The AANS Research Foundation awardees who were coming to the conclusion of their funding cycle, as of June 1996, were honored at the Annual Meeting in Minneapolis. If history is any guide, they will go on to great success, as evidenced by the experience of previous grant recipients (please see accompanying article on page 32).
The AANS membership, through its generous donations, may claim much of the responsibility for making these awards a reality. We are pleased, therefore, to share with you brief summaries of these researchers’ work.
1994 Research Fellow
William D. Hunter, MD
Georgetown University Hospital
Sponsor and Chairman: Robert Martuza, MD
Project Title: Genetically Engineered Herpes Simplex Virus Containing G IL-4, IL-6, and Antisense IGF-I and Its Effects on Glioblastoma Cells
G207 is a double mutant virus of herpes simplex virus type 1 which has been constructed in our laboratory. Subcutaneous or intracranial injection of G207 has shown prolonged survival of mice compared with controls. The aim of my project has been
- to further test the abilities of G207 to mediate an antitumor response to gliomas in nude mice,
- to test G207 for safety prior to human use,
- to study the acute and long term neuropathology of G207, and
- to establish a method to enhance delivery of the mutant virus.
The focus of my research has been to perform safety experiments with G207. G207 was intracranially injected into 12 Aotus nancymae monkeys. This species is extremely sensitive to HSV. So far, no toxicity has been seen from the mutated virus. At different times post injection of G207, monkeys were sacrificed and the organs tested for evidence of HSV infection using standard pathological techniques, H&E staining, immunocy tochemistry, and currently, PCR. Animals were injected over one year ago without adverse effects. MRIs have been per formed preoperatively and postoperatively which show no abnormalities consistent with herpes infection.
Other mutant viruses have been shown to have detrimental effects when placed in the cerebral spinal fluid. After developing conditions for stereotactic intraventricular injections of either G207 and control substance into mice, survivals were noted to be no different between the two groups.
There are two additional issues for evaluation of G207 which I am currently examining. One is the possibility of reactivation of an existing latent virus after inocula tion of G207. Experiments were performed to obtain and intracra nial LD50 for wild type herpes virus, in order to establish a dose required to obtain latent HSV in the cortex. Inoculation of G207 intracranially and evaluating the animals for reactivation of the latent wild type virus is ongoing.
The last issue which my project entails is enhancing the delivery of G207. After developing a surgical procedure for disrupting the blood brain barrier in mice, ongoing experiments may show better distribution of G207 throughout the tumor compared to stereotactic inoculation.
1994 Research Fellow
James D. Guest, MD, PhD
Miami Project to Cure Paralysis
Sponsor: Richard P. Bunge, MD
Chairman: F.A. Durity, MD Co-funded with the Joint Section on Spine
Project Title: Demyelination in the Human and Primate
Neuropathological Study — The distribution of peripheral and central myelin in human contusive spinal cord injury epicenters has been studied. Evidence was obtained that human Schwann cells which enter the spinal cord following trauma may contribute to remyelination of CNS axons. Immunohistochemical evidence has also obtained that demyelination at the injury epicenter may persist following human spinal cord contusion for at least two years.
Experimental Study — The ability of purified, expanded human Schwann cells (HSC) to influence neuronal regeneration following lower thoracic spinal cord transection was investigated in the nude rat. Tissue cable composed of HSC and Matrigel within PAN/PVC guidance channels 10 mm in length were apposed to spinal cord stumps following T8-T11 resection. The survival of transplanted Schwann cells was confirmed for up to 40 days. The cables were able to unite the two ends of the transected spinal cord and provide a highly linear substrate for the regeneration of several thousand axons, some of which were enveloped by immunohistochemically specific human myelin.
Extensive anterograde and retrograde tracing studies were performed to elucidate the response of various neuronal systems to the grafts and were supplemented with immunohistochemical evidence. Unexpectedly, a significant regenerative response from brainstem neurons of the vestibular, raphe and reticulospinal systems as well as from propriospinal and sensory neurons was found. Identical control experiments employed nude rat Schwann cells instead of human Schwann cells. The response from brainstem neurons was not significantly different in these experiments.
Propriospinal neurons respond strongly to the grafts and were retro gradely labeled as much as 12 segments rostral to the graft. Propriospinal neurons can regenerate throughout the entire length of the graft and then re-enter the host spinal cord for up to 2 mm. The adrenergic and dopaminergic systems regenerate fibers into the graft and such fibers have been detected up to 2 mm distal to the graft. Transected corticospinal characterized by end bulbs between 1 and 1.5 mm above the grafts.
Ongoing investigations involve subarachnoid delivery of tissue culture suprnatant containing an antibody known to reduce inhibitory properties of adult white matter in the rat in conjunction with open-ended human Schwann cell grafts. An improved response from the sensory and corticospinal systems has been observed.
1995 Young Clinician Investigator
Lawrence S. Chin, MD
University of Maryland
Sponsor: J. Marc Simard, MD, PhD
Chairman: Howard Eisenberg, MD
Project Title: Potassium Channel Activity in Malignant Astrocttoma
The role of ion channels and pumps in malignant brain tumors is largely un known. Preliminary
results from our lab suggested that the K+ channel blocker, 4 -aminopyridine (4-AP), inhibited cell proliferation. Examination of this hypoth esis in greater detail has occurred over the past year. Detection of the presence of a delayed outward rectifier K+ channel that is sensitive to 4-AP (ED 50 =3.87 mM) in the gliomas cell lines U87 and A172 has been found. This 4-AP effect may be therapeutically relevant because the channel effects are observed at the resting membrane potential of these tumor cells. Cell proliferation in three glioma cell lines was measured using the MTS assay in 96-well microtiter plates. A dose-dependent inhibition of cell proliferation was observed in U87, A172, and T98G cells following treatment with 4-AP; a half-maximal inhibition of cell growth was observed at 2-3 mM. The terminal dUTP transfease end-labeling (TUNEL) technique was used to determine that the mode of cell death was apoptosis. These results are being submitted for publica tion. Future studies will examine other ion channels and their role in the regula tion of apoptosis in gliomas. Results from these studies will provide a scientific basis for new treatment approaches to malignant gliomas.
1995 Young Clinician Investigator
Mark Dias, MD
State University of New York/Buffalo
Sponsor and Chairman: L.N. Hopkins, MD
Co-Funded with the Section on Pediatric Surgery
Project Title: Neuronal Proliferation and Programmed Cell Death of Spinal Motoneurons in a Chick Embryo Model of Neural Tube Defects
Neural tube defects (NTDs) such as myelomeningocele constitute the most common developmental central nervous system (CNS) abnormality and afflict approximately 1:1000 newborns annually. While the thrust of research efforts have thus far been directed toward understanding both the embryogenesis and preven tion of the disorder, remarkably little is known about the impact of the NTD once it has occurred, on the subsequent development of the nervous system. A number of clinical and experimental observations suggest that:
- anatomical and functional connections from the placode to the lower limb musculature are remarkably preserved in many children with complete muscular paralysis,
- some spinal cord function may well be retained until late in gestation, and
- some evidence of spinal cord function is present even post-natally in many children with this disorder.
Despite these exciting observations, the anatomical and physiological sub strate for sensorimotor paralysis in these children remains an important and uncharted area of investigation. In contrast, the normal development of the spinal cord has been extensively studied in several animal models; how these normal processes are altered in the face of an NTD is unknown.
Currently, the project continues by examining the histogenesis of the spinal cord in a chick mode of NTD. Initial studies are focusing on both proliferation and programmed, or opoptotic, cell death in chick spinal cord motoneurons. A simple mechanical model of NTD in chick embryos was employed, and are currently obtaining embryos with NTDs that we will be examining at various embryonic stages to determine the extent of both neuronal proliferation and apoptosis in these embryos. Ultimately, it is hoped to examine the directed outgrowth of axons from spinal cord moto neurons to lower limb muscle targets. These studies will provide a much better understanding of neuronal growth and development in the face of an NTD, and hopefully will lead to experimental and, ultimately, clinical interventions whose goal is to improve motoneuron survival and spinal cord function for children with these disorders.