Acta Neuropathologica
Acta neuropath. (Berl.) 38, 4 9 - 52 (1977)
9 by Springer-Verlag I977
Retrograde Axonal Transport of Horseradish Peroxidase from Cornea to Trigeminal Ganglion Bj6rn Arvidson
Neuropathological Laboratory, Institute of Pathology, University of Uppsala, Box 553, S-75122 Uppsala, Sweden
S u m m a r y . Horseradish peroxidase (HRP) was dripped
on the scarified left cornea of adult mice. Twentyfour hours later the animals were fixed by vascular perfusion and frozen sections cut from both trigeminal ganglia. After incubation for peroxidase activity labelled nerve cells were restricted to the medial ophthalmic part of the ganglion ipsilateral to HRP administration. If the scarification was omitted no neuronal labelling was observed. This labelling of the neurons is most probably the result from axonal uptake and subsequent retrograde axonal transport of the tracer. The similarity in distribution of peroxidase labelled nerve cells and the first ganglionic lesions occurring after instillation of herpes simplex virus in the cornea is pointed out. Key words: Cornea - Trigeminal ganglion trograde axonat transport.
Re-
Introduction
Different methods have been used in investigations concerning a somatotopical organization of nerve cell bodies in the trigeminal ganglion. Physiological studies of this ganglion in the cat indicate that cell bodies of the mandibular nerve are located posterolaterally, those of the ophthalmic nerve anteromedially and those of the maxillary nerve in the area b e t w e e n these two locations (Darian-Smith et al., Beaudreau and Jerge). Anatomical studies of chromatolytic cell changes in the trigeminal ganglion after nerve section have been made in several species including rat, rabbit and cat (Allen; Strassburg; Mazza and Dixon; Sakai). After mandibular nerve section in the rat chromatolytic nerve cells were concentrated laterally in the ganglion. The ophthalmic neurons seemed to be located mostly
medially and the neurons of the maxillary nerve b e t w e e n these cell groups (Sakai). In the cat a large cephalic and median portion was made up only of ophthalmic-maxillary cells, while a smaller lateral and caudal division consisted entirely of mandibular cells (Allen). After unilateral maxillary dental extraction in rabbits chromatotytic nerve cell changes were observed in a clearly determinable region of the central maxillary ganglion portion (Strassburg). The ophthalmic division of the trigeminal nerve has been used for several experimental studies on viral transmission along peripheral sensory nerves. After corneal inoculation of herpes simplex virus (HSV) the virus is capable of travelling in the nerve to produce a primary infection in neurons of the Gasserian ganglion (Marinesco; Baringer and Griffith; Stevens et al. ; Baringer and Swoveland; Knotts et al.). Intranasal inoculation of HSV also resulted in an infection of neurons in the trigeminal ganglion (Rabin et al.). The retrograde horseradish peroxidase method (Kristensson and Olsson) has been used extensively for mapping neuroanatomical pathways both in the central (La Vail and La Vail; La Vail) and peripheral (Kristensson and Olsson; Kristensson) nervous system. With regard to the trigeminal nerve this method has previously been used for tracing the cell bodies in the trigeminal ganglion innervating the dental pulp of different teeth (Furstman et al. ; Arvidsson). Within the motor division of the fifth nerve a topographical localization of neurons supplying the masticatory muscles has been described with use of the same method (Mizuno et al.). The present study was undertaken for several reasons. It was of interest to find out if a foreign protein such as H R P is taken up in axonal branches in the ophthalmic nerve and transported back to the trigeminal ganglion as has been suggested for certain viral infections (Baringer and Swoveland; Cooks et
50
Acta neuropath. (Berl.) 38 (1977)
~ant lat Fig. 1
Peroxidase labelled nerve cell in the trigeminal ganglion, x 800 Fig. 2
I ,,-I
i
al.). In addition this study is part o f a project studying the cellular localization o f foreign proteins in peripheral ganglia following various modes o f administration (Arvidson, to be published).
M a t e r i a l s and M e t h o d s
The experiments were performed on fourteen adult male albino mice. After local anesthesia of the cornea with 4 700lidocain and general ether anesthesia the left cornea was scratched with a needle in a checked pattern. About 5 gl of a 40 % HRP solution in isotonic saline was then dripped in the left eye and the eyelid sutured to prevent escape of the tracer. In a few animals the scarification of the cornea was omitted prior to HRP administration. In some of these animals the eyelid was also left without suture. After 24 h the animals were perfused through the heart with 2.0 % glutaraldehyde in S6rensens phosphate buffer, pH 7.4. Both trigeminal ganglia were dissected out and placed in cold fixative for additional 4 h. Overnight they were washed in phosphate buffer with 5 sucrose at 4~C. The following day both ganglia were placed horizontally and cut into 40 gm thick serial sections on a freezing microtome. Every second section was sampled. The sections were preincubated for 15 min in 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in Tris-buffer at pH 7.5. Then followed incubation in the full medium of Graham and Karnovsky with the addition of hydrogen peroxide for 45 min at room temperature. They were mounted without counterstaining and examined by light microscopy.
Results
HRP-labelled nerve cells were identified by the presence o f small darkstaining granules in the cytoplasm (Fig. 1). Such cells were only f o u n d in the ganglion ipsilateral to the administration o f H R P . Labelled neurons were limited to the anteromedial p o r t i o n
Schematic drawing of the left trigeminal ganglion of one experimental animal showing the distribution of peroxidase labelled nerve cells, m mandibular nerve; o m opthalmic-maxillary nerve
2
o f the ganglion and were often located superficially u n d e r the ganglionic capsule (Fig. 2). Both large and small neurons were labelled. In all animals given H R P after corneal scarification labelled cells were f o u n d but the n u m b e r varied between individual mice with a m a x i m u m o f 80 and a m i n i m u m o f 16 labelled cells per ganglion. The reason for this variation in the n u m b e r o f labelled cells is difficult to understand but m a y be explained by variations in the technical procedure o f H R P administration. The average n u m b e r o f labelled cells was 44 per ganglion. In animals where the corneal scarification was omitted and the eyelid sutured only two or three m a r k e d nerve cells were identified in the ipsilateral ganglion. U p t a k e o f peroxidase in sensory nerve endings injured by the lid suture m a y explain these findings. If the eyelid was also left without suture no n e u r o n a l labelling t o o k place. Discussion
The nervous innervation o f the cornea has been extensively investigated. Studies on the rabbit cornea have shown that nerve bundles f r o m the ciliary nerves enter the cornea in a radial m a n n e r and ultimately give rise to a plexiform aggregation o f nerve fibers which pervades the substantia propria (Zander and Weddell). In the mouse this plexus is arranged in three main layers; a deep, midstromal and subepithelial plexus. Myelinated fibers are f o u n d in the deep and midstromal plexus while only unmyelinated axons pass into the subepithelial plexus (Whitear). The corneal epithelium acts as a barrier against penetration o f foreign substances into the eye. In vitro studies o f rabbit cornea have demonstrated that
B, Arvidson: Transport of HRP from Cornea to Trigeminal Ganglion
HRP does not penetrate the surface epithelium after local application (T~bnjum). The imperviousness of the corneal epithelium can be ascribed to tight junctions between adjacent surface epithelial cells (Pei and Rhodin). This barrier apparently has to be broken before an uptake of HRP in nerve endings takes place since no labelled neurons are observed after application of HRP to the intact cornea. Following uptake in normal or injured nerve branches in the cornea HRP is most probably transported retrogradely in axons to the neuronal cell bodies in the trigeminal ganglion. Such a transport has previously been described in peripheral motor (Kristensson and Olsson) and sensory (Furstman et al. ; Arvidsson) neurons as well as in various central neurons (La Vail and La Vail; La Vail). Following such a transport HRP accumulates in a granular way close to the nucleus in the same way as in the present experiments and is in contrast to vascular administration of tracer when HRP initially accumulates in macrophages in the ganglia (Jacobs et al. ; Arvidson, to be published). Following vascular infusion of large doses of HRP in mice there is a retrograde axonal transport of this protein to neurons in neurosecretory, motor, sensory and autonomic systems (Broadwell and Brightman). The possibility of a retrograde neuronal labelling due to blood reabsorption of HRP injected into the tissues in microliter quantities has been discussed. This mechanism of neuronal labelling is not likely to occur in the present experiments since marked nerve ceils are only observed in a restricted area of the ganglion ipsilateral to the injection of peroxidase. Furthermore the cornea is avascular. The number of labelled nerve cells in the Gasserian ganglion in the present experiments should not be considered as the real number of neurons innervating the cornea. Variations in uptake of HRP in normal and injured nerves possibly resulting from failure to reach all sensory nerves may contribute to the low number of labelled neurons. In the present study marked nerve cells are only observed in the anteromedial part of the ganglion. This distribution is in agreement with earlier studies using physiological and degenerative methods. The present experiments can also be considered as a model for studies on routes taken by foreign substances after instillation in the cornea. In this connection spread of viral particles to the Gasserian ganglion is of special interest. After corneal inoculation of HSV in mice nerve cells in the Gasserian ganglion show morphological signs of infection. Only the medial portion of the ganglion corresponding to the ophthalmic branch is markedly involved (Knotts et al.). The distribution of infected neurons is thus
51
in agreement with the location of peroxidase labelled nerve cells after corneal instillation of HRP. The route taken by HSV after corneal inoculation has been debated although recent reports (Baringer and Swoveland; Knotts et al.) show that an intraaxonal spread is probable. The demonstration of an apparently intraaxonat transport of HRP in the same nerves and the similarity in distribution of infected and peroxidase labelled neurons in the trigeminal ganglion should give further support to this mode of virus spread. Acknowledgement. Supported by grants from the Swedish Medical Research Council, Project No. B77-17X-03020-08C from Riksf6reningen f6r trafik- och polioskadade. The author is indebted to Prof. Yngve Olsson for his advice and helpful suggestions with regard to the manuscript.
References Allen, W. F. : Localization in the ganglion semilunare of the cat. J. comp. Neurol. 38, 1 - 2 5 (1924) Arvidsson, J. : Location of cat trigeminal ganglion cells innervating dental pulp of upper and lower canines studied by retrograde transport of horseradish peroxidase. Brain Res. 99, 135-139 (1975) Baringer, J. R., Swoveland, P.: Persistent herpes simplex virus infection in rabbit trigeminal ganglia. Lab. Invest. 30, 230- 240 (1974) Baringer, J.R., Griffith, J . F . : Experimental herpes simplex encephalitis: early neuropathologic changes. J. Neuropath. exp. Neurol. 29, 89-104 (1970) Beaudreau, D. E., Jerge, C. R. : Somatotopic representation in the Gasserian ganglion of tactile peripheral fields in the cat. Arch. oral Biol. 13,247-256 (1968) Broadwell, R. D., Brightman, M.W.: Entry of peroxidase into neurons of the central and peripheral nervous systems from extracerebral and cerebral blood. J. comp. Neurol. 166, 257- 284 (1976) Darian-Smith, I., Mutton, P., Proctor, R.: Functional organization of tactile cutaneous afferents within the semilunar ganglion and trigeminal spinal tract of the cat. J. Neurophysiol. 28, 682-- 694 (1965) Furstman, L., Saporta, S., Kruger, L. : Retrograde axonal transport of horseradish peroxidase in sensory nerves and ganglion cells of the rat. Brain Res. 84, 320- 324 (1975) Graham, R. C., Karnovsky, M. J. : The early stages of absorption of injected horseradish peroxidase in the proximal tubules o mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291 - 302 (1966) Jacobs, J. M., MacFarlane, R. M., Cavanagh, I. B. : Vascular leakage in the dorsal root ganglia of the rat studied with horseradish peroxidase. J. neurol. Sci. 29, 95-107 (1976) Knotts, F. B., Cook, M. L., Stevens, J. G. : Pathogenesis of herpetic encephalitis in mice after ophthalmic inoculation. J. infect. Dis. 130, 16-27 (1974) Kristensson, K. : Retrograde axonal transport of protein tracers. In: The use of axonal transport for studies of neuronal connectivity (eds. W. M. Cowan and M. Cu6nod), pp. 70-82. Amsterdam: Elsevier 1975 Kristensson, K., Olsson, Y. : Retrograde axonal transport of protein. Brain Res. 29, 363-365 (1971)
52 La Vail, J. I-I. : Retrograde cell degeneration and retrograde transport techniques. In: The use of axonal transport for studies of neuronal connectivity (eds. W. M. Cowan and M. Cu6nod), pp. 217-248. Amsterdam: Elsevier 1975 La Vail, J. H., La Vail, M. M. : Retrograde axonal transport in the central nervous system. Science 176, 1416-1417 (1972) Marinesco, G. : Recherches sur la pathologic de certaines enc6phalomy61ites a ultravirus. Rev. neurol. 1, 1 - 36 (1932) Mazza, J. P., Dixon, A. D. : A histological study of chromatolytic cell groups in the trigeminal ganglion of the rat. Arch. oral Biol. 17, 377- 387 (1972) Mizuno, N., Konishi, A., Sato, M. : Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase. J. comp. Neurol. 164, 105-116 (1975) Pei, Y. F., Rhodin, J. A. G.: Electron microscopic study of the development of the mouse corneal epithelium. Invest. Ophthal. 10, 81I - 8 2 5 (1971) Rabin, E. R., Bennett Jenson, A., Melnick, J. L. : Herpes simplex virus in mice: Electron microscopy of neural spread. Science 162, 126-127 (1968)
Acta neuropath. (Bed.) 38 (1977) Sakai, A. : Innervation and afferent fiber projection of rat incisor pulp. Bull. Tokyo med. dent. Univ. 21 (Suppl.), 22-24 (1974) Stevens, J. G., Nesburn, A. B., Cook, M. L. : Latent herpes simplex virus from trigeminal ganglia of rabbits with recurrent eye infection. Nature (Lond.) 235, 216-217 (1972) Strassburg, M. : Morphologic reaction of the trigeminal ganglion after experimental surgery on the maxillodental region. J. Oral Surg. 25, 107-114 (1967) TCnjum, A. M.: Permeability of horseradish peroxidase in the rabbit corneal epithelium. Acta ophthal. (Kbh.) 52, 650-658 (1974) Whitear, M. : An electron microscope study of' the cornea in mice with special reference to the innervation. J. Anat. (Lond.) 94, 387-409 (1960) Zander, E., Weddell, G.: Observations on the innervation of the cornea. J. Anat. (Lond.) 85, 68-99 (1951)
Received October 27, 1976/Accepted November 5, 1976
Acta neuropath. (Berl.) 38, 4 9 - 52 (1977)
9 by Springer-Verlag I977
Retrograde Axonal Transport of Horseradish Peroxidase from Cornea to Trigeminal Ganglion Bj6rn Arvidson
Neuropathological Laboratory, Institute of Pathology, University of Uppsala, Box 553, S-75122 Uppsala, Sweden
S u m m a r y . Horseradish peroxidase (HRP) was dripped
on the scarified left cornea of adult mice. Twentyfour hours later the animals were fixed by vascular perfusion and frozen sections cut from both trigeminal ganglia. After incubation for peroxidase activity labelled nerve cells were restricted to the medial ophthalmic part of the ganglion ipsilateral to HRP administration. If the scarification was omitted no neuronal labelling was observed. This labelling of the neurons is most probably the result from axonal uptake and subsequent retrograde axonal transport of the tracer. The similarity in distribution of peroxidase labelled nerve cells and the first ganglionic lesions occurring after instillation of herpes simplex virus in the cornea is pointed out. Key words: Cornea - Trigeminal ganglion trograde axonat transport.
Re-
Introduction
Different methods have been used in investigations concerning a somatotopical organization of nerve cell bodies in the trigeminal ganglion. Physiological studies of this ganglion in the cat indicate that cell bodies of the mandibular nerve are located posterolaterally, those of the ophthalmic nerve anteromedially and those of the maxillary nerve in the area b e t w e e n these two locations (Darian-Smith et al., Beaudreau and Jerge). Anatomical studies of chromatolytic cell changes in the trigeminal ganglion after nerve section have been made in several species including rat, rabbit and cat (Allen; Strassburg; Mazza and Dixon; Sakai). After mandibular nerve section in the rat chromatolytic nerve cells were concentrated laterally in the ganglion. The ophthalmic neurons seemed to be located mostly
medially and the neurons of the maxillary nerve b e t w e e n these cell groups (Sakai). In the cat a large cephalic and median portion was made up only of ophthalmic-maxillary cells, while a smaller lateral and caudal division consisted entirely of mandibular cells (Allen). After unilateral maxillary dental extraction in rabbits chromatotytic nerve cell changes were observed in a clearly determinable region of the central maxillary ganglion portion (Strassburg). The ophthalmic division of the trigeminal nerve has been used for several experimental studies on viral transmission along peripheral sensory nerves. After corneal inoculation of herpes simplex virus (HSV) the virus is capable of travelling in the nerve to produce a primary infection in neurons of the Gasserian ganglion (Marinesco; Baringer and Griffith; Stevens et al. ; Baringer and Swoveland; Knotts et al.). Intranasal inoculation of HSV also resulted in an infection of neurons in the trigeminal ganglion (Rabin et al.). The retrograde horseradish peroxidase method (Kristensson and Olsson) has been used extensively for mapping neuroanatomical pathways both in the central (La Vail and La Vail; La Vail) and peripheral (Kristensson and Olsson; Kristensson) nervous system. With regard to the trigeminal nerve this method has previously been used for tracing the cell bodies in the trigeminal ganglion innervating the dental pulp of different teeth (Furstman et al. ; Arvidsson). Within the motor division of the fifth nerve a topographical localization of neurons supplying the masticatory muscles has been described with use of the same method (Mizuno et al.). The present study was undertaken for several reasons. It was of interest to find out if a foreign protein such as H R P is taken up in axonal branches in the ophthalmic nerve and transported back to the trigeminal ganglion as has been suggested for certain viral infections (Baringer and Swoveland; Cooks et
50
Acta neuropath. (Berl.) 38 (1977)
~ant lat Fig. 1
Peroxidase labelled nerve cell in the trigeminal ganglion, x 800 Fig. 2
I ,,-I
i
al.). In addition this study is part o f a project studying the cellular localization o f foreign proteins in peripheral ganglia following various modes o f administration (Arvidson, to be published).
M a t e r i a l s and M e t h o d s
The experiments were performed on fourteen adult male albino mice. After local anesthesia of the cornea with 4 700lidocain and general ether anesthesia the left cornea was scratched with a needle in a checked pattern. About 5 gl of a 40 % HRP solution in isotonic saline was then dripped in the left eye and the eyelid sutured to prevent escape of the tracer. In a few animals the scarification of the cornea was omitted prior to HRP administration. In some of these animals the eyelid was also left without suture. After 24 h the animals were perfused through the heart with 2.0 % glutaraldehyde in S6rensens phosphate buffer, pH 7.4. Both trigeminal ganglia were dissected out and placed in cold fixative for additional 4 h. Overnight they were washed in phosphate buffer with 5 sucrose at 4~C. The following day both ganglia were placed horizontally and cut into 40 gm thick serial sections on a freezing microtome. Every second section was sampled. The sections were preincubated for 15 min in 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in Tris-buffer at pH 7.5. Then followed incubation in the full medium of Graham and Karnovsky with the addition of hydrogen peroxide for 45 min at room temperature. They were mounted without counterstaining and examined by light microscopy.
Results
HRP-labelled nerve cells were identified by the presence o f small darkstaining granules in the cytoplasm (Fig. 1). Such cells were only f o u n d in the ganglion ipsilateral to the administration o f H R P . Labelled neurons were limited to the anteromedial p o r t i o n
Schematic drawing of the left trigeminal ganglion of one experimental animal showing the distribution of peroxidase labelled nerve cells, m mandibular nerve; o m opthalmic-maxillary nerve
2
o f the ganglion and were often located superficially u n d e r the ganglionic capsule (Fig. 2). Both large and small neurons were labelled. In all animals given H R P after corneal scarification labelled cells were f o u n d but the n u m b e r varied between individual mice with a m a x i m u m o f 80 and a m i n i m u m o f 16 labelled cells per ganglion. The reason for this variation in the n u m b e r o f labelled cells is difficult to understand but m a y be explained by variations in the technical procedure o f H R P administration. The average n u m b e r o f labelled cells was 44 per ganglion. In animals where the corneal scarification was omitted and the eyelid sutured only two or three m a r k e d nerve cells were identified in the ipsilateral ganglion. U p t a k e o f peroxidase in sensory nerve endings injured by the lid suture m a y explain these findings. If the eyelid was also left without suture no n e u r o n a l labelling t o o k place. Discussion
The nervous innervation o f the cornea has been extensively investigated. Studies on the rabbit cornea have shown that nerve bundles f r o m the ciliary nerves enter the cornea in a radial m a n n e r and ultimately give rise to a plexiform aggregation o f nerve fibers which pervades the substantia propria (Zander and Weddell). In the mouse this plexus is arranged in three main layers; a deep, midstromal and subepithelial plexus. Myelinated fibers are f o u n d in the deep and midstromal plexus while only unmyelinated axons pass into the subepithelial plexus (Whitear). The corneal epithelium acts as a barrier against penetration o f foreign substances into the eye. In vitro studies o f rabbit cornea have demonstrated that
B, Arvidson: Transport of HRP from Cornea to Trigeminal Ganglion
HRP does not penetrate the surface epithelium after local application (T~bnjum). The imperviousness of the corneal epithelium can be ascribed to tight junctions between adjacent surface epithelial cells (Pei and Rhodin). This barrier apparently has to be broken before an uptake of HRP in nerve endings takes place since no labelled neurons are observed after application of HRP to the intact cornea. Following uptake in normal or injured nerve branches in the cornea HRP is most probably transported retrogradely in axons to the neuronal cell bodies in the trigeminal ganglion. Such a transport has previously been described in peripheral motor (Kristensson and Olsson) and sensory (Furstman et al. ; Arvidsson) neurons as well as in various central neurons (La Vail and La Vail; La Vail). Following such a transport HRP accumulates in a granular way close to the nucleus in the same way as in the present experiments and is in contrast to vascular administration of tracer when HRP initially accumulates in macrophages in the ganglia (Jacobs et al. ; Arvidson, to be published). Following vascular infusion of large doses of HRP in mice there is a retrograde axonal transport of this protein to neurons in neurosecretory, motor, sensory and autonomic systems (Broadwell and Brightman). The possibility of a retrograde neuronal labelling due to blood reabsorption of HRP injected into the tissues in microliter quantities has been discussed. This mechanism of neuronal labelling is not likely to occur in the present experiments since marked nerve ceils are only observed in a restricted area of the ganglion ipsilateral to the injection of peroxidase. Furthermore the cornea is avascular. The number of labelled nerve cells in the Gasserian ganglion in the present experiments should not be considered as the real number of neurons innervating the cornea. Variations in uptake of HRP in normal and injured nerves possibly resulting from failure to reach all sensory nerves may contribute to the low number of labelled neurons. In the present study marked nerve cells are only observed in the anteromedial part of the ganglion. This distribution is in agreement with earlier studies using physiological and degenerative methods. The present experiments can also be considered as a model for studies on routes taken by foreign substances after instillation in the cornea. In this connection spread of viral particles to the Gasserian ganglion is of special interest. After corneal inoculation of HSV in mice nerve cells in the Gasserian ganglion show morphological signs of infection. Only the medial portion of the ganglion corresponding to the ophthalmic branch is markedly involved (Knotts et al.). The distribution of infected neurons is thus
51
in agreement with the location of peroxidase labelled nerve cells after corneal instillation of HRP. The route taken by HSV after corneal inoculation has been debated although recent reports (Baringer and Swoveland; Knotts et al.) show that an intraaxonal spread is probable. The demonstration of an apparently intraaxonat transport of HRP in the same nerves and the similarity in distribution of infected and peroxidase labelled neurons in the trigeminal ganglion should give further support to this mode of virus spread. Acknowledgement. Supported by grants from the Swedish Medical Research Council, Project No. B77-17X-03020-08C from Riksf6reningen f6r trafik- och polioskadade. The author is indebted to Prof. Yngve Olsson for his advice and helpful suggestions with regard to the manuscript.
References Allen, W. F. : Localization in the ganglion semilunare of the cat. J. comp. Neurol. 38, 1 - 2 5 (1924) Arvidsson, J. : Location of cat trigeminal ganglion cells innervating dental pulp of upper and lower canines studied by retrograde transport of horseradish peroxidase. Brain Res. 99, 135-139 (1975) Baringer, J. R., Swoveland, P.: Persistent herpes simplex virus infection in rabbit trigeminal ganglia. Lab. Invest. 30, 230- 240 (1974) Baringer, J.R., Griffith, J . F . : Experimental herpes simplex encephalitis: early neuropathologic changes. J. Neuropath. exp. Neurol. 29, 89-104 (1970) Beaudreau, D. E., Jerge, C. R. : Somatotopic representation in the Gasserian ganglion of tactile peripheral fields in the cat. Arch. oral Biol. 13,247-256 (1968) Broadwell, R. D., Brightman, M.W.: Entry of peroxidase into neurons of the central and peripheral nervous systems from extracerebral and cerebral blood. J. comp. Neurol. 166, 257- 284 (1976) Darian-Smith, I., Mutton, P., Proctor, R.: Functional organization of tactile cutaneous afferents within the semilunar ganglion and trigeminal spinal tract of the cat. J. Neurophysiol. 28, 682-- 694 (1965) Furstman, L., Saporta, S., Kruger, L. : Retrograde axonal transport of horseradish peroxidase in sensory nerves and ganglion cells of the rat. Brain Res. 84, 320- 324 (1975) Graham, R. C., Karnovsky, M. J. : The early stages of absorption of injected horseradish peroxidase in the proximal tubules o mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291 - 302 (1966) Jacobs, J. M., MacFarlane, R. M., Cavanagh, I. B. : Vascular leakage in the dorsal root ganglia of the rat studied with horseradish peroxidase. J. neurol. Sci. 29, 95-107 (1976) Knotts, F. B., Cook, M. L., Stevens, J. G. : Pathogenesis of herpetic encephalitis in mice after ophthalmic inoculation. J. infect. Dis. 130, 16-27 (1974) Kristensson, K. : Retrograde axonal transport of protein tracers. In: The use of axonal transport for studies of neuronal connectivity (eds. W. M. Cowan and M. Cu6nod), pp. 70-82. Amsterdam: Elsevier 1975 Kristensson, K., Olsson, Y. : Retrograde axonal transport of protein. Brain Res. 29, 363-365 (1971)
52 La Vail, J. I-I. : Retrograde cell degeneration and retrograde transport techniques. In: The use of axonal transport for studies of neuronal connectivity (eds. W. M. Cowan and M. Cu6nod), pp. 217-248. Amsterdam: Elsevier 1975 La Vail, J. H., La Vail, M. M. : Retrograde axonal transport in the central nervous system. Science 176, 1416-1417 (1972) Marinesco, G. : Recherches sur la pathologic de certaines enc6phalomy61ites a ultravirus. Rev. neurol. 1, 1 - 36 (1932) Mazza, J. P., Dixon, A. D. : A histological study of chromatolytic cell groups in the trigeminal ganglion of the rat. Arch. oral Biol. 17, 377- 387 (1972) Mizuno, N., Konishi, A., Sato, M. : Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase. J. comp. Neurol. 164, 105-116 (1975) Pei, Y. F., Rhodin, J. A. G.: Electron microscopic study of the development of the mouse corneal epithelium. Invest. Ophthal. 10, 81I - 8 2 5 (1971) Rabin, E. R., Bennett Jenson, A., Melnick, J. L. : Herpes simplex virus in mice: Electron microscopy of neural spread. Science 162, 126-127 (1968)
Acta neuropath. (Bed.) 38 (1977) Sakai, A. : Innervation and afferent fiber projection of rat incisor pulp. Bull. Tokyo med. dent. Univ. 21 (Suppl.), 22-24 (1974) Stevens, J. G., Nesburn, A. B., Cook, M. L. : Latent herpes simplex virus from trigeminal ganglia of rabbits with recurrent eye infection. Nature (Lond.) 235, 216-217 (1972) Strassburg, M. : Morphologic reaction of the trigeminal ganglion after experimental surgery on the maxillodental region. J. Oral Surg. 25, 107-114 (1967) TCnjum, A. M.: Permeability of horseradish peroxidase in the rabbit corneal epithelium. Acta ophthal. (Kbh.) 52, 650-658 (1974) Whitear, M. : An electron microscope study of' the cornea in mice with special reference to the innervation. J. Anat. (Lond.) 94, 387-409 (1960) Zander, E., Weddell, G.: Observations on the innervation of the cornea. J. Anat. (Lond.) 85, 68-99 (1951)
Received October 27, 1976/Accepted November 5, 1976
