed and allowed to sink in formalin-sucrose before cryostat sectioning. Twenty-eight-micrometer sections were cut at 18°C, collected at 5% formol, and mounted on a dust-free incubator at 37°C. Slides were stained with hematoxylin and eosin. sucrose.
Brains
were
—
109
HADANI ET AL.
mié* *
i
i
1¿¿
^s*
Mt.
FIG. 3. H&E-stained coronal section 28 days after transplantation. The graft (arrow) is located at an area relatively remote from the center of ischemia (stereotactic point A). Note that the neurons are mature and appear normal.
Experimental Groups The rats were divided into four groups. Group A. Eight animals received a transplant 3 h after the onset of left middle cerebral artery occlusion
(MCAo). Group B. Eight animals received a transplant 24 h after left MCAo. Group C. Eight animals received a transplant 7 days after MCAo. Group D. Control: Six intact animals received a transplant. All the animals
were
killed 28
days after transplantation. RESULTS
Cortical grafts survived in 23 of 26 animals who lived for the duration of the experiment. All grafts survived in the control animals. In the MCAo group, grafts survived in 17 of 20 animals (85%). Four animals from the MCAo groups died at or shortly after the stereotactic procedure (2 from group A, 1 from group B, and 1 from group C). Transplant survival was evaluated by light microscopy. The presence of clusters of cells at the target of implantation indicated viable grafts. No attempt was made to evaluate the total number of surviving cells. Grafts were considered viable only when found in both stereotactic points A and B in each animal. Transplant survival was 66% when grafted 3 h after MCAo (group A), 86% when grafted 24 hours after MCAo (group B), and 100% when grafted 7 days after MCAo (group C). The difference in survival in these three groups is not significant (Chi-square test). In all surviving grafts, the cells matured to normal-appearing neurons and glial cells, with profound capillary proliferation. The neuropil looked lighter in the graft than in the surrounding tissue (Fig. 2). In those animals where the graft was not identified as viable, the site of the injection showed only scattered gliosis. 110
FETAL CORTICAL CELLS IN FOCAL CEREBRAL INFARCT
FIG. 4.
H&E-stained coronal section 28 days after transplantation. The graft is located at the border of the necroticcyst (stereotactic point B) in the region of cerebral tissue that has lost its normal cellular arrangement due to the ischémie insult.
Those grafts transplanted in stereotactic point A appeared as clusters of cells embedded in otherwise normal brain tissue (Fig. 3). The grafts transplanted to stereotactic point B, located adjacent to the necrotic cyst, were identified within cerebral tissue that had lost its normal cellular appearance due to the ischémie insult (Fig 4). There was no evidence that transplant affected either the size or the shape of the ischémie lesion.
DISCUSSION This study demonstrates that fetal cortical cells can survive and mature in cerebral tissue involved in a recent ischémie insult. The fetal cells survived at the edge of the ischémie territory created by the permanent occlusion of the middle cerebral artery. At this region, ischémie changes are profound due to reduction in regional cerebral blood flow to 20% of the normal value (Tamura et al., 1981b). The hallmarks of these changes are calcium entry into the cells and profound electrolyte derangement (Hadani et al., 1988). The grafted cells survived when transplanted 3 h after MCAo, when blood flow is severely affected and causes acceleration in the derangement of ions and water balance in and around cerebral cells. Grafts survived also at 24 h after MCAo when electrolyte and water changes are at their peak, with calcium accumulating to about twice its normal value (Young et al., 1986). Graft survival was not significantly different when transplanted at the acute phase or at 7 days after MCAo, when metabolic changes are already minimal and regional cerebral blood flow is partially restored at the penumbra (Tamura et al., 1981b). It is evident from the present study that fetal cortical cells are capable of surviving and growing in cerebral tissue undergoing severe metabolic change. Whether growth factors, either from the ischémie recipient tissue, or from the transplanted fetal cells play a role in graft survival is unknown and is still a matter of speculation. Neuronotrophic factors had been shown to affect graft survival after denervation or tissue cavitation (Nieto-Sampedro et al., 1983; Gage and Bjorklund, 1986). These trophic factors may be released by ischémie tissue. However, it seems unlikely that such factors are present shortly 111
HADANI ET AL. after the onset of ischemia, that is, 3 h after arterial occlusion. Trophic factors may be present in the graft itself to maintain growth and protect the cell in the unfavorable conditions related to ischemia. The effect of such fetal factors on the recipient injured tissue is also attractive. Alexandrova (1985) demonstrated that fetal cortical transplants exerted a protective and reparative effect on cortical neurons after transient global cerebral anoxia. Studies have demonstrated that embryonic CNS tissue is endowed with the ability to induce a regeneration response in severed adult CNS tissue (Hadani et al., 1984). Indeed, transplantation of fetal brain grafts resulted in promotion of regeneration of axons in the brain (Floeter and Jones, 1984) and in recovery of function in rats with ablated frontal cortex (Labbe et al., 1983). The presence of fetal cells at the edge of the ischémie cerebral tissue may facilitate the process of regeneration of cells and fibers shortly after the injury, before degeneration becomes irreversible (Kromer, 1985).
REFERENCES ALEXANDROVA, M.A. (1985). Transplantation of embryo brain tissue into the brain of adult rats, intact and exposed to hypoxia, in: Neural Grafting in the Mammalian CNS. A. Bjorklund and U Steveni (eds). Elsevier Science Publishers: Amsterdam.
FLOETER, M.K., and JONES, E.J. (1984). Connections made by transplants to the cerebral cortex of rat brains damaged in utero. J. Neurosci.
4, 141-150.
GAGE, F.H., and BJORKLUND, A. (1986). Enhanced graft survival in the hippocampus following selective denervation. Neuroscience 17, 89-98.
HADANI, M., FREEMAN, T., PEARSON, J., et al. Embryonic cortical transplants survive in middle cerebral artery territory after permanent arterial occlusion in adult rats. Ann. NY Acad. Sei. 495, 711-714.
HADANI, M., HAREL, A., SOLOMON, A., etal. (1984). Substances originating from the optic nerve of neonatal rabbit induce regeneration-associated response in the injured optic nerve of adult rabbit. Proc. Nati. Acad. Sei. USA 81, 7965-7969.
HADANI, M., YOUNG, W., and FLAMM, E.S. (1988). Nicardipine reduces calcium accumulation and electrolytes
derangement in regional cerebral
ischemia in rats. Stroke
19, 1125-1132.
KROMER, L.F. (1985). Factors in neural transplants which influence regeneration in the mature mammalian central nervous system, in: Neural Grafting in the Mammalian CNS. A. Bjorklund and U. Stenevi (eds). Elsevier Science Publishers, Amsterdam, pp. 309-318.
LABBE, R., FIRL, A.. MUFSON, E.J., and STEIN, D.G. (1983). Fetal brain transplants: Reduction of cognitive deficit
221, 470-472. NIETO-SAMPEDRO, M., MANTROPE, M., BARBIN, G., et al. (1984). Injury induced neuronotrophic activity in adult rat brain: Correlation with survival of delayed implants in the wound cavity. J. Neurosci. 3, 2219-2229. PAUL, J. (1975). In Cell and Tissue Culture, 5th ed. Churchill Livingstone, New York, pp. 367-368. TAMURA, A., GRAHAM, D. I., McCULLOCH, J., and TEASDALE, G. M. ( 1981 a). Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab. 1, 53-60. TAMURA, A., GRAHAM, D.I., McCULLOCH, J., andTEASDALE, G.M. (1981b). Focal cerebral ischemia in the rat: 2. Regional cerebral blood flow changes. J. Cereb. Blood Flow Metab. 1, 61-69. in rats with frontal cortex lesions. Science
YOUNG, W., HADANI, M., RAPPAPORT, H., ischemia: Their measurement and
interpretation.
et al. (1986). Tissue Na, K, and Ca CNS Trauma 3, 215-234.
changes
in
regional
cerebral
Address reprint requests to: Moshe Hadani, M.D.
Department of Neurosurgery Sheba Medical Center Tel Hasomer 52621 Israel 112
Fetal Cortical Cells Survive in Focal Cerebral Infarct After Permanent Occlusion of the Middle Cerebral Artery in Adult Rats MOSHE
HADANI,1
THOMAS
FREEMAN,2 AMAR MUNSIFF,2 WISE YOUNG,2 and EUGENE
FLAMM3
ABSTRACT Fetal rat cortical cells have been shown previously to survive at the periphery of cerebral infarction. The present study was designed to examine the ability of fetal cells to survive at the edge of the central core of ischemia. In three groups of 8 adult Sprague-Dawley rats, fetal cortical cells from ED 16 were stereotactically transplanted at 3 h, 24 h, and 7 days after unilateral middle cerebral artery occlusion. In 6 rats, fetal cells were transplanted by using the same coordinates, without arterial occlusion, for control. In the ischémie groups, overall graft survival was 85%, and in the control group, all grafts survived. Graft survival was determined by light microscopy. No significant difference was found in the survival of grafts transplanted at different intervals after middle cerebral artery occlusion. It is concluded that fetal cortical cells can survive in cerebral tissue undergoing severe ischémie change.
INTRODUCTION iscHEMic insult to cerebral tissue is considered an end die, and recovery. The cells at the center of the ischémie
Focal
point in regard to structural and functional
cerebral tissue is lost. However, cells at region These territory only partially damaged. injured neurons may still have the periphery conditions the and are to signals present. potential regenerate, provided appropriate In a previous study (Hadani et al., 1987), we demonstrated that embryonic cortical grafts survived when transplanted at the periphery of a focus of cerebral infarction. Grafts, however, did not survive in any animal when transplanted into the core of the infarct, which was subsequently transformed into a necrotic cyst. In the present study, we extend these studies in the same model by now transplanting fetal cells into the penumbra just at the edge of the central core of infarction, where damaged but yet potentially viable neurons
the
of the ischémie
are
'Department of Neurosurgery, Sheba Medical Center, Tel Hashomer, Israel.
2The Neurosurgical Research Laboratories, Department of Neurosurgery, New York University Medical Center, New York.
^Division of Neurosurgery Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania. 107
HADANI ET AL. are
present. Further to complement these studies,
tissue
we
also examined the
subjected to acute ischémie insult.
potential of fetal cells to survive in
MATERIALS AND METHODS Middle Cerebral Artery Occlusion (MCAo)
Sprague-Dawley rats weighing 290-300 g were anesthetized with intramuscular injections of ketamine HC1 mg/kg and xylazine (Rumpon) 6 mg/kg. The left middle cerebral artery (MCA) was occluded as previously described by Tamura et al. (1981a). In brief, the left temporal muscle was excised, and the left mandible was retracted to expose the temporal bone. A subtemporal craniectomy (2 mm x 3 mm) was made with a microdrill anterolateral to the foramen ovale and posteromedial to the optic foramen. The dura was opened with a hooked 25-gauge needle, and the main trunk of the MCA was exposed. The arachnoid membrane above the artery was opened. The MCA was coagulated by a bipolar current and divided distal to the lateral striate branch at its junction with the olfactory stria. The wound was irrigated and closed with metal clips. The animals were housed in separate cages and were fed postoperatively with high-calorie liquid food (Ensure, vanilla flavor) 300 cal/kg. Tetracycline (1.5 g/1) was added to the drinking water for 7 days. 30
Graft Preparation harvested from the anterolateral aspect of the telencephalic vesicle of outbred 16. The developing basal ganglia were not included in the graft. Tissue from 12-14 fetuses was collected in plastic dishes containing PBS/glucose and washed at room temperature. The tissue was dissociated mechanically by repeated gentle pipetting in trypsin and subsequently washed in PBS/glucose with fetal calf serum. The suspension was centrifuged at 500g for 5 min for debris removal and then diluted in PBS/glucose to 40,000 cells per microliter. Fetal cortical cells
were
Sprague-Dawley embryos at embryonic day (ED)
FIG. 1.
H&E-stained coronal section of a rat brain 24 h after MCAo. The region of ischemia is pale. The coordinates for mm, lateral 4.0 mm, vertical, 2.0 mm, and (B) anterior 2.1 mm, lateral, 4.1 mm, vertical, 4.0 mm.
transplantation are (A) anterior 2.1
108
FETAL CORTICAL CELLS IN FOCAL CEREBRAL INFARCT
FIG. 2. H&E-stained section of the graft 28 show a normal pattern of arrangement.
at
A trypan blue exclusion test (Paul, the time of transplantation.
days after transplantation.
Note that the cells
are
mature, yet
they do not
1975) was used to ensure that donor tissue showed at least 75% viability
Transplantation Technique The host rat was anesthetized with ketamine and Rumpon. A single injection of cefamezine 500 mg/kg was The rat was placed in a stereotactic frame (Kopf), and the head was fixed. The bregma was exposed, and a craniectomy was drilled anterior to the coronal suture at the left frontal bone. The caudal-rostral zero point was bregma. The tooth bar was 5.0 mm above the interaural line. The coordinates for transplantation were decided according to the location of the infarcted cyst and the penumbra demonstrated in our previous experiments (Young et al., 1986; Hadani et al., 1987). The coordinates for points A and B were as follows (Fig. 1).
given.
A. Anterior 2.1 mm Lateral 4.0 mm Vertical 2.0 mm
B. Anterior 2.1 mm Lateral 4.1 mm Vertical 4.0 mm
Three microliters of the cell suspension (120,000 cells) were injected into each target through a blunt needle connected to a Hamilton syringe.
Histologie Preparation Rats
were
perfused transcardially under nembutal
anesthesia with 50 ml of 10%
formaldehyde
and 30%
removed and allowed to sink in formalin-sucrose before cryostat sectioning. Twenty-eight-micrometer sections were cut at 18°C, collected at 5% formol, and mounted on a dust-free incubator at 37°C. Slides were stained with hematoxylin and eosin. sucrose.
Brains
were
—
109
HADANI ET AL.
mié* *
i
i
1¿¿
^s*
Mt.
FIG. 3. H&E-stained coronal section 28 days after transplantation. The graft (arrow) is located at an area relatively remote from the center of ischemia (stereotactic point A). Note that the neurons are mature and appear normal.
Experimental Groups The rats were divided into four groups. Group A. Eight animals received a transplant 3 h after the onset of left middle cerebral artery occlusion
(MCAo). Group B. Eight animals received a transplant 24 h after left MCAo. Group C. Eight animals received a transplant 7 days after MCAo. Group D. Control: Six intact animals received a transplant. All the animals
were
killed 28
days after transplantation. RESULTS
Cortical grafts survived in 23 of 26 animals who lived for the duration of the experiment. All grafts survived in the control animals. In the MCAo group, grafts survived in 17 of 20 animals (85%). Four animals from the MCAo groups died at or shortly after the stereotactic procedure (2 from group A, 1 from group B, and 1 from group C). Transplant survival was evaluated by light microscopy. The presence of clusters of cells at the target of implantation indicated viable grafts. No attempt was made to evaluate the total number of surviving cells. Grafts were considered viable only when found in both stereotactic points A and B in each animal. Transplant survival was 66% when grafted 3 h after MCAo (group A), 86% when grafted 24 hours after MCAo (group B), and 100% when grafted 7 days after MCAo (group C). The difference in survival in these three groups is not significant (Chi-square test). In all surviving grafts, the cells matured to normal-appearing neurons and glial cells, with profound capillary proliferation. The neuropil looked lighter in the graft than in the surrounding tissue (Fig. 2). In those animals where the graft was not identified as viable, the site of the injection showed only scattered gliosis. 110
FETAL CORTICAL CELLS IN FOCAL CEREBRAL INFARCT
FIG. 4.
H&E-stained coronal section 28 days after transplantation. The graft is located at the border of the necroticcyst (stereotactic point B) in the region of cerebral tissue that has lost its normal cellular arrangement due to the ischémie insult.
Those grafts transplanted in stereotactic point A appeared as clusters of cells embedded in otherwise normal brain tissue (Fig. 3). The grafts transplanted to stereotactic point B, located adjacent to the necrotic cyst, were identified within cerebral tissue that had lost its normal cellular appearance due to the ischémie insult (Fig 4). There was no evidence that transplant affected either the size or the shape of the ischémie lesion.
DISCUSSION This study demonstrates that fetal cortical cells can survive and mature in cerebral tissue involved in a recent ischémie insult. The fetal cells survived at the edge of the ischémie territory created by the permanent occlusion of the middle cerebral artery. At this region, ischémie changes are profound due to reduction in regional cerebral blood flow to 20% of the normal value (Tamura et al., 1981b). The hallmarks of these changes are calcium entry into the cells and profound electrolyte derangement (Hadani et al., 1988). The grafted cells survived when transplanted 3 h after MCAo, when blood flow is severely affected and causes acceleration in the derangement of ions and water balance in and around cerebral cells. Grafts survived also at 24 h after MCAo when electrolyte and water changes are at their peak, with calcium accumulating to about twice its normal value (Young et al., 1986). Graft survival was not significantly different when transplanted at the acute phase or at 7 days after MCAo, when metabolic changes are already minimal and regional cerebral blood flow is partially restored at the penumbra (Tamura et al., 1981b). It is evident from the present study that fetal cortical cells are capable of surviving and growing in cerebral tissue undergoing severe metabolic change. Whether growth factors, either from the ischémie recipient tissue, or from the transplanted fetal cells play a role in graft survival is unknown and is still a matter of speculation. Neuronotrophic factors had been shown to affect graft survival after denervation or tissue cavitation (Nieto-Sampedro et al., 1983; Gage and Bjorklund, 1986). These trophic factors may be released by ischémie tissue. However, it seems unlikely that such factors are present shortly 111
HADANI ET AL. after the onset of ischemia, that is, 3 h after arterial occlusion. Trophic factors may be present in the graft itself to maintain growth and protect the cell in the unfavorable conditions related to ischemia. The effect of such fetal factors on the recipient injured tissue is also attractive. Alexandrova (1985) demonstrated that fetal cortical transplants exerted a protective and reparative effect on cortical neurons after transient global cerebral anoxia. Studies have demonstrated that embryonic CNS tissue is endowed with the ability to induce a regeneration response in severed adult CNS tissue (Hadani et al., 1984). Indeed, transplantation of fetal brain grafts resulted in promotion of regeneration of axons in the brain (Floeter and Jones, 1984) and in recovery of function in rats with ablated frontal cortex (Labbe et al., 1983). The presence of fetal cells at the edge of the ischémie cerebral tissue may facilitate the process of regeneration of cells and fibers shortly after the injury, before degeneration becomes irreversible (Kromer, 1985).
REFERENCES ALEXANDROVA, M.A. (1985). Transplantation of embryo brain tissue into the brain of adult rats, intact and exposed to hypoxia, in: Neural Grafting in the Mammalian CNS. A. Bjorklund and U Steveni (eds). Elsevier Science Publishers: Amsterdam.
FLOETER, M.K., and JONES, E.J. (1984). Connections made by transplants to the cerebral cortex of rat brains damaged in utero. J. Neurosci.
4, 141-150.
GAGE, F.H., and BJORKLUND, A. (1986). Enhanced graft survival in the hippocampus following selective denervation. Neuroscience 17, 89-98.
HADANI, M., FREEMAN, T., PEARSON, J., et al. Embryonic cortical transplants survive in middle cerebral artery territory after permanent arterial occlusion in adult rats. Ann. NY Acad. Sei. 495, 711-714.
HADANI, M., HAREL, A., SOLOMON, A., etal. (1984). Substances originating from the optic nerve of neonatal rabbit induce regeneration-associated response in the injured optic nerve of adult rabbit. Proc. Nati. Acad. Sei. USA 81, 7965-7969.
HADANI, M., YOUNG, W., and FLAMM, E.S. (1988). Nicardipine reduces calcium accumulation and electrolytes
derangement in regional cerebral
ischemia in rats. Stroke
19, 1125-1132.
KROMER, L.F. (1985). Factors in neural transplants which influence regeneration in the mature mammalian central nervous system, in: Neural Grafting in the Mammalian CNS. A. Bjorklund and U. Stenevi (eds). Elsevier Science Publishers, Amsterdam, pp. 309-318.
LABBE, R., FIRL, A.. MUFSON, E.J., and STEIN, D.G. (1983). Fetal brain transplants: Reduction of cognitive deficit
221, 470-472. NIETO-SAMPEDRO, M., MANTROPE, M., BARBIN, G., et al. (1984). Injury induced neuronotrophic activity in adult rat brain: Correlation with survival of delayed implants in the wound cavity. J. Neurosci. 3, 2219-2229. PAUL, J. (1975). In Cell and Tissue Culture, 5th ed. Churchill Livingstone, New York, pp. 367-368. TAMURA, A., GRAHAM, D. I., McCULLOCH, J., and TEASDALE, G. M. ( 1981 a). Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab. 1, 53-60. TAMURA, A., GRAHAM, D.I., McCULLOCH, J., andTEASDALE, G.M. (1981b). Focal cerebral ischemia in the rat: 2. Regional cerebral blood flow changes. J. Cereb. Blood Flow Metab. 1, 61-69. in rats with frontal cortex lesions. Science
YOUNG, W., HADANI, M., RAPPAPORT, H., ischemia: Their measurement and
interpretation.
et al. (1986). Tissue Na, K, and Ca CNS Trauma 3, 215-234.
changes
in
regional
cerebral
Address reprint requests to: Moshe Hadani, M.D.
Department of Neurosurgery Sheba Medical Center Tel Hasomer 52621 Israel 112
