neural grafting to ischemic and excitotoxic, · and extensive ingrowth of cholinergic host...

ACTA NEUROBIOL. EXP. 1990, 50: 367-380

Symposium "Recovery from brain damage: behavioral and neurochemical approaches" 4-7 July, 1989, Warsaw, Poland

NEURAL GRAFTING TO ISCHEMIC AND EXCITOTOXIC, HIPPOCAMPAL LESIONS IN THE ADULT RAT

Niels TPINDER, Torben SBRENSEN, Flemming Fryd JOHANSEN and Jens ZIMMER

PharmaBiotec, Institute of Neurobiolagy, University of Aarhus, DK-8000 Aarhus C. Denmark

Key words: transplantation, regeneration, fascia dentata, nerve cannect,ions, ibotenic acid

Abstract. Results of neural grafting to excitobxic and ischemic lesions of the adult rat hippocampus is reviewed with particular emphasis an the exchange of host-transplant nerve ~ a e c t i o n s . Bawd on observations obtained by a variety of tracing techniques a b u t 6 weeks after - (i) grafting d pieces of neonatal fascia dentata to one week old ibotenic acid lesions of the adult rat fascia dentata, (ii) grafting of suspensions of late fetal CA3 cells to one week old ibotenic acid lesions of the adult rat CA3 - and (iii) grafting of suspensions of late fetal CAI cells to one week old ischemic lesions of the adult rat CAI, we conclude that axon-sparing lesions of the mentioned types enhance the growth of adult host brain axons of the so called point-to-point type into neural grafts. We interpret these findings as the result of an h- proved capability of adult host brain axans to participate in the graft hast interaxonal competition for synaptic sites in the developing neural grafts. At the same time an extensive growth of graft CA3 and CAI axons into the host brain was demonstrated.

INTRODUCTION

Grafting of immature central nervous tissue is now a well-established tool in experimental studies of the growth, plasticity and regeneration

18 - Acta Neurobiol. Exp. 4-5/90

of nerve cells and nerve connections in the brain and spinal cord. We have previously used grafting of immature hippocampal and fascia dentata tissue to the brain of newborn and adult rats with this purpose (25, 26, 30-33, 42-47). In m e set of studies we found that homotopic grafting of pieces of nelonatal fascia dentata to rats with an X-ray induced maldevelopment of the fascia dentata could restore the damaged hippocampal neuronal circuitry. The recipient age did, however, clearly influence the extent of this restoration (25, 27). The exchange of host- graft nerve connections was thus much more extensive after grafting to developing one to 21 day old recipient rats than after grafting to adult, 5 week to 10 month old rats (27). The importance of nerve type f o r the exchange of host-graft nerve connections was also demonstrated. There was thus no apparent decrease in the ingrowth of cholinergic fibers from the host septum into the hippocampal grafts with increasing maturity of the recipient rats. In this presentation we shall review results from some of our recent studies, showing that extensive exchange of nerve connections is possible a h after grafting to adult rats, provided that the grafts are placed in so-called axon-sparing lesions. In such lesions, induced by local intracerebral injectim of excitotoxins like kainic or ibotenic acid or transient cerebral ischemia, the neurons degene- rate, while the extrinsic nerve fibers remain for several months (cp. axon-sparing), despite the absence of normal target structures (5) (Fig. 1).

AXON-SPARING LESIONS

Adult rats were subjected to axon-sparing lesioning of the hippocampus or fascia dentata by - (1) injectim of the glutamic acid ana- logue, ibotenic acid (IA) into either the CA3 area (35) or the fascia dm- tata (34) of the dorsal hippwampal region, - or (2) subjecting the rats to transient cerebral ischemia by the 4-vessel occlusim technique, which causes degeneration of CAI pyramidal cells in the dorsal hippocampus (36).

For the IA-lesions a dose of 5 pg IA dissolved in 0.5 p1 phosphate buffer (pH 7.4) was injected stereotaxically. Depending on the injection site this resulted in - (1) degeneration of the fascia dentata, including the dentate granule cells, the adjacent hilar (CA4) neurons and some- times the pyramidal cells in the most adjacent parts of CA3, - or (2) neuronal degeneration in the dorsal CA3 area. The axon-sparing property of the IA-lesion has previously been demonstrated by showing the persistence of cholinergic, noradrenergic and serotonergic fibers from the septum, locus coeruleus and the raphe nuclei, respectively, in the lesion area (11, 12, 24). In our studies the persistence of cholinergic septohippo-

campal fibers was shown by acetylcholinesterase (AChE) staining. Other spared dentate and hippocampal afferents were dem,oastrated by histochemical, Timm sulphide-silver staining for nerve terminal-related hea- vy metals (zinc), silver staining of induced anterograde axonal degeneration, or immunocytochemistry for neuropeptides (34, 35).

The ischemic lesions were induced by transient cerebral ischemia, using the 4-vessel occlusion technique of Pulsinelli and Brierley (23). Under controlled circumstances transient (15-20 min) interruption of the cerebral blood flolw results in a selective degeneration of CAI pyramidal cells (1, 9, 23) and somatastatinergic ,neurons in the dentate hilus (CA4) (8) at the septo-dorsal levels of the hippocampus. Electron micruscopical demonstration of surviving presynaptic structures in the lesion area together with the presence of terminal related high-affinity uptake of glutamate a t 4 days after the ischemic insult has previously been used to show the axon-sparing nature of the lesion (7). In our study cholinergic AChE-positive septohippwampal fibers and Timm-positive and immunocytochemically reactive neuropeptidergic afferents were found to he present in the CA1 lesions for several weeks after the ischemic insult (36).

THE AXON-SPARING LESION AS A FAVOURABLE GRAFTING SITE?

Although the excitotoxin injectiorns and the ischemic insults spare the afferent nerve fibers of extrinsic origin in the lesion area, long-term reorganization of these is likely to take place, just as the lesion areas *lay long-term atrophy of the neuxopil (Fig. 1) (5, 17, 34-36). In our studies the neural grafting was performed one week after lesiorning. At this time the lesioned areas were characterized by dense reactive gliosis and persistence of afferent nerve fibres originating from neurons outside the lesion (and in case of the ischemic lesions some surviving, local non- pyramidal CAI neurons). Without actually having been traumatically damaged these afferents had lost their normal target cells. One week after the insult i t might therefore be anticipated that they would be in a primed, reactive state, which would increase their ability, compared to both intact and acutely transected nerve fibers, to innervate new nerve cells grafted into the area.

TRANSPLANTATION

Neanatal dentate tissue or late embryonic CA3 or CAI hippocampal tissue were homotopically grafted to the respective, one week old lesions (34-36). For grafting to the IA-lesioned fascia dentata tissue blocks con- taining the fascia dentata with adjacent hilar (CA4) tissue were dissected

IBOTENIC ACID INJECTION /CEREBRAL ISCHEMIA

Focal Degeneration & Gliosis .

Reorganization > lrnplantation

/ L A t r o ~ h v Regenerat ion

&

Fig. 1. The axon-sparing lesion-transplantation model. a, ibotenic acidlischemia induced neuronal degeneration and reactive gliosis. b, reorganization and atrophy of the lesioned area. c, connective integration of grafted neurons with the host

brain. Mo'dified from Coyle and Schwarcz (5).

from newborn rats. For the hippocampal grafts we used cell suspensions made from embryonic (E18-20) CAI and CA3 tissue, respectively. Six weeks or more a f t e ~ grafting the histological o~ganizatiom of the grafts and the structural and connective integration with the host brains were studied wing wdinary cell staining, AChE and Timm sulphide-silver staining, immunocytochemical staining for neuropeptides (cholecystoki- nin, enkephalin, ~om~atostatin) and astroglia, and fiber tracing by either retrograde axonal transport of the stereotaxically injected fluorescent dye Fluoro-Gold, or light or electron microscopy of induced anterograde axonal degeneration.

GRAFT SURVIVAL AND EiISTOLOGICAL ORGANIZATION

The dentate and hippocampal grafts survived well in their respective homotopic lesion areas. After 6 weeks or more the grafts did not display any significant supranormal increase in imrnunoreactivity for the astroglial marker, glial fibrillary acidic protein, GFAP. In this way the grafts stood out in contrast to the heavily gliotic, damaged parts of the surrounding host brain. Instead of being harmful, the host brain astro- gliosis might in fact have been beneficial to the grafts when it is con- sidered that neurotrophic factojrs are produced by reactive glial cells (4, 6, 10, 13, 18, 37).

Fig. 2. Cell stain of fascia dentata transplant 6 weeks after grafting into 1 week old IA-lesion of the dorsal fascia dentata of adult rat. The graft with a preserved, but extraordinary folded, organotypic organization occupies the entire lesion area. f, fimbria; g, granule cell layer; h , hilus (CA4);

rn, molecular layer; p, pyramidal cell layer. x 36.

Fig. 3. CA3 cell suspension transplant (T) 6 weeks after grafting to 1 week old IA-lesion of adult rat CA3 area. In the graft clusters of CA3 pyramidal cells are seen. Ordinary cell stained section. FD,

fascia dentata. x 33.

The individual grafts contained the neurons typical of the origin of the donor tissue. The dentate grafts, which were transplanted as tissue blocks, had in general retained an orgallotypical appearance with a distinct molecular and granule cell layer surrounding a typical hilar area (Fig. 2). The CA3 and CAI transplants, which were grafted as cell suspensims with disruption of the original organization at the time of grafting, were dominated by their respective pyramildal cells (Fig. 3). The pyramidal cells were typically arranged in clusters or short bands, suggestive of reaggregation of the suspended cells. In addition to the respective area-specific, main types of neurons, CCK and somatostatin irnmunoreactive moa-pyramidal neuxcms were present in all transplants. The same was shown by Zimmer and Sunde (45) for both homo- and heterotopieally located solid grafts d hippocampal and dentate tissue in normal newborn and adult recipients, and by Mudrick e t al. (14) after homotopic grafting of CAI cell suspemsions to ischemic CAI lesions of adult rats.

NERVE CONNECTIONS

Dentate grafts

The dentate grafts placed in IA-lesions of the adult rat fascia dentata were innervated by host septohippocampal fibers, demonstrated by histochemical AChE staining. This staining has previously been used to monitor cholinergic innervation of hippocampal and dentate transplants (26, 42), and in accordance with these studies the AChE staining was mast dense around the cell layers. In grafts innervated by host perforant path fibers in a homotypic, lamimar fashion (see below), the AChE- positive fibers adapted to the laminar termination of these normal, major afferent pathways, as also shown by Zimmer e t al. (46). The consistent and extensive ingrowth of cholinergic host septohippocampal fibers was expected. These fibers belong to the so-called global, and in functional terms "level setting" systems. These systems also include the central nmadrenergic and se~otonergic projections, and they are all known to passess a high regenerative c,apacity even in the mature brain (2, 26, 46).

The perforant path projection from the entorhinal cortex to the dentate molecular layer belongs to the so-called "point-to-point" system. These systems typically have a very precise and limited distribution and cellular terminatian, and nerve fibers of this type, which typically use aminoacid transmitters, normally possess a very low regenerative ability in the adult brain. The tissue blocks of neonatal fascia dentata grafted to the IA-lesions in the adult rats were, however, heavily and extensively innervated by host perforant path fibers, as demonstrated

by Timm staining and traced by anterograde axonal degeneration and silver staining or electron microscopy. In well-placed grafts the host perforant path fibers thus innervated the outer 1/2 to 213 of the graft dentate molecular layer with a laminar distribution and density resem- bling that of the normal perforant path. Comparisons with dentate grafts placed in adult rats without other preceding or acute lesioning than the one caused by the actual insertion of the grafts (25, 42, 48), made it evident that the IA-lesion had enhanced the extent and density of the host perforant path ingrowth (Fig. 4). The innervation of dmtate grafts in the IA-lesioned fascia dentata was more like the innervation observed after grafting to immature normal crr X-irradiated newborn rats (25, 27-29). From these results we conclude that a preceding, axon-sparing lesion can stimulate anid enhance the regenerative axcmal growth of mature central neurons of the "point-to-point" type, monitored by their ability to innervate grafts of immature brain tissue.

Fig. 4. Density and distribution of degenerating perforant path terminals plotted from columns of electronmicrographs covering the dentate molecular layer of normal (11, and grafted (2-4) fascia dentata. 1, normal adult ra t with predominantly medial perforant path degeneration. 2, fascia dentata grafted to intact adult fascia dentata, showing distribution of degenerating host perforant path terminals close to host-graft interface. 3, same case as in 2), demonstrat4g rapid decline in innervation density only 200 km further away from interface. 4, dentate graft in IA-lesion of adult fascia dentata, displaying dense host perforant path innervation

far from host-graft interface and entry of host fibers, cf. Tender et al. (35).

Growth of dentate graft mossy fibers into the adult, IA-lesioned recipient brain was not oibserved in our study (34). Two likely rewins were the lack of orientation of the graft hilar area towards the host

CA3 region (which locally had been deprived of its mossy fiber input due to the host dentate lesion), and the presence of gliotic host dentate and CA3 tissue between the graft hilus and intact parts of the host CA3. It should be mentioned, hawever, that growth of dentate graft mossy fibers to the host CA3 has been demonstrated both after grafting to the normal adult ra t hippocampus (29) and after grafting to colchicine-induced lesions of the adult rat fascia dentata (21).

Hippocampal CA3 grafts

Several types of nerve connections were exchanged between homotopically placed CA3 grafts and the IA-lesi'aned host brain (35). The c ~ e c t i m s are shawn schematically in Fig. 5, together with the normal main connections of CA3.

NORMAL IA LESION TRANSPLANT contralat.

hip.

ipsiiat . hip.

contralat. hip.

I I

com \ I I

ipsilat. hip.

Fig. 5. Schematic illustration of the connective integration of CA3 transplants in IA-induced, axon-sparing lesions of the adult rat CA3 region. assoc, CA3 asso- ciational fibers; corn, comissural fibers; mf, mossy fibers; pp, entorhinal perforant

path fibers; septal, cholinergic septohippocarnpal fibers.

The afferent connections f rom the host brain to the CA3 grafts in- cluded a homotypic innervation by AChE-positive septohippocampal fibers evidenced by the presence of a dense AChE-staining in all CA3 grafts, in particular around the cell clusters.

In T i m stained sections the hippocampal mossy fibers are characterized by their large terminals and the very dense, jet black staining of these terminals. Such characteristically stained, mossy fiber terminals were present in several of the CA3 grafts, placed in the axon and hence also mossy fiber-sparing IA-lesi.ons of the host CA3. The mossy fiber

h e r v a t i o n was confirmed by electron microscopy, demmstrating typical large terminals in the mormal position close to the CA3 pyramidal cell bodies. As no~mally each mossy fiber terminal formed multiple asymmetric synapses with dendritic shafts and complex spines. By their specific, homotypic distribution and ultrastructural characteristics the mossy fibers provided another example of increased regene~ative axcmal growth of a so-called "point-toipoint" system in the mature brain, induced by a preceding axon-sparing lesion.

Innervation of the CA3 grafts by commissural fibers arising in the contralateral host hippoicampus was demonstrated by anterograde axomal degeneration and electron micrloscopy. The commissural fibers were traced even to1 the central parts of the grafts and f m e d asymmetrical synaptic contacts with dendritic spines and shafts of the graft CA3 neurons.

Regarding efferent connections from hippocampal grafts to the host brain, we have recently s h o w that hippocampal CA3 and hilar (CA4) neurons can establish colmmissural connections to the contralateral host hippocampus after grafting to the newborn (33) and the adult rat hippocampus (unpublished results). In accordance with the expected effects of increasing recipient age (see above) the number of projecting neurons were, however, few after grafting to the intact, adult hippocampus.

CA3 graft efferent connections to the IA-lesimed host brain were traced, using retrograde axanal transport of the fluorescent dye, Fluoro- Gold, from injection sites in the hast brain (35). In this way graft CA3 pyramidal cells were found to project to at least two normal CA.7 projection areas in the host brain, namely intact parts of the ipsilateral hippocampus (Schaffer collaterals) and the contralateral hippocampus (commissural fibers). Ipsilateral projections have previously been demonstrated by Zhou et al. (39) by immunocytochemical staining of mouse hippocampal grafts placed in the hippocampus of intact, adult mice. In view of the recent observations that white matter oligodendrocytes impedes fiber growth (3), it is noteworthy that the CA3 graft fibers at least during part of their quite lengthy trajectory to the contralateral host hippocampus must traverse mature white matter in the host fimbria and hippocampal commissure.

Hippocampal CA 1 grafts

The exchange of nerve cmnectims between CAI grafts placed in ischemic CAI lesions and the adult recipient rat brain is sholm rxhei- matically in Fig. 6.

Like the other grafts the CAI grafts also received a cholinergic AChE-positive host septohippocampal input.

NORMAL ISCHEMIC LESION TRANSPLANT

SEPTUM SEPTUM

Sch

PP P ipsilat. ipsilat.

hip. , hip.

Fig. 6. Schematic illustration of the oomective integration of CAI transplants in ischemia-induced axon-sparing lesions of the adult rat CAI region. com, commissural fibers; pp, entorhinal perforant path fibers; Sch, CA3 Schaffer collaterals;

septal, cholinergic septohippocampal fibers.

Schaffer collaterals and commissural fibers from the ipsilateral and cmkalateral host hippocampus were shown to innervate the CAI grafts by anterograde axonal degeneration after lesioning of the ipsilateral CA3 and the contralateral fimbria. Electron microscopy of such cases revealed that the afferent host fibers made asymmetric synaptic coin- tacts with in particular demdritic spines of graft neurons.

Tracing of efferent CAI graft connections into the host brain was successfully perf o r m d by demonstrating retrograde axonal transport of Fluoro-Gold from injections placed in the ipsilateral host CAlIsubicu- lum at distances of 2 mm or more away from the graft (36).

CONCLUDING REMARKS

The results reviewed here demonstrate that excitotoxic and ischemic l e s i m in the adult brain provide a suitable and apparently even favourable environment for the survival and growth oif grafted, immature central neurons. In this way our results support and extend observations made in other studies (15, 16, 19, 20, 22, 38-40). Through a supply of glial derived neurotrophic factors the lesion-induced reactive gliosis might accordingly be beneficial for the grafted neuroms (4, 6, 10, 13, 18, 37). The axon-sparing properties of the excitotoxic, ibotenic acid and ischemic lesions moreover significantly enhanced the growth of mature hwt brain

nerve f ibe~s of the "point-to-point" type into the developing grafts. This apparent priming effect of the preceding, axon-sparing lesion, which deprives the host fibers of their normal target neurons without traumatically injuring the fibers, obviously allowed the mature host fibers to compete more successfully with the developing, intrinsic graft fibers for available and potential synaptic sites in the grafts. The axonal growth capacity of graft projection neurons, like the CAI and CA3 pyramidal cells, was demonstrated by the formation of long, efferent projections from the grafts to the adult recipient brain. The observation that some of these fibers must have grown through host white matter to get to their host brain target areas suggests that white matter in the mature brain may not form an absolute barrier for neurite outgrowth, cp. Ca- roni et al. (3). At present we do not know, however, to what extent the graft fibers were growing through intact white matter or along lesioned host fibers.

The possible functional effects of the dentate and hippocampal grafts placed in the ibotenic-acid lesions are not 'known at present. From other studies (15, 16) we know, however, that efferent projections from CAI cell suspensim grafts placed in the ischemic CAI lesions are electrophy- siologically functional. Attempts to examine the behavioral effects of these and the CA3 and dentate grafts are in progress.

This study was supported by the Aarhus University Research Foundation, the Danish MRC, the Lundbeck Foundation, the NOVO Foundation and the Danish State Biotechnology Program.

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neural grafting to ischemic and excitotoxic, · and extensive ingrowth of cholinergic host...

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