Host Hippocampus Research Articles

Freshly Thawed Cryobanked Human Neural Stem Cells Engraft within Endogenous Neurogenic Niches and Restore Cognitive Function after Chronic Traumatic Brain Injury.

Human neural stem cells (hNSCs) have potential as a cell therapy after traumatic brain injury (TBI). While various studies have demonstrated the efficacy of NSCs from ongoing culture, there is a significant gap in our understanding of freshly thawed cells from cryobanked stocks-a more clinically relevant source. To address these shortfalls, the therapeutic potential of our previously validated Shef-6.0 human embryonic stem cell (hESC)-derived hNSC line was tested after long-term cryostorage and thawing before transplant. Immunodeficient athymic nude rats received a moderate unilateral controlled cortical impact (CCI) injury. At four weeks post-injury, 6 × 105 freshly thawed hNSCs were transplanted into six injection sites (two ipsi- and four contra-lateral) with 53.4% of cells surviving three months post-transplant. Interestingly, most hNSCs were engrafted in the meninges and the lining of lateral ventricles, associated with high CXCR4 expression and a chemotactic response to SDF1alpha (CXCL12). While some expressed markers of neuron, astrocyte, and oligodendrocyte lineages, the majority remained progenitors, identified through doublecortin expression (78.1%). Importantly, transplantation resulted in improved spatial learning and memory in Morris water maze navigation and reduced risk taking in an elevated plus maze. Investigating potential mechanisms of action, we identified an increase in ipsilateral host hippocampus cornu ammonis (CA) neuron survival, contralateral dentate gyrus (DG) volume, and DG neural progenitor morphology as well as a reduction in neuroinflammation. Together, these findings validate the potential of hNSCs to improve function after TBI and demonstrate that long-term biobanking of cells and thawing aliquots before use may be suitable for clinical deployment.

Human Neural Stem Cells Reinforce Hippocampal Synaptic Network and Rescue Cognitive Deficits in a Mouse Model of Alzheimer's Disease.

SummaryAlzheimer's disease (AD) is characterized by memory impairments in its earliest clinical phase. The synaptic loss and dysfunction leading to failures of synaptic networks in AD brain directly cause cognitive deficits of patient. However, it remains unclear whether the synaptic networks in AD brain could be repaired. In this study, we generated functional human induced neural progenitor/stem cells (iNPCs) that had been transplanted into the hippocampus of immunodeficient wild-type and AD mice. The grafted human iNPCs efficiently differentiated into neurons that displayed long-term survival, progressively acquired mature membrane properties, formed graft-host synaptic connections with mouse neurons and functionally integrated into local synaptic circuits, which eventually reinforced and repaired the neural networks of host hippocampus. Consequently, AD mice with human iNPCs exhibited enhanced synaptic plasticity and improved cognitive abilities. Together, our results suggest that restoring synaptic failures by stem cells might provide new directions for the development of novel treatments for human AD.

Neural Stem Cell or Human Induced Pluripotent Stem Cell-Derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy.

Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE-like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE-NSCs and hiPSC-derived MGE-like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post-grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft-derived cells, and the effects of grafts on the host hippocampus. © 2016 by John Wiley & Sons, Inc.

Defining the optimal window for cranial transplantation of human induced pluripotent stem cell-derived cells to ameliorate radiation-induced cognitive impairment.

Past preclinical studies have demonstrated the capability of using human stem cell transplantation in the irradiated brain to ameliorate radiation-induced cognitive dysfunction. Intrahippocampal transplantation of human embryonic stem cells and human neural stem cells (hNSCs) was found to functionally restore cognition in rats 1 and 4 months after cranial irradiation. To optimize the potential therapeutic benefits of human stem cell transplantation, we have further defined optimal transplantation windows for maximizing cognitive benefits after irradiation and used induced pluripotent stem cell-derived hNSCs (iPSC-hNSCs) that may eventually help minimize graft rejection in the host brain. For these studies, animals given an acute head-only dose of 10 Gy were grafted with iPSC-hNSCs at 2 days, 2 weeks, or 4 weeks following irradiation. Animals receiving stem cell grafts showed improved hippocampal spatial memory and contextual fear-conditioning performance compared with irradiated sham-surgery controls when analyzed 1 month after transplantation surgery. Importantly, superior performance was evident when stem cell grafting was delayed by 4 weeks following irradiation compared with animals grafted at earlier times. Analysis of the 4-week cohort showed that the surviving grafted cells migrated throughout the CA1 and CA3 subfields of the host hippocampus and differentiated into neuronal (∼39%) and astroglial (∼14%) subtypes. Furthermore, radiation-induced inflammation was significantly attenuated across multiple hippocampal subfields in animals receiving iPSC-hNSCs at 4 weeks after irradiation. These studies expand our prior findings to demonstrate that protracted stem cell grafting provides improved cognitive benefits following irradiation that are associated with reduced neuroinflammation.

Transplantation of Human Fetal-Derived Neural Stem Cells Improves Cognitive Function following Cranial Irradiation

Treatment of central nervous system (CNS) malignancies typically involves radiotherapy to forestall tumor growth and recurrence following surgical resection. Despite the many benefits of cranial radiotherapy, survivors often suffer from a wide range of debilitating and progressive cognitive deficits. Thus, while patients afflicted with primary and secondary malignancies of the CNS now experience longer local regional control and progression-free survival, there remains no clinical recourse for the unintended neurocognitive sequelae associated with their cancer treatments. Multiple mechanisms contribute to disrupted cognition following irradiation, including the depletion of radiosensitive populations of stem and progenitor cells in the hippocampus. We have explored the potential of using intrahippocampal transplantation of human stem cells to ameliorate radiation-induced cognitive dysfunction. Past studies demonstrated the capability of cranially transplanted human embryonic (hESCs) and neural (hNSCs) stem cells to functionally restore cognition in rats 1 and 4 months after cranial irradiation. The present study employed an FDA-approved fetal-derived hNSC line capable of large scale-up under good manufacturing practice (GMP). Animals receiving cranial transplantation of these cells 1 month following irradiation showed improved hippocampal spatial memory and contextual fear conditioning performance compared to irradiated, sham surgery controls. Significant newly born (doublecortin positive) neurons and a smaller fraction of glial subtypes were observed within and nearby the transplantation core. Engrafted cells migrated and differentiated into neuronal and glial subtypes throughout the CA1 and CA3 subfields of the host hippocampus. These studies expand our prior findings to demonstrate that transplantation of fetal-derived hNSCs improves cognitive deficits in irradiated animals, as assessed by two separate cognitive tasks.

Subventricular Zone-Derived Neural Stem Cell Grafts Protect Against Hippocampal Degeneration and Restore Cognitive Function in the Mouse Following Intrahippocampal Kainic Acid Administration

Temporal lobe epilepsy (TLE) is a major neurological disease, often associated with cognitive decline. Since approximately 30% of patients are resistant to antiepileptic drugs, TLE is being considered as a possible clinical target for alternative stem cell-based therapies. Given that insulin-like growth factor I (IGF-I) is neuroprotective following a number of experimental insults to the nervous system, we investigated the therapeutic potential of neural stem/precursor cells (NSCs) transduced, or not, with a lentiviral vector for overexpression of IGF-I after transplantation in a mouse model of kainic acid (KA)-induced hippocampal degeneration, which represents an animal model of TLE. Exposure of mice to the Morris water maze task revealed that unilateral intrahippocampal NSC transplantation significantly prevented the KA-induced cognitive decline. Moreover, NSC grafting protected against neurodegeneration at the cellular level, reduced astrogliosis, and maintained endogenous granule cell proliferation at normal levels. In some cases, as in the reduction of hippocampal cell loss and the reversal of the characteristic KA-induced granule cell dispersal, the beneficial effects of transplanted NSCs were manifested earlier and were more pronounced when these were transduced to express IGF-I. However, differences became less pronounced by 2 months postgrafting, since similar amounts of IGF-I were detected in the hippocampi of both groups of mice that received cell transplants. Grafted NSCs survived, migrated, and differentiated into neurons-including glutamatergic cells-and not glia, in the host hippocampus. Our results demonstrate that transplantation of IGF-I producing NSCs is neuroprotective and restores cognitive function following KA-induced hippocampal degeneration.

Expression of FMRP by Neurosphere Derived Cells Following Transplantation in the Fragile X Mouse Model

Fragile X Syndrome, characterized by intellectual impairment and learning disabilities is caused by a trinucleotide repeat expansion in the x-linked FMR1 gene leading to a complete lack of Fragile X Mental Retardation Protein (FMRP). Neural stem/progenitor cells (NSCs) cultured as floating aggregates of cells commonly referred to as neurospheres can survive, differentiate and integrate into the host environment when transplanted to the rodent CNS. Neurospheres were established from early postnatal wild-type mice and labeled with the cytoplasmic dye CFDA-SE and then stereotaxically injected as cell suspensions into the hippocampus of young adult (3-6 week old) FMR1 knockout mice. Immunocytochemical analysis of the brains revealed the presence of FMRP-expressing cells up to two weeks after transplantation. Some transplanted cells migrated within the host hippocampus and formed dendrite-like processes. A small subpopulation of the transplanted cells expressed markers of neuronal or glial phenotypes, although many cells did not stain for any of the markers that we probed. These experiments constitute an attempt to restore FMRP-expression in the brains of FMR1 knockout mice using cell suspension transplants of stem and progenitor cells.

Colloids as Mobile Substrates for the Implantation and Integration of Differentiated Neurons into the Mammalian Brain

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.

Neural Stem Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy

Neural stem cell (NSC) transplantation into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts ∼30% of mesial temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs). However, to provide a comprehensive methodology involved in testing the efficacy of transplantation of NSCs in a rat model of chronic TLE, NSCs derived from the embryonic medial ganglionic eminence (MGE) are taken as an example in this unit. The topics comprise description of the required materials, reagents and equipment, and protocols for expanding MGE-NSCs in culture, generating chronically epileptic rats, the intrahippocampal grafting, post-grafting evaluation of the effects of NSC grafts on spontaneous recurrent seizures and cognitive impairments, analyses of the yield and the fate of graft-derived cells, and the effects of NSC grafts on the host hippocampus.

Effects of NT-4 gene modified fibroblasts transplanted into AD rats

It is well known that fibroblasts can act as a cell vector to express functional protein, like neurotrophin-4 (NT-4). The present study evaluated the effect of NT-4 gene modified fibroblasts grafted into the hippocampus of AD rat model. AD rats were reproduced by bilateral transection of the hippocampal fimbria-fornix. The transplanted fibroblasts steadily expressed NT-4 proteins at least 2 months after transplantation. This correlated with a significant rescue in the number of cholinergic neurons in the host hippocampus. Morris water maze tests demonstrated significant improvements in learning and memory, especially in rats receiving NT-4-modified fibroblasts. The present results showed that NT-4 gene modified fibroblasts could provide a long-term and steady expression of NT-4, and it significantly improved the behavior of AD rats. These findings should have important clinical applications in providing a long-term NT-4 Secretion for the treatment of AD.

Grafting of striatal precursor cells into hippocampus shortly after status epilepticus restrains chronic temporal lobe epilepsy

Status epilepticus (SE) typically progresses into temporal lobe epilepsy (TLE) typified by complex partial seizures. Because sizable fraction of patients with TLE exhibit chronic seizures that are resistant to antiepileptic drugs, alternative therapies that are efficient for diminishing SE-induced chronic epilepsy have great significance. We hypothesize that bilateral grafting of appropriately treated striatal precursor cells into hippocampi shortly after SE is efficacious for diminishing SE-induced chronic epilepsy through long-term survival and differentiation into GABA-ergic neurons. We induced SE in adult rats via graded intraperitoneal injections of kainic acid, bilaterally placed grafts of striatal precursors (pre-treated with fibroblast growth factor-2 and caspase inhibitor) into hippocampi at 4 days post-SE, and examined long-term effects of grafting on spontaneous recurrent motor seizures (SRMS). Analyses at 9–12 months post-grafting revealed that, the overall frequency of SRMS was 67–89% less than that observed in SE-rats that underwent sham-grafting surgery and epilepsy-only controls. Graft cell survival was ~ 33% of injected cells and ~ 69% of surviving cells differentiated into GABA-ergic neurons, which comprised subclasses expressing calbindin, parvalbumin, calretinin and neuropeptide Y. Grafting considerably preserved hippocampal calbindin but had no effects on aberrant mossy fiber sprouting. The results provide novel evidence that bilateral grafting of appropriately treated striatal precursor cells into hippocampi shortly after SE is proficient for greatly reducing the frequency of SRMS on a long-term basis in the chronic epilepsy period. Presence of a large number of GABA-ergic neurons in grafts further suggests that strengthening of the inhibitory control in host hippocampi likely underlies the beneficial effects mediated by grafts.

Pretreatment of donor cells with FGF-2 enhances survival of fetal hippocampal CA3 cell transplants in the chronically lesioned young adult hippocampus

The lesioned CA3 region of the young adult hippocampus is very conducive for robust survival and integration of fetal hippocampal CA3 cell grafts when transplanted at an early postlesion delay of 4 days. However, similar CA3 cell grafts placed at 45 days postlesion display significantly diminished cell survival, implying that the receptivity of the lesioned young adult host hippocampus to grafts decreases considerably with a prolonged postlesion transplantation delay. We hypothesize that decreased cell survival in grafts placed into the chronically lesioned hippocampus is due to a reduced level of host neurotrophic factors that support fetal hippocampal cells; hence, pretreatment and grafting of donor fetal CA3 cells with fibroblast growth factor-2 (FGF-2) considerably enhances graft neuronal integration into the chronically lesioned young adult hippocampus. We employed the optical fractionator cell counting method and rigorously quantified the number of surviving cells and neurons derived from 5′-bromodeoxyuridine-labeled Embryonic Day 19 CA3 cell grafts pretreated and transplanted with FGF-2 into the lesioned CA3 region of the young adult rat hippocampus, at a delay of 60 days after a unilateral intracerebroventricular administration of the kainic acid. For comparison, we also analyzed the survival of standard fetal CA3 cell grafts (i.e., without FGF-2 treatment) after similar transplantation. Pre treatment and transplantation of CA3 cell grafts with FGF-2 resulted in a robust yield of surviving cells (115% of injected cells) and neurons (100% of injected cells) from grafts. In contrast, standard CA3 cell grafts exhibited a reduced yield of surviving cells (29%) and neurons (25%). Thus, the yield of neurons from fetal hippocampal CA3 cell grafts placed into the chronically lesioned young adult hippocampus can be greatly enhanced by a simple pretreatment and grafting of donor fetal CA3 cells with FGF-2. These results have significance toward advancement of clinically feasible cell grafting strategies for repair of the damaged young adult hippocampus, particularly at extended periods after the injury or the onset of neurodegenerative diseases.

Fetal Hippocampal CA3 Cell Grafts Transplanted to Lesioned CA3 Region of the Adult Hippocampus Exhibit Long-Term Survival in a Rat Model of Temporal Lobe Epilepsy

Intracerebroventricular administration of kainic acid in the adult rat, a widely used model for studying human temporal lobe epilepsy, results in widespread degeneration of CA3-pyramidal neurons. Transplantation of specific fetal hippocampal CA3 cell grafts into the lesioned CA3-region at a prolonged post lesion delay of 45-day leads to 31% graft cell survival at 1 month postgrafting and significantly facilitates appropriate recovery of the lesioned host hippocampus. However, the capability of hippocampal CA3 cell grafts for enduring survival in this model is unknown. We hypothesize that a significant fraction of fetal CA3 cells grafted into the lesioned CA3 region of the adult hippocampus at 45-days postlesion exhibit long-term survival. We measured the extent of cell survival within 5′-bromodeoxyuridine-labeled CA3 cell grafts at 1 year postgrafting, following their transplantation at 45 days postlesion into the lesioned CA3-region. Quantification of absolute graft cell survival using BrdU immunostaining and the optical fractionator counting method revealed survival of 36% of grafted cells at 1 year postgrafting. Thus, over a third of fetal hippocampal CA3 cells transplanted to the lesioned CA3-region at 45 days postlesion exhibit long-term survival. Further, the extent of cell survival in these grafts is highly analogous to the degree of cell survival in CA3 grafts analyzed earlier at 1 month postgrafting, suggesting that specific fetal cells that survive the first month of grafting into the lesioned CNS area are capable of exhibiting enduring survival.

Serotonin Release from Mesencephalic Raphe Neurons Grafted to the 5,7-Dihydroxytryptamine-Lesioned Rat Hippocampus: Effects of Behavioral Activation and Stress

Transplants of fetal midbrain raphe neurons into the adult brain have been shown to promote recovery of complex behavioral deficits in several experimental models, but the mechanisms underlying these effects are only partially understood. In the present study, we have used a well-characterized model system to ascertain whether midbrain raphe graft can display behaviorally relevant changes in transmitter release and/or metabolism. Fetal mesencephalic raphe neurons were grafted unilaterally into the hippocampus previously deprived of its innate serotonergic innervation by intraventricular injections of 5,7-dihydroxytryptamine. The contralateral hippocampus remained as a nongrafted, lesioned control. Microdialysis probes were implanted in the hippocampus 5–7 months postgrafting. Under baseline conditions, extracellular levels of serotonin were similar to normal in the grafted hippocampi, but undetectable on the contralateral, nongrafted side. Levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were markedly higher than normal in the grafted hippocampi, but dramatically reduced on the contralateral nongrafted side. Handling stimulation (gentle stroking of a rat's fur and tail for 15 min) induced a 64% increase in serotonin output in the intact rats and a small but significant 12% increase in the grafted animals. Nonnoxious tail-pinch (15 min) enhanced serotonin release by 86% in the intact rats and 28% in the grafted ones. Extracellular 5-HIAA levels remained unchanged during both handling and tail-pinch in both the intact and the grafted rats. Forced immobilization of the rats for 15 min induced a transient 124% increase in extracellular serotonin levels in the intact rats and a significant 19% increase in the grafted animals, whereas swimming in temperate water (25–30°C; 15 min) induced no detectable changes in serotonin output in any of the groups. 5-HIAA levels remained unchanged during forced immobilization, but were significantly reduced during the swimming session in both the intact (−38%) and grafted (−15%) animals. The present results indicate that median raphe grafts can become functionally integrated in the denervated host hippocampus and respond by altered indole output when the animal is exposed to different types of environmental challenges.

Transplanted embryonic entorhinal neurons make functional synapses in adult host hippocampus

Grafts of embryonic entorhinal cortex (EC) or non-entorhinal cortex (NEC) were placed into the hippocampus of adult rats with transection of the perforant paths. Graft–host connectivity was investigated at 4–6 months post-transplantation by recording extracellular evoked responses in hippocampal slice preparations. Electrical stimulation of the grafts evoked excitatory postsynaptic potentials (EPSPs) in the outer molecular layer of the dentate gyrus, and the stratum lacunosum moleculare of CA1, CA3, and elicited population spikes in the granule cell layer and the pyramidal cell layer of CA1, but not CA3. While the latencies and the forms of these evoked response were similar to those in matched control slices from the normal animals, the amplitudes were smaller than normal controls. However, in the slices with NEC grafts, no such responses were recorded when stimulus was applied in similar position in the grafts. The findings suggest that grafted entorhinal neurons make viable synaptic connections with the host hippocampus.

Development of long-distance efferent projections from fetal hippocampal grafts depends upon pathway specificity and graft location in kainate-lesioned adult hippocampus

Fetal hippocampal cells grafted into the excitotoxically lesioned hippocampus of adult rats are capable of extending axonal projections into the host brain. We hypothesize that the axonal growth of grafted fetal cells into specific host targets, and the establishment of robust long-distance efferent graft projections, require placement of fetal cells in close proximity to appropriate axon guidance pathways. Intracerebroventricular administration of kainic acid in adult rats leads to a specific loss of hippocampal CA3 pyramidal neurons. We grafted 5′-bromodeoxyuridine-labeled embryonic day 19 hippocampal cells into adult hippocampus at four days post-kainic acid lesion, and quantitatively measured the projection of grafted cells into the contralateral hippocampus and the septum after three to four months survival using Fluoro-Gold and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) tracing. Grafts located in or near the degenerated CA3 cell layer exhibited numerous neurons which established commissural projections with the contralateral hippocampus. However, such projections did not occur in intrahippocampal grafts located away from the CA3 cell layer. In contrast, neurons in all grafts established robust projections into the septum regardless of location within hippocampus although grafts located near the degenerated CA3 cell layer displayed more neurons with such projections.Location of grafted cells clearly influences the development of efferent graft projections into distant targets in the adult host brain, particularly access to axon guidance pathways to facilitate the formation of projections. The establishment of robust long-distance commissural projections of fetal hippocampal grafts is clearly dependent on their placement in or near the degenerated CA3 cell layer, suggesting that appropriate axon guidance pathways for commissural pathways are tightly focussed near this cell layer. However, the establishment of septal projections of these grafts was not dependent on specific location within the CA3 cell layer, suggesting that axonal guidance mechanisms to the septum are more diffuse and not limited to the CA3 dendritic layers.The results underscore that fetal hippocampal grafts are capable of partly restoring lesioned hippocampal circuitry in adult animals when appropriately placed in the host hippocampus.

DEVELOPMENT OF FETAL HIPPOCAMPAL GRAFTS IN INTACT AND LESIONED HIPPOCAMPUS

Functional recovery observed in Parkinson's disease patients following grafting of fetal substantia nigra has encouraged the development of similar grafting therapy for other neurological disorders. Fetal hippocampal grafting paradigms are of considerable significance because of their potential to treat neurological disorders affecting primarily hippocampus, including temporal lobe epilepsy, cerebral ischemia, stroke, and head injury. Since many recent studies of hippocampal transplants were carried out with an aim of laying the foundation for future clinical applications, an overview of the development of fetal hippocampal transplants, and their capability for inducing functional recovery under different host conditions is timely. In this review, we will summarize recent developments in hippocampal transplants, especially the anatomical and/or functional integration of grafts within the host brain under specific host conditions, including a comparison of intact hippocampus with various types of hippocampal lesions or injury. Improvements in grafting techniques, methods for analysis of graft integration and graft function will be summarized, in addition to critical factors which enhance the survival and integration of grafted cells and alternative sources of donor cells currently being tested or considered for hippocampal transplantation. Viewed collectively, hippocampal grafting studies show that fetal hippocampal tissue/cells survive grafting, establish both afferent and efferent connections with the host brain, and are also capable of ameliorating certain learning and memory deficits in some models. However, the efficacy of intracerebral fetal hippocampal grafts varies considerably in different animal models, depending on several factors: the mode of donor tissue preparation, the method of grafting, the state of host hippocampus at the time of grafting, and the placement of grafts within the hippocampus. Functional improvement in many models appeared to be caused partially by re-establishment of damaged circuitry and partially by a trophic action of grafts. However, exact mechanisms of graft-mediated behavioral recovery remain to be clarified due to the lack of correlative analysis in the same animal between the degree of graft integration and behavioral recovery. Issues of mechanisms of action, degree of restoration of host circuitry and amelioration of host pathological conditions will need to be sorted out clearly prior to clinical use of fetal hippocampal transplants for susceptible neurological conditions. Copyright © 1996 Elsevier Science Ltd.

Extensive reinnervation of the hippocampus by embryonic basal forebrain cholinergic neurons grafted into the septum of neonatal rats with selective cholinergic lesions.

Reconstruction of the septohippocampal pathways by axons extending from embryonic cholinergic neuroblasts grafted into the neuron-depleted septum has been explored in the neonatal rat by using a novel lesioning and grafting protocol. Neonatal ablation of the basal forebrain cholinergic projection neurons, accompanied by extensive bilateral cholinergic denervation of the hippocampus and neocortex, was produced at postnatal day (PD) 4 by 192 immunoglobulin (IgG)-saporin intraventricularly. Four days later, cholinergic neuroblasts (from embryonic day 14 rats) were implanted bilaterally into the neuron-depleted septum by using a microtransplantation approach. The results show that homotopically implanted septal neurons survive and integrate well into the developing septal area, extending axons caudally along the myelinated fimbria-fornix and supracallosal pathways that are able to reach the appropriate targets in the denervated hippocampus and cingulate cortex as early as 4 weeks postgrafting. Moreover, the laminar innervation patterns established by the graft-derived axons closely resembled the normal ones and remained essentially unchanged up to at least 6 months, which was the longest postoperative time studied. The reinnervating fibers restored tissue choline acetyltransferase activity (up to 50% of normal) in the dorsal hippocampus and the parietooccipital cortex. Retrograde labeling with Fluoro-Gold from the host hippocampus combined with immunocytochemistry confirmed that most of the projecting neurons, indeed, were cholinergic. The results suggest that the graft-host interactions that are necessary for target-directed axon growth are present in the septohippocampal system during early postnatal maturation. Thus, the present approach may contribute to overcome the functional limitations inherent in the use of ectopically placed intrahippocampal transplants.

Extensive reinnervation of the hippocampus by embryonic basal forebrain cholinergic neurons grafted into the septum of neonatal rats with selective cholinergic lesions

Reconstruction of the septohippocampal pathways by axons extending from embryonic cholinergic neuroblasts grafted into the neuron-depleted septum has been explored in the neonatal rat by using a novel lesioning and grafting protocol. Neonatal ablation of the basal forebrain cholinergic projection neurons, accompanied by extensive bilateral cholinergic denervation of the hippocampus and neocortex, was produced at postnatal day (PD) 4 by 192 immunoglobulin (IgG)-saporin intraventricularly. Four days later, cholinergic neuroblasts (from embryonic day 14 rats) were implanted bilaterally into the neuron-depleted septum by using a microtransplantation approach. The results show that homotopically implanted septal neurons survive and integrate well into the developing septal area, extending axons caudally along the myelinated fimbria-fornix and supracallosal pathways that are able to reach the appropriate targets in the denervated hippocampus and cingulate cortex as early as 4 weeks postgrafting. Moreover, the laminar innervation patterns established by the graft-derived axons closely resembled the normal ones and remained essentially unchanged up to at least 6 months, which was the longest postoperative time studied. The reinnervating fibers restored tissue choline acetyltransferase activity (up to 50% of normal) in the dorsal hippocampus and the parietooccipital cortex. Retrograde labeling with Fluoro-Gold from the host hippocampus combined with immunocytochemistry confirmed that most of the projecting neurons, indeed, were cholinergic. The results suggest that the graft-host interactions that are necessary for target-directed axon growth are present in the septohippocampal system during early postnatal maturation. Thus, the present approach may contribute to overcome the functional limitations inherent in the use of ectopically placed intrahippocampal transplants. © 1996 Wiley-Liss, Inc.

Use of Fetal Cortical Grafts in Hypoxic-Ischemic Brain Injury in Neonatal Rats

In view of numerous studies demonstrating that intracerebral implants of fetal neural tissue can promote functional recovery and structural repair in the damaged brain, the present study examined the potential use of neocortical transplantation in newborn rats that sustained hypoxic-ischemic brain injury. Ischemic insult was induced in Long-Evans, black-hooded 1-week-old rats by unilateral common carotid artery occlusion followed by 2.5 h of hypoxia in 8% O2. One week later, animals received neocortical block transplants. At 2–6 weeks posttransplantation, animals were sacrificed and their brains examined histologically. Transplants survived in over 80% of the animals and the presence of acetylcholinesterase-positive fibers crossing the host-transplant interface provided evidence of transplant integration with the host brain. However, morphometric measurements revealed that the transplants were unable to reduce the hypoxia-ischemia-induced degeneration in the host hippocampus, caudate-putamen, or thalamus. Nonetheless the demonstrated survival of grafts in the neonatal hypoxia-ischemia model suggests a potential therapeutic effect.