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Olfactory ensheathing glia and platelet-derived growth factor B-chain reactivity in the transplanted rat olfactory bulb
hr. I Ded hkuroscimcr,
0736~5748(94)E0011-P
Vol. 12. Na. 4, pp. 31.5-323. 1994
Elsevier Science Ltd ISDN Printed in Great Britain
OLFACTORY ENS~AT~ING GLIA AND PLATELET-D~RI~D GROWTH FACTOR B-CHAIN REACTIVITY IN THE TRANSPLANTED RAT OLFACTORY BULB J. N. KO’rr,* Departments
L. E. WESTRUM,
E.
W. RAINES, M. SASAHARA and R. Ross
of Neurological Surgery, Biological Structure and Pathology, Unive~ity of Washington, Seattle, WA 98195, U.S.A. (Received 23 Augusl 1993: in revised form 21 January 1994; accepted 25 /anuury 1994)
Abstract-Using a monoclonal antibody against the B-chain of platelet-derived growth factor as a marker, we have examined the behavior of olfactory ensheathing glia in the normal and transpi~ted rat olfactory bulb. In the normal postnatal olfacto~ bulb, these glia are found to ensheatb the bundles of incoming primary olfactory nerve fibers as well as those in the olfactory nerve layer. Olfactory marker protein antibody was used to identify the olfactory nerve proper. Within the transplant. the same glia: (I) ensheath bundles of bath primary olfactory and non-primary olfactory axons, (2) ensheath axonal bundles deep within the donor tissue, and (3) eventually permit radiation of individual axons from bundles to surrounding neuropil. We believe that ensheathing glia (being rich in growth-related factors and extracellular matrix motec&es) may be usefui in providing trophic support and guidance for the reconst~ction of dev~io~ent~ly or traumati~liy damaged neuronal pathways not dire&y related to the olfactory system. The evidence presented here indicates that ensheathing glia are capable of existing in deep brain areas and ensheathing other than primary olfactory axons. The special molecular characteristics of these glia along with the morphological findings presented here provide a foundation for further studies of these unique glia and their potential utility in the restoration of damaged neural pathways. Iyq~ words: platelet-derive marker protein.
growth factor, transplant, olfactory, regencrati~n, ensheathing glia, olfactory
Mental retardation may be related to perinatal trauma to the brain and the level of recovery from such trauma may be useful in predicting functional recovery. *l Thus, repair, reorganization or remodeling of an injured nervous system are events important to evaluation of resultant behavior.2.6 A knowledge of the capacity of the brain to reconstruct or reconstitute a given damaged area, pathway or system should be essential to our unde~tanding of the basis for mental retardation. One method to encourage or facilitate such regeneration or reconstitution of a damaged system is the use of transplantation protocols (see e.g. Ref. 8). We are using the olfactory system as a biological model for the study of not only injury-related regeneration in the system, but also the usefulness of transplantation protocols for reconstructing the damaged pathway(s). 124*27As a part of this larger investigation of neuropl~ti~ty, transplantation and recovery of this system when damaged, we are analysing the changes in expression of selected growth-related factors in discrete loci of the pathway following injury and with transplantation to enhance repair. The olfactory system is being used here for several reasons. The primary portion of the system, the olfactory mucosa, contains receptor neurons which uniquely regenerate from stem cells throughout life, including in aduith~.~ Upon contact with their appropriate target, these neurons express a unique protein (olfactory marker protein, OMP)‘O which provides a convenient immunocytochemical marker for their identification. They have axons which synapse in a highly organized fashion in the well-studied and uniquely laminated target organ, the olfactory bulb. The second order neurons of the olfactory system, mitral and tufted cells in the olfactory bulb, then project in a consistent and comp~m~nta~ way to the target cerebral cortical area, including the pylon or olfacto~ cortex. Our previous studies after olfactory bulb lesions and su~equent graEting of donor (fetal) olfactory bulbs have shown that viable grafted olfactory bulbs develop and *To whom all correspondence should be addressed. E, embryonic day; EC, ensheathing cell; OMP, olfactory marker protein; ~75 NGFR, nerve growth factor receptor: PBS, phosphate-buffered saline: PDGF-B, platelet-derive growth factor-B chain: PN, postnatal day. Abbreviufions:
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become reconnected within the system.‘jM2” Although we have shown that these transplants have many of the features of normal olfactory bulb. 7.23albeit in an often disorganized fashion, the role and involvement of growth factors in the maturation and final organization of this area have only recently been considered.” Doucette4 and Raisman14 described a specializedglial subtype that ensheaths the incoming axons of sensory neurons of the nasal and vomeronasal epithelia, respectively. At that time. both suggested (based solely on morphological grounds) that these specialized glia might be responsible for the unique ability of newly generated olfactory and vomeronasal receptor neurons to reinnervate the olfactory and accessory olfactory bulb. As these glia do not normally myelinate single axons, but rather ‘ensheath’ large fascicles of axons, they are often referred to as ensheathing cells (ECs). Previous work has shown that ~75 nerve growth factor-receptor (~75 NGFR; the shared, low-affinity receptor subunit for several neurotrophic factors) is heavily expressed in the developing olfactory nerve layer of the olfactory bulb,*” and specifically in the ECs” of that layer.** These same ECs re-express ~75 NGFR during the process of regeneration of the olfactory nerve after injury.” Recently, Devon and Doucette,3 have shown that ECs, which do not normally produce myelin, may be induced to do so under irz vitro conditions. To supplement our studies on ~75 NGFR, we are presently using antibodies to the B-chain of platelet-derived growth factor (PDGF-B). The distribution of this factor has recently been fully described throughout the adult and developing brain of the normal rat, and has been shown to be particularly selective to the olfactory system.15.16 PDGF-B is transiently present in the mitral cell layer of the normal olfactory bulb during development and also in the perikaryal region of most, if not all, mature neurons.t5 On the other hand, PDGF-B is continuously expressed in the olfactory nerve layer of the olfactory bulb. beginning during the third week of gestation and persisting through adulthood. Clearly, during early development, PDGF-B is localized to specialized glial cells, the ECs of the olfactory nerve 1ayer.s.” The occurrence of PDGF-B in mature neuronal perikarya may represent a potential confound in the localization of ECs in more mature animals; however, the antibody against this growth factor still provides an excellent marker for olfactory ECs. In addition to p7S NGFR and PDGF-B. ECs also express extracellular matrix molecules, t2 which provide a substrate for extending axons. Based on all of the above characteristics, it has been hypothesized that it is the unique properties of these ECs that may account for the continuous repenetration of newly generated olfactory axons from the periphery into the central nervous system (i.e. olfactory bulb).’ The delineation of the precise behavior of the ECs, as demonstrated by PDGF-B immunoreactivity, during the maturation of a transplant and its reconnectivity is the objective of this study. In addition, the possible implications for a more general role for ECs permitting repenetration of other types of regenerating axons within or into the central nervous system may be directly relevant to understanding general repair in the brain and possibly mental retardation. EXPERIMENTAL
PROCEDURES
Both host animals and donor olfactory bulbs were derived from time-mated female rats of the Sprague-Dawley strain (Tyler Laboratories, Bellevue, WA). Tritiated thymidine injections into time-mated dams was used to label fetal olfactory bulbs. Injections were carried out (5 l&i/g body wt, s.q.) at embryonic days (E) 13 and 14. At E15, under deep anesthesia (Ketamine-xylazine. 3:1, 0.05 ml/g body wt, i.m.), fetuses were removed and the olfactory bulbs dissected out in cold (4°C) medium. The olfactory bulbs were immediately transplanted into the site vacated by aspiration of an olfactory bulb in a I- to 2-day-old postnatal (PN) host according to previously described methods.24,25 Deep inhalation anesthesia and hypothermia were used for postnatal host surgery and transplantation. Host or experimental animals were killed after survival times of I, 2 or at least 8 weeks (the latter is hereafter referred to as adult). Control animals for 1 week transplants were killed at PNl and those for 2 week transplants were taken at PN7. This was to permit comparisons of control and transplant bulbs of similar chronological age. Animals were deeply anesthetized using pentobarbital(11 mg/lOO body wt, i.m.), and after a thoracotomy were perfused through the heart with phosphate-buffered saline (PBS). After perfusion, brains were rapidly removed and fixed by
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immersion in methyl Camoy’s solution for at least 18 hr at 4°C. Tissue was subsequently trimmed to include the areas of interest and embedded in low melting point (56°C) paraffin. Serial sagittal sections (8 p.m thick) were cut on a rotary microtome and mounted singly on Probe Op Plus slides (Fisher). Every sixth section was processed for autoradiography.18 Intervening sections were immunocytochemically processed for OMP and PDGF-B. Adjacent or near adjacent sections (separated by no more than 16 Frn) were used for comparison of OMP and PDGF-B immunoreactivity. Immunocytochemical
processing
Sections were de-paraffinized with xylene and rehydrated through graded ethanol solutions. Endogenous peroxidases were blocked by incubating slides with 0.3% hydrogen peroxide plus 0.1% sodium azide in PBS for 10 min. Incubations in primary antibodies [PDGF-007 (417 ng/ml; kindly provided by Mochida Pharmaceutical, Tokyo, Japan); anti-OMP (2 p,l/ml; kindly provided by Dr Frank Margolis)] were performed at 4°C overnight. Biotinylated secondary antibodies (rabbit anti-mouse for PDGF-B and rabbit anti-goat for OMP) were applied for 30 min at room temperature in dilutions of 1:400. Horseradish peroxidase-conjugated streptavidin solution at a 1:5OOOdilution was then applied for 40 min. At least three 5-min rinses with PBS were carried out after each of the preceding steps. Finally, sections were reacted for 6 min at room temperature with diaminobenzidine (0.25 mg/ml) and 3% hydrogen peroxide (1.25 $/ml) dissolved in Tris buffer. Following immunocytochemical reactions, all sections were counterstained with Methyl Green, then dehydrated, cleared in xylene and coverslipped using Permount. Finished slides were examined and photographed with a Leitz Diaplan microscope using a no. 58 green filter (subtractive for green counterstain). Control tissue for both antibodies from both normal and transplant olfactory bulbs was carried through each of the above steps except that the primary antibody was deleted from the incubation. In an earlier experiment PDGF-007 was neutralized by incubating with a five-fold excess of the immunizing peptide at 37°C for 1 hr.
RESULTS One week transplantdPN1 control Normal OMPpatterns. The normal pattern of OMP immunoreactivity in the l- day-old olfactory bulb is characterized by dense label within bundles of olfactory nerve fibers over nearly the entire surface of the olfactory bulb (Fig. 1A). Individual olfactory nerve fibers radiate from these densely labeled bundles to form diffusely labeled immature glomeruli (Fig. 1A). These poorly defined glomeruli do not occur in the regular laminae seen in more mature tissue but are sporadically spaced just deep to the heavily labeled fascicles and bundles of fibers in the olfactory nerve layer (Fig. 1A). Normal PDGF-B patterns. The PDGF-B immunoreactivity pattern in the normal olfactory bulb at PNl contrasts with that of the OMP immunoreactivity (compare Fig. 1B with Fig. 1A). The densest label for PDGF-B is in the olfactory nerve layer (Fig. lB, which is known to contain both olfactory nerve bundles and ECs. This pattern of reactivity is quite similar to that seen in the OMP material. However, in contrast to the OMP pattern, which labels the olfactory glomeruli, PDGF-B label is absent from the glomeruli but does extend to their peripheral borders (Fig. 1B). Transplant OMP patterns. The transplanted olfactory bulb is considerably less well-organized than the normal olfactory bulb, but certain patterns of OMP reactivity are identifiably common to both (Fig. 1C). In the transplant, immunoreactive fibers, occurring as patches on the surface of the olfactory bulb, course in irregular fascicles or bundles within the transplant, often penetrating into deep regions (Fig. 1C). The bundles vary in thickness but are usually well circumscribed with clean borders. In some cases, however, individual fibers may radiate out from the bundles into the adjacent neuropil and appear in a diffuse pattern without clear borders (Fig. 1C). Transplant PDGF-Bpatterns. Within the transplanted olfactory bulb, the PDGF-B-reactive cells and fibers are less well-defined than normal (compare Fig. 1B and D), occurring in irregular areas sometimes near the surface and especially deep within the olfactory bulb. The immunoreactivity is somewhat less dense than normal, but is clearly located in fascicles or bundles and may be associated
with both OMP-positive and OMP-negative fiber bundles (compare Fig. 1C and D). The PDGF-H immunoreactivity is especially absent in areas where OMP-positive fibers radiate into diffuse fields (Fig. 1D). Also, OMP-positive fiber bundles mav occur in the absence of PDGF-B-reactive fascicles (Fig. lC, D). Thus, when OMP-positive fibeis radiate from dense reactive bundles. PDGF- R immunoreactivity is generally absent.
Fig. 1. (A) A normal control olfactory bulb (OB) from a postnatal (PN) day 1 rat pup immunoreacted from OMP. Fascicles of reactive axons of the olfactory nerve (ON) are located superficial to the olfactory nerve layer (ONL), which is also reactive. The latter seemingly encircle the olfactory glomeruli (G), which show only minimal to light reactivity (see inset for detail). (B) A preparation similar and adjacent to that in A of a PNI control OB immunoreacted with antiserum to PDGF-B. Strongly reactive ON and ONL are seen but the glomeruli, which are partly encircled by the reactive ONL. are clearly non-reactive (see inset). (C) A sagittal section of an OB transplant (TX) in a PN7 rat (see Experimental Procedures) reacted with anti-OMP. Lightly immunoreactive patches (small arrow) and strongly reactive fascicles (arrowheads) of axons are seen. the latter located at deeper areas of the transplant. An example of immunoreactive ‘fiber radiations’ out of a bundle (asterisk) is seen in the inset. (D) A preparation adjacent to that in C of a PNl transplanted OB (TX OB) immunoreacted with antiserum to PDGF-B. Small arrow indicates lightly reactive surface areas and arrowheads point out deeper. more heavily reactive fascicles. An area (asterisk) here is non-reactive (see inset), w*hereas the same site in the OMP material (see C. asterisk) shows reactive ‘fiber radiations’. Eight-micrometer paraffin sections. Scale bars _ 100 pm.
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Two week transplant’PIV7 control iVormal0MPpanern.s. The control PN? olfactory bulb shows immunoreactivity characteristics generally similar to that seen in the PNl material (compare Figs 1A and 2A). I~unoreactive fibers form bundles over the surface of the olfactory bulb and radiate into the underly~gglome~~. Within the now more fully developed glomeruli, replete with periglomerular cells, the immunoreactivity
Fig. 2. (A) A sa&tal section through a normal olfactory bulb (OB) from a control PN7 rat immunoreacted for OMP. The olfactory nerve layer (ONL) is strongly reactive and individual fibers disperse un~ormly (see inset for details) through the underlving glomeruli (G). (B) an adiacent section to that in A immunoreacted for PRGF-BOlfactory nerve layer fibers {dk) are heavily immunoreactive (see inset) but the olfactory glomeruh (G) subjacent to the ONL are non-reactive. (C) A portion of a sagittal section from a 2 week olfactory bulb transplant (TX OB) immunoreacted for OMP. Strongly reactive fascicles or bundles (see inset) of fibers (arrowheads) are seen, often in groupings, as well as possible ‘fiber radiations’ (asterisks) deep within the TX. (D) An adjacent section to C immunoreacted for PDGF-B. Reactive bundles of fibers are seen deep in the TX OB, often ensheathing (see inset) the OMP bundles (compare to C; arrowheads). Eight micrometer paraffin sections. Scale bars=100 pm. DN 12:4-D
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takes on a uniformly diffuse appearance (Fig. 2A). Thus, OMP-positive glomeruli are clearly present by this stage and immunoreactivity changes from the coarse quality seen in the peripheral fibers to the homogeneously fine and diffuse quality within the glomeruli (Fig. 2A). Normal PDGF-Bpatterns. Fibers and cells reactive for PDGF-B are limited to the olfactory nerve layer, especially its deeper portions (Fig. 2B). These fascicles of fibers and cells penetrate the olfactory bulb, coursing around the outer portions of the glomerular layer (Fig. 2B) and occasionally deep to the glomeruli. The glomeruli themselves are clearly PDGF-B non-reactive (Fig. 2B). Therefore, PDGF-B-reactive fascicles are associated with tightly bound OMP-reactive bundles and absent when olfactory nerves penetrate and radiate into the glomeruli themselves. Transplant OMP patterns. Within the 2 week transplant, OMP immunoreactivity is clearly identifiable, but irregular in distribution. Well-delineated OMP-reactive bundles occur throughout the transplant, often in large groupings, clusters or masses (Fig. 2C). Areas of possible fiber radiations out of the bundles are also seen. The overall picture is one of larger fields of fiber radiation than seen earlier but still with areas of well circumscribed fiber bundles reactive for OMP (Fig. 2C). Transplant PDGF-B patterns. Here, the PDGF-B immunoreactivity occurs as dense bundles of fibers, sometimes complimentary to OMP reactive structures (Fig. 2D). The PDGF-B-reactive fascicles often ensheath non-reactive fiber bundles which correspond to OMP-positive bundles in adjacent sections (compare Fig. 2C and D). Regions showing OMP-reactive radiation (Fig. 2C) are non-reactive. In other areas (not depicted) PDGF-B-positive fascicles may partially ensheath bundles of fibers that are not OMP reactive. Adult transplant/adult control Normal OMPpatterns. The normal OMP immunoreactivity is restricted to the olfactory nerve layer over the surface of the olfactory bulb and the bundles of fibers that course between glomeruli (Fig. 3A). The nerve layer and coursing fibers that are reactive are similar to that of the 2 week control but differ somewhat in the glomeruli labeling. The adult shows larger, more heavily reactive glomeruli that are more clearly defined by well-delineated layers of periglomerular cells (Fig. 3A). The OMP reactivity within glomeruli is uniform and diffuse, as in 2 week material (PN7). Normal PDGF-B patterns. In the adult, the PDGF-B immunoreactivity is also quite similar to that in the 2 week PDGF-B preparations (Fig. 3B). The olfactory nerve layer is densely positive with some reactivity penetrating between glomeruli. The olfactory glomeruli themselves are usually non-reactive, but occasionally they do show a minimal amount of immunoreactivity (Fig. 3B). A few immunoreactive fascicles of fibers and cells may be seen penetrating a glomerulus. As reported previously15 most, if not all, mature neuronal perikarya exhibit PDGF-B immunoreactivity (Fig. 3B). Transplant OMP patterns. In the fully developed transplant, OMP-reactive fiber bundles with well-defined boundaries are apparent, but many diffuse areas of reactivity are also seen (Fig. 3C). As in more immature transplants, both well-defined bundles and fields of diffuse reactivity may be found throughout the transplant superficially and deeper. The bundles of OMP-positive fibers are irregularly spaced, often occurring in clusters or groups (Fig. 3C). Transplant PDGF-Bpatterns. The PDGF-B-reactive fascicles sometimes partially ensheath areas non-reactive for PDGF-B, but which correspond to the same OMP-positive areas seen in adjacent OMP-reacted sections (compare Fig. 3C and D). In addition, some areas, not imrnunoreactive for OMP, are also partially surrounded by PDGF-B-positive fibers and cells (Fig. 3D). Varying degrees of PDGF-B immunoreactivity occur within OMP-positive fiber bundles (compare Fig. 3C and D). Notably, radiation of individual OMP-positive fibers into uniformly reactive fields appears to be favored where PDGF-B ensheathment is limited or absent (compare Fig. 3C and D, asterisk). Thus, PDGF-B-positive structures surround areas that are OMP-positive and are usually absent from the OMP fiber radiations.
DISCUSSION We are examining the behavior of olfactory ECs (as identified by their strong PDGF-B immunoreactivity) to assess the growth promoting and axonal guidance potential of these growth
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factor-rich cells. Since these cells produce extracellular matrix molecules12 and show high expression of both PDGF-B15 and at least one of the nerve growth factor receptor antigens (~75 NGFR)‘Y~~ they are likely candidates for facilitation of axonal growth into and through the nervous system. One of these is the pathway of the primary olfactory nerve into the olfactory bulb of the central nervous system. By using a transplantation model (replacing a host olfactory bulb with one
Fig. 3. (A) A portion of the sagittal section from a normal adult olfactory bulb (OB) immunoreacted for OMP. Olfactory nerve layer fibers (ONE) are strongly reactive, as are the subjacent olfactory glomeruli (G) (see inset for details). (B) A portion of a section adjacent to A reactive for PDGF-B. Olfactory nerve layer fibers (ONE) are immunoreactive and partially encircle the underlying lightly reactive (see inset) olfactory glomeruli (G). Note, at this maturity, many neurons are now immunoreactive (arrowheads). (C) A portion of a sagittal section from an adult olfactory bulb transplant (TX OB) ~unoreaued for OMP. Heavily reactive bundles of fibers (a~owheads) on the surface and deeper are seen. Some deeper bundles have surrounding diffuse hnmunoreactivity (see inset) suggestive of a ‘fiber radiation’ (asterisks). (D) A near adjacent section to C immunoreacted for PDGF-B. Reactive patches and fibers are seen deep within the TX OB (arrowheads), some of which are in OMP-negative areas while others surround and infiltrate OMP-immunoreactive bundles (see C). Other areas show PDGF-B-reactive bundles (small arrows) at sites devoid of OMPreactivity. Upper regions positive for OMP are negative for PDGF-B (asterisks), especially (see inset) the ‘fiber radiation’ regions. Eight micrometer paraffin sections. Scale bars=100 p,m.
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from a fetal donor) we have created a situation in which the activity and distribution of ECs is not constrained by the normal growth patterns of the olfactory bulb. We have identified several characteristics of ECs that suggest they may be useful in repair and regeneration of injured nervous system and, thus, in the recovery of function of such damaged tissue. First, in addition to ensheathing olfactory nerve fibers and axons (as shown by OMP reactivity), ECs are also capable of ensheathing non-olfactory nerve fibers/axons within fascicles. Second, EC’s may operate to ensheath fascicles of axons deep within the transplant as opposed to their exclusive localization near the surface in their normal situation. Third, diffusion or radiation of OMP-positive fibers/axons from bundles or fascicles is favored in the absence of PDGF-B-positive ECs. Fourth, this radiation of OMP-positive fibers first seen in the 1 week transplant increases in frequency and distribution at 2 weeks and in the adult. Although there is overlap in some cases, there is a striking complimentary pattern involving the EC expression of PDGF-B and that of OMP by the olfactory nerve. To be of clinical interest, it is necessary that ECs survive and provide guidance as well as growth-related factors for fiber systems other than the primary olfactory nerve. By combining immunoc~ochemistry for OMP and PDGF-B, we have demonstrated that the PDGF-B-rich ECs become associated with OMP-negative (and therefore non-primary olfactory) nerve fascicles within the transplant. Earlier work also demonstrated that ECs retain their ~75 NGFR reactivity within the transplant.23 The findings in the present study of EC involvement in repair following injury and transplantation need to be confirmed in other systems where ECs are not normally found. For example, will introduction of ECs to injured dorsal root fibers facilitate outgrowth of partially damaged or adjacent uninjured dorsal root fibers? In relation to mental retardation, it would be helpful to know if ECs could play a role in re-establishing damaged hippocampal pathways involved in memory.“~‘7 Our finding here of OMP-negative (therefore non-olfactory nerve) fibers, which were immunoreactive for PDGF-B, suggests there may be a potential for ECs to facilitate growth of fiber systems other than olfactory nerve. In order to be effective in repair, the ECs must be able to survive and function not only in peripheral or superficial regions of the brain, but also in deeper areas. Here, we have shown that ECs can penetrate and exist through adulthood, not only on the surface of the olfactory bulb transplant (their usual position), but also within its depths. The EC ensheathment of both olfactory nerve fiber bundles (OMP-positive) and non-olfactory nerve (OMP-negative) fiber bundles at deeper brain regions, in addition to the previous demonstration of p7.5 NGFR reactivity in the same regions of similar transplants, 23 all point to a possible role for these cells in the growth of regenerating fiber bundles into deep brain. Thus, there may be a potential for enhancement of transplant outgrowth and internal reconnectivity in brain. Despite outgrowth, even for great distances in deep brain areas, complete repair and recovery of function after injury or transplantation will ultimately depend on re-establishment of contacts or synapses with an appropriate target area. I9 In addition to initiating outgrowth, facilitation of guidance and ‘nou~shment’ of the pathway, ECs need to allow all of these roles to culminate in a functional reconnectivity in some ordered pattern, with an effective target. The ECs need to permit a ‘branching out’, for example, of axons from the bundles into a terminal field where synaptic contacts can be made. Examples of these events might be identified in the visual system where terminal arbors of undamaged optic fibers sprout into colliculus or thalamic fields after damage.” Atypical reinnervation patterns have been reported in such cases as this and other systems, resulting in altered function, but this often depends on such factors as extent of lesion and distances for fiber regrowth.” The ECs’ effectiveness in bringing the ‘regenerating’ fiber bundles as near as possible to the appropriate target, and then allowing sprouting and synapse formation, needs to be determined. We have shown that, in the transplant situation, ensheathed olfactory nerve fiber bundles do eventually undergo branching. Whether or not EC ensheathed bundles will behave in the same manner when transplanted into another system is unclear. Other than electrophysiology, electron microscopy in conjunction with transpfant markers (such as horseradish peroxidase) or appropriate neurotransmitter localization will be necessary to determine such re-establishment of contacts. We have not addressed the function of the ECs in systems other than olfactory transplants. Our present intent was to fully describe the anatomical, structural details of the patterns of EC
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distribution and activity in relation to the architecture of a maturing transplant (a model for repair after tissue damage). This should form a basis to elucidate possible functional features of these patterns and considerations of potential or limitations for success of our approach. The findings presented here have suggested that ECs are attractive candidates for facilitation of axonal penetration from the periphery into the central nervous system. Further, ECs also seem to act in manipulated guidance of nerve fascicles through areas of injury within the brain. The actual mechanism by which these cells accomplish this remains to be elucidated. Much work is required to demonstrate not only how these events are functionally limited, but whether isolated ECs can actually participate in the processes in cases of human brain injury, disease or malformations. Acknowledgements-This study was supported by N.I.H. Research Grant nos. NS09678 and HL18645. The authors are indebted to Ren6e Czarapata for secretarial help, to Paul Schwartz and Janet Clardy for preparation of photographic plates, and to Jeff Bedell and Mary Gross for technical assistance. L. E. W. is an affiliate of the Child Development and Mental Retardation Center of the University of Washington.
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0736~5748(94)E0011-P
Vol. 12. Na. 4, pp. 31.5-323. 1994
Elsevier Science Ltd ISDN Printed in Great Britain
OLFACTORY ENS~AT~ING GLIA AND PLATELET-D~RI~D GROWTH FACTOR B-CHAIN REACTIVITY IN THE TRANSPLANTED RAT OLFACTORY BULB J. N. KO’rr,* Departments
L. E. WESTRUM,
E.
W. RAINES, M. SASAHARA and R. Ross
of Neurological Surgery, Biological Structure and Pathology, Unive~ity of Washington, Seattle, WA 98195, U.S.A. (Received 23 Augusl 1993: in revised form 21 January 1994; accepted 25 /anuury 1994)
Abstract-Using a monoclonal antibody against the B-chain of platelet-derived growth factor as a marker, we have examined the behavior of olfactory ensheathing glia in the normal and transpi~ted rat olfactory bulb. In the normal postnatal olfacto~ bulb, these glia are found to ensheatb the bundles of incoming primary olfactory nerve fibers as well as those in the olfactory nerve layer. Olfactory marker protein antibody was used to identify the olfactory nerve proper. Within the transplant. the same glia: (I) ensheath bundles of bath primary olfactory and non-primary olfactory axons, (2) ensheath axonal bundles deep within the donor tissue, and (3) eventually permit radiation of individual axons from bundles to surrounding neuropil. We believe that ensheathing glia (being rich in growth-related factors and extracellular matrix motec&es) may be usefui in providing trophic support and guidance for the reconst~ction of dev~io~ent~ly or traumati~liy damaged neuronal pathways not dire&y related to the olfactory system. The evidence presented here indicates that ensheathing glia are capable of existing in deep brain areas and ensheathing other than primary olfactory axons. The special molecular characteristics of these glia along with the morphological findings presented here provide a foundation for further studies of these unique glia and their potential utility in the restoration of damaged neural pathways. Iyq~ words: platelet-derive marker protein.
growth factor, transplant, olfactory, regencrati~n, ensheathing glia, olfactory
Mental retardation may be related to perinatal trauma to the brain and the level of recovery from such trauma may be useful in predicting functional recovery. *l Thus, repair, reorganization or remodeling of an injured nervous system are events important to evaluation of resultant behavior.2.6 A knowledge of the capacity of the brain to reconstruct or reconstitute a given damaged area, pathway or system should be essential to our unde~tanding of the basis for mental retardation. One method to encourage or facilitate such regeneration or reconstitution of a damaged system is the use of transplantation protocols (see e.g. Ref. 8). We are using the olfactory system as a biological model for the study of not only injury-related regeneration in the system, but also the usefulness of transplantation protocols for reconstructing the damaged pathway(s). 124*27As a part of this larger investigation of neuropl~ti~ty, transplantation and recovery of this system when damaged, we are analysing the changes in expression of selected growth-related factors in discrete loci of the pathway following injury and with transplantation to enhance repair. The olfactory system is being used here for several reasons. The primary portion of the system, the olfactory mucosa, contains receptor neurons which uniquely regenerate from stem cells throughout life, including in aduith~.~ Upon contact with their appropriate target, these neurons express a unique protein (olfactory marker protein, OMP)‘O which provides a convenient immunocytochemical marker for their identification. They have axons which synapse in a highly organized fashion in the well-studied and uniquely laminated target organ, the olfactory bulb. The second order neurons of the olfactory system, mitral and tufted cells in the olfactory bulb, then project in a consistent and comp~m~nta~ way to the target cerebral cortical area, including the pylon or olfacto~ cortex. Our previous studies after olfactory bulb lesions and su~equent graEting of donor (fetal) olfactory bulbs have shown that viable grafted olfactory bulbs develop and *To whom all correspondence should be addressed. E, embryonic day; EC, ensheathing cell; OMP, olfactory marker protein; ~75 NGFR, nerve growth factor receptor: PBS, phosphate-buffered saline: PDGF-B, platelet-derive growth factor-B chain: PN, postnatal day. Abbreviufions:
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become reconnected within the system.‘jM2” Although we have shown that these transplants have many of the features of normal olfactory bulb. 7.23albeit in an often disorganized fashion, the role and involvement of growth factors in the maturation and final organization of this area have only recently been considered.” Doucette4 and Raisman14 described a specializedglial subtype that ensheaths the incoming axons of sensory neurons of the nasal and vomeronasal epithelia, respectively. At that time. both suggested (based solely on morphological grounds) that these specialized glia might be responsible for the unique ability of newly generated olfactory and vomeronasal receptor neurons to reinnervate the olfactory and accessory olfactory bulb. As these glia do not normally myelinate single axons, but rather ‘ensheath’ large fascicles of axons, they are often referred to as ensheathing cells (ECs). Previous work has shown that ~75 nerve growth factor-receptor (~75 NGFR; the shared, low-affinity receptor subunit for several neurotrophic factors) is heavily expressed in the developing olfactory nerve layer of the olfactory bulb,*” and specifically in the ECs” of that layer.** These same ECs re-express ~75 NGFR during the process of regeneration of the olfactory nerve after injury.” Recently, Devon and Doucette,3 have shown that ECs, which do not normally produce myelin, may be induced to do so under irz vitro conditions. To supplement our studies on ~75 NGFR, we are presently using antibodies to the B-chain of platelet-derived growth factor (PDGF-B). The distribution of this factor has recently been fully described throughout the adult and developing brain of the normal rat, and has been shown to be particularly selective to the olfactory system.15.16 PDGF-B is transiently present in the mitral cell layer of the normal olfactory bulb during development and also in the perikaryal region of most, if not all, mature neurons.t5 On the other hand, PDGF-B is continuously expressed in the olfactory nerve layer of the olfactory bulb. beginning during the third week of gestation and persisting through adulthood. Clearly, during early development, PDGF-B is localized to specialized glial cells, the ECs of the olfactory nerve 1ayer.s.” The occurrence of PDGF-B in mature neuronal perikarya may represent a potential confound in the localization of ECs in more mature animals; however, the antibody against this growth factor still provides an excellent marker for olfactory ECs. In addition to p7S NGFR and PDGF-B. ECs also express extracellular matrix molecules, t2 which provide a substrate for extending axons. Based on all of the above characteristics, it has been hypothesized that it is the unique properties of these ECs that may account for the continuous repenetration of newly generated olfactory axons from the periphery into the central nervous system (i.e. olfactory bulb).’ The delineation of the precise behavior of the ECs, as demonstrated by PDGF-B immunoreactivity, during the maturation of a transplant and its reconnectivity is the objective of this study. In addition, the possible implications for a more general role for ECs permitting repenetration of other types of regenerating axons within or into the central nervous system may be directly relevant to understanding general repair in the brain and possibly mental retardation. EXPERIMENTAL
PROCEDURES
Both host animals and donor olfactory bulbs were derived from time-mated female rats of the Sprague-Dawley strain (Tyler Laboratories, Bellevue, WA). Tritiated thymidine injections into time-mated dams was used to label fetal olfactory bulbs. Injections were carried out (5 l&i/g body wt, s.q.) at embryonic days (E) 13 and 14. At E15, under deep anesthesia (Ketamine-xylazine. 3:1, 0.05 ml/g body wt, i.m.), fetuses were removed and the olfactory bulbs dissected out in cold (4°C) medium. The olfactory bulbs were immediately transplanted into the site vacated by aspiration of an olfactory bulb in a I- to 2-day-old postnatal (PN) host according to previously described methods.24,25 Deep inhalation anesthesia and hypothermia were used for postnatal host surgery and transplantation. Host or experimental animals were killed after survival times of I, 2 or at least 8 weeks (the latter is hereafter referred to as adult). Control animals for 1 week transplants were killed at PNl and those for 2 week transplants were taken at PN7. This was to permit comparisons of control and transplant bulbs of similar chronological age. Animals were deeply anesthetized using pentobarbital(11 mg/lOO body wt, i.m.), and after a thoracotomy were perfused through the heart with phosphate-buffered saline (PBS). After perfusion, brains were rapidly removed and fixed by
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immersion in methyl Camoy’s solution for at least 18 hr at 4°C. Tissue was subsequently trimmed to include the areas of interest and embedded in low melting point (56°C) paraffin. Serial sagittal sections (8 p.m thick) were cut on a rotary microtome and mounted singly on Probe Op Plus slides (Fisher). Every sixth section was processed for autoradiography.18 Intervening sections were immunocytochemically processed for OMP and PDGF-B. Adjacent or near adjacent sections (separated by no more than 16 Frn) were used for comparison of OMP and PDGF-B immunoreactivity. Immunocytochemical
processing
Sections were de-paraffinized with xylene and rehydrated through graded ethanol solutions. Endogenous peroxidases were blocked by incubating slides with 0.3% hydrogen peroxide plus 0.1% sodium azide in PBS for 10 min. Incubations in primary antibodies [PDGF-007 (417 ng/ml; kindly provided by Mochida Pharmaceutical, Tokyo, Japan); anti-OMP (2 p,l/ml; kindly provided by Dr Frank Margolis)] were performed at 4°C overnight. Biotinylated secondary antibodies (rabbit anti-mouse for PDGF-B and rabbit anti-goat for OMP) were applied for 30 min at room temperature in dilutions of 1:400. Horseradish peroxidase-conjugated streptavidin solution at a 1:5OOOdilution was then applied for 40 min. At least three 5-min rinses with PBS were carried out after each of the preceding steps. Finally, sections were reacted for 6 min at room temperature with diaminobenzidine (0.25 mg/ml) and 3% hydrogen peroxide (1.25 $/ml) dissolved in Tris buffer. Following immunocytochemical reactions, all sections were counterstained with Methyl Green, then dehydrated, cleared in xylene and coverslipped using Permount. Finished slides were examined and photographed with a Leitz Diaplan microscope using a no. 58 green filter (subtractive for green counterstain). Control tissue for both antibodies from both normal and transplant olfactory bulbs was carried through each of the above steps except that the primary antibody was deleted from the incubation. In an earlier experiment PDGF-007 was neutralized by incubating with a five-fold excess of the immunizing peptide at 37°C for 1 hr.
RESULTS One week transplantdPN1 control Normal OMPpatterns. The normal pattern of OMP immunoreactivity in the l- day-old olfactory bulb is characterized by dense label within bundles of olfactory nerve fibers over nearly the entire surface of the olfactory bulb (Fig. 1A). Individual olfactory nerve fibers radiate from these densely labeled bundles to form diffusely labeled immature glomeruli (Fig. 1A). These poorly defined glomeruli do not occur in the regular laminae seen in more mature tissue but are sporadically spaced just deep to the heavily labeled fascicles and bundles of fibers in the olfactory nerve layer (Fig. 1A). Normal PDGF-B patterns. The PDGF-B immunoreactivity pattern in the normal olfactory bulb at PNl contrasts with that of the OMP immunoreactivity (compare Fig. 1B with Fig. 1A). The densest label for PDGF-B is in the olfactory nerve layer (Fig. lB, which is known to contain both olfactory nerve bundles and ECs. This pattern of reactivity is quite similar to that seen in the OMP material. However, in contrast to the OMP pattern, which labels the olfactory glomeruli, PDGF-B label is absent from the glomeruli but does extend to their peripheral borders (Fig. 1B). Transplant OMP patterns. The transplanted olfactory bulb is considerably less well-organized than the normal olfactory bulb, but certain patterns of OMP reactivity are identifiably common to both (Fig. 1C). In the transplant, immunoreactive fibers, occurring as patches on the surface of the olfactory bulb, course in irregular fascicles or bundles within the transplant, often penetrating into deep regions (Fig. 1C). The bundles vary in thickness but are usually well circumscribed with clean borders. In some cases, however, individual fibers may radiate out from the bundles into the adjacent neuropil and appear in a diffuse pattern without clear borders (Fig. 1C). Transplant PDGF-Bpatterns. Within the transplanted olfactory bulb, the PDGF-B-reactive cells and fibers are less well-defined than normal (compare Fig. 1B and D), occurring in irregular areas sometimes near the surface and especially deep within the olfactory bulb. The immunoreactivity is somewhat less dense than normal, but is clearly located in fascicles or bundles and may be associated
with both OMP-positive and OMP-negative fiber bundles (compare Fig. 1C and D). The PDGF-H immunoreactivity is especially absent in areas where OMP-positive fibers radiate into diffuse fields (Fig. 1D). Also, OMP-positive fiber bundles mav occur in the absence of PDGF-B-reactive fascicles (Fig. lC, D). Thus, when OMP-positive fibeis radiate from dense reactive bundles. PDGF- R immunoreactivity is generally absent.
Fig. 1. (A) A normal control olfactory bulb (OB) from a postnatal (PN) day 1 rat pup immunoreacted from OMP. Fascicles of reactive axons of the olfactory nerve (ON) are located superficial to the olfactory nerve layer (ONL), which is also reactive. The latter seemingly encircle the olfactory glomeruli (G), which show only minimal to light reactivity (see inset for detail). (B) A preparation similar and adjacent to that in A of a PNI control OB immunoreacted with antiserum to PDGF-B. Strongly reactive ON and ONL are seen but the glomeruli, which are partly encircled by the reactive ONL. are clearly non-reactive (see inset). (C) A sagittal section of an OB transplant (TX) in a PN7 rat (see Experimental Procedures) reacted with anti-OMP. Lightly immunoreactive patches (small arrow) and strongly reactive fascicles (arrowheads) of axons are seen. the latter located at deeper areas of the transplant. An example of immunoreactive ‘fiber radiations’ out of a bundle (asterisk) is seen in the inset. (D) A preparation adjacent to that in C of a PNl transplanted OB (TX OB) immunoreacted with antiserum to PDGF-B. Small arrow indicates lightly reactive surface areas and arrowheads point out deeper. more heavily reactive fascicles. An area (asterisk) here is non-reactive (see inset), w*hereas the same site in the OMP material (see C. asterisk) shows reactive ‘fiber radiations’. Eight-micrometer paraffin sections. Scale bars _ 100 pm.
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Two week transplant’PIV7 control iVormal0MPpanern.s. The control PN? olfactory bulb shows immunoreactivity characteristics generally similar to that seen in the PNl material (compare Figs 1A and 2A). I~unoreactive fibers form bundles over the surface of the olfactory bulb and radiate into the underly~gglome~~. Within the now more fully developed glomeruli, replete with periglomerular cells, the immunoreactivity
Fig. 2. (A) A sa&tal section through a normal olfactory bulb (OB) from a control PN7 rat immunoreacted for OMP. The olfactory nerve layer (ONL) is strongly reactive and individual fibers disperse un~ormly (see inset for details) through the underlving glomeruli (G). (B) an adiacent section to that in A immunoreacted for PRGF-BOlfactory nerve layer fibers {dk) are heavily immunoreactive (see inset) but the olfactory glomeruh (G) subjacent to the ONL are non-reactive. (C) A portion of a sagittal section from a 2 week olfactory bulb transplant (TX OB) immunoreacted for OMP. Strongly reactive fascicles or bundles (see inset) of fibers (arrowheads) are seen, often in groupings, as well as possible ‘fiber radiations’ (asterisks) deep within the TX. (D) An adjacent section to C immunoreacted for PDGF-B. Reactive bundles of fibers are seen deep in the TX OB, often ensheathing (see inset) the OMP bundles (compare to C; arrowheads). Eight micrometer paraffin sections. Scale bars=100 pm. DN 12:4-D
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takes on a uniformly diffuse appearance (Fig. 2A). Thus, OMP-positive glomeruli are clearly present by this stage and immunoreactivity changes from the coarse quality seen in the peripheral fibers to the homogeneously fine and diffuse quality within the glomeruli (Fig. 2A). Normal PDGF-Bpatterns. Fibers and cells reactive for PDGF-B are limited to the olfactory nerve layer, especially its deeper portions (Fig. 2B). These fascicles of fibers and cells penetrate the olfactory bulb, coursing around the outer portions of the glomerular layer (Fig. 2B) and occasionally deep to the glomeruli. The glomeruli themselves are clearly PDGF-B non-reactive (Fig. 2B). Therefore, PDGF-B-reactive fascicles are associated with tightly bound OMP-reactive bundles and absent when olfactory nerves penetrate and radiate into the glomeruli themselves. Transplant OMP patterns. Within the 2 week transplant, OMP immunoreactivity is clearly identifiable, but irregular in distribution. Well-delineated OMP-reactive bundles occur throughout the transplant, often in large groupings, clusters or masses (Fig. 2C). Areas of possible fiber radiations out of the bundles are also seen. The overall picture is one of larger fields of fiber radiation than seen earlier but still with areas of well circumscribed fiber bundles reactive for OMP (Fig. 2C). Transplant PDGF-B patterns. Here, the PDGF-B immunoreactivity occurs as dense bundles of fibers, sometimes complimentary to OMP reactive structures (Fig. 2D). The PDGF-B-reactive fascicles often ensheath non-reactive fiber bundles which correspond to OMP-positive bundles in adjacent sections (compare Fig. 2C and D). Regions showing OMP-reactive radiation (Fig. 2C) are non-reactive. In other areas (not depicted) PDGF-B-positive fascicles may partially ensheath bundles of fibers that are not OMP reactive. Adult transplant/adult control Normal OMPpatterns. The normal OMP immunoreactivity is restricted to the olfactory nerve layer over the surface of the olfactory bulb and the bundles of fibers that course between glomeruli (Fig. 3A). The nerve layer and coursing fibers that are reactive are similar to that of the 2 week control but differ somewhat in the glomeruli labeling. The adult shows larger, more heavily reactive glomeruli that are more clearly defined by well-delineated layers of periglomerular cells (Fig. 3A). The OMP reactivity within glomeruli is uniform and diffuse, as in 2 week material (PN7). Normal PDGF-B patterns. In the adult, the PDGF-B immunoreactivity is also quite similar to that in the 2 week PDGF-B preparations (Fig. 3B). The olfactory nerve layer is densely positive with some reactivity penetrating between glomeruli. The olfactory glomeruli themselves are usually non-reactive, but occasionally they do show a minimal amount of immunoreactivity (Fig. 3B). A few immunoreactive fascicles of fibers and cells may be seen penetrating a glomerulus. As reported previously15 most, if not all, mature neuronal perikarya exhibit PDGF-B immunoreactivity (Fig. 3B). Transplant OMP patterns. In the fully developed transplant, OMP-reactive fiber bundles with well-defined boundaries are apparent, but many diffuse areas of reactivity are also seen (Fig. 3C). As in more immature transplants, both well-defined bundles and fields of diffuse reactivity may be found throughout the transplant superficially and deeper. The bundles of OMP-positive fibers are irregularly spaced, often occurring in clusters or groups (Fig. 3C). Transplant PDGF-Bpatterns. The PDGF-B-reactive fascicles sometimes partially ensheath areas non-reactive for PDGF-B, but which correspond to the same OMP-positive areas seen in adjacent OMP-reacted sections (compare Fig. 3C and D). In addition, some areas, not imrnunoreactive for OMP, are also partially surrounded by PDGF-B-positive fibers and cells (Fig. 3D). Varying degrees of PDGF-B immunoreactivity occur within OMP-positive fiber bundles (compare Fig. 3C and D). Notably, radiation of individual OMP-positive fibers into uniformly reactive fields appears to be favored where PDGF-B ensheathment is limited or absent (compare Fig. 3C and D, asterisk). Thus, PDGF-B-positive structures surround areas that are OMP-positive and are usually absent from the OMP fiber radiations.
DISCUSSION We are examining the behavior of olfactory ECs (as identified by their strong PDGF-B immunoreactivity) to assess the growth promoting and axonal guidance potential of these growth
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factor-rich cells. Since these cells produce extracellular matrix molecules12 and show high expression of both PDGF-B15 and at least one of the nerve growth factor receptor antigens (~75 NGFR)‘Y~~ they are likely candidates for facilitation of axonal growth into and through the nervous system. One of these is the pathway of the primary olfactory nerve into the olfactory bulb of the central nervous system. By using a transplantation model (replacing a host olfactory bulb with one
Fig. 3. (A) A portion of the sagittal section from a normal adult olfactory bulb (OB) immunoreacted for OMP. Olfactory nerve layer fibers (ONE) are strongly reactive, as are the subjacent olfactory glomeruli (G) (see inset for details). (B) A portion of a section adjacent to A reactive for PDGF-B. Olfactory nerve layer fibers (ONE) are immunoreactive and partially encircle the underlying lightly reactive (see inset) olfactory glomeruli (G). Note, at this maturity, many neurons are now immunoreactive (arrowheads). (C) A portion of a sagittal section from an adult olfactory bulb transplant (TX OB) ~unoreaued for OMP. Heavily reactive bundles of fibers (a~owheads) on the surface and deeper are seen. Some deeper bundles have surrounding diffuse hnmunoreactivity (see inset) suggestive of a ‘fiber radiation’ (asterisks). (D) A near adjacent section to C immunoreacted for PDGF-B. Reactive patches and fibers are seen deep within the TX OB (arrowheads), some of which are in OMP-negative areas while others surround and infiltrate OMP-immunoreactive bundles (see C). Other areas show PDGF-B-reactive bundles (small arrows) at sites devoid of OMPreactivity. Upper regions positive for OMP are negative for PDGF-B (asterisks), especially (see inset) the ‘fiber radiation’ regions. Eight micrometer paraffin sections. Scale bars=100 p,m.
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from a fetal donor) we have created a situation in which the activity and distribution of ECs is not constrained by the normal growth patterns of the olfactory bulb. We have identified several characteristics of ECs that suggest they may be useful in repair and regeneration of injured nervous system and, thus, in the recovery of function of such damaged tissue. First, in addition to ensheathing olfactory nerve fibers and axons (as shown by OMP reactivity), ECs are also capable of ensheathing non-olfactory nerve fibers/axons within fascicles. Second, EC’s may operate to ensheath fascicles of axons deep within the transplant as opposed to their exclusive localization near the surface in their normal situation. Third, diffusion or radiation of OMP-positive fibers/axons from bundles or fascicles is favored in the absence of PDGF-B-positive ECs. Fourth, this radiation of OMP-positive fibers first seen in the 1 week transplant increases in frequency and distribution at 2 weeks and in the adult. Although there is overlap in some cases, there is a striking complimentary pattern involving the EC expression of PDGF-B and that of OMP by the olfactory nerve. To be of clinical interest, it is necessary that ECs survive and provide guidance as well as growth-related factors for fiber systems other than the primary olfactory nerve. By combining immunoc~ochemistry for OMP and PDGF-B, we have demonstrated that the PDGF-B-rich ECs become associated with OMP-negative (and therefore non-primary olfactory) nerve fascicles within the transplant. Earlier work also demonstrated that ECs retain their ~75 NGFR reactivity within the transplant.23 The findings in the present study of EC involvement in repair following injury and transplantation need to be confirmed in other systems where ECs are not normally found. For example, will introduction of ECs to injured dorsal root fibers facilitate outgrowth of partially damaged or adjacent uninjured dorsal root fibers? In relation to mental retardation, it would be helpful to know if ECs could play a role in re-establishing damaged hippocampal pathways involved in memory.“~‘7 Our finding here of OMP-negative (therefore non-olfactory nerve) fibers, which were immunoreactive for PDGF-B, suggests there may be a potential for ECs to facilitate growth of fiber systems other than olfactory nerve. In order to be effective in repair, the ECs must be able to survive and function not only in peripheral or superficial regions of the brain, but also in deeper areas. Here, we have shown that ECs can penetrate and exist through adulthood, not only on the surface of the olfactory bulb transplant (their usual position), but also within its depths. The EC ensheathment of both olfactory nerve fiber bundles (OMP-positive) and non-olfactory nerve (OMP-negative) fiber bundles at deeper brain regions, in addition to the previous demonstration of p7.5 NGFR reactivity in the same regions of similar transplants, 23 all point to a possible role for these cells in the growth of regenerating fiber bundles into deep brain. Thus, there may be a potential for enhancement of transplant outgrowth and internal reconnectivity in brain. Despite outgrowth, even for great distances in deep brain areas, complete repair and recovery of function after injury or transplantation will ultimately depend on re-establishment of contacts or synapses with an appropriate target area. I9 In addition to initiating outgrowth, facilitation of guidance and ‘nou~shment’ of the pathway, ECs need to allow all of these roles to culminate in a functional reconnectivity in some ordered pattern, with an effective target. The ECs need to permit a ‘branching out’, for example, of axons from the bundles into a terminal field where synaptic contacts can be made. Examples of these events might be identified in the visual system where terminal arbors of undamaged optic fibers sprout into colliculus or thalamic fields after damage.” Atypical reinnervation patterns have been reported in such cases as this and other systems, resulting in altered function, but this often depends on such factors as extent of lesion and distances for fiber regrowth.” The ECs’ effectiveness in bringing the ‘regenerating’ fiber bundles as near as possible to the appropriate target, and then allowing sprouting and synapse formation, needs to be determined. We have shown that, in the transplant situation, ensheathed olfactory nerve fiber bundles do eventually undergo branching. Whether or not EC ensheathed bundles will behave in the same manner when transplanted into another system is unclear. Other than electrophysiology, electron microscopy in conjunction with transpfant markers (such as horseradish peroxidase) or appropriate neurotransmitter localization will be necessary to determine such re-establishment of contacts. We have not addressed the function of the ECs in systems other than olfactory transplants. Our present intent was to fully describe the anatomical, structural details of the patterns of EC
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distribution and activity in relation to the architecture of a maturing transplant (a model for repair after tissue damage). This should form a basis to elucidate possible functional features of these patterns and considerations of potential or limitations for success of our approach. The findings presented here have suggested that ECs are attractive candidates for facilitation of axonal penetration from the periphery into the central nervous system. Further, ECs also seem to act in manipulated guidance of nerve fascicles through areas of injury within the brain. The actual mechanism by which these cells accomplish this remains to be elucidated. Much work is required to demonstrate not only how these events are functionally limited, but whether isolated ECs can actually participate in the processes in cases of human brain injury, disease or malformations. Acknowledgements-This study was supported by N.I.H. Research Grant nos. NS09678 and HL18645. The authors are indebted to Ren6e Czarapata for secretarial help, to Paul Schwartz and Janet Clardy for preparation of photographic plates, and to Jeff Bedell and Mary Gross for technical assistance. L. E. W. is an affiliate of the Child Development and Mental Retardation Center of the University of Washington.
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