Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs.

BACKGROUND: There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. METHODS: The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. RESULTS: Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. CONCLUSIONS: This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

Hereditary multi-infarct dementia of the Swedish type is a novel disorder different from NOTCH3 causing CADASIL.

Several hereditary small vessel diseases (SVDs) of the brain have been reported in recent years. In 1977, Sourander and Wålinder described hereditary multi-infarct dementia (MID) in a Swedish family. In the same year, Stevens and colleagues reported chronic familial vascular encephalopathy in an English family bearing a similar phenotype. These disorders have invariably been suggested to be cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) but their genetic identities remain unknown. We used molecular, radiological and neuropathological methods to characterize these disorders. Direct DNA sequencing unexpectedly confirmed that affected members of the English family carried the R141C mutation in the NOTCH3 gene diagnostic of CADASIL. However, we did not detect any pathogenic mutations in the entire 8091 bp reading frame of NOTCH3 or find clear evidence for NOTCH3 gene linkage in the Swedish DNA. This was consistent with the lack of hyperintense signals in the anterior temporal pole and external capsule in Swedish subjects upon magnetic resonance imaging. We further found no evidence for granular osmiophilic material in skin biopsy or post-mortem brain samples of affected members in the Swedish family. In addition, there was distinct lack of NOTCH3 N-terminal fragments in the cerebral microvasculature of the Swedish hereditary MID subjects compared to the intense accumulation in the English family afflicted with CADASIL. Several differences in arteriosclerotic changes in both the grey and white matter were also noted between the disorders. The sclerotic index values, density of collagen IV immunoreactivity in the microvasculature and number of perivascular macrophages were greater in the English CADASIL samples compared to those from the Swedish brains. Multiple approaches suggest that the Swedish family with hereditary MID suspected to be CADASIL has a different novel disorder with dissimilar pathological features and belongs to the growing number of genetically uncharacterized familial SVDs.

Transposon-based interferon gamma gene transfer overcomes limitations of episomal plasmid for immunogene therapy of glioblastoma.

Despite improvements in gene delivery technology, transient expression of plasmid DNA has limited the efficacy of nonviral vectors applied to cancer gene therapy. We previously developed plasmid DNA vectors capable of transgene integration and long-term expression in human glioblastoma cells by utilizing the Sleeping Beauty (SB) transposable element. In this study, we compared the efficacy of interferon gamma (IFN-gamma) immunogene therapy using episomal or SB vectors in a syngeneic GL261 glioma model. Gene delivery was achieved by intratumoral convection-enhanced delivery of DNA/polyethylenimine complexes. Only mice treated with SB transposase-encoding DNA to facilitate chromosomal integration exhibited a significant increase in survival (P<0.05). SB-mediated intratumoral gene transfer caused sustained IFN-gamma expression assessed by reverse transcription-polymerase chain reaction, of both vector-derived and endogenous IFN-gamma, whereas expression following episomal plasmid gene transfer was undetectable within 2 weeks. Median survival was enhanced further when SB-mediated IFN-gamma gene transfer was combined with CpG oligodeoxynucleotides as adjuvant therapy. Prolonged survival positively correlated with tumor regression measured by in vivo bioluminescent imaging, and enhanced T-cell activation revealed by the ELISPOT assay. SB appears to improve the efficacy of cytokine gene therapy using nonviral vectors by enhancing the duration of transgene expression.

Laminin peptide ameliorates brain injury by inhibiting leukocyte accumulation in a rat model of transient focal cerebral ischemia.

Postischemic cerebral inflammation has been reported to contribute to ischemic brain damage. During inflammation, constituents of the extracellular matrix such as fibronectin and laminin are recognized by certain integrins or proteoglycans and play an important role in the cell adhesion process. The purpose of this study was to evaluate the efficacy of peptides derived from laminin on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Forty-four animals were included in this study: transient ischemia without treatment (Group I), treatment with TG-1 peptide (Group II), GD-1 peptide (Group III), and GD-6 peptide (Group IV). Group II showed a significant reduction of the leukocyte accumulation (p < 0.001) and infarct size (p = 0.015) when compared with Group I. The neurological grade of Group II was also significantly better than in Group I at 48 h after reperfusion (p = 0.012). Based on these data, which are the first to explore the therapeutic potential of this peptide in cerebral ischemia, laminin peptide may offer a novel therapeutic approach to allaying injury in ischemic stroke.

Neuronal protection from cerebral ischemia by synthetic fibronectin peptides to leukocyte adhesion molecules.

Leukocytes play an important role in the development of ischemia/reperfusion injury. Recent work in our laboratory has demonstrated that a mixture of synthetic fibronectin peptides to leukocyte adhesion molecules reduces ischemic brain damage after transient focal cerebral ischemia. The purpose of this study was to evaluate the efficacy of the individual peptides on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Thirty-five animals were divided into five groups: transient ischemia without treatment (Group I), treatment with arginyl-glycyl-aspartic acid (RGD) peptide (Group II), connecting segment (CS)-1 peptide (Group III), fibronectin (FN)-C/H-V peptide (Group IV), and scrambled FN-C/H-V peptide (Group V). Groups III and IV showed a significant decrease in the degree of leukocyte infiltration in the lesion and in the infarct size (p < 0.05) when compared to Groups I, II, and V. The neurological grade of Groups III and IV was significantly better than in Groups I, II, and V at 48 h after reperfusion (p < 0.01). Thus, in addition to demonstrating the potential efficacy of synthetic peptides as therapeutic agents for ischemia-reperfusion, these results also offer new insights into the mechanisms of leukocyte arrest and recruitment in ischemia/reperfusion injury.

Rat nigral xenografts survive in the brain of MHC class II-, but not class I-deficient mice.

We have examined the role of the indirect pathway of antigen recognition and T cells in neural xenografts rejection by using major histocompatibility complex (MHC) class II-deficient mice as xenograft recipients. Dissociated embryonic ventral mesencephalic tissue from Sprague-Dawley rats was stereotaxically injected as a cell suspension into the striatum of MHC class II-deficient adult mice as well as MHC class I-deficient and wild-type mice as controls. All of the MHC class II-deficient mice had surviving grafts in the striatum 4 weeks post-grafting. In contrast, only a few of the MHC class I-deficient mice exhibited very small grafts and none of the wild-type mice had any surviving grafts. The mean number of surviving transplanted dopamine neurons in the MHC class II-deficient group was significantly larger than that observed in the other two groups. Moderate levels of MHC class I antigen expression were seen in the transplantation sites of some animals in the MHC class II-deficient group. No helper or cytotoxic T cells were observed infiltrating into the graft sites of this group. However, there were markedly increased levels of expression of MHC class I and class II antigens, and a number of T cells infiltrating in the graft sites in both the MHC class I-deficient and wild-type groups. These results show that rat embryonic nigral tissue can survive transplantation in the brain of the MHC class II-deficient mice for at least 4 weeks without any overt signs of rejection, suggesting that the indirect pathway of foreign antigen recognition mediated by host MHC class II molecules and helper T cells plays an important role in the rejection responses to intracerebral xenografts.

Creatine-supplemented diet extends Purkinje cell survival in spinocerebellar ataxia type 1 transgenic mice but does not prevent the ataxic phenotype.

It is not known why expression of a protein with an expanded polyglutamine region is pathogenic in spinocerebellar ataxia, Huntington's disease and several other neurodegenerative diseases. Dietary supplementation with creatine improves survival and motor performance and delays neuronal atrophy in the R6/2 transgenic mouse model of Huntington's disease. These effects may be due to improved energy and calcium homeostasis, enhanced presynaptic glutamate uptake, or protection of mitochondria from the mitochondrial permeability transition. We tested the effects of a 2% creatine-supplemented diet and treatment with taurine-conjugated ursodeoxycholic acid, a bile constituent that can inhibit the mitochondrial permeability transition, on ataxia and Purkinje cell survival in a transgenic model of spinocerebellar ataxia type 1. After 24 weeks, transgenic mice on the 2% creatine diet had cerebellar phosphocreatine levels that were 72.5% of wildtype controls, compared to 26.8% in transgenic mice fed a control diet. The creatine diet resulted in maintenance of Purkinje cell numbers in these transgenic mice at levels comparable to wildtype controls, while transgenic mice fed a control diet lost over 25% of their Purkinje cell population. Nevertheless, the ataxic phenotype was neither improved nor delayed. Repeated s.c. ursodeoxycholic acid injections markedly elevated ursodeoxycholic acid levels in the brain without adverse effects, but provided no improvement in phenotype or cell survival in spinocerebellar ataxia type 1 mice. These results demonstrate that preserving neurons from degeneration is insufficient to prevent a behavioral phenotype in this transgenic model of polyglutamine disease. In addition, we suggest that the means by which creatine mitigates against the neurodegenerative effects of an ataxin-1 protein containing an expanded polyglutamine region is through mechanisms other than stabilization of mitochondrial membranes.

Enhancement of nigral graft survival in rat brain with the systemic administration of synthetic fibronectin peptide V.

A major obstacle in neural transplantation is a severe loss of neurons in grafts soon after implantation. In the present study, we have investigated whether the systemic administration of synthetic fibronectin peptide V can increase the survival of neural grafts. Synthetic fibronectin peptide V is derived from the 33,000 mol. wt carboxyl-terminal heparin-binding domain of fibronectin. Previous studies have shown that these polypeptides possess anti-inflammatory properties. However, it is currently unknown whether this peptide has anti-apoptotic properties. Dissociated neural grafts were prepared from the ventral mesencephalon of pregnant Sprague-Dawley rats and were stereotaxically injected as a cell suspension into the striatum of adult Sprague-Dawley rats. A group of recipient rats received i.v. injections of peptide V (5mg/kg, dissolved in saline) at 24 and 4h prior to transplantation, at the time of transplantation, and 24, 48 and 72h post-transplantation. Saline-treated rats served as controls. The rats were killed at two, four and 42 days post-grafting and the brain tissue was immunologically processed for tyrosine-hydroxylase, major histocompatibility complex class I and class II antigens, complement receptor type 3 and leukocyte common antigen immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. We found a significant increase (approximately twofold) in the number of dopamine neurons in the grafts for the peptide-treated group at four and 42 days compared with the controls. In contrast, there was no significant difference in the patterns of inflammation using different immunocytochemical markers in the two different groups. The levels of expression for these markers, however, were reduced over time. Interestingly, the number of apoptotic cells in the graft areas was significantly smaller in the peptide-treated group than in the control group two days after grafting. The results demonstrate that the systemic administration of synthetic fibronectin peptide V can dramatically increase the survival of nigral grafts in the brain and substantially reduce the number of apoptotic cells in the graft site, suggesting that this peptide may exert a beneficial effect on survival of nigral grafts through an anti-apoptotic mechanism.

Specificity of fiber innervation by cholinergic neurons transplanted into the retrosplenial cortex of adult rats as revealed by choline acetyltransferase immunocytochemistry.

The retrosplenial cortex (RSC) is a target of the forebrain cholinergic projecting system. It receives extensive cholinergic innervation from the medial septal nucleus and the diagonal band of Broca. These cholinergic afferents travel along the paths of cingulate bundle and fornix. In the present study we investigated the ability of cholinergic fetal septal grafts to reinnervate the deafferented RSC. Four groups of rats were used: (1) normal control rats (NC); (2) rats with bilateral transections of the cingulate bundle (CgX); (3) rats with simultaneous lesions of both the cingulate bundle and the fornix (FX), and (4) rats with intra-retrosplenial fetal septal grafts and lesions in both cingulate bundle and the fornix (RSCsep-TPL). We found that lesions in the cingulate bundle alone produced a modest reduction of cholinergic innervation whereas lesions in both the fornix and cingulate bundle resulted in a complete loss of cholinergic inputs in this area, indicating that both the cingulate bundle and the fornix are involved in mediating cholinergic projections from the septal-diagonal area to the RSC. Transplantation of cholinergic fetal septal neurons into the RSC of animals with simultaneous lesions in both the fornix and cingulate bundle restored the cholinergic innervation pattern to that which is typical of the normal septo-retrosplenial inputs. These results provide the neuroanatomical basis for behavioral studies which have documented graft-mediated recovery of spatial memory function in rats with lesions of the cholinergic septo-retrosplenial pathways.

Intraretrosplenial cortical grafts of fetal cholinergic neurons and the restoration of spatial memory function.

The retrosplenial cortex (RSC) receives cholinergic afferent fibers from the medial septal nucleus and diagonal band of Broca (DBB) by way of the cingulate bundle and the fornix. Bilateral lesions of both the cingulate and fornix pathways result in a complete depletion of cholinergic input to the RSC. In the present study we have examined the effects of transplanting cholinergic neurons from fetal rat pups to the RSC of adult rats following lesions of the cingulate bundle and fornix. The animals with lesions exhibited severe spatial memory impairments with a complete loss of extrinsic cholinergic afferents to the RSC. Animals with intraretrosplenial cortical transplants exhibited significant improvements in learning and memory performance as revealed by decreased escape latencies in spatial reference memory tests, increased numbers of platform crossings in spatial navigation tests, and a higher percentage of correct choices in a spatial working memory task. These improvements appeared to be cholinergically mediated because atropine administration significantly disrupted spatial navigation performance. The survival of the transplanted cholinergic neurons and their innervation of the RSC were characterized using a monoclonal antibody to choline acetyltransferase (ChAT). The staining of graft-derived ChAT-positive fibers also revealed a pattern of innervation that mimicked that of the cholinergic input in normal animals. These results indicate that intraretrosplenial cortical transplants of cholinergic neurons can rectify spatial memory deficits produced by the loss of intrinsic cholinergic afferents from the medial septal nucleus.

Organization and histochemical phenotype of human fetal cerebellar cells following transplantation into the cerebellum of nude mice.

Previous rodent studies have demonstrated the capacity of cerebellar transplants to organize into trilaminar cell layers typically observed in the normal cerebellum. In Purkinje Cell (PC)-deficient animals, PCs will migrate into the host and form synaptic connections. Recently, fetal cerebellar grafts transplanted into the Purkinje cell degeneration (pcd) mutant mouse were shown to result in an improvement of motor behaviors. These studies indicate the potential therapeutic use of neural transplantation in patients with cerebellar degeneration. In the present study, human fetal cerebellar tissue (8.5 wk postconception) was dissociated and transplanted into the normal cerebellum of nude mice. Six months following transplantation, histological analysis revealed donor cells in recipient mice. Immunostaining for the 28 kDa calcium-binding protein (calbindin) revealed the presence of donor PCs that were organized in discrete cellular layers within the transplant neuropil. In most cases the dendritic processes were oriented in a planar fashion perpendicular to the transplant cell layer. Human neurofilament immunostaining revealed bundles of donor fibers within the core of the transplant and/or at the periphery. These bundles were found to be calbindin positive (PC fibers). Three animals provided evidence of donor PC axon growth ventrally into host white matter, and in one case, this ventral migration reached the deep cerebellar nuclei. Most notable was the development of a pronounced folia-like organization by the implanted cell suspensions. Glial processes within the grafts were aligned perpendicular to the long axis of the transplant folia. These results demonstrate the capacity of human fetal cerebellar cell suspension to reorganize into cell layers typical of the normal cerebellum following transplantation into the rodent cerebellum, and develop an organotypic folia-like organization.

Transplantation of human fetal tissue from spontaneous abortions to a rodent model of Parkinson's disease.

The use of human fetal tissue from elective abortions for cell transplantation therapies has been the subject of considerable controversy. Because of concerns regarding the use of tissue from elective abortions, tissue from spontaneous abortions has been suggested as an alternate donor source. In the present study we have evaluated human fetal tissue from spontaneous abortions to assess its viability, growth potential, and functional expression. Viable cells (Grade I) from a donor (7 wk postconception) were transplanted as a suspension into the striatum of rats with unilateral 6-OHDA lesions of the nigrostriatal pathway. A second group of animals received solid grafts of tissue from a Grade I donor 14 wk postconception. Tissue from Grade II and III specimens were not sufficiently viable for transplantation. Locomotor responses were monitored over a period of 15 wk and revealed an amelioration of rotational asymmetry by animals that received tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor showed no functional improvement. We found numerous graft-derived tyrosine hydroxylase (TH) immunopositive neurons contained within the transplantation site, and a rich plexus of TH-immunopositive fibers extending into the striatum of animals receiving tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor exhibited tissue necrosis at the transplant site and were devoid of TH-immunopositive neurons. These results suggest that human fetal ventral mesencephalic cells from spontaneous abortions can survive and develop after transplantation, and rectify locomotor deficits associated with experimental parkinsonism if the donor tissue is of the appropriate gestational age at the time of implantation. Our study further suggests, however, that the availability of tissue from spontaneous abortions of sufficient viability is quite limited and may thus restrict its potential use in cell transplantation therapies for Parkinson's disease.

Development of human fetal ventral mesencephalic grafts in rats with 6-OHDA lesions of the nigrostriatal pathway.

Neuronal transplantation is an approach that can be exploited to study the development of the human central nervous system as well as being used in attempts to restore neurological function. In the present study, we have examined cellular events that appear to precede the development of dopamine nerve fiber extension by neurons from the human fetal ventral mesencephalon. These cellular events were examined using neuronal cell suspensions from human fetal ventral mesencephalic tissue (gestational ages 7-10 weeks) transplanted into the striatum of unilaterally lesioned 6-hydroxydopamine (6-OHDA) rats. Animals were sacrificed for immunohistochemistry 9-10 weeks after the transplantation prior to the manifestation of behavioral recovery. Histological analysis revealed tyrosine hydroxylase (TH) immunoreactive neurons in the grafts. The majority of these neurons had very short TH positive processes (60-70 microns), indicating that the maturation of grafted dopaminergic neurons was still incomplete. Immunostaining for the human specific intermediate neurofilament (hNF, clone: BF-10) showed dense neuronal fibers in the grafts. These fibers extended deeper into the host brain than the TH positive neuronal processes. The whole striatum, particularly the medial part of the striatum, exhibited long NF positive processes. Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed fine astrocytic processes inside the grafts, which were clearly different from host reactive glial cells surrounding the grafts. These graft-derived glial processes tended to extend into the host brain deeper than the TH positive neuronal processes from the grafts. These early histological findings of the grafted human fetal ventral mesencephalon suggest that the graft-derived NF positive neuronal processes, as well as the glial processes, radiate from the grafted tissue and extend into the host brain prior to the extension of TH positive processes. These results further suggest that human-to-rat xenografts can be used to study the neural development of human fetal brain tissue.

Transplantation of human fetal striatum into a rodent model of Huntington's disease ameliorates locomotor deficits.

Previous studies have demonstrated that syngeneic transplants of striatal tissue can ameliorate locomotor deficits in rodent models of Huntington's disease (HD). In the present study, we have examined whether human to rat xenografts of fetal striatal tissue can exert a similar recovery of function. Rodents with unilateral striatal lesions were transplanted with human striatal cells from a donor 14 weeks post-conception, and subsequently displayed a progressive decrease in rotational asymmetry in comparison to sham (saline) transplanted animals. Histological analysis revealed acetylcholinesterase (AChE)-positive fibers and NADPH-diaphorase (NADPH-d)-positive neurons within transplanted tissue. These results suggest that human fetal striatum at a gestational age of 14 weeks may potentially be useful as a source of donor tissue for transplantation in the treatment of HD.

Localization of dopamine receptors and associated mRNA in transplants of human fetal striatal tissue in rodents with experimental Huntington's disease.

Huntington's Disease (HD) is characterized by deficits in motor and cognitive functions. This neurodegenerative disease shows an extensive loss of medium-sized spiny projection neurons (GABAergic) within the neostriatum. With the loss of these neurons, there is a concomitant loss of associated receptors, such as those for GABA, glutamate, and dopamine. In the present study, we have addressed the question of whether dopamine receptors are re-established in the lesioned rodent striatum following the transplantation of human striatal cells. Human striatal cell suspension or saline (transplant controls) was injected into the striatum of rats previously lesioned with quinolinic acid (QA). Three nine months following transplantation, the animals were sacrificed and the brains were processed for receptor autoradiography and in situ hybridization of dopamine D1 and D2 receptor subtypes. Our results demonstrate that animals transplanted with human striatal cells show a significant increase in D1 receptors following transplantation when compared to the lesion area in control animals, while D1 receptor mRNA remains unchanged. In contrast to D1 receptor binding, D2 receptor levels are not increased in the lesioned and transplanted area of the striatum when compared to controls; however, D2 receptor mRNA levels are significantly increased. These results demonstrate that at the times the animals were examined, D1 and D2 receptors were differentially regulated. Our results further indicate that human striatal primordium will survive following transplantation and will express D1 receptors and D2 receptor mRNA that are depleted in the QA lesioned rodent striatum. This study compliments and extends previous findings on human striatal cell transplantation in rodent models of HD.

Long-term effects of neonatal ischemic-hypoxic brain injury on sensorimotor and locomotor tasks in rats.

Perinatal ischemia and/or hypoxia in humans are major risk factors for neurologic injury that often manifest as sensorimotor and locomotor deficits throughout development and into maturity. In these studies, we utilized an established model of neonatal ischemic-hypoxia that creates unilateral striatal, cortical, and hippocampal damage (Rice III, J.E., Vanucci, R.C. and Brierley, J.B., Ann. Neurol., 9 (1981) 131-141) to investigate sensorimotor and locomotor deficits in these animals during development and as adults. Sensorimotor deficits were examined by measuring the amount of time that the animals were able to remain on a rotating treadmill. Locomotor abnormalities were assessed by measuring apomorphine-induced rotational asymmetry. Following the neonatal ischemic-hypoxic episode, at 3-9 weeks of age, animals were not able to remain on the treadmill as long as their normal littermate controls. In addition, these animals demonstrated an abnormal, ipsiversive rotational asymmetry in response to systemic administration of apomorphine. When these animals reached adulthood, the degree of atrophy in specific regions of the damaged hemisphere was quantified using measurements of cross-sectional area. The mean cross-sectional area of the striatum was decreased by 29%, the sensorimotor cortex area by 26%, and the dorsal hippocampus cross-sectional area was approximately 6% of its normal size. These data suggest that this rodent model of neonatal ischemic-hypoxic brain injury results in cerebral atrophy and long-lasting sensorimotor and locomotor deficits. These particular behavioral tasks may be used in future studies to assess locomotor and sensorimotor deficits following neonatal ischemic-hypoxic brain injury.

Neural grafting of cholinergic neurons in the hippocampal formation.

The cholinergic septohippocampal system plays an important role in spatial learning and memory functions. Transections of the septohippocampal pathway have been shown to result in a near complete loss of cholinergic innervation in the hippocampus and induce severe spatial memory impairments. In this article, we have reviewed the studies which demonstrate the ability of intrahippocampal septal grafts to reinnervate the hippocampal formation and ameliorate spatial learning and memory deficits. Neuroanatomical studies suggest that grafts of cholinergic tissue can innervate the host hippocampal formation in a pattern that mimics that of the normal septohippocampal pathway. This innervation, in turn, is associated with the formation of graft-to-host synaptic connections. Neurochemical studies reveal that intrahippocampal grafts of septal cells can restore choline acetyltransferase activity, acetylcholine synthesis, and high affinity choline uptake in presynaptic terminals of grafted neurons. In addition, these grafts can normalize the upregulation of cholinergic muscarinic receptors seen postsynaptically in the hippocampus following lesions of the septohippocampal pathway. The functional nature of these grafts is also substantiated by electrophysiological recordings which demonstrate stimulus-evoked graft-to-host synaptic transmission as well as the reinstatement of EEG activity typical of septohippocampal connectivity. In addition to graft-to-host connections, behavioral and neurochemical studies also provide evidence for host-to-graft connections that can regulate the activity of grafted cholinergic neurons during the performance of specific behavioral tasks requiring spatial memory function. Together, these studies suggest that grafts of cholinergic neurons from the medial septal nucleus can become anatomically and functionally incorporated into the circuitry of the host hippocampal formation.

Quantitative analysis of contralateral hemisphere hypertrophy and sensorimotor performance in adult rats following unilateral neonatal ischemic-hypoxic brain injury.

The immature nervous system is capable of considerable compensatory reorganization following injury. This has been studied extensively following many different types of injury in humans and laboratory animals. One common risk factor associated with perinatal brain injury that has been associated with such reorganization is an ischemic-hypoxic event. Using the established Levine model of neonatal ischemic-hypoxia (IH) to create unilateral striatal, cortical and hippocampal damage, we investigated anatomical changes in the undamaged hemisphere contralateral to the injury. Specifically, we measured cross-sectional area (mm2) of brain sections at the level of +1.20 and -2.12 mm from bregma. In addition, we examined sensorimotor deficits in these animals during development and as adults by measuring the amount of time that the animals were able to remain on a rotating treadmill. Our results show that some animals exhibited hypertrophy in the hemisphere contralateral to the lesion as compared to measurements taken from normal control animals. Additionally, we have demonstrated that, following IH, animals that showed significant contralateral whole-hemisphere hypertrophy were able to remain on the Rota-Rod treadmill significantly longer than the animals that did not exhibit this hypertrophy. We conclude that there are compensatory reorganizational changes that occur in the undamaged hemisphere contralateral to injury in some animals following neonatal ischemic-hypoxic brain injury. Furthermore, our data suggest that this plasticity in the contralateral hemisphere may be functionally advantageous.

The fate of human glial cells following transplantation in normal rodents and rodent models of neurodegenerative disease.

Investigations on xenografting in the brain have previously focused on the anatomical and functional integration of the transplanted neurons. More recently, astrocytes are being implicated as having complex functions following transplantation, and are being investigated to determine their role(s) in transplantation. The present study was undertaken to investigate the migration of human astrocytes following transplantation of thalamic, striatal, and mesencephalic tissue into the rodent striatum. Human donor fetuses (9-16 weeks in gestation) obtained through elective and spontaneous abortions were utilized in this study. Following transplantation, donor astrocytes were labeled with an antiserum directed against human glial fibrillary acidic protein. Our results demonstrate that astrocytic elements from all three tissue types are capable of incorporating into the host brain, and have a tendency to follow white matter tracts (such as the corpus callosum, internal capsule, and fiber bundles in the striatum). Human astrocytes, originating from the striatum and thalamus exhibited extensive migration, while migration was more limited in animals with ventral mesencephalon transplants. Ventral mesencephalon transplanted animal demonstrated positive astrocytes within the transplant, with processes (very few cell bodies) extending into white matter of adjacent host striatum. Astrocytes demonstrating immature morphology were observed with all transplant types, but were most prevalent in the striatal transplanted animals. The extent of astrocyte migration and the morphologies observed in this study reflect regional differences of the developing human brain. These results confirm and extend previous investigations on glial cell migration following transplantation in the brain.

Differences in transmission through the dorsal column nuclei in spontaneously hypertensive and Wistar Kyoto rats.

The medical lemniscal evoked potential in response to a range of footshock intensities was recorded in spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). Input-output (I-O) relationships were constructed as the percent of maximum response at each intensity. The SHR had a steeper I-O relationship than did the WKY. This difference was also evident when SHR maintained normotensive from weaning with hydralazine were compared with identically treated WKY. The treatment itself steepened the I-O relationship of the SHR while leaving that of the WKY unchanged. These results indicate an inherent hyperresponsiveness in the SHR dorsal column nuclei and an inhibitory effect of elevated blood pressure on transmission through these nuclei.