Morphologic features of embryonic neocortex grafts in adult rats following frontal cortical ablation

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Brain Research, 40 ~ (1987) 162-167

Elsevier BRE 21963

Morphologic features of embryonic neocortex grafts in adult rats following frontal cortical ablation E.J. Mufson 1, R. L a b b e 2 a n d D . G . Stein 2 1Division of Neuroscience and Behavioral Neurology of the Harvard Neurology Department and the Charles A. Dana Research Institute of Beth Israel Hospital, Boston, MA 02215 (U.S.A.) and 2Brain Research Laboratory, Clark University, Worcester, MA 01610 ( U.S.A. )

(Accepted 26 August 1986) Key words: Fetal transplants: Frontal cortex; Acetylcholinesterase: Choline acetyltransferase; Cytochrome oxidase; Morphology

Embryonic cortex from 19-day fetuses was transplanted in a medial frontal cortex wound cavity of 105-day-old male rats. Nisslstained tissue revealed little internal laminar organization. Graft sections impregnated by the Loyez method exhibited bands of myelinated fibers surrounding implants as well as long-interconnecting and swirl-like fiber fascicles within the implant. Tissue processed histochemically for acetylcholinesterase and choline acetyltransferase revealed enzyme-positive fibers and cell bodies within the grafts. Cytochrome oxidase histochemistry revealed regional variations in the metabolic activity of the grafts. In summary, although our frontal cortex grafts exhibit many of the morphological features seen in intact frontal cortex, the organization of these components within the implant is dissimilar to normal cortical tissue.

During the last several years, there has been a resurgence of interest in the ability of implanted fetal central nervous system (CNS) tissue to promote anatomic restoration and return of function to brain-damaged mammals 3'4'6-11A6'20. However, the parameters underlying functional recovery are not well defined. One important question is whether brain grafts develop morphological characteristics seen in normal intact tissue, since it may be that such features are a requisite for functional restitution. Thus, the purpose of this study was to analyze the histologic features of fetal neocortex grafts which we have previously shown to enhance behavioral recovery 3°'31. Specifically, grafted fetal cortex was processed for the demonstration of cell types using Nissl stains, myelinated fibers using the Loyez method, acetylcholinesterase (ACHE) using A C h E histochemistry, choline acetyltransferase (CHAT) using antibody immunohistochemistry and metabolic activity demonstrated by cytochrome oxidase histochemistry. The present observations are based on male Sprag u e - D a w l e y (Charles River: CD) rats, approximately 105 days of age which sustained bilateral medial

frontal cortex aspiration lesions prior to receiving fetal cortex implants. Seven days after the initial aspiration surgery, the rats were anesthetized and, on this day, according to procedures described previously 20 received transplants of frontal cortex taken from S p r a g u e - D a w l e y (Charles River: CD) donors on day 19 of gestation. With a metal rod (tip diameter 1.0 mm) attached to a 1.0 ktl syringe, 2 pieces of the donor tissue representing frontal cortex from each hemisphere (6 mm3), bathed in cold ( 3 - 5 °C) Ringer's solution, were extruded from the syringe approximately 0.5 mm into the wound cavity. Two months postimplantation, rats (n = 7) were perfused with either 0.1% glutaraldehyde ( T A B B ) - 4 % paraformaldehyde (Fisher) or 0.1% glutaraldehyde (Kodak)-2% paraformaldehyde (Aldrich). The latter fixative combination was used to enhance immunohistochemical staining performed with the rat monoclonal antibody AB8 against ChAT 23-25'28. Additional animals (n = 5) were perfused with a solution of 1% paraformaldehyd e - 1 . 2 5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for cytochrome oxidase staining 34. Follow-

Correspondence: E.J. Mufson, Department of Neurology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, U.S.A.

0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

163 ing fixation, each brain was cut on a freezing microtome into 6 series of matching 40 p m thick sections. Individual series of sections were then processed according to the following procedures. Cyto- and myelo-architectonic information was obtained from sections stained with cresyl violet or thionin for Nissl substance and with the Loyez method for myelin. Additional cytoarchitectonic observations were made on animals (n -- 2) perfused with 10% formaldehyde and embedded in celloidin and cut serially at 40/xm thickness. Acetylcholinesterase histochemistry was performed with acetylthiocholine as the substrate according to the procedure of Hedreen et al. 13. This AChE reaction product yields sharply defined axons and cell bodies which appear black 2'13. Control material was obtained by using butyrylthiocholine as the substrate. Some sections processed for AChE were also counterstained with thionin in order to observe the relationship between AChE-positive neurons

Fig. 1. Dorsal surface view of two rat brains showing bilateral (A; x5) and unilateral (B; ×4) cortex implants (arrows) positioned within the damaged frontal cortex of the host brain.

and other perikarya of the transplants. Choline acetyltransferase immunohistochemistry was done using rat monoclonal antibody AB8 and the peroxidase-antiperoxidase method of Sternberger 33. Monoclonal antibody AB8 binds active ChAT in a specific double antibody assay, binds specifically to ChAT using the Western blotting system and localizes known cholinergic neurons immunocytochemically21,22,24,25,28. Control material was obtained using rat IgG instead of AB8. Cytochrome oxidase histochemistry was performed according to the procedure of Wong-Riley 3~. Formation of the wound cavity was confined mainly to the medial frontal cortex, although there was some damage to the caudate nucleus and the olfactory bulb (Fig. 1). However, there was no apparent involvement of the septal region. The transplants from embryonic frontal cortex seen in the implantation cavity were either embedded in the parenchyma of the host brain (Fig. 2A), forming a continuous bridge connecting the injured hemispheres, or came to lie in the subarachnoid space connected by a tissue stalk to the host cortex. Light microscopic examination of the host-transplant interface revealed continuity as well as regions marked by apparent glial proliferation (Fig. 2A, B). Border regions separating the graft and host were often observed adjacent to a zone of low neuronal cell density (Fig. 2B). We also found marked variation in the internal organization of the grafts. In some cases, the transplant was composed of several cellular islands with no preferential positioning or lamination of neurons (Fig. 2A). In contrast, grafts which were more homogeneous in composition had the appearance of a pseudolaminar organization (Fig. 2B). In these grafts, separate cell aggregates were surrounded by a cellular zone resembling the molecular layer of intact cortex. Nissl-stained tissue demonstrated that the grafts contained various size neurons as well as binuclear-like perikarya (Fig. 2C), neurons with multiple nucleoli, and glia. In grafted tissue processed with the Loyez stain we observed bundles of myelinated fibers. The most consistent location of myelinated fiber tracts was along either the interface separating graft subsectors or the host and the graft (Fig. 2G). Some areas of the grafts contained narrow fiber tracts which coursed parallel to the long axis of the implant, whereas in other regions fascicles formed swirl-like fiber arrays (Fig. 2G).

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Fig. 2. A: photomicrograph of cresyl violet-stained coronal section of fetal cortex implant partially embedded within the parenchyma of the host brain consisting of several lobules (open circles). Note the non-laminar arrangement of the graft as compared to the adjacent host cortex (HC). Dorsal is to the left and medial is towards the top. x25. B: photomicrograph of cresyl violet stained transplant (TP) containing a cluster of neurons (small black arrows) surrounded by a relatively cell-free molecular-like zone. Virtually no gila scarring is seen along the host-transplant interface, x50. C: binuclear-like neuron, x 100. D: intense AChE-positive fiber staining in both the host cortex (HC) and adjacent transplant (TP). Positive axons (arrows) are seen crossing the border (dashed line) between the host and the transplant, x50. E: fetal cortex grafted tissue stained for AChE showing AChE-reactive fibers (curved arrows) and neurons (small black arrows) as well as butyrylcholinesterase-positive blood vessels (open arrows), x50. F: grafted cortical neuron immunohistochemistry stained for CHAT. x 100. G: photomicrograph showing myelinated fibers (large black arrow) along the exterior surface of an implant. Note the bifurcation of a bundle of myelinated fibers (arrowhead) prior to encircling a piece of the implant. Swirls (open arrow) and pencil thin bundles (curved arrow) of myelinated fibers seen coursing within a transplant, x25. H: coronal section of grafted fetal cortex showing variations in endogenous cytochrome oxidase activity. Note the dark patches of enzymatic activity (open arrows) located in the lateral as compared to the lower levels of activity in the more medial portions of the graft. × 5. Dorsal is left, ventral is right, lateral is up and medial is down. The dark strip across the left is a fold in the tissue, x5.

165 Furthermore, all implants stained for the enzyme ACHE, exhibited positive fibers and cell bodies (Fig. 2D, E). Control sections treated with butyrylthiocholine were negative for cellular or axonal staining. Unlike normal adult rat cortex, there was no clear laminar organization of AChE and the staining was not uniform within or between grafts (Fig. 2D, E). In addition, enzyme-containing fibers were observed crossing the host-graft interface (Fig. 2D) where the staining was most intense as well as within tissue stalks connecting the host cortex and the implant. Thionin-counterstained, AChE-reacted, grafted tissue revealed that only a small number of neurons were AChE-positive within the implant. AChE-stained fibers were also observed adjacent to esterase positive blood vessels (Fig, 2E). This vascular staining was seen in tissue reacted with butyrylthiocholine and acetylthiocholine, indicating that these vessels were butyrylcholinesterase-positive. Tissue stained with the monoclonal antibody AB8 for the cholinergic enzyme, CHAT, revealed only a few positive neurons and axons. These grafted ChAT immunoreactive neurons were small bipolar cell bodies located primarily in the periphery of each implant (Fig. 2F). Control sections employing non-specific rat IgG in place of the AB8 were negative. Staining for the mitochondrial enzyme, cytochrome oxidase, which has been suggested to correlate with cell metabolism 34, revealed that graft metabolic activity was not homogeneous. For example, in the graft shown in Fig. 2H, areas of intense activity were located in its lateral portion as compared to the less intense more homogeneous reactivity seen in its medial sector. Since grafted fetal cortex does not appear uniformly active, the zones of highest metabolism may have a greater concentration of neurons or receive greater synaptic input as suggested by Sharp and Gonzalez 29. It is also possible that these areas of greater physiological activity may produce a neurotrophic substance(s) critical for behavioral recovery. In the present study, E l 9 frontal cortex implanted into a bilateral frontal cortex wound cavity developed adjacent to the damaged cortex and contained many of the morphologic features characteristic of mature cortex. Although the grafts contained normal-appearing neurons, long myelinated axons, ACHE- and ChAT-positive fibers and perikarya as well as distinct regions of metabolic activity, these

morphologic components were not displayed in a fashion similar to that observed in normal brain tissue. Furthermore, distributed among these normallooking perikarya were cell bodies with multiple nucleoli as well as binuclear appearing neurons similar to those described by Alexandrova and Polezhaev 1. Since most mature neurons have a single nucleolus as compared to the multiple nucleoli which are often seen in developing neurons 15, it is possible that some .of the neuronal perikarya in the grafts may not have responded to or lacked the signals that guide differentiation of young neurons. We also demonstrated bands of myelinated fibers located primarily along the external regions of the graft similar to that reported in brainstem implants ~8 suggesting that the mechanism(s) guiding the development of myelinated axons in such grafts may be potentially similar. In general, the overall arrangement of myelinated axons within the implant was unlike that found in normal frontal cortex. These observations may, however, provide clues as to the processes underlying the normal maturation of long axonal pathways in the central nervous system of mammals. ChAT and AChE staining revealed positive fibers and cell bodies distributed within the grafts. CHATreacted tissue revealed only a very few positive perikarya located mainly at the periphery as compared to the more numerous and widely distributed ACHEpositive neurons seen in grafts. Interestingly, the distribution of AChE-reactive perikarya has been shown to be widespread throughout all cortical lamina, whereas ChAT-positive neurons are found mainly in the more superficial cortical layers 23. Perhaps the differential normal cortical distribution of these cell types are only partially reflected in our cortical grafts. The AChE activity observed in the cortical grafts may have developed intrinsically after implantation or originated from extrinsic sources. Since histochemical investigations of rodent cortical AChE indicate that there are very few positive fibers in the newborn ~7, it is more likely that there was fiber ingrowth arising from extra-transplant areas. The major extrinsic source of cortical cholinergic input arises from the cholinergic neurons located within the nucleus basalis (Ch4) 24,25, whereas a minor cortical cholinergic innervation originates from the brainstem cholinergic neurons of the pedunculo-pontine (Ch5) or the

166 lateral dorsal t e g m e n t a l (Ch6) nuclei 27. It is possible, therefore, that the cholinergic axons originating from these cholinergic n e u r o n groups grew to innervate the maturing cortical transplant. Interestingly, reinnervation of d e n e r v a t e d h i p p o c a m p u s has been d e m o n s t r a t e d by A C h E and ChAT histochemistry following implantation of embryonic cholinergic septal neurons into animals with damage to the septoh i p p o c a m p a l cholinergic pathways 5'1s'32. The reinnervation of h i p p o c a m p u s appears in a laminar fashion as o p p o s e d to the non-laminar p a t t e r n seen in our neocortical grafts. Perhaps, differences in the transplantation p r o c e d u r e s used may account for these observations. Such results have led to the suggestion that regeneration of a x o n t o m i z e d brain perikarya can be initiated by a piece of deafferented embryonic CNS 32. H o w e v e r , it has also been shown that trophic factors can influence the growth and survival of various types of neurons and non-neuronal tissue including cholinergic p e r i k a r y a 14'26. Therefore, it is possible that grafted fetal cortex releases a t r o p h i c substance which may p r o m o t e axonal sprouting or sparing of neurons that would ordinarily die as a result of the injury. With respect to behavior after frontal cortex grafts, the present as well as previous results 2°'3°'31 in-

1 Alexandrova, M.A. and Polezhaev, L.V., Transplantation of various regions of embryonic brain tissue into the brain of adult rats, J. Hirnforsch., 25 (1984) 89-97. 2 Bakst, I. and Amaral, D.G.. The distribution of acetylcholinesterase in the hippocampal formation of the monkey, J. Cornp. Neurol., 225 (1984) 344-371. 3 Bjorklund, A. and Stenevi, U., Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants, Brain Research. 177 (1979) 555-560. 4 Bjorklund, A. and Stenevi, U., Intracerebral nigral implants: neuronal replacement and reconstruction of damaged circuitries, Annu. Rev. Neurosci., 7 (1984) 279-308. 5 Cotman, C.W., Specificity of termination fields formed in developing hippocampus by fibers from transplants. In J. Sladek, Jr. and D. Gash (Eds.), Neural Transplants, Plenum Press, New York, 1984, pp. 305-324. 6 Das, G,D., Hallas, B.H. and Das, K.G., Transplantation of brain tissue in the brain of rat. I. Growth characteristics of transplants from embryos of different ages, Am. J. Anat., 158 (1980) 135-145. 7 Dunnett, S.B., Low, W.C., Iversen, S.D., Stenevi, U. and Bjorklund, A., Septal transplants restore maze learning in rats with fornix-fimbria lesions, Brain Research, 251 (1982) 335-348. 8 Fine, A., Dunnett, S.B., Bjorklund, A. and Iversen, S.D., Cholinergic ventral forebrain grafts into the neocortex im-

dicate that n o r m a l m o r p h o l o g i c organization of implanted cortex may not be a necessary prerequisite for restoration of behavioral performance. F o r example, we have found that fetal frontal cortex implants also are capable of reducing visual learning impairments in animals with occipital cortex d a m a g e , while transplanted occipital cortex tissue was less effective in producing recovery of brightness discrimination c o m p a r e d to frontal cortex grafts 3°'31. These investigations suggest that behavioral i m p r o v e m e n t m a y be the result of non-structural factors related to the grafted tissue. Perhaps fetal cortex grafts induce functional e n h a n c e m e n t by the p r o d u c t i o n of some as yet unknown trophic substance. W e wish to thank L e a h Christie, Chris Palatucci, D i a n n a K a h m Terry Martin and R i c h a r d Plourde for expert secretarial, histological and p h o t o g r a p h i c assistance. W e are grateful to Dr. B, W a i n e r for supplying the A B 8 antibody and to Dr, J. Rogers for helpful comments on the manuscript. S u p p o r t e d in part by N I M H 1339514, A m e r i c a n Paralysis Association Contract 169184001, an A D R D A Faculty Scholar A w a r d ( E . J . M . ) , an A l z h e i m e r ' s R e s e a r c h C e n t e r N I A AG-05134, a Javits Neuroscience Investigator A w a r d , and by Clark University.

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