Andrew Freese

Analysis of harvest morbidity and radiographic outcome using autograft for anterior cervical fusion

Study Design. Retrospective study of 184 autologous iliac crest bone grafts used for anterior cervical fusion in 144 procedures. Objectives. To evaluate the effect of autologous iliac crest bone graft harvest site on operation and recovery and to identify patients at risk for harvest morbidity. Summary of Background Data. Although autologous iliac crest bone graft is considered the most successful grafting material, concerns about harvest morbidity provide a rationale for considering allograft. Data about the use of autograft therefore would assist spinal surgeons in selecting the appropriate substrates for fusion after anterior cervical decompression. Methods. Statistical analysis based on patient gender, smoking history, obesity, and medical or pharmacologic risk factors for wound healing was used to evaluate morbidity after patient interviews and examinations. Limited assessment of radiographic outcome also was performed. Results. A second operation because of donor site morbidity was performed in four patients (2.8%), but only one (0.7%) with meralgia paresthetica had permanent sequelae. Superficial wound infection or dehiscence occurred in 5.6% of patients, with a disproportionate number of women, obese patients, and those with medical risk represented. Protracted wound symptoms of pain and poor cosmesis were reported in 2.8% and 3.5% of patients, respectively, and also were found in a significant number of female and obese patients. Evidence of fusion was present in 97% of cases. Conclusion. Autologous iliac crest bone graft harvest results in minimal major morbidity when regional anatomy is respected and careful technique is observed. The identification of patients at risk for minor complications suggests that allograft may be appropriate in these patients; however, prospective comparison is required to identify whether graft material or technical factors determine fusion success and relative benefit.

Gene Therapy of Canavan Disease: AAV-2 Vector for Neurosurgical Delivery of Aspartoacylase Gene (ASPA) to the Human Brain

This clinical protocol describes virus-based gene transfer for Canavan disease, a childhood leukodystrophy. Canavan disease, also known as Van Bogaert-Bertrand disease, is a monogeneic, autosomal recessive disease in which the gene coding for the enzyme aspartoacylase (ASPA) is defective. The lack of functional enzyme leads to an increase in the central nervous system of the substrate molecule, N-acetyl-aspartate (NAA), which impairs normal myelination and results in spongiform degeneration of the brain. No effective treatment currently exists; however, virus-based gene transfer has the potential to arrest or reverse the course of this otherwise fatal condition. This procedure involves neurosurgical administration of approximately 900 billion genomic particles (approximately 10 billion infectious particles) of recombinant adeno-associated virus (AAV) containing the aspartoacylase gene (ASPA) directly to affected regions of the brain in each of 21 patients with Canavan disease. Pre- and post-delivery assessments include a battery of noninvasive biochemical, radiological, and neurological tests. This gene transfer study represents the first clinical use of AAV in the human brain and the first instance of viral gene transfer for a neurodegenerative disease.

Immune responses to AAV in a phase I study for Canavan disease

Canavan disease is a rare leukodystrophy with no current treatment. rAAV‐ASPA has been developed for gene delivery to the central nervous system (CNS) for Canavan disease. This study represents the first use of a viral vector in an attempt to ameliorate a neurodegenerative disorder.

In vivo expression of therapeutic human genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector

An adeno-associated virus (AAV) vector, expressing genes for human tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC), demonstrated significantly increased production of dopamine in 293 (human embryonic kidney) cells. This bicistronic vector was used to transduce striatal cells of six asymptomatic but dopamine-depleted monkeys which had been treated with the neurotoxin MPTP. Striatal cells were immunoreactive for the vector-encoded TH after stereotactic injection for periods up to 134 days, with biochemical effects consistent with dopamine biosynthetic enzyme expression. A subsequent experiment was carried out in six more severely depleted and parkinsonian monkeys. Several TH/aadc-treated monkeys showed elevated levels of dopamine near injection tracts after 2.5 months. Two monkeys that received a β-galactosidase expressing vector showed no change in striatal dopamine. Behavioral changes could not be statistically related to the vector treatment groups. Toxicity was limited to transient fever in several animals and severe hyperactivity in one animal in the first days after injection with no associated histological evidence of inflammation. This study shows the successful transfection of primate neurons over a period up to 2.5 months with suggestive evidence of biochemical phenotypic effects and without significant toxicity. While supporting the idea of an in vivo gene therapy for Parkinson’s disease, more consistent and longer lasting biochemical and behavioral effects will be necessary to establish the feasibility of this approach in a primate model of parkinsonism.

Basic fibroblast growth factor protects striatal neurons in vitro from NMDA-receptor mediated excitotoxicity.

Basic fibroblast growth factor (bFGF) promotes the survival and outgrowth of neurons. In this study the neuroprotective effects of bFGF were examined in 12-18-day-old cultured striatal neurons exposed to glutamic acid, kainic acid (KA), and quinolinic acid (QA), an N-methyl-D-aspartate (NMDA)-receptor agonist. Results showed that preincubation with bFGF (6 pM) from the day of plating significantly increased the survival of striatal neurons treated for 3 h with glutamate (3 mM) or QA (1 mM), but had little effect on KA (1 mM) induced toxicity. Moreover, maximum protection by bFGF against glutamate neurotoxicity was observed in cultures treated as little as 2 h before glutamate exposure. These results show that bFGF markedly protects striatal neurons from NMDA-receptor induced neurotoxicity.

Long-Term Follow-Up After Gene Therapy for Canavan Disease

Gene therapy for Canavan disease results in a decrease in pathologically elevated N-acetyl-aspartate concentrations in the brain and long-term clinical stabilization. Gene Therapy for Canavan Disease Canavan disease is a fatal childhood neurodegenerative disorder for which there is no effective treatment. It is caused by a defect in a single gene (ASPA) that results in a deleterious buildup of N-acetyl-aspartate in the brain. This process starts at birth and is accompanied by a failure to form and maintain myelin, the protective sheath surrounding nerves. As a brain-specific disorder with simple Mendelian inheritance, Canavan disease represents an excellent target for enzyme replacement using gene therapy. Leone et al. now report the long-term results of gene therapy in 13 Canavan disease patients using adeno-associated viral vector delivery of the ASPA gene. The investigators found that gene therapy was safe and led to a decrease in N-acetyl-aspartate in the brain, together with decreased seizure frequency and clinical stabilization. Clinical stabilization was greatest in the youngest patients. Early detection and treatment with gene therapy–mediated enzyme replacement in the neonatal period may offer the best opportunity for a reduction in symptoms and long-term stabilization in patients with Canavan disease. Canavan disease is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and increased concentrations of the substrate N-acetyl-aspartate (NAA) in the brain. Accumulation of NAA results in spongiform degeneration of white matter and severe impairment of psychomotor development. The goal of this prospective cohort study was to assess long-term safety and preliminary efficacy measures after gene therapy with an adeno-associated viral vector carrying the ASPA gene (AAV2-ASPA). Using noninvasive magnetic resonance imaging and standardized clinical rating scales, we observed Canavan disease in 28 patients, with a subset of 13 patients being treated with AAV2-ASPA. Each patient received 9 × 1011 vector genomes via intraparenchymal delivery at six brain infusion sites. Safety data collected over a minimum 5-year follow-up period showed a lack of long-term adverse events related to the AAV2 vector. Posttreatment effects were analyzed using a generalized linear mixed model, which showed changes in predefined surrogate markers of disease progression and clinical assessment subscores. AAV2-ASPA gene therapy resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy, with some improvement in seizure frequency and with stabilization of overall clinical status.

An HSV-1 Vector Expressing Tyrosine Hydroxylase Causes Production and Release of l-DOPA from Cultured Rat Striatal Cells

Abstract: In this report we demonstrate that a defective herpes simplex virus type one (HSV‐1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into l‐DOPA‐producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV‐1 vector (pHSVth) and packaged into virus particles using an HSV‐1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production of l‐DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release of l‐DOPA and low levels of dopamine were observed from cells in pHSVth‐infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week after infection.

Controlled release of dopamine from a polymeric brain implant: In vitro characterization

A biocompatible polymeric matrix system for the long-term controlled release of dopamine has been developed. Solid particles of this bioactive agent were encapsulated in ethylene-vinyl acetate copolymer (EVAc). Following immersion in an aqueous buffer solution, the release rate of dopamine from the polymer matrix was found to depend on the initial concentration of dopamine in the polymer. After coating the matrix devices with an additional impermeable layer of EVAc, constant rates of release were obtained by creating a cavity in this impermeable layer. The observed experiments are consistent with a diffusion-limited model of dopamine release; all the in vitro experimental results were therefore correlated by the effective diffusion coefficient of dopamine through the porous polymer network. These results are discussed in terms of potential design modifications to achieve desired release characteristics for a variety of neuroactive substances, including neurotransmitters or their precursors.

Characterization and mechanism of glutamate neurotoxicity in primary striatal cultures

Excitatory amino acids may play a role in the pathogenesis of cell death in neurodegenerative diseases, including Huntingtons disease (HD). In an attempt to develop a tissue culture model for HD, the toxicity of glutamate was examined in primary striatal cultures derived from newborn rats. Morphological criteria were used to determine the toxic effects of glutamate in 6-, 12-, and 18-day-old cultures which were examined before and after 1-3 h of exposure to glutamate. Although younger cultures demonstrated little susceptibility to glutamate relative to controls, the number of neurons in older cultures was significantly depleted in the presence of glutamate. Glutamate toxicity was dose-dependent, with an ED50 of approximately 300 microns glutamate, and a maximal effect was observed within 3 h of initial exposure. Affected neurons demonstrated somal swelling within 1 h of glutamate exposure and disruption of neuritic processes and somal integrity within 3 h. Cell death was significantly increased by raising the extracellular calcium concentration and could be decreased by the addition of magnesium to the incubation medium. Moreover, the N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid, showed a toxicity profile similar to that of glutamate. The NMDA receptor competitive antagonist, 2-amino-5-phosphonovalerate (APV) significantly reduced toxicity, albeit incompletely. An additional component of glutamate mediated toxicity in striatal cultures could be explained by activation of non-NMDA receptor subtypes. These in vitro studies indicate that glutamate is toxic to a subset of mature striatal neurons in the absence of a glutamatergic afferent input, and that this toxicity is mediated partially by the NMDA receptor, with an additional component due to non-NMDA receptors.

Kynurenine metabolites of tryptophan: Implications for neurologic diseases

Over the past 2 decades, a number of studies have demonstrated that amino acids act as precursors for the biosynthesis of a variety of neuroactive compounds, including catecholamines and indoleamines. For example, the aromatic amino acid l-tryptophan is a precursor for serotonin biosynthesis. Based on this observed precursor relationship, dietary tryptophan supplementation is used to treat a number of neurologic disorders attributed to alterations in serotoninergic neurotransmission. Recent studies have revealed that, in addition to serotonin, a number of neuroactive compounds, the kynurenines, are metabolities of tryptophan. Of these, perhaps the most important is quinolinic acid, a neurotoxin that acts at the N-methyl-d-aspartate (NMDA) receptor and whose precursor responsiveness to tryptophan far exceeds that of serotonin. In the central nervous system, kynurenines, and in particular quinolinic acid, may modulate excitatory amino acid transmission, and may act as neurotoxic agents implicated in the pathogenesis of several neurologic diseases.