- Email: [email protected]
Regeneration of ganglion cell axons in the cat retina
$39
HEPATOCYTE CONDITIONED MEDIUM PROMOTES NEIIRITE REGENERATION FROM RETINA EXPLANT$ OF ADULT MICE IN VITRO. HIDENORI HORIE~rMASAHIKO TAKANO 2 AND TOSIIIFUMI TAKENAKA a, Department of 'Physiology and Ophthalmolo~y2, School of MedicinerYokohama City University, 3-9 Fukuura, Kanazawaku, Yokohama, 236 Japan, The optic nerve of 3-4-month-old mice was mechanically crushed by fine forceps at a distance of 2-3 mm from the optic disc. Seven days later the retinae were removed from their eyecups and divided into fragments. They were cultured in collagen gel or on poly.lysine-laminin coated dishes. Neurites appeared 24 h after in culture and the processes were positive to antibodies against neurofilament and Thy-1. These results indicated that the neurites grew from ganglion cells in the explants. Hepatocytes dissociated from livers of 3-4-month-old mice by the collagenase perfusion method were cultured and the cultured medium was apllied to the explants. The number of regenerating neurites was increased and their survival was prolonged by the hepatocyte condition medium. The factor secreted from hepatocyte not only improved regeneration of adult peripheral nervous tissues but also the central nervous system in adult mice.
RETINAL GANGLION CE.I J.S SURVIVED LONG AFTER THE OPTIC NERVE SECTION IN ADULT RATS. MASAKI TAUCHI AND YUTAKA FUKUDA, Res. Inst. National, Rehab, C~nter for the Disabled.Tokorozawa, 369, and Dept. of Physiol., O~aka Univ, Med. Seh., Kita-ku, Osaka 530. Japan, A great majority of retinal ganglion cells in adult mammals gradually die after axotomy. However, a small proportion of ganglion cells has been reported to survive in both rats and cats retinas. In the present study we identified these surviving ganglion cells with retrogradely transported granular blue (GB) and investigated their morphological characteristics by intracellular injection of Lucifer Yellow (LY). Under anesthesia with chloral hydrate the optic nerve of the adult Wistar rats (70 days old) was sectioned behind the eye ball and GB was applied to the cut edge of the optic nerve to retrogradely label the ganglion cells. The animals were anesthetized again 8 months after the surgery and the eye was removed from the orbit and the retina was whole-mounted in aeriated (95% 0 2 and 5%CO2) Ames medium. Some of the BG-labeled ganglion cells were injected with LY under epifluorescent microscope. In one such retina a total of 683 ganglion cells were observed to survive at various locations in whole mount retina; 361 were in ganglion cell layer whereas 322 in inner nuclear layer. Soma size of 284 cells, 78%of the total GB-labeled cells in ganglion cell layer, exceeded 20 um in diameter. In the retina of intact rats such large ganglion cells correspond to alpha cells. After intracellular dye injection most of the large ganglion cells showed dendritic morphologies similar to those of intact alpha cells. Since alpha cells in normal retina count approximately 1000, about one third of the alpha cells could have survived after axotomy. Intmcellular LY-injections also revealed that some of these large cells had markedly simpler dendritic branchings as compared with the dendrites of intact alpha cells in normal rats. Furthermore a group of large cells revealed very asymmetric dendritic structures and thin dendritic branches taking curved and complicated courses. These latter dendrites may reflect the regrowth of dendrites after degeneration of original dendrites during a long surviving period after axotomy.
REGENERATION OF GANGLION CELL AXONS IN THE CAT RETINA..MASAMI WATANABE 1, AND YUTAKA FUKUDA 2, 1Department of Physiology, Institute for Developmental Research, Aichi Prefectural Colony. Kasugai, Aichi 480-0~, and 2Department of Physiology, Osaka University Medical School, Kitaku, Osaka 530, Japan... Retinal ganglion cells in adult rodents regenerate their axon along autografted peripheral nerve after transection (Aguayo, '85). We were able to show that retinal ganglion cells in cats also regenerated their axon along the peripheral nerve graft. Adult cats were anesthetized with halothane (1%) and nitrous oxide (0.81/min), the left optic nerve was exposed and totally cut close to the eyeball. The autologous sciatic nerve, 30-50 mm in length, was transplanted to the stump. After 2 months a mixture of 15% HRP and about 10 pl of 10% dextran-rhodamine was injected into the graft to retrogradely label regenerating ganglion cells. After 48hr the cats were perfused with 0.5% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer. The dissected retinas were observed under a fluorescence microscope, then were reacted with DAB. Numbers of regenerating ganglion cells varied from 513 to 993. Regenerating cells were distributed most densely in the area nasal to the area centralis dorsal to the optic disk. SomaI diameters of regenerating ganglion cells ranged from 8 to 55 pm, tended to be larger than those in the normal retina, and did not show increase as a function of eccentricity from the presumed area centralis. Ganglion cells that projected to the lateral geniculate nucleus (LGN) were labeled with DiI seven days prior to transplantation of the peripheral nerve. After 60 days of transplantation the nerve graft received 10% dextranfluorescein. We found substantial numbers of fluorescein-labeled cells were labeled with DiI also, hence were able to demonstrate that LGN-projecting ganglion cells regenerated axons. Dendritic morphology of regenerating ganglion cells was visualized with the intracellular HRP injection, and alpha, beta, and other cell types were recognized.
HEPATOCYTE CONDITIONED MEDIUM PROMOTES NEIIRITE REGENERATION FROM RETINA EXPLANT$ OF ADULT MICE IN VITRO. HIDENORI HORIE~rMASAHIKO TAKANO 2 AND TOSIIIFUMI TAKENAKA a, Department of 'Physiology and Ophthalmolo~y2, School of MedicinerYokohama City University, 3-9 Fukuura, Kanazawaku, Yokohama, 236 Japan, The optic nerve of 3-4-month-old mice was mechanically crushed by fine forceps at a distance of 2-3 mm from the optic disc. Seven days later the retinae were removed from their eyecups and divided into fragments. They were cultured in collagen gel or on poly.lysine-laminin coated dishes. Neurites appeared 24 h after in culture and the processes were positive to antibodies against neurofilament and Thy-1. These results indicated that the neurites grew from ganglion cells in the explants. Hepatocytes dissociated from livers of 3-4-month-old mice by the collagenase perfusion method were cultured and the cultured medium was apllied to the explants. The number of regenerating neurites was increased and their survival was prolonged by the hepatocyte condition medium. The factor secreted from hepatocyte not only improved regeneration of adult peripheral nervous tissues but also the central nervous system in adult mice.
RETINAL GANGLION CE.I J.S SURVIVED LONG AFTER THE OPTIC NERVE SECTION IN ADULT RATS. MASAKI TAUCHI AND YUTAKA FUKUDA, Res. Inst. National, Rehab, C~nter for the Disabled.Tokorozawa, 369, and Dept. of Physiol., O~aka Univ, Med. Seh., Kita-ku, Osaka 530. Japan, A great majority of retinal ganglion cells in adult mammals gradually die after axotomy. However, a small proportion of ganglion cells has been reported to survive in both rats and cats retinas. In the present study we identified these surviving ganglion cells with retrogradely transported granular blue (GB) and investigated their morphological characteristics by intracellular injection of Lucifer Yellow (LY). Under anesthesia with chloral hydrate the optic nerve of the adult Wistar rats (70 days old) was sectioned behind the eye ball and GB was applied to the cut edge of the optic nerve to retrogradely label the ganglion cells. The animals were anesthetized again 8 months after the surgery and the eye was removed from the orbit and the retina was whole-mounted in aeriated (95% 0 2 and 5%CO2) Ames medium. Some of the BG-labeled ganglion cells were injected with LY under epifluorescent microscope. In one such retina a total of 683 ganglion cells were observed to survive at various locations in whole mount retina; 361 were in ganglion cell layer whereas 322 in inner nuclear layer. Soma size of 284 cells, 78%of the total GB-labeled cells in ganglion cell layer, exceeded 20 um in diameter. In the retina of intact rats such large ganglion cells correspond to alpha cells. After intracellular dye injection most of the large ganglion cells showed dendritic morphologies similar to those of intact alpha cells. Since alpha cells in normal retina count approximately 1000, about one third of the alpha cells could have survived after axotomy. Intmcellular LY-injections also revealed that some of these large cells had markedly simpler dendritic branchings as compared with the dendrites of intact alpha cells in normal rats. Furthermore a group of large cells revealed very asymmetric dendritic structures and thin dendritic branches taking curved and complicated courses. These latter dendrites may reflect the regrowth of dendrites after degeneration of original dendrites during a long surviving period after axotomy.
REGENERATION OF GANGLION CELL AXONS IN THE CAT RETINA..MASAMI WATANABE 1, AND YUTAKA FUKUDA 2, 1Department of Physiology, Institute for Developmental Research, Aichi Prefectural Colony. Kasugai, Aichi 480-0~, and 2Department of Physiology, Osaka University Medical School, Kitaku, Osaka 530, Japan... Retinal ganglion cells in adult rodents regenerate their axon along autografted peripheral nerve after transection (Aguayo, '85). We were able to show that retinal ganglion cells in cats also regenerated their axon along the peripheral nerve graft. Adult cats were anesthetized with halothane (1%) and nitrous oxide (0.81/min), the left optic nerve was exposed and totally cut close to the eyeball. The autologous sciatic nerve, 30-50 mm in length, was transplanted to the stump. After 2 months a mixture of 15% HRP and about 10 pl of 10% dextran-rhodamine was injected into the graft to retrogradely label regenerating ganglion cells. After 48hr the cats were perfused with 0.5% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer. The dissected retinas were observed under a fluorescence microscope, then were reacted with DAB. Numbers of regenerating ganglion cells varied from 513 to 993. Regenerating cells were distributed most densely in the area nasal to the area centralis dorsal to the optic disk. SomaI diameters of regenerating ganglion cells ranged from 8 to 55 pm, tended to be larger than those in the normal retina, and did not show increase as a function of eccentricity from the presumed area centralis. Ganglion cells that projected to the lateral geniculate nucleus (LGN) were labeled with DiI seven days prior to transplantation of the peripheral nerve. After 60 days of transplantation the nerve graft received 10% dextranfluorescein. We found substantial numbers of fluorescein-labeled cells were labeled with DiI also, hence were able to demonstrate that LGN-projecting ganglion cells regenerated axons. Dendritic morphology of regenerating ganglion cells was visualized with the intracellular HRP injection, and alpha, beta, and other cell types were recognized.