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Intraocular transplants of olfactory neuroepithelium in rat
Int. J. Devl. Neuroscience,Vol. 1, No. 415,pp. 27%287,1983
0736-.5748/83 $3.~+0.~ Pergamon Press Ltd. @ 1983ISDN
Printed in Great Britain.
INTRAOCULAR TRANSPLANTS OF OLFACTORY NEUROEPITHELIUM IN RAT JOHN
A. HECKROTH,G. A. MONTIGRAZIADEIand PASQUALEP. C. GRAZIADEI*
Department of Biological Sciences, Florida State University, Tallahassee, FL 32306, U.S.A. (Accepted 20 April 1983)
Abstract-Olfactory neuro~pitheliumof neonatal rat pupshas been transplanted in the anterior chamber of the eye of adult rats. Structural and ultrast~ctural observations at 5, IO, 30,50,90 and 120 days show that mature neurons degenerate rapidly in the explant (before 5 days) while the basal elements proliferate and produce a new population of young neurons between 1 and 10 days. At longer survivals (30-120 days) it is seen that the neurons acquire morphologicat maturity, and positivity to the olfactory marker protein, as demonstrated by immunohistochemical methods. Our observations show that new neurons can be generated in the transplanted neuroepithelium and that their generation and maturation occurs in the absence of connections with a brain target. Key words: Transplantation,
Olfactory neuroepithelium.
The olfactory neurons are located in the neuroepithelium lining the sensory area of the nasal cavity of mammals.‘~‘4 Replacement of the olfactory neurons is assured by the presence, at the base of the neuroepithelium, of stem cells (basal cells)’ which mature and reconstitute a viable population of neurons after chemical’5*‘9 or mechani~a15~9~1’~1*~*1 d amage has induced their degeneration. Interestingly, replacement of the olfactory neurons occurs in normal animals, even independently of experimental lesions.637*8 Because the replacement of neurons is an exceptional phenomenon in adult mammals, possibly regulated by a variety of factors (some intrinsic to the neuroepithelium and the nasal cavity, some of more systemic nature), we have begun to study the behavior of the olfactory neurons and of their matrix when the neuroepithelium is transplanted from the nasal cavity to the anterior chamber of the eye. In this experimental framework, we intend to explore the capacity of the olfactory matrix to manufacture neurons when removed from its normal environment, and to explore the ability of these neurons to maintain their morpholo~cal characteristics and to produce olfactory marker protein (OMP).
METHODS Thirty Charles River albino rats of various ages (300-500 g) were used as hosts in this experiment. Pups, 5-8 days of age, served as donors. ._ Following decapitation, fragments of nasal neuroepithelium were removed from the septum and turbinates of donor pups and placed in Ringer’s lactate at room temperature. A 1% atropine solution was applied to the eyes of Nembutal anesthetized hosts to dilate the pupils. Epithelial fragments were introduced into the anterior chamber of both eyes of each host following the technique of Olson et al. 2o At 5 (n = 5), 10 (n = 5), 50 (n = 6), 90 (n = 5) and 120 (n = 4) days postoperatively, hosts were killed by Nembutal overdose and perfused through the heart with Ringer’s saline with procaine, followed by either Bouin’s fixative (for light microscopy: IZ= 15) or a mixture of aldehydes (for electron microscopy: n = 15). Material for light microscopy was embedded in paraffin, sectioned at 12 km, and stained with iron hematoxylin. Aldehyde fixed material was post-fixed in 0~04 and embedded in Araldite 506. These specimens were sectioned at 1 pm thickness and stained with methylene blue-Azur B for light microscopy. Appropriate areas were thin sectioned and stained with uranyl acetate and lead citrate for electron microscopic observations.
* To whom reprint requests should be addressed. 273
274
J. A. Heckroth et al.
The OMP was identified in the mature neurons with the peroxidase-antiperoxidase method.” The anti-OMP. used at a dilution of 1:40 was prepared by one of us (GAMG) according to the procedure described by Margolisi’ for the rat.
RESULTS Fragments of olfactory neuroepithelium with attached lamina propria, sometimes adherent to small bone lamellae from the nasal septum, survive well in the anterior chamber of the eye. A survival rate of 95% was obtained, discounting transplants lost through the cornea before the incision had sealed (during the first 12 h postoperatively). Only 60% of surviving transplants contained viable olfactory epithelium, however, the rest having epithelium only of the respiratory variety. At 5 days survival, the transplant is already richly vascularized with the epithelial lining, of both sensory and respiratory nature, tending to form vesicular or cystic structures. In light microscope preparations the thickness of the neuroepithelium is in the range of 20-30 pm (reduced from control values of 60-70 pm). Mature neurons have disappeared, while supporting cells and basal cells remain as the only cellular components. Among the basal cells there are many mitotic figures (only occasionally seen in controls). At the ultrastructural level, olfactory vesicles and cilia have disappeared from the surface, confirming the absence of mature neurons. The supporting cells form a non-interrupted layer at the surface of the epithelium and their morphology is similar to controls. Close to the basal lamina, cells with the ultrastructural characteristics of basal and globose basal cells as well as mitotic figures can be identified. Due to the use of young donors. the neuroepithelium contains a large number of immature elements which have survived the transplantation and continued their maturation process. At 10 days survival, the sensory epithelium shows regions of increased thickness up to about 40 pm. At the ultrastructural level the globose basal cells are increased in number and show further development exemplified by dendritic processes, some of which terminate close to or at the free epithelial surface. A few olfactory vesicles emerge from the epithelial surface. At longer survivals the process of maturation of the neural elements continues and by 30 days post-transplantation the epithelium has acquired stable characteristics unique to this experimental environment (Fig. 1). The sensory l~euroepithelium has a variable thickness in different regions of the same vesicle with values ranging from 20 to 60 p+rn(Fig. 2). Bowman’s glands are present in the lamina propria (Fig. I) and they open at the surface of the epithelium. Semi-thin sections of Araldite embedded material show the irregular outline of the deep boundaries of the neuroepithelium (Figs 2-4). Capillary loops deeply penetrate into the epithelial layer forming finger-like papillae not normally observed in the olfactory neuroepithelium lining the nasal cavity. In the lamina propria, where an extensive network of blood vessels is obvious (Figs 2-5) there are myelinated fibers running singly or in small bundles (Figs 3-5). At times these fibers are located only a few microns from the base of the epithelium. Several cellular types contribute to the sensory epithelium. Small, densely stained basal cells lie close to the basal lamina (Figs 4 and 5). Among and above the basal cells there are large, globose lightly stained cells (Figs 4 and 5) grouped in nests. Solid cords of these cells often protrude downwards into the lamina propria (Figs 3 and 4). In some instances the large, lightly stained cells have a pear-shaped outline and a distal process resembling a dendrite. A few mature neurons with a dense cytoplasm and a dendrite extending to the epithelial surface can also be recognized. Among the mature neurons pyknotic nuclei can be observed. The cytoplasm of the supporting celis forms a continuous layer in the upper third of the neuroepithelium. However, their nuclei have lost the regular arrangement in a discrete band (Fig. 4), an obvious characteristic in normal neuroepithelia. Frequently, in the transplanted neuroepithelium, cells with a very clear, unstained cytoplasm are present. They are found at all depths and their shape varies from irregularly globose to pear-shaped (Figs 2-S). Not uncommonly, a dendrite-like process reaching the surface can be observed. At the ultrastructural level, in the lamina propria, in addition to myelinated fibers, relatively large, unmyelinated axons run individually, invaginated in the Schwann cell cytoplasm (Fig. 6). Nerve bundles, formed by small, unmyelinated axons can also be observed (Fig. 7). The origin of the small, uIlmyelinated fibers can be traced from the base of the neuroepithelium (Fig. 8) and their olfactory sensory nature is likely. These small axons are characteristically arranged in large pockets
Fig. 1. Histological section of transplanted olfactory neuroepithelium (ne) provided with Bowman’s glands (bg). Below the epithelium, a loose, irregular connective tissue (Iamina propria, Im) adheres to bone lamellae (bl). 30 days survival. Hematoxylin-eosin stain. The bar indicates 100 p,m.
Fig. 2. Figures 2-5 are from semi-thin sections of Araldite embedded material at 50 days survival, stained with Methylene blue-Azur II. Figure 2 shows the rich vasculature in the lamina propria (Im), the uneven thickness of the neuroepitheiium (see points * and ** for thin and thick zones) and groups of globose, lightly stained cells (gb). Cells undergoing translucent degeneration (see text) are indicated (td). The amorphous material filling the cavity (am) contains cellular debris (cd). The bar indicates 100 pm. Fig. 3. Myelinated nerve fibers (my) run in the lamina propria. A capillary loop (ca) penetrates deep into the epithelial layer. The deep boundaries of the neuroepithelium are irregular and groups of basal and globose basal cells protrude in irregular masses into the lamina propria (at arrows). The section is not perpendicular to the epithelial surface; however, the groups of large, globose cells (gb) can be seen to form compact groups close to the epithelial base. The bar indicates 50 pm.
275
Fig. 4. Several cells with the characteristics of lamina propria. In the neuroepithelium many elements undergoing translucent degeneration (my). The
lightly stained globose elements (gb) protrude into the dendrites (d) are seen above the neuronal layer. Two (td) lie close to the epithelial surface Myelinated fiber bar indicates 50 pm
Fig. 5. Groups of globose cells (gb) are numerous and obvious in the center of the figure while mature neurons (n) are sparse. Myelinated fibers (my); elements undergoing translucent degeneration (td); basal cells (bc); blood vessels (bv). The bar indicates 50 pm.
276
Fig. 6. In the lamina propria of the olfactory mucosa there is a large myelinated fiber (my). In its vicinity there run several unmyehnated axons (ax) singularly enwrapped in Schwann cell pockets. 50 days survival. The bar indicates 1 pm. Fig. 7. An olfactory nerve fasicle composed of several subunits runs in the lamina propria. Notice that the sensory axons (ax) do not form a compact bundle in each of the sub-units. Electron clear spaces (s) possibly indicate a considerable remanagement in the organization of these bundles due to the degeneration-regeneration phenomena induced by the transplantation (see text). 30 days survival. The bar indicates 10 pm.
277
Fig. 8. On the right of the figure is the basal layer of the neuroepithelium with basal (bc) and globose basal (gb) cells. A small fasicle of axons exits from the neuroepithelium (at arrows). In the vicinity of a blood vessel (bv) there are globose basal cells (gb’) as p reviously indicated in Fig. 4. A large unmyelinated axon runs singularly (ax) between the blood vessel and the globose cells. Figure 10 includes a detail of gb’. 50 days survival. The bar indicates 10 pm.
27X
Fig. 9. In the basal region of the neuroepitheIium, there are basal (bc) and giobose basal cells (gb). The globose cells show a clear cytopfasm with many free ribosomes and a paucity of other organelles. Another cell, recognized as a young neuron (n) shows rER and a denser nucleus. Colfagenous fibers (c) invaginate into the epithelial layer in papillary structures (see text). 50 days survival. The bar indicates 1 pm. Fig, 10. A group of globose basal ceils, under the epithelium, in the Iamina propria are in close apposition to a blood vessel (bv). This figure is from the same section of Fig. 8 and represents a detail. The globose cells (gb) show the characteristics of similar elements in the neuroepith~I~um. The unmyel~nated axon (ax) is clearly seen invaginated into the Schwann ceil cytoplasm. The bar indicates 1 brn.
279
Fig. Il. A mitotic figure of a stem cell at the base of the neuroepitheiium shows a centriole (CT), chromosomes (ch) and the typical cytoplasm of the mitotic stem cells. 30 days survival. The bar indicates 1 km. Fig. It. Detail of the neuroepitheIja1 surface showing supporting cells (SC) with microvilli (mv). Several profiles of dendrites (d), some of them containing a number of basal bodies (bb) run between the supporting cells. Olfactory vesicles (ov) with cilia (ci) reach the epithelial surface. 30 days survival. The bar indicates 1 pm.
2x0
Fig. 13. Thin portion of neuroepithelium whose total thickness is 25 pm. Cllobose basal ceils (gb) and supporting cells (SC) are the only constituents of this epithelial segment. An element undergong translucent degeneration (td) occupies a midepithelial position. At the surface of the epithelium there are a few elements in advanced stages of degeneration (dg). 50 days survival. The bar indicates 5 pm. Fig. 14. Three elements undergoing translucent degeneration (td) are surrounded by the cytoplasm of supporting cells (SC). Many dendritic profiles (d), one of them filled with basal bodies (bb), indicate that morphologically mature neurons are present in this epithelial segment. 50 days survival. The bar indicates 5 p.m.
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Fig. IS. A cell undergoing translucent degeneration No other neural element is present in this portion degenerating cell (putative neuron) is indicated
(td) is directly apposed to the epithehal basal lamina. of the epithelium. Supporting cells (SC); profile of a at (d). 90 days survival. The bar indicates IO urn.
Fig. 16. A capillary loop (c) reaches close to the epithclial surface where olfactory vesicles (w) arc surrounded by microvilli (mi) of the supporting cells (SC). A basal lamina (hl) and collagenous tibcrs (cf) surround the capillary vessel. 90 days survival. The har indicates 1 pm.
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Fig. 17. A segment of neuroepithelium shows a capillary (c), a small nerve bundle (nb), supporting cells (SC)and putative neural elements in different stages of translucent degeneration (td). A young differentiating neuron has an obvious cilium (ci). This figure well represents the disorderly arrangement of the epithelial components which fail to be regularly layered in the fashion typical of the common olfactory neuroepithelium before transplantation. 90 days survival. The bar indicates 10 pm.
Fig. 18. The base of the neuroepithelium is indicated by arrowheads. A few OMP positive neurons (II) with their dendrite reaching the epithelial surface (arrows) contrast with the negative immature elements at the base of the epithelium. In the lamina propria there are positive nerve bundles (nb). 50 days survival. The bar indicates 100 pm.
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of the sheath cells. The pockets are not homogeneously filled by the unmyelinated axons (Fig. 7), and empty spaces can be observed among areas closely packed with fibers. In the neuroepithelium, the large, lightly stained cells observed in the light microscope preparations can be identified with the globose basal cells previously described in the normal epithelium.8 The globose cells have a characteristic nucleus with a relatively homogeneous cromatin pattern and obvious nucleoli. The cytoplasmic matrix is electron clear and filled with free ribosomes. Mitochondria, cisternae of the ER system and other organelles are sparse (Figs 8-10). In the epithelium the globose basal cells are the most numerous and noticeable elements as observed in low power light microscope preparations (Figs 2 and 5) and mitotic figures among them are common (Fig. 11). The same morphological characteristics of the globose basal cells apply to those cells displaced in cords, protruding downwards from the epithelium into the lamina propria (Figs 8 and 10; cf. 4 at LM). Mature neurons characterized by an electron dense cytoplasm with numerous profiles of rER and Golgi are present above the globose basal cells; however, their number is greatly reduced when compared with the number of similar cells in the normal neuroepithelium of rats. Many dendrites terminate with an olfactory knob at the surface of the neuroepithelium; others apparently terminate with a bulbous enlargement filled with basal bodies below the surface (Fig. 12). Their morphological appearance is typical of young dendrites (i.e. sparse number of cilia, numerous basal bodies located both in the olfactory knob and along the dendritic shaft). Degenerative figures of mature neurons, characterized by increased density of their cytoplasm, numerous lysosomes and vacuoles, and partial disintegration of the nuclear material are not uncommon in long survival preparations. These degenerating neurons are localized in the upper layer of the epithelium, at times close to its free surface. These elements can be recognized in a more advanced stage of degeneration inside the lumen of the vesicles, embedded in the amorphous matter (Fig. 13). At the ultrastructural level, the clear cells observed with the light microscope show an electronlucent cytoplasm and swollen organelles indicative of an ongoing degenerative process (translucent degeneration) different from the one just described for the mature neurons. These elements, showing on occasion a pear-shaped outline or a long apical process resembling a dendrite, are present in all layers of the epithelium; they may be found in intimate contact with the basal lamina or close to the epithelial surface (Figs 13-15). The blood vessels described above at the LM level as being contained in papillary, finger-like structures inside the epithelium are, at the ultrastructural level, seen to be surrounded by a thin layer of collagenous fibers and a basal lamina. We have observed them, at instances, only a few microns from the free epithelial surface (Figs 16 and 17). With immunohistochemical methods, at more than 30 days survival, only a small fraction of the neuronal population contains OMP (Fig. 18). The positive elements have the characteristic profile of mature neurons whose dendrite reaches the epithelial surface terminating with the bulbous olfactory vesicle. In the lamina propria there are numerous bundles of OMP positive axons. These bundles, whose origin can be traced to the epithelium are often observed to terminate in large neuromata.
DISCUSSION Our results indicate that the olfactory neuroepithelium of postnatal rats can be successfully grafted into the anterior chamber of the eye and that even in this altered environment new neurons can differentiate and mature. The occurrence of an active neurogenetic process is indicated by the early degeneration of the mature neurons, due to the severance of their axons during the transplantation procedure, and by the appearance of mitotic figures, numerous neural elements in various stages of differentiation, and the reappearance of mature OMP positive neurons whose axons form large nerve bundles terminating in neuromata. The frequent observation of mitotic figures and young developmental stages at long survival times (50-120 days) as well as of degenerating neurons is indicative of an ongoing process of neuronal renewal which is presently being investigated by means of [3H]thymidine autoradiography. Although the stem cells’ ability to produce new neurons apparently remains relatively unimpaired, the observed invagination of immature receptors into the lamina propria and the penetration of capillary loops deep among epithelial elements points to an altered capacity of the sensory
J. A. Heckroth et al.
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layer to maintain its regular organization. In the anterior chamber of the eye, moreover, the neuro~p~thelium does not organize in an open layer, typical of the nasal cavity, even when the presence of lamellar bone could provide a supportive structure. The intercommunicating vesicles containing amorphous material and cellular debris seem to be the characteristic adaptation of the neuroepithelium to the new environment. Two morphologically distinguishable types of degeneration have been noted in the olfactory epithelium. The more common form under normal circumstances is characterized by increased density of the cytoplasmic matrix, numerous lysosomes, and condensation of the nuclear pattern; it represents the terminal stage of the life cycle of the olfactory neurons8 The other type of degeneration, which we will refer to as translucent degeneration, involves a clearing of the cytoplasm and a swelling of membrane bound organelles. Translucent degeneration is observed only occasionally in normal material but appears with a greatly increased frequency in the intraocular transplants. Autoradiographic observations of the olfactory mucosa in situ indicate that those elements undergoing translucent degeneration originate from the neurogenetic matrix and that they acquire degenerative characteristics at early stages in their development (unpublished observations). Consequently, we tentatively interpret translucent degeneration as a manifestation of early neuronal death. Why this type of degeneration should be so prominent in the transplanted epithelium is not certain at present. Myelinated and large unmyelinated nerve fibers present in the transplants beyond 30 days survival must have invaded the transplant from the host’s iris, as any such fibers present in the tissue after removal from the donor would have degenerated over this time period. It is likely that the myelinated fibers are of trigeminal origin. ’ ’ Their functional impact is difficult to assess at present. The possibility that they replace the fibers of trigeminal origin normally present in the nasal sensory area’*’ in some trophic function must not be discounted. The large unmyelinated fibers” (probably of autonomic origin) may also play a role in the survival of the transplants. The presence of OMP positive neurons in the transplant beyond 30 days survival testifies to the ability of the transplanted neuronal elements to manufacture this protein upon reaching morphological maturity. l6 The neurons app arently do not need their target nor their natural environment of the nasal cavity to become OMP positive. It seems likely, on the basis of our so far limited data, that the matrix is predetermined to manufacture its neurons independently of environmental factors present in the nasal cavity and normal synaptic relations. Parallel transplantation experiments in amphibians, as well as developmental studies, show the capacity of the olfactory anlage to differentiate into a nasal organ even when transplanted far from its normal location. 23Recent observations in Xenopus indicate that the olfactory neurons reach morphological maturity well before contact with the brain is established during development.‘” Experimental embryological studies” also indicate that the olfatory anlage is specified at an early deveIopmenta1 stage, before the appearance of the neural plate. Our observations of intraocular grafting provide the opportunity to study the differentiation of the olfactory neurons outside the environment of the nasal cavity where they demonstrate the ability to migrate from the epithelial environment, a property which is characteristic of many neurons during development. The survival and maturation of olfactory neurons in the anterior chamber of the eye could provide an ideal material for electrophysiological studies, as suggested for other neuronal populations by the work of Hoffer ef al.‘” Ack,l(lwlec~~emmts-This
work was supported
by a grant from the National
Institutes
of Health,
ROl-MS16421.
REFERENCES 1. Allison A. C. (1953) The morphology of the olfactory system in the vertebrates. &of. Kev. 2, 195-244. Anatomie der menschlichen Nasenhonle. Arch. mikrosk. Anat. 39, 2. Brunn A. von (1892) Beitrage zur mikroskopischen 63245 1. E. (1957) The head pattern in amblystoma studied by vital staining and transplantation methods. J. exp. 3. Carpenter Bid. 75, 103-129. of olfactory epithelium. Z. Zellforsch. mikrosk. Anaf. 4. Graziadei P. P. C. &LGagne H. T. (1973) Extrinsic innervation 138,315-326. sensoryneurons in 5. Graziadei P. P. C., Karian M. S., Monti Graziadei G. A. & Bernstein J. J. (1980) Neurogenesis the primate olfactory system after section of the fita olfactoria. Bruin Rex 1,289-300.
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6. Graziadei P. P. C. & Metcalf J. F. (1971) Autoradiographic and ultrastructural observations of the frog’s olfactory mucosa. 2. Zellfarsch. mikrosk. Anat. 116,305-318. 7. Graziadei P. P. C. & Monti Graziadei G. A. (1978) Continuous nerve cell renewal in the olfactory system. In Handbook of Sensory Physiology (ed. Jacobson M.), Vol. 9, pp. 55-82. Springer Verlag, New York. 8. Graziadei P. P. C. & Monti Graziadei G. A. (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons. J. Neurocytot. 8,1-l& 9. Harding J., Graziadei P. P. C., Monti Graziadei G. A. & Margolis F. L. (1977) Denervation
in the primary olfactory pathway of mice. 3min Ref. f32,1 l-28. 10. Hoffer 3., Seiger A., Freedman R., Olsen L. &Taylor D. (1977) Ele~rophysiolo~ and cytology of hjppocampal formation transplants in the anterior chamber of the eye. II. Cholinergic mechanisms. Bruin Res. 119,107-132. 11. Huhtala A. (1976) Origin of myelinated nerves in the rat iris. Expl. Eye Res. 22,259-265. 12. Keller A. & Margolis F. L. (1976) Isolation and characterization of rat olfactory marker protein. J. biuf. Chem. 251, 6232. 13. Klein S. L. & Graziadei P. P. C. (1983) The differentiation of the olfactory placode in Xenupus iaevis: a light and electron microscope study. .I. camp. Neural. 217, 17-30. 14. Kolmer W. (1927) Geruchsorgan. In Havtdbuch der Mikroskopbche Anatomie des Menschen, Vol. 3, pp. 2-249. Springer-Verlag, Berlin. 1.5. Matulionis D. H. (1975) Ultrastructural study of mouse olfactory epithelium following destruction by ZnS04 and its subsequent regeneration. Am. J. Anat. l&,87-90. 16. Miraeall F. & Monti Graziadei G. A. (1982) Exoerimental studies on the olfactorv marker motein, II. Appearance of the $actory marker protein during difiereniiati& of the olfactory sensory neuronsof mousd: an immunol%tochemical and autoradiographic study. Brain Res. 239,245-250. 17. Monti Graziadei G. A. & Graziadei P. P. C. (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reinstitution of the olfactory sensory neurons after axotomy. b. ~~~?o~~ro~. 8,197213. 18. Monti Graziadei G. A., Karlan M. S., Bernstein J. J. & Graziadei P. P. C. (1980) Reinnervation of the olfactory bulb after section of the olfactory nerve in monkey (Suimiri sciureus). Brain Res. 189,343-354. 19. Mulvaney B. D. & Heist H. E. (1971) Regeneration of rabbit olfactory epithelium. Am. J. An& 131,241~252. 20. Olson L. & Seiger A. (1972) Brain tissue transplanted to the anterior chamber of the eye. 1. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons reinnervating the rat iris. 2. Zelvorsch. 135, 175-194. 21. Schultz E. W. (1960) Repair of the olfactory mucosa with special reference to regeneration of the olfactory cells (sensory neurons). Am. J. Path. 37, I-19. 22. Sternberger L. A,, Hardy P. H. Jr., Cuculis J. J. & Meyer H. G. (1970) The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antige-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J. Htitochevn. Cytochem. 18,315. 23. Stout R. P. & Graziadei P. P. C. (1980) Influence of the olfactory placode on the development of the brain in Xenopus iaevis (daudin). 1. Axonal growth and connections of the transplanted olfactory p&code. Neuroscience 5,21’?5-2186.
0736-.5748/83 $3.~+0.~ Pergamon Press Ltd. @ 1983ISDN
Printed in Great Britain.
INTRAOCULAR TRANSPLANTS OF OLFACTORY NEUROEPITHELIUM IN RAT JOHN
A. HECKROTH,G. A. MONTIGRAZIADEIand PASQUALEP. C. GRAZIADEI*
Department of Biological Sciences, Florida State University, Tallahassee, FL 32306, U.S.A. (Accepted 20 April 1983)
Abstract-Olfactory neuro~pitheliumof neonatal rat pupshas been transplanted in the anterior chamber of the eye of adult rats. Structural and ultrast~ctural observations at 5, IO, 30,50,90 and 120 days show that mature neurons degenerate rapidly in the explant (before 5 days) while the basal elements proliferate and produce a new population of young neurons between 1 and 10 days. At longer survivals (30-120 days) it is seen that the neurons acquire morphologicat maturity, and positivity to the olfactory marker protein, as demonstrated by immunohistochemical methods. Our observations show that new neurons can be generated in the transplanted neuroepithelium and that their generation and maturation occurs in the absence of connections with a brain target. Key words: Transplantation,
Olfactory neuroepithelium.
The olfactory neurons are located in the neuroepithelium lining the sensory area of the nasal cavity of mammals.‘~‘4 Replacement of the olfactory neurons is assured by the presence, at the base of the neuroepithelium, of stem cells (basal cells)’ which mature and reconstitute a viable population of neurons after chemical’5*‘9 or mechani~a15~9~1’~1*~*1 d amage has induced their degeneration. Interestingly, replacement of the olfactory neurons occurs in normal animals, even independently of experimental lesions.637*8 Because the replacement of neurons is an exceptional phenomenon in adult mammals, possibly regulated by a variety of factors (some intrinsic to the neuroepithelium and the nasal cavity, some of more systemic nature), we have begun to study the behavior of the olfactory neurons and of their matrix when the neuroepithelium is transplanted from the nasal cavity to the anterior chamber of the eye. In this experimental framework, we intend to explore the capacity of the olfactory matrix to manufacture neurons when removed from its normal environment, and to explore the ability of these neurons to maintain their morpholo~cal characteristics and to produce olfactory marker protein (OMP).
METHODS Thirty Charles River albino rats of various ages (300-500 g) were used as hosts in this experiment. Pups, 5-8 days of age, served as donors. ._ Following decapitation, fragments of nasal neuroepithelium were removed from the septum and turbinates of donor pups and placed in Ringer’s lactate at room temperature. A 1% atropine solution was applied to the eyes of Nembutal anesthetized hosts to dilate the pupils. Epithelial fragments were introduced into the anterior chamber of both eyes of each host following the technique of Olson et al. 2o At 5 (n = 5), 10 (n = 5), 50 (n = 6), 90 (n = 5) and 120 (n = 4) days postoperatively, hosts were killed by Nembutal overdose and perfused through the heart with Ringer’s saline with procaine, followed by either Bouin’s fixative (for light microscopy: IZ= 15) or a mixture of aldehydes (for electron microscopy: n = 15). Material for light microscopy was embedded in paraffin, sectioned at 12 km, and stained with iron hematoxylin. Aldehyde fixed material was post-fixed in 0~04 and embedded in Araldite 506. These specimens were sectioned at 1 pm thickness and stained with methylene blue-Azur B for light microscopy. Appropriate areas were thin sectioned and stained with uranyl acetate and lead citrate for electron microscopic observations.
* To whom reprint requests should be addressed. 273
274
J. A. Heckroth et al.
The OMP was identified in the mature neurons with the peroxidase-antiperoxidase method.” The anti-OMP. used at a dilution of 1:40 was prepared by one of us (GAMG) according to the procedure described by Margolisi’ for the rat.
RESULTS Fragments of olfactory neuroepithelium with attached lamina propria, sometimes adherent to small bone lamellae from the nasal septum, survive well in the anterior chamber of the eye. A survival rate of 95% was obtained, discounting transplants lost through the cornea before the incision had sealed (during the first 12 h postoperatively). Only 60% of surviving transplants contained viable olfactory epithelium, however, the rest having epithelium only of the respiratory variety. At 5 days survival, the transplant is already richly vascularized with the epithelial lining, of both sensory and respiratory nature, tending to form vesicular or cystic structures. In light microscope preparations the thickness of the neuroepithelium is in the range of 20-30 pm (reduced from control values of 60-70 pm). Mature neurons have disappeared, while supporting cells and basal cells remain as the only cellular components. Among the basal cells there are many mitotic figures (only occasionally seen in controls). At the ultrastructural level, olfactory vesicles and cilia have disappeared from the surface, confirming the absence of mature neurons. The supporting cells form a non-interrupted layer at the surface of the epithelium and their morphology is similar to controls. Close to the basal lamina, cells with the ultrastructural characteristics of basal and globose basal cells as well as mitotic figures can be identified. Due to the use of young donors. the neuroepithelium contains a large number of immature elements which have survived the transplantation and continued their maturation process. At 10 days survival, the sensory epithelium shows regions of increased thickness up to about 40 pm. At the ultrastructural level the globose basal cells are increased in number and show further development exemplified by dendritic processes, some of which terminate close to or at the free epithelial surface. A few olfactory vesicles emerge from the epithelial surface. At longer survivals the process of maturation of the neural elements continues and by 30 days post-transplantation the epithelium has acquired stable characteristics unique to this experimental environment (Fig. 1). The sensory l~euroepithelium has a variable thickness in different regions of the same vesicle with values ranging from 20 to 60 p+rn(Fig. 2). Bowman’s glands are present in the lamina propria (Fig. I) and they open at the surface of the epithelium. Semi-thin sections of Araldite embedded material show the irregular outline of the deep boundaries of the neuroepithelium (Figs 2-4). Capillary loops deeply penetrate into the epithelial layer forming finger-like papillae not normally observed in the olfactory neuroepithelium lining the nasal cavity. In the lamina propria, where an extensive network of blood vessels is obvious (Figs 2-5) there are myelinated fibers running singly or in small bundles (Figs 3-5). At times these fibers are located only a few microns from the base of the epithelium. Several cellular types contribute to the sensory epithelium. Small, densely stained basal cells lie close to the basal lamina (Figs 4 and 5). Among and above the basal cells there are large, globose lightly stained cells (Figs 4 and 5) grouped in nests. Solid cords of these cells often protrude downwards into the lamina propria (Figs 3 and 4). In some instances the large, lightly stained cells have a pear-shaped outline and a distal process resembling a dendrite. A few mature neurons with a dense cytoplasm and a dendrite extending to the epithelial surface can also be recognized. Among the mature neurons pyknotic nuclei can be observed. The cytoplasm of the supporting celis forms a continuous layer in the upper third of the neuroepithelium. However, their nuclei have lost the regular arrangement in a discrete band (Fig. 4), an obvious characteristic in normal neuroepithelia. Frequently, in the transplanted neuroepithelium, cells with a very clear, unstained cytoplasm are present. They are found at all depths and their shape varies from irregularly globose to pear-shaped (Figs 2-S). Not uncommonly, a dendrite-like process reaching the surface can be observed. At the ultrastructural level, in the lamina propria, in addition to myelinated fibers, relatively large, unmyelinated axons run individually, invaginated in the Schwann cell cytoplasm (Fig. 6). Nerve bundles, formed by small, unmyelinated axons can also be observed (Fig. 7). The origin of the small, uIlmyelinated fibers can be traced from the base of the neuroepithelium (Fig. 8) and their olfactory sensory nature is likely. These small axons are characteristically arranged in large pockets
Fig. 1. Histological section of transplanted olfactory neuroepithelium (ne) provided with Bowman’s glands (bg). Below the epithelium, a loose, irregular connective tissue (Iamina propria, Im) adheres to bone lamellae (bl). 30 days survival. Hematoxylin-eosin stain. The bar indicates 100 p,m.
Fig. 2. Figures 2-5 are from semi-thin sections of Araldite embedded material at 50 days survival, stained with Methylene blue-Azur II. Figure 2 shows the rich vasculature in the lamina propria (Im), the uneven thickness of the neuroepitheiium (see points * and ** for thin and thick zones) and groups of globose, lightly stained cells (gb). Cells undergoing translucent degeneration (see text) are indicated (td). The amorphous material filling the cavity (am) contains cellular debris (cd). The bar indicates 100 pm. Fig. 3. Myelinated nerve fibers (my) run in the lamina propria. A capillary loop (ca) penetrates deep into the epithelial layer. The deep boundaries of the neuroepithelium are irregular and groups of basal and globose basal cells protrude in irregular masses into the lamina propria (at arrows). The section is not perpendicular to the epithelial surface; however, the groups of large, globose cells (gb) can be seen to form compact groups close to the epithelial base. The bar indicates 50 pm.
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Fig. 4. Several cells with the characteristics of lamina propria. In the neuroepithelium many elements undergoing translucent degeneration (my). The
lightly stained globose elements (gb) protrude into the dendrites (d) are seen above the neuronal layer. Two (td) lie close to the epithelial surface Myelinated fiber bar indicates 50 pm
Fig. 5. Groups of globose cells (gb) are numerous and obvious in the center of the figure while mature neurons (n) are sparse. Myelinated fibers (my); elements undergoing translucent degeneration (td); basal cells (bc); blood vessels (bv). The bar indicates 50 pm.
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Fig. 6. In the lamina propria of the olfactory mucosa there is a large myelinated fiber (my). In its vicinity there run several unmyehnated axons (ax) singularly enwrapped in Schwann cell pockets. 50 days survival. The bar indicates 1 pm. Fig. 7. An olfactory nerve fasicle composed of several subunits runs in the lamina propria. Notice that the sensory axons (ax) do not form a compact bundle in each of the sub-units. Electron clear spaces (s) possibly indicate a considerable remanagement in the organization of these bundles due to the degeneration-regeneration phenomena induced by the transplantation (see text). 30 days survival. The bar indicates 10 pm.
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Fig. 8. On the right of the figure is the basal layer of the neuroepithelium with basal (bc) and globose basal (gb) cells. A small fasicle of axons exits from the neuroepithelium (at arrows). In the vicinity of a blood vessel (bv) there are globose basal cells (gb’) as p reviously indicated in Fig. 4. A large unmyelinated axon runs singularly (ax) between the blood vessel and the globose cells. Figure 10 includes a detail of gb’. 50 days survival. The bar indicates 10 pm.
27X
Fig. 9. In the basal region of the neuroepitheIium, there are basal (bc) and giobose basal cells (gb). The globose cells show a clear cytopfasm with many free ribosomes and a paucity of other organelles. Another cell, recognized as a young neuron (n) shows rER and a denser nucleus. Colfagenous fibers (c) invaginate into the epithelial layer in papillary structures (see text). 50 days survival. The bar indicates 1 pm. Fig, 10. A group of globose basal ceils, under the epithelium, in the Iamina propria are in close apposition to a blood vessel (bv). This figure is from the same section of Fig. 8 and represents a detail. The globose cells (gb) show the characteristics of similar elements in the neuroepith~I~um. The unmyel~nated axon (ax) is clearly seen invaginated into the Schwann ceil cytoplasm. The bar indicates 1 brn.
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Fig. Il. A mitotic figure of a stem cell at the base of the neuroepitheiium shows a centriole (CT), chromosomes (ch) and the typical cytoplasm of the mitotic stem cells. 30 days survival. The bar indicates 1 km. Fig. It. Detail of the neuroepitheIja1 surface showing supporting cells (SC) with microvilli (mv). Several profiles of dendrites (d), some of them containing a number of basal bodies (bb) run between the supporting cells. Olfactory vesicles (ov) with cilia (ci) reach the epithelial surface. 30 days survival. The bar indicates 1 pm.
2x0
Fig. 13. Thin portion of neuroepithelium whose total thickness is 25 pm. Cllobose basal ceils (gb) and supporting cells (SC) are the only constituents of this epithelial segment. An element undergong translucent degeneration (td) occupies a midepithelial position. At the surface of the epithelium there are a few elements in advanced stages of degeneration (dg). 50 days survival. The bar indicates 5 pm. Fig. 14. Three elements undergoing translucent degeneration (td) are surrounded by the cytoplasm of supporting cells (SC). Many dendritic profiles (d), one of them filled with basal bodies (bb), indicate that morphologically mature neurons are present in this epithelial segment. 50 days survival. The bar indicates 5 p.m.
281
Fig. IS. A cell undergoing translucent degeneration No other neural element is present in this portion degenerating cell (putative neuron) is indicated
(td) is directly apposed to the epithehal basal lamina. of the epithelium. Supporting cells (SC); profile of a at (d). 90 days survival. The bar indicates IO urn.
Fig. 16. A capillary loop (c) reaches close to the epithclial surface where olfactory vesicles (w) arc surrounded by microvilli (mi) of the supporting cells (SC). A basal lamina (hl) and collagenous tibcrs (cf) surround the capillary vessel. 90 days survival. The har indicates 1 pm.
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Fig. 17. A segment of neuroepithelium shows a capillary (c), a small nerve bundle (nb), supporting cells (SC)and putative neural elements in different stages of translucent degeneration (td). A young differentiating neuron has an obvious cilium (ci). This figure well represents the disorderly arrangement of the epithelial components which fail to be regularly layered in the fashion typical of the common olfactory neuroepithelium before transplantation. 90 days survival. The bar indicates 10 pm.
Fig. 18. The base of the neuroepithelium is indicated by arrowheads. A few OMP positive neurons (II) with their dendrite reaching the epithelial surface (arrows) contrast with the negative immature elements at the base of the epithelium. In the lamina propria there are positive nerve bundles (nb). 50 days survival. The bar indicates 100 pm.
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of the sheath cells. The pockets are not homogeneously filled by the unmyelinated axons (Fig. 7), and empty spaces can be observed among areas closely packed with fibers. In the neuroepithelium, the large, lightly stained cells observed in the light microscope preparations can be identified with the globose basal cells previously described in the normal epithelium.8 The globose cells have a characteristic nucleus with a relatively homogeneous cromatin pattern and obvious nucleoli. The cytoplasmic matrix is electron clear and filled with free ribosomes. Mitochondria, cisternae of the ER system and other organelles are sparse (Figs 8-10). In the epithelium the globose basal cells are the most numerous and noticeable elements as observed in low power light microscope preparations (Figs 2 and 5) and mitotic figures among them are common (Fig. 11). The same morphological characteristics of the globose basal cells apply to those cells displaced in cords, protruding downwards from the epithelium into the lamina propria (Figs 8 and 10; cf. 4 at LM). Mature neurons characterized by an electron dense cytoplasm with numerous profiles of rER and Golgi are present above the globose basal cells; however, their number is greatly reduced when compared with the number of similar cells in the normal neuroepithelium of rats. Many dendrites terminate with an olfactory knob at the surface of the neuroepithelium; others apparently terminate with a bulbous enlargement filled with basal bodies below the surface (Fig. 12). Their morphological appearance is typical of young dendrites (i.e. sparse number of cilia, numerous basal bodies located both in the olfactory knob and along the dendritic shaft). Degenerative figures of mature neurons, characterized by increased density of their cytoplasm, numerous lysosomes and vacuoles, and partial disintegration of the nuclear material are not uncommon in long survival preparations. These degenerating neurons are localized in the upper layer of the epithelium, at times close to its free surface. These elements can be recognized in a more advanced stage of degeneration inside the lumen of the vesicles, embedded in the amorphous matter (Fig. 13). At the ultrastructural level, the clear cells observed with the light microscope show an electronlucent cytoplasm and swollen organelles indicative of an ongoing degenerative process (translucent degeneration) different from the one just described for the mature neurons. These elements, showing on occasion a pear-shaped outline or a long apical process resembling a dendrite, are present in all layers of the epithelium; they may be found in intimate contact with the basal lamina or close to the epithelial surface (Figs 13-15). The blood vessels described above at the LM level as being contained in papillary, finger-like structures inside the epithelium are, at the ultrastructural level, seen to be surrounded by a thin layer of collagenous fibers and a basal lamina. We have observed them, at instances, only a few microns from the free epithelial surface (Figs 16 and 17). With immunohistochemical methods, at more than 30 days survival, only a small fraction of the neuronal population contains OMP (Fig. 18). The positive elements have the characteristic profile of mature neurons whose dendrite reaches the epithelial surface terminating with the bulbous olfactory vesicle. In the lamina propria there are numerous bundles of OMP positive axons. These bundles, whose origin can be traced to the epithelium are often observed to terminate in large neuromata.
DISCUSSION Our results indicate that the olfactory neuroepithelium of postnatal rats can be successfully grafted into the anterior chamber of the eye and that even in this altered environment new neurons can differentiate and mature. The occurrence of an active neurogenetic process is indicated by the early degeneration of the mature neurons, due to the severance of their axons during the transplantation procedure, and by the appearance of mitotic figures, numerous neural elements in various stages of differentiation, and the reappearance of mature OMP positive neurons whose axons form large nerve bundles terminating in neuromata. The frequent observation of mitotic figures and young developmental stages at long survival times (50-120 days) as well as of degenerating neurons is indicative of an ongoing process of neuronal renewal which is presently being investigated by means of [3H]thymidine autoradiography. Although the stem cells’ ability to produce new neurons apparently remains relatively unimpaired, the observed invagination of immature receptors into the lamina propria and the penetration of capillary loops deep among epithelial elements points to an altered capacity of the sensory
J. A. Heckroth et al.
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layer to maintain its regular organization. In the anterior chamber of the eye, moreover, the neuro~p~thelium does not organize in an open layer, typical of the nasal cavity, even when the presence of lamellar bone could provide a supportive structure. The intercommunicating vesicles containing amorphous material and cellular debris seem to be the characteristic adaptation of the neuroepithelium to the new environment. Two morphologically distinguishable types of degeneration have been noted in the olfactory epithelium. The more common form under normal circumstances is characterized by increased density of the cytoplasmic matrix, numerous lysosomes, and condensation of the nuclear pattern; it represents the terminal stage of the life cycle of the olfactory neurons8 The other type of degeneration, which we will refer to as translucent degeneration, involves a clearing of the cytoplasm and a swelling of membrane bound organelles. Translucent degeneration is observed only occasionally in normal material but appears with a greatly increased frequency in the intraocular transplants. Autoradiographic observations of the olfactory mucosa in situ indicate that those elements undergoing translucent degeneration originate from the neurogenetic matrix and that they acquire degenerative characteristics at early stages in their development (unpublished observations). Consequently, we tentatively interpret translucent degeneration as a manifestation of early neuronal death. Why this type of degeneration should be so prominent in the transplanted epithelium is not certain at present. Myelinated and large unmyelinated nerve fibers present in the transplants beyond 30 days survival must have invaded the transplant from the host’s iris, as any such fibers present in the tissue after removal from the donor would have degenerated over this time period. It is likely that the myelinated fibers are of trigeminal origin. ’ ’ Their functional impact is difficult to assess at present. The possibility that they replace the fibers of trigeminal origin normally present in the nasal sensory area’*’ in some trophic function must not be discounted. The large unmyelinated fibers” (probably of autonomic origin) may also play a role in the survival of the transplants. The presence of OMP positive neurons in the transplant beyond 30 days survival testifies to the ability of the transplanted neuronal elements to manufacture this protein upon reaching morphological maturity. l6 The neurons app arently do not need their target nor their natural environment of the nasal cavity to become OMP positive. It seems likely, on the basis of our so far limited data, that the matrix is predetermined to manufacture its neurons independently of environmental factors present in the nasal cavity and normal synaptic relations. Parallel transplantation experiments in amphibians, as well as developmental studies, show the capacity of the olfactory anlage to differentiate into a nasal organ even when transplanted far from its normal location. 23Recent observations in Xenopus indicate that the olfactory neurons reach morphological maturity well before contact with the brain is established during development.‘” Experimental embryological studies” also indicate that the olfatory anlage is specified at an early deveIopmenta1 stage, before the appearance of the neural plate. Our observations of intraocular grafting provide the opportunity to study the differentiation of the olfactory neurons outside the environment of the nasal cavity where they demonstrate the ability to migrate from the epithelial environment, a property which is characteristic of many neurons during development. The survival and maturation of olfactory neurons in the anterior chamber of the eye could provide an ideal material for electrophysiological studies, as suggested for other neuronal populations by the work of Hoffer ef al.‘” Ack,l(lwlec~~emmts-This
work was supported
by a grant from the National
Institutes
of Health,
ROl-MS16421.
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