Elisabet Åkesson

Motor Performance Score: A New Algorithm for Accurate Behavioral Testing of Spinal Cord Injury in Rats

To evaluate the usefulness of standard neurological tests in predictin g the neurological outcome after photochemically induced spinal cord lesions in rats, we inflicted injuries of different severity to adult female rats. The behavior of the rats was followed for 6 weeks and the results of the behavioral tests were correlated with morphological indicators of tissue destruction at the end of this period. We found many behavioral tests to be highly correlated with the loss of tissue, whereas some tests were inaccurate in correlating with degree of tissue destruction. Motor score, beam walk, and righting reflect were all highly correlated with the volume of the lesion as well as the depth of the lesion cavity at its epicenter. We propose a protocol for neurological evaluation of this type of spinal cord injury consisting of six individual tests, hierarchally organized such that injured rats can be divided into 11 groups ofn eurological deficit, scored from 10 to 0. This so-called motor performance score is fast and easy to perform and shows high correlation with the lesion volume, and is thus suitable for neurological evaluation of photochemically induced spnial cord injury.

Cellular composition of long-term human spinal cord- and forebrain-derived neurosphere cultures.

In vitro expanded neural precursor cells (NPCs) may provide a stable source for cell therapy. In search of the optimal cell source for spinal cord repair, we investigated influences of gestational age, regional heterogeneity, and long‐term in vitro propagation. The cellular content of neurosphere cultures prior to and after in vitro differentiation was studied by immunocytochemistry and flow cytometry. Human forebrain and spinal cord NPCs deriving from first‐trimester tissue were cultured as neurospheres in the presence of epidermal growth factor, basic fibroblast growth factor, and ciliary neurotrophic factor. Proteins characteristic for embryonic stem cells, i.e., Tra‐1‐60, Tra‐1‐81, and SSEA‐4, were present in ≈0.5% of the cells in donor tissues and neurospheres. The proportions of nestin‐ and proliferating cell nuclear antigen‐immunoreactive (IR) cells were also maintained, whereas the CD133‐IR population increased in vitro. Glial fibrillary acidic protein‐IR cells increased in number, and in contrast the fraction of β‐tubulin III‐IR cells decreased, at and beyond passage 5 in spinal cord but not forebrain cultures. However, dissociated and in vitro‐differentiated forebrain‐ and spinal cord‐derived neurospheres generated similar proportions of neurons, astrocytes, and oligodendrocytes. Gestational age of the donor tissue, which ranged from 4.5 to 12 weeks for forebrain and from 4.5 to 9.5 weeks for spinal cord, did not affect the proportion of cells with different phenotypes in culture. Thus, cellular composition of human neurosphere cultures differs as a result of long‐term in vitro propagation and regional heterogeneity of source tissue, despite expansion under equal culture conditions. This could in turn imply that human spinal cord and forebrain NPCs present different repair potentials in in vivo settings.

Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres.

There is heterogeneity in neural stem and progenitor cell characteristics depending on their species and regional origin. In search for potent in vitro-expanded human neural precursor cells and cell therapy methods to repair the injured human spinal cord, the possible influence exerted by intrinsic cellular heterogeneity has to be considered. Data available on in vitro-expanded human spinal cord-derived cells are sparse and it has previously been difficult to establish long-term neurosphere cultures showing multipotentiality. In the present paper, human spinal cord-derived neurospheres were cultured in the presence of EGF, bFGF and CNTF for up to 25 passages (>350 days) in vitro. In contrast to the human first trimester subcortical forebrain, spinal cord tissue>9.5 weeks of gestation could not serve as a source for long-term neurosphere cultures under the present conditions. After withdrawal of mitogens, cultured neurospheres (at 18 passages) gave rise to cells with neuronal, astrocytic and oligodendrocytic phenotypes in vitro. After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immuno-deficient adult rats, large numbers of cells survived at least up to 6 weeks, expressing neuronal and astrocytic phenotypes. These results demonstrate that it is possible to expand and maintain multipotent human spinal cord-derived neurospheres in vitro for extended time-periods and that they have promising in vivo potential after engraftment to the injured spinal cord.

Cloning and expression of the human N-methyl-d-aspartate receptor subunit NR3A

Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding to a 93% homology at the amino acid level. Based on available data from human genome databases, we localized the gene to chromosome 9. The transcript could be detected by in situ hybridization in human fetal spinal cord and forebrain. Two splice variants of NR3A have been reported in rat brain, the longer of the two containing a 60 bp insert in the intracellular domain. We were unable to detect this 60 bp insert in fetal or adult human brain, suggesting that only the short variant is expressed in humans.

Markers of pluripotency and differentiation in human neural precursor cells derived from embryonic stem cells and CNS tissue.

Cell transplantation therapies for central nervous system (CNS) deficits such as spinal cord injury (SCI) have been shown to be effective in several animal models. One cell type that has been transplanted is neural precursor cells (NPCs), for which there are several possible sources. We have studied NPCs derived from human embryonic stem cells (hESCs) and human fetal CNS tissue (hfNPCs), cultured as neurospheres, and the expression of pluripotency and neural genes during neural induction and in vitro differentiation. mRNA for the pluripotency markers Nanog, Oct-4, Gdf3, and DNMT3b were downregulated during neural differentiation of hESCs. mRNA for these markers was found in nonpluripotent hfNPC at higher levels compared to hESC-NPCs. However, Oct-4 protein was found in hESC-NPCs after 8 weeks of culture, but not in hfNPCs. Similarly, SSEA-4 and CD326 were only found in hESC-NPCs. NPCs from both sources differentiated as expected to cells with typical features of neurons and astrocytes. The expressions of neuronal markers in hESC-NPCs were affected by the composition of cell culture medium, while this did not affect hfNPCs. Transplantation of hESC-NPC or hfNPC neurospheres into immunodeficient mouse testis or subcutaneous tissue did not result in tumor formation. In contrast, typical teratomas appeared in all animals after transplantation of hESC-NPCs to injured or noninjured spinal cords of immunodeficient rats. Our data show that transplantation to the subcutaneous tissue or the testes of immunodeficient mice is not a reliable method for evaluation of the tumor risk of remaining pluripotent cells in grafts.

First Trimester Development of the Human Nigrostriatal Dopamine System

The aim of the present study was to characterize the morphological and neurochemical differentiation of mesencephalic dopaminergic neurons in human embryos, derived from elective first trimester abortions. Embryonic brain tissue was taken for analysis of tyrosine hydroxylase (TH) by immunohistochemistry and Western blot, and for analysis of endogenous dopamine (A) content using HPLC-ED. TH expression was first detected at 3.5 weeks of gestational age (Carnegie stage 11) by immunohistochemical staining of the primordial sympathetic trunk along both sides of the neural tube. In sagittal sections of the intact 4.5-week-old embryo, a small, distinct population of rounded, densely packed TH-immunoreactive perikarya with short primary processes was seen in the midbrain. During the latter half of the first trimester, the number of TH-stained cells as well as the length and number of axonal processes projecting toward and into the developing neostriatum increased rapidly. At the end of the first trimester, varicose fibers could be detected in the striatal anlage. In order to verify that TH was the antigen recognized by the antibodies used for immunohistochemistry on human tissue specimens, mesencephalic tissue of 5-10 weeks gestation was analyzed by Western blot technique. A single, homogeneous band with the apparent molecular weight of approximately 60 kDa was clearly detected at 5 weeks of age. The amount of TH/mg total protein increased at least 10-fold between 5-10 weeks of gestation. For comparison, the mesencephalon and the forebrain/basal ganglia were analyzed for endogenous DA content using HPLC-ED. DA was first detected at 5.5 weeks of gestational age in both mid- and forebrain, and DA levels were found to increase exponentially from 7 to 7.5 weeks of age, reaching 4-5.5 ng DA/mesencephalon and 50-75 ng DA/g caudate nucleus-putamen anlage at the end of the first trimester. Together, morphological and biochemical data presented here constitute evidence for a very early appearance, migration, and differentiation as well as functional development of human mesencephalic dopaminergic neurons and their projections into target areas during the first trimester.

Solid human embryonic spinal cord xenografts in acute and chronic spinal cord cavities: a morphological and functional study.

While therapeutic spinal cord grafting procedures are of interest in the chronic spinal cord injury stage, previous experimental grafting studies, including human spinal cord tissue, have mainly focused on the acute stage. Therefore, solid human embryonic spinal cord grafts were implanted in acute or chronic spinal cord aspiration cavities of immunodeficient rats to compare the morphological and locomotor outcome to that of lesion alone cases. Locomotor function was assessed using the Basso, Beattie, and Bresnahan open-field locomotor rating scale up to 6 months, while the morphological evaluation of graft survival, growth, and integration was performed at 6 weeks or 6 months after implantation. Graft survival was 94% in both lesion models, while graft growth was enhanced in the chronic compared to the acute cavity group. Human specific Thy-1 and neurofilament immunoreactive fibers were observed up to 7 mm into host white matter, while aminergic fibers were observed up to 1 mm into the grafts. Abundant calcitonin gene-related peptide immunoreactive fibers in the grafts in the absence both of immunoreactive cell bodies and colocalized human-specific neurofilament immunoreactivity, suggested host fiber ingrowth. At 6 months, the grafted cases presented less central canal deformation and lower glial fibrillary acidic protein immunoreactivity at the host cavity border compared to that of the nongrafted cases. The strong compensatory regain of locomotor function after unilateral spinal cord lesions was not affected by the human spinal cord grafts. In conclusion, solid human embryonic spinal cord tissue transplanted to a cavity in the adult injured spinal cord results in beneficial morphological effects in both the acute and chronic spinal cord lesion.

Analysis of NR3A receptor subunits in human native NMDA receptors

NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2A/B-NR3A assemblies in adult human CNS. While NR1 and NR2A could only be efficiently solubilized by deoxycholate, NR3A was extracted by all detergents, suggesting that a large fraction is weakly anchored to cell membranes and other proteins. Using size exclusion chromatography we found that just as for NR1, a large fraction of NR3A exists as monomers and dimers, suggesting that these two glycine binding subunits behave similarly with regard to receptor assembly and trafficking.

Human Embryonic Spinal Cord Grafts in Adult Rat Spinal Cord Cavities: Survival, Growth, and Interactions with the Host☆

The ability of solid pieces of transplanted human embryonic spinal cord to survive, grow, and integrate with adult rat host spinal cord tissue was investigated. Unilateral cavities were surgically created at vertebral level T12-T13 in 10 athymic nude rats and 5 regular Sprague-Dawley rats. Seven of the athymic rats acutely received a human spinal cord graft, while the remaining 8 rats served as controls, with cavities alone. After 6 months the morphological outcome was evaluated with cresyl violet and with immunohistochemistry using antibodies toward human-specific neurofilament (hNF), human-specific Thy-1 (Thy-1), neurofilament, glial fibrillary acidic protein, serotonin (5-HT), and tyrosine hydroxylase (TH). The in situ morphology of the human embryonic spinal cord was also investigated and compared with grafts that were six months older. Solid human embryonic spinal cord grafts showed a 100% survival rate, grew to fill the volume of the cavity in a noninvasive manner, and expressed human specific antigens 6 months postgrafting. Thy-1 immunoreactivity (IR) was demonstrated up to 8 mm rostral to the graft suggestive of graft-derived fiber outgrowth. hNF-IR fibers and 5-HT- and TH-IR fibers traversed the graft-host border for a few hundred micrometers, respectively. Finally, our findings suggest that grafted solid pieces of human embryonic spinal cord minimize cystic deformations seen in the adult rat spinal cord with a unilateral cavity.

Human neural stem cells and astrocytes, but not neurons, suppress an allogeneic lymphocyte response

Transplantation of human neural stem cells (NSCs) and their derivatives is a promising future treatment for neurodegenerative disease and traumatic nervous system lesions. An important issue is what kind of immunological reaction the cellular transplant and host interaction will result in. Previously, we reported that human NSCs, despite expressing MHC class I and class II molecules, do not trigger an allogeneic T cell response. Here, the immunocompetence of human NSCs, as well as differentiated neural cells, was further studied. Astrocytes expressed both MHC class I and class II molecules to a degree equivalent to that of the NSCs, whereas neurons expressed only MHC class I molecules. Neither the NSCs nor the differentiated cells triggered an allogeneic lymphocyte response. Instead, these potential donor NSCs and astrocytes, but not the neurons, exhibited a suppressive effect on an allogeneic immune response. The suppressive effect mediated by NSCs most likely involves cell-cell interaction. When the immunogenicity of human NSCs was tested in an acute spinal cord injury model in rodent, a xenogeneic rejection response was triggered. Thus, human NSCs and their derived astrocytes do not initiate, but instead suppress, an allogeneic response, while they cannot block a graft rejection in a xenogeneic setting.