Vaccinia virus complement control protein modulates inflammation following spinal cord injury.

The vaccinia virus complement control protein (VCP) possesses multiple modulatory functions. Functioning as a complement inhibitory protein, VCP reduces production of proinflammatory chemotactic factors produced during complement activation. Additionally, VCP binds heparin and heparan sulfate proteoglycans, resulting in added functions shown to block monocyte chemotaxis in vitro. Using an in vivo spinal cord contusive injury model in rats, the inflammation-modulating abilities of VCP were evaluated. The results of both myeloperoxidase assaying and H&E stained section counts of spinal tissue reveal that neutrophil infiltration to the area of the lesion was reduced in animals that received VCP as compared to saline-injected controls.

Traumatic intracranial aneurysms.

Traumatic intracranial aneurysms are rare, occurring in fewer than 1% of patients with cerebral aneurysms. They can occur following blunt or penetrating head trauma and are more common in the pediatric population. Traumatic aneurysms can be categorized histologically as true, false, or mixed, with false aneurysms being the most common. These aneurysms can present in a variety of ways, but are typically associated with an acute episode of delayed intracranial hemorrhage with an average time from initial trauma to aneurysm hemorrhage of approximately 21 days. The mortality rate for patients harboring these aneurysms may be as high as 50%. Prompt diagnosis based on arteriography and aggressive surgical management are associated with better outcome than conservative treatment. The authors describe a classification scheme for traumatic aneurysms based on their anatomical location and conclude that 1) posttraumatic aneurysm must be considered in patients with acute neurological deterioration following closed head injury; 2) they can occur following mild closed head injury; 3) they occur more commonly in children than in adults; and 4) surgical clipping and/or endovascular occlusion is the definitive treatment.

Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.

Electrophysiological properties of mitogen-expanded adult rat spinal cord and subventricular zone neural precursor cells.

Growth factor-expanded neural precursor cells isolated from the mammalian central nervous system can differentiate into neurons and glia. Although the morphological and neurochemical development of these neural precursor cells has been investigated, little attention has been paid to their electrophysiology. This study examined the electrophysiological properties of neurons and glia derived from neural precursor cells isolated from the adult rat spinal cord (SC) and subventricular zone (SVZ). Cells were cultured in medium containing epidermal growth factor and/or fibroblast growth factor-2. After at least two passages, spheres of neural precursor cells were plated on coated coverslips and maintained in culture for up to 6 weeks. Whole-cell patch recordings were made using standard current clamp techniques. Immature action potentials were observed within hours of plating for both SC and SVZ cells. Input resistance and time constants decreased over the first week after plating and no further changes were found at later times. At similar times following plating, however, SVZ cells had a lower input resistance and shorter time constant compared to SC cells. SVZ cells also had higher resting membrane potentials and smaller after hyperpolarizations than those of SC cells, despite no significant difference in the amplitude of action potentials. Neither the SC nor the SVZ cells were capable of eliciting more than a single action potential in response to injected current. While all SC cells tested were depolarized by glutamate, the response of SVZ cells to glutamate varied considerably. This study revealed that neural precursor cells from SC and SVZ differ in both active and passive membrane properties. It appears also that the electrophysiological development of SC and SVZ precursor-derived neurons is incomplete under the conditions used. These observations suggest that the neural precursor cells from different anatomical locations may be physiologically diverse and may exhibit some differences in commitment toward neuronal or glial phenotypes.

Comparing deficits following excitotoxic and contusion injuries in the thoracic and lumbar spinal cord of the adult rat.

The majority of human spinal cord injuries involve gray matter loss from the cervical or lumbar enlargements. However, the deficits that arise from gray matter damage are largely masked by the severe deficits due to associated white matter damage. We have developed a model to examine gray matter-specific deficits and therapeutic strategies that uses intraspinal injections of the excitotoxin kainic acid into the T9 and L2 regions of the spinal cord. The resulting deficits have been compared to those from standard contusion injuries at the same levels. Injuries were assessed histologically and functional deficits were determined using the Basso, Beattie, and Bresnahan (BBB) 21-point open field locomotor scale and transcranial magnetic motor evoked potentials (tcMMEPs). Kainic acid injections into T9 resulted in substantial gray matter damage; however, BBB scores and tcMMEP response latencies were not different from those of controls. In contrast, kainic acid injections into L2 resulted in paraplegia with BBB scores similar to those following contusion injuries at either T9 or L2, without affecting tcMMEP response latencies. These observations demonstrate that gray matter loss can result in significant functional deficits, including paraplegia, in the absence of a disruption of major descending pathways.

Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.

It has been established that the adult mouse forebrain contains multipotential (neuronal/glial) progenitor cells that can be induced to proliferate in vitro when epidermal growth factor is provided. These cells are found within the subventricular zone of the lateral ventricles, together with other progenitor cell populations, whose requirements for proliferation remain undefined. Using basic fibroblast growth factor (bFGF), we have isolated multipotential progenitors from adult mouse striatum. These progenitors proliferate and can differentiate into cells displaying the antigenic properties of astrocytes, oligodendrocytes, and neurons. The neuron-like cells possess neuronal features, exhibit neuronal electrophysiological properties, and are immunoreactive for GABA, substance P, choline acetyl-transferase, and glutamate. Clonal analysis confirmed the multipotency of these bFGF-dependent cells. Most significantly, subcloning experiments demonstrated that they were capable of self-renewal, which led to a progressive increase in population size over serial passaging. These results demonstrate that bFGF is mitogenic for multipotential cells from adult mammalian forebrain that possess stem cell properties.

Neurons derived from P19 embryonal carcinoma cells develop responses to excitatory and inhibitory neurotransmitters.

Cells of the P19 line of embryonal carcinoma cells differentiate into neurons, astrocytes and oligodendrocytes following treatment with retinoic acid. The neurons from these differentiating P19 cultures synthesize a pattern of neurotransmitters that resembles that of neurons of the forebrain. We treated P19 cells with retinoic acid and then implanted them into the striatum of adult rats. After times ranging from 1 to 15 weeks post-implantation, brain slices containing the implanted tissue were prepared and used for intracellular recording of electrical activity and responsiveness to application of neurotransmitters. Within 2 weeks of implantation, the P19-derived neurons had developed responsiveness to the excitatory neurotransmitter glutamate and the inhibitory transmitters gamma-aminobutyric acid and glycine. These neurons also exhibited spontaneous synaptic potentials. The responses to glutamate appear to be mediated by N-methyl-D-aspartic acid as well as non-N-methyl-D-aspartic acid receptor subtypes. Gamma-aminobutyric acid evoked bicuculline-sensitive depolarizing responses in the younger grafts and biphasic depolarizing/hyperpolarizing responses in older ones. Responses to glycine were strychnine sensitive and also showed age-related changes from depolarizing to biphasic character. Synaptic potentials in the younger grafts were exclusively depolarizing, but in older ones both depolarizing and hyperpolarizing events were observed. The synaptic potentials appear to arise from synaptic connections between P19-derived neurons within the grafts. Many of the features of P19-derived neurons are similar to those of neurons in the developing forebrain.

In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line.

The multipotent embryonal carcinoma (EC) P19 cell line differentiates into neurons, glia and smooth muscle following exposure to retinoic acid (RA). RA-induced differentiation is irreversible and the neurons that develop are abundant, post-mitotic, and survive for prolonged periods in culture or when grafted into the CNS of adult rats. Striatal slices containing grafted P19 cells were studied with intracellular recording and labelling techniques to examine the development of electrophysiological and morphological properties of P19-derived neurons over a period of 6 to 120 days after grafting into ibotenic acid lesioned striatum. Cells from 1-week-old grafts had a range of immature electrophysiological characteristics including unstable resting membrane potentials (RMP's) and very high membrane input resistances (Rin's). Many were not able to produce action potentials (AP's). In contrast, the majority of cells recorded from 2- and 3-week-old grafts had stable RMP's, moderate Rin's, and were able to produce regenerative AP's. In grafts over 4 weeks of age, the majority of P19-derived neurons had mature neuronal electrophysiological characteristics including RMP's of -60 mV, Rin's of 100-300 M omega, and overshooting AP's. Morphologically, P19 derived neurons increase in soma size from 12-15 mu in diameter in 7-14-day-old grafts, to 25-35 mu in diameter in grafts 50-120 days old. Developing neurons exhibited a variety of morphotypes with increasingly complex processes and lengths of process extension. Our results demonstrate a developmental progression of the electrophysiology of P19-derived neurons, culminating in mature characteristics closely resembling those of adult rodent hippocampal or cortical pyramidal neurons. The ability to easily alter these cells genetically provides a powerful model for addressing issues specific to neuronal development.

Neurons derived from P19 embryonal carcinoma cells have varied morphologies and neurotransmitters.

Treatment of P19 embryonal carcinoma cells with retinoic acid induces their differentiation into a population of cells consisting of neurons and other cell types normally derived from neuroectoderm. We used immunohistological and histochemical techniques to identify some of the neurotransmitters in the P19-derived neurons. The majority of neurons contained GABA, glutamic acid decarboxylase, and GABA-transaminase. Neuropeptide Y and somatostatin were less frequently found and both were partially co-expressed with GABA and with one another. Smaller numbers of cells were positive for tyrosine hydroxylase, DOPA decarboxylase, serotonin, calcitonin gene-related peptide, galanin and substance P. The variety and proportions of cells with different transmitter types were reproducible from one experiment to the next and varied very little over 40 days in culture except for cells containing enkephalin, which were abundant only in mature cultures of 32 days or more. Synapses formed between neurons and some contained both small clear and large dense-core vesicles within the presynaptic bouton. Because GABA, neuropeptide Y and somatostatin are abundant in P19-derived neurons as well as in embryonic neurons in rostral regions of the mammalian CNS, we suggest that the developmental events occurring in P19 cell cultures closely resemble those of the embryonic neuroectoderm.

Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum.

P19 embryonal carcinoma cells are pluripotent and can be efficiently induced to differentiate in culture into neurons and astroglia by brief treatment with retinoic acid. Retinoic acid-treated P19 cells survive after grafting into the adult rat striatum and differentiate into neurons and glia within the transplantation site. No tumours develop from the grafted cells which continue to express foreign genes that had been transfected into the parental P19 cells. The neurons in these grafts express a variety of neurotransmitters similar to those formed in retinoic acid-treated P19 cell cultures and they mature to acquire the electrophysiological properties expected of fully developed neurons. These results suggest that P19 cells may be used for studies related to neuronal cell development and maturation and that P19 cells may be considered for cell replacement strategies in neurodegenerative disorders of the central nervous system.

New aspects of neurotransplantation.

We have examined the possibility of promoting axonal regeneration within lesioned neural tissue using grafted artificial gel matrices. Polymeric matrices which feature a three-dimensional crosslinked macromolecular network were implanted into preformed lesions of the central nervous system (CNS). The host response consisted of matrix invasion by glial elements and the deposition of newly synthesized extracellular molecules. This rearrangement of the brain scarring process into an organized cellular coating promoted axonal regeneration into the gels. Entrapment of embryonic neurons and embryonal carcinoma (EC)-derived neurons, within the gels, was performed to explore the possibility of using polymer brain implants as neural graft microcarriers. Our results suggest that this approach will be useful for the delivery of cells and the promotion of axonal elongation required for successful neurotransplantation.

SYNTHETIC POLYMER MATRICES FOR NEURAL CELL TRANSPLANTATION.

This study proposes a strategy to promote the integration of a neural graft into the host brain tissue. It involves the attachment of donor cells to a polymeric matrix, and the implantation of this cell-polymer matrix. We have synthesized hydrogels based on N-(2-hydroxypropyl)-methacrylamide (HPMA) to produce highly porous matrices. As preliminary steps, we have examined: 1) The response of the brain tissue to the implantation of PHPMA/collagen hydrogels; 2) adhesion, growth, differentiation, and viability of embryonic neuronal cells, and embryonal carcinoma-derived neurons seeded onto PHPMA substrates containing hexosamine residues (glucosamine and N-acetylglucosamine), and after entrapment of cells within the hydrogels. Histological analysis seven wk after implantation showed the tolerance of PHPMA hydrogels, and the penetration of host cells into the pore structures. However, cellular ingrowth requires the presence of collagen, and is dependent upon porosity. In vitro data showed that PHPMA substrates supported neuronal cell attachment and neuritic growth, but the biocompatibility of the substrate was enhanced after incorporation of N-acetylglucosamine into the hydrogel. The data also showed the feasibility of entrapping cells into the polymer matrices, and that these "cellular" hydrogel matrices could be maintained in vitro with preservation of cell viability and differentiation. These findings suggest that PHPMA-based hydrogels can serve as carriers for neural transplant, and as a support to guide tissue ingrowth and organization.