Paul J. Reier

Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters

In this study, we have evaluated the capacity of recombinant adeno-associated virus (rAAV) vectors, containing cell type-specific promoters, to transduce neurons in vivo in the normal adult rat spinal cord. The neuron-specific enolase (NSE) promoter and the platelet-derived growth factor (PDGF) B-chain promoter were used to direct expression of a ‘humanized’ form of the gene for green fluorescent protein (GFP). Neuron-specific rAAVs were injected into the mid-cervical regions of adult rat spinal cords. At 10–14 days, expression was detected in all animals and persisted for up to 15 weeks. Immunocytochemical and morphological profiles of transduced cells were consistently neuronal, and there was no evidence of transgene expression in glial elements. Transduction efficiencies for the NSE and PDGF rAAVs were estimated at 15 and 45 infectious particles per GFP-positive neuron, respectively, in the absence of detectable adenovirus. This study strongly supports a role for rAAV vectors in CNS gene therapy and lays the groundwork for delivery of more functional genes to spinal cord neurons as a possible way to enhance spinal cord repair following injury.

Cellular Transplantation Strategies for Spinal Cord Injury and Translational Neurobiology

SummaryBasic science advances in spinal cord injury and regeneration research have led to a variety of novel experimental therapeutics designed to promote functionally effective axonal regrowth and sprouting. Among these interventions are cell-based approaches involving transplantation of neural and non-neural tissue elements that have potential for restoring damaged neural pathways or reconstructing intraspinal synaptic circuitries by either regeneration or neuronal/glial replacement. Notably, some of these strategies (e.g., grafts of peripheral nerve tissue, olfactory ensheathing glia, activated macrophages, marrow stromal cells, myelin-forming oligodendrocyte precursors or stem cells, and fetal spinal cord tissue) have already been translated to the clinical arena, whereas others have imminent likelihood of bench-to-bedside application. Although this progress has generated considerable enthusiasm about treating what once was thought to be a totally incurable condition, there are many issues to be considered relative to treatment safety and efficacy. The following review reflects on different experimental applications of intraspinal transplantation with consideration of the underlying pathological, pathophysiological, functional, and neuroplastic responses to spinal trauma that such treatments may target along with related issues of procedural and biological safety. The discussion then moves to an overview of ongoing and completed clinical trials to date. The pros and cons of these endeavors are considered, as well as what has been learned from them. Attention is primarily directed at preclinical animal modeling and the importance of patterning clinical trials, as much as possible, according to laboratory experiences.

Fetal grafts alter chronic behavioral outcome after contusion damage to the adult rat spinal cord.

In the present experiments, we have examined the capacity of intraspinal transplants to effect alterations in certain locomotor behaviors after spinal contusion injuries. An electromechanical impactor that was sensitive to tissue biomechanical characteristics was used to produce rapid (20 ms) compression injuries to the thoracic spinal cord (T8). Suspensions of fetal spinal tissue (14-day) were placed at 10 days postinjury into the intraspinal cavity created by these reproducible spinal injuries. In the pre- and postinjury period, a number of general and sensitive motor behaviors were used to characterize the immediate and long-term progress of hindlimb behavioral recovery over an extended period of time (73 days). Our data reveal that a lasting alteration in some motor behaviors can be achieved by suspension grafts. While little improvement in some generalized motor tasks (inclined plane analysis, grid walking) takes place, fetal transplants precipitate a rapid and enduring change in certain motivated fine motor behaviors (gait analysis). The base of support and stride length of the hindlimbs were improved by 7 days post-transplantation and the effect was stable over time. The angle of rotation was, however, not altered. The lasting effect in two gait parameters noted was accompanied by the presence of well-developed spinal grafts that often fused with the host spinal parenchyma. These results provide the first documentation of an influence of fetal transplants on motivated locomotor capacity in a well-characterized spinal injury model that mimics lesions seen in the contused adult human spinal cord.

Fetal cell grafts into resection and contusion/compression injuries of the rat and cat spinal cord

This article reviews recent findings concerning the feasibility, basic neurobiology, and potential functional benefits of fetal CNS tissue grafts into acute and chronic lesions of the adult spinal cord. In the rat, neuro-anatomical observations suggest that transplants into resection cavities establish neuritic projections that could functionally reunite separated rostral and caudal segments of the host spinal cord. Furthermore, some complementary electrophysiological evidence has been obtained for synaptic connectivity between host and graft neurons. In these studies, extracellular single-unit activity was evoked in fetal spinal cord (FSC) transplants by stimulating host dorsal roots that had been juxtaposed to donor tissue at the time of transplantation. In other investigations, we examined whether grafts could also establish axonal projections to appropriate areas of gray matter in the chronically injured spinal cord. For this purpose, fetal serotoninergic (5-HT) neurons were injected caudal to complete spinal cord transections that had been made 1-3 months earlier. Immunocytochemistry revealed that these cells projected their axons into gray matter regions normally innervated by bulbospinal 5-HT neurons. To investigate transplantation in a more clinically relevant lesion model, a third group of experiments involved injection of dissociated cell suspensions into acute [less than 24 h postinjury (p.i.)]), subchronic (7-10 days p.i), and chronic (greater than or equal to one month, p.i.) contusion lesions. Such grafts routinely filled areas that otherwise would have been regions of cavitation extending rostral-caudal distances of approximately 7 mm. FSC transplants in such injuries also appeared to influence some aspects of motoneuron excitability and hindlimb locomotion. More recent studies of the cat spinal cord have extended these findings in the rat by showing long-term survival (greater than 2 years) of fetal CNS allografts in recipients with either subtotal transection or compression lesions. Preliminary studies of connectivity have also shown host-graft projection patterns similar to those seen in the rat. Behavioral analyses are currently underway to examine the effects of fetal grafts in cats with chronic postcompression lesions. These observations in the rat and cat are discussed in the general context of basic biological and clinical issues relevant to the long-term objective of promoting functional improvement in the damaged spinal cord.

Astrocyte activation and fibrous gliosis: glial fibrillary acidic protein immunostaining of astrocytes following intraspinal cord grafting of fetal CNS tissue

Publisher Summary This chapter highlights some basic facts pertaining to glial intermediate filaments with emphasis on the chemistry and metabolism of their major constituent protein, glial fibrillary acidic protein (GFAP). Gliosis is usually characterized by extensive astroglial proliferation and hypertrophy. In addition, reactive astrocytes undergo numerous cytological and histochemical features, including increases in nuclear diameter, elevated DNA levels, an accumulation of intermediate filaments, heightened oxidoreductive enzyme activity, and increased synthesis of GFAP, vimentin, glutamine synthetase, and glycogen. In principle, fibrous gliosis can be considered as a part of an important healing response to a central nervous system (CNS) injury because astrocytes are thought to actively monitor and control the contents of the extracellular space of the CNS, including the amounts of ions, transmitters, trophic factors, nutrients, and waste materials. They also play a role in the removal of myelin and neuronal debris and encapsulate the regions of the CNS that are exposed to non-CNS tissue environments following trauma. The most prominent characteristic of fibrous gliosis is an extensive synthesis of intermediate filaments.

Spontaneous Functional Recovery in a Paralyzed Hemidiaphragm Following Upper Cervical Spinal Cord Injury in Adult Rats

Previous studies have shown that latent respiratory pathways can be activated by as phyxia or systemic theophylline administration to restore function to a hemidiaphragm paralyzed by C2 spinal cord hemisection in adult female rats. Based on this premise, electrophysiologic recording techniques were employed in the present investigation to first determine qualitatively whether latent respiratory pathways are activated spon taneously following prolonged post hemisection periods (4-16 weeks) without any therapeutic intervention. Our second objective in a separate group of hemisected an imals was to quantitate any documented functional recovery under the following stan dardized recording conditions: bilateral vagotomy, paralysis with pancuronium bro mide, artificial ventilation, and constant PCO 2 (maintained at 25 mmHg).

Cervical prephrenic interneurons in the normal and lesioned spinal cord of the adult rat

Although monosynaptic bulbospinal projections to phrenic motoneurons have been extensively described, little is known about the organization of phrenic premotor neurons in the adult rat spinal cord. Because interneurons may play an important role in normal breathing and recovery following spinal cord injury, the present study has used anterograde and transneuronal retrograde tracing to study their distribution and synaptic relations. Exclusive unilateral, first‐order labeling of the phrenic motoneuron pool with pseudorabies virus demonstrated a substantial number of second‐order, bilaterally distributed cervical interneurons predominantly in the dorsal horn and around the central canal. Combined transneuronal and anterograde tracing revealed ventral respiratory column projections to prephrenic interneurons, suggesting that some propriospinal relays exist between medullary neurons and the phrenic nucleus. Dual‐labeling studies with pseudorabies virus recombinants also showed prephrenic interneurons integrated with either contralateral phrenic or intercostal motoneuron pools. The stability of interneuronal pseudorabies virus labeling patterns following lateral cervical hemisection was then addressed. Except for fewer infected contralateral interneurons at the level of the central canal, the number and distribution of phrenic‐associated interneurons was not significantly altered 2 weeks posthemisection (i.e., the point at which the earliest postinjury recovery of phrenic activity has been reported). These results demonstrate a heterogeneous population of phrenic‐related interneurons. Their connectivity and relative stability after cervical hemisection raise speculation for potentially diverse roles in modulating phrenic function normally and postinjury. J. Comp. Neurol. 511:692–709, 2008.

Feasibility and Safety of Neural Tissue Transplantation in Patients with Syringomyelia

Transplantation of fetal spinal cord (FSC) tissue has demonstrated significant potential in animal models for achieving partial anatomical and functional restoration following spinal cord injury (SCI). To determine whether this strategy can eventually be translated to humans with SCI, a pilot safety and feasibility study was initiated in patients with progressive posttraumatic syringomyelia (PPTS). A total of eight patients with PPTS have been enrolled to date, and this report presents findings for the first two patients through 18 months postoperative. The study design included detailed assessments of each subject at multiple pre- and postoperative time points. Outcome data were then compared with each subjects own baseline. The surgical protocol included detethering, cyst drainage, and implantation of 6-9-week postconception human FSC tissue. Immunosuppression with cyclosporine was initiated a few days prior to surgery and continued for 6 months postoperatively. Key outcome measures included: serial magnetic resonance imaging (MRI) exams, standardized measures of neurological impairment and functional disability, detailed pain assessment, and extensive neurophysiological testing. Through 18 months, the first two patients have been stable neurologically and the MRIs have shown evidence of solid tissue at the graft sites, without evidence of donor tissue overgrowth. Although it is still too soon to draw any firm conclusions, the findings from the initial two patients in this study suggest that intraspinal grafting of human FSC tissue is both feasible and safe.

Neural deficits contribute to respiratory insufficiency in Pompe disease

Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid α-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease—the Gaa−/− mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa−/− mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa−/− mice vs. isogenic controls, and glycogen was observed in Gaa−/− phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa−/− mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa−/− mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa−/− and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa−/− mice, and therapies targeting muscle alone may be ineffective in Pompe disease.

Altered Respiratory Motor Drive after Spinal Cord Injury: Supraspinal and Bilateral Effects of a Unilateral Lesion

Because some bulbospinal respiratory premotor neurons have bilateral projections to the phrenic nuclei, we investigated whether changes in contralateral phrenic motoneuron function would occur after unilateral axotomy via C2 hemisection. Phrenic neurograms were recorded under baseline conditions and during hypercapnic and hypoxic challenge in C2 hemisected, normal, and sham-operated rats at 1 and 2 months after injury. The rats were anesthetized, vagotomized, and mechanically ventilated. No group differences were seen in contralateral neurograms at 1 month after injury. At 2 months, however, there was a statistically significant decrease in respiratory rate (RR) at normocapnia, an elevated RR during hypoxia, and an attenuated increase in phrenic neurogram amplitude during hypercapnia in the C2-hemisected animals. To test whether C2 hemisection had induced a supraspinal change in respiratory motor drive, we recorded ipsilateral and contralateral hypoglossal neurograms during hypercapnia. As with the phrenic motor function data, no change in hypoglossal output was evident until 2 months had elapsed when hypoglossal amplitudes were significantly decreased bilaterally. Last, the influence of serotonin-containing neurons on the injury-induced change in phrenic motoneuron function was examined in rats treated with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Pretreatment with 5,7-dihydroxytryptamine prevented the effects of C2 hemisection on contralateral phrenic neurogram amplitude and normalized the change in RR during hypoxia. The results of this study show novel neuroplastic changes in segmental and brainstem respiratory motor output after C2hemisection that coincided with the spontaneous recovery of some ipsilateral phrenic function. Some of these effects may be modulated by serotonin-containing neurons.