The spinal cord research foundation: two decades of progress.

Over the course of the past 24 years, the Paralyzed Veterans of America's Spinal Cord Research Foundation (SCRF) has provided support for more than 400 research grants in a wide range of areas, from improved wheelchair design to axon pathfinding in Drosophila. The Founders of SCRF, as well as its current trustees, believe that it is imperative to target a broad range of research areas to maximize the quality of life for people, both veterans and nonveterans, with paralysis. This approach has involved the support of basic science and clinical research directed towards repair of the spinal cord, as well as research into improved treatments for complications of spinal cord dysfunction and other projects, including engineering grants and conferences, that may enhance the quality of life for people with paralysis within the immediate future.

Rapid regulation of cytoskeletal proteins and their mRNAs following afferent deprivation in the avian cochlear nucleus.

During development, removal of neuronal input can lead to profound changes in postsynaptic cells, including atrophy and cell death. In the chicken brainstem cochlear nucleus, the nucleus magnocellularis (NM), deprivation of auditory input via unilateral cochlea removal or silencing the eighth nerve with tetrodotoxin leads to a loss of 25-30% of the neurons and the atrophy of surviving neurons. One intracellular component that may be involved in both cell atrophy and cell death is the cytoskeleton. The degradation of the cytoskeleton following deafferentation could potentially lead to either atrophy or death of NM neurons. However, little is known regarding the role of neuronal input on the cytoskeletal structure of NM neurons and whether changes in the cytoskeleton are responsible for cell death following deafferentation. The present study examined whether changes in the cytoskeleton of NM neurons occurred following cochlea removal. Several components of the cytoskeleton were analyzed following unilateral afferent deprivation. Levels of immunostaining for tubulin, actin, and microtubule-associated protein 2 (MAP-2), and levels of beta-tubulin and beta-actin mRNAs were assessed in NM neurons following cochlea removal. Our results revealed that afferent deprivation results in a rapid decrease in immunostaining for all three cytoskeletal proteins examined. These decreases were observed as early as 3 hours after cochlea removal and persisted for up to 4 days. In addition, these changes occurred in all deafferented NM neurons at the early time points, indicating that both dying and surviving NM neurons undergo a similar change in their cytoskeletons. In contrast to the decreases in immunostaining, levels of beta-tubulin and beta-actin mRNAs were not noticeably altered by deafferentation. Our findings indicate that the cytoskeleton is altered or degraded following deafferentation but that this process is not regulated at the transcriptional level.

Injury-induced physiological events that may modulate gene expression in neurons and glia.

Damage to the brain triggers a host of reactive responses in neurons and glia which are seen at sites of focal injury as well as at sites that are at a distance from the injury. Although many of these responses have been studied extensively, the signals that initiate the different responses have not been fully characterized, and it is still not understood how focal injury affects neurons and glia in distant sites. The present review summarizes recent findings that suggest that physiological events that occur at the time of the injury or during the early postlesion period can play an important and variable role in modulating neuronal and glial responses to injury. We focus on the events that occur in the hippocampal formation following unilateral lesions of the entorhinal cortex - a model system that has been used extensively for studies of cellular responses following focal brain injury. This lesion destroys the cells of origin of a massive excitatory projection to the dentate gyrus and hippocampus proper. Over time, the denervated neurons in the hippocampal formation are almost completely reinnervated as a result of local sprouting of systems that survive the lesion. Thus, this model system has been useful for studying cellular responses to both denervation and reinnervation. We summarize the information that this injury triggers physiological events that can strongly modulate gene expression in neurons and glia, including episodes of spreading depression that occur at the time of the injury, seizures that occur during the early postlesion period, the loss of afferent drive which leads to decreases in postsynaptic activity, and the restoration of activity that occurs in conjunction with reinnervation. We describe recent studies which suggest that some of these physiological events occur to a variable extent in different animals, especially the episodes of spreading depression and the recurrent seizures. Thus, the spatial pattern and temporal dynamics of altered gene expression following this "model" experimental injury may vary from animal to animal. The fact that physiological events strongly modulate the reactive changes in gene expression that occur following injury has important implications for understanding the sequelae of injury, and offers new opportunities for experimental and therapeutic interventions that may improve cellular repair, regeneration, and recovery of function.

Prevention of stressor-induced disturbances of self-stimulation by desmethylimipramine.

Following exposure to uncontrollable footshock mice displayed a pronounced reduction of responding for electrical brain stimulation from the nucleus accumbens. In mice that received repeated treatment with the tricyclic antidepressant, desmethylimipramine, the shock induced reduction of responding was attenuated. It was suggested that, among other things, uncontrollable stressors result in disturbances of motivational/reward processes, which are antagonized by repeated treatment with desmethylimipramine.

The role of neuronal activity in upregulating GFAP mRNA levels after electrolytic lesions of the entorhinal cortex.

This study evaluates whether the rapid transient increases in glial fibrillary acidic protein (GFAP) mRNA in the hippocampus after electrolytic lesions of the entorhinal cortex (EC) are triggered by lesion-induced changes in hippocampal neuronal activity (either the decreases that result from loss of afferent drive or transient increases that occur during lesion production). To evaluate the role of activity, we carried out four experiments: (1) tetrodotoxin (TTX) was injected into the EC to mimic the decreases in afferent drive that occur after lesion; (2) TTX was injected into the EC or hippocampus before producing electrolytic lesions to block any abnormal activity induced during lesion production; (3) the EC was destroyed by aspiration, thus creating a lesion comparable in size to the electrolytic lesion, without passing direct current; (4) seizures were elicited by stimulating the EC of anesthetized rats, to examine whether electrographic seizures alone can induce the same type of increases in GFAP mRNA as lesions. Our results demonstrated that: (1) TTX injections into the EC did not induce the same increases in GFAP mRNA levels that occurred after EC lesions; (2) animals that received TTX injections into the EC prior to lesions exhibited increases in hippocampal GFAP mRNA that were nearly as great as following EC lesions alone; (3) aspiration lesions of the EC resulted in increases in GFAP mRNA that were comparable to those observed after electrolytic lesions; and (4) seizure-inducing stimulation of the EC resulted in 2-fold increases in GFAP mRNA in the hippocampus 24 hr after stimulation rather than the 5-13-fold increases observed after lesions. These results suggest that lesion-induced changes in hippocampal neuronal activity are not solely responsible for inducing the rapid transient increases in GFAP mRNA levels in the hippocampus ipsilateral to EC lesions.

The structure of D-erythro-C18 ceramide at the air-water interface.

X-ray reflectivity (XR) and diffraction at grazing angles of incidence (GID) were conducted to determine the structure of synthetic D-erythro C18-ceramide films at the air-water interface at various surface pressures (pi). Analysis of the GID reveals that the monomolecular film, at the crystalline phase (pi > 0 mN/m), is predominantly hexagonal. In this crystalline phase, the analysis of the reflectivity yields an electron density profile that consists of three distinct homogeneous slabs, one associated with the headgroup region and the other two with the hydrocarbon chains. At large molecular areas (pi approximately 0), isolated crystalline domains coexist with two-dimensional gas phase. Within the crystalline domains, we find an orthorhombic arrangement of the chains that coexists with the hexagonal symmetry. It is argued that the two-dimensional orthorhombic crystals are induced by hydrogen bonding between headgroups even at very low surface pressures. Although their structure is incommensurate with the simple hexagonal arrangement, they act as nucleation centers for the conventional hexagonal phase which dominates at high pi.

The process of reinnervation in the dentate gyrus of adult rats: physiological events at the time of the lesion and during the early postlesion period.

Destruction of the entorhinal cortex (EC) triggers a number of cellular and molecular responses in the denervated hippocampus and dentate gyrus. The signals that trigger these changes are not known but could include physiological events that occur during the production of the injury or during the early postlesion period. Of particular interest is whether experimental lesions induce seizures and/or spreading depression (SD), both of which have been shown to dramatically alter neuronal and glial gene expression. In the present study, acute neurophysiological techniques were used to evaluate whether seizures or SD occur during the production of EC lesions. Chronic recording techniques were used to monitor electroencephalographic (EEG) activity during the first 24 h after the injury in order to evaluate the extent of postlesion seizures. One or more episodes of SD occurred in 9 of 13 animals during the production of electrolytic EC lesions. However, hippocampal seizures were not observed except for very brief episodes of seizure activity at the onset of an episode of SD. Chronic recordings of postlesion EEG activity revealed that spontaneous electrographic seizures occurred during the first 24 h postlesion in all animals. The spontaneous electrographic seizures were approximately 30 s in duration and were not accompanied by motor convulsions. The first seizures occurred within several hours after the lesion, and seizures continued to occur periodically (at an average frequency of 0.42 per hour) over the 24-h recording period. Seizures occurred on the side of the brain ipsilateral to the lesion in all animals and occurred on the side contralateral to the lesion in 3 of 5 animals. These results indicate that EC lesions produce physiological events that occur variably in different animals; these processes may account for some of the variability in the cellular responses to this "standardized" injury.

The role of postlesion seizures and spreading depression in the upregulation of glial fibrillary acidic protein mRNA after entorhinal cortex lesions.

Unilateral lesions of the entorhinal cortex have been shown to lead to dramatic increases in GFAP mRNA levels in denervated zones in the hippocampus and dentate gyrus and sometimes (but not always) in nondenervated zones in the contralateral hippocampus and dentate gyrus. The variable distribution of the increases in GFAP mRNA expression suggests that the events which trigger changes in GFAP mRNA levels occur to a variable extent in individual animals. The companion paper characterizes two candidate triggering events: spreading depression (SD) that occurs to a variable extent at the time of the lesion and recurrent seizures that occur during the early postlesion interval. The goal of the present study was to evaluate whether individual differences in the extent or spatial distribution of lesion-induced increases in GFAP mRNA are related to the occurrence of either SD or seizures. We quantified the increases in GFAP mRNA levels in individual animals that had been monitored physiologically to define the incidence of SD and postlesion seizures. The results revealed that the quantitative extent of the increases in GFAP mRNA in denervated zones and was not related to either SD or postlesion seizures. The increases in GFAP mRNA in nondenervated zones also were not related to episodes of spreading depression that occurred at the time of lesion production but were related to the spontaneous seizures that developed during the first 24 h postlesion after the animals had recovered from the surgical anesthesia. Taken together, these data indicate that physiological events that occur during the early postlesion interval can play an important role in determining the pattern and extent of altered cellular gene expression in response to an injury.

Signals that regulate astroglial gene expression: induction of GFAP mRNA following seizures or injury is blocked by protein synthesis inhibitors.

Previous studies have revealed that a single electroconvulsive seizure (ECS) strongly induces glial fibrillary acidic protein (GFAP) expression in astrocytes in the hippocampal dentate gyrus. The signals that trigger this induction are not known, but circumstantial evidence suggests the hypothesis that GFAP expression may be induced as a result of the induction of growth factor expression by dentate granule cells that also occurs as a result of the ECS and other types of seizures. The present study tests one prediction of this hypothesis by evaluating whether increases in GFAP mRNA levels after ECS are blocked by inhibiting protein synthesis at various times after the ECS. We report that the upregulation of GFAP expression following ECS is blocked by protein synthesis inhibitors given 5 min before or up to 12 h after a single ECS. This temporal gradient suggests an intermediate step involving the increased expression of a protein growth factor.

The process of reinnervation in the dentate gyrus of adult rats: temporal relationship between changes in the levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA in reactive astrocytes.

The present study evaluates the temporal relationships between increases in glial fibrillary acidic protein (GFAP) mRNA, GFA protein levels, and GFAP immunostaining in the hippocampus of adult rats following unilateral lesions of the entorhinal cortex (EC). GFAP mRNA levels were assessed at 12 h, 1, 2, 4, 6, 8, 10, 12, 14, and 30 days postlesion by dot blot assays using 35S-labeled cRNA probes against the mRNA. Animals were also prepared for in situ hybridization during the peak of GFAP mRNA expression (2 days postlesion) to explore the nature of individual differences in the spatial extent of the increases. GFA protein levels were assessed by Western blot and dot immunoblot techniques in a separate group of animals prepared at 1, 2, 4, 6, 8, and 10 days postlesion and by immunostaining at 1, 2, 4, 6, and 8 days postlesion. The dot blot analyses of GFAP mRNA levels confirmed previous studies, in that we observed dramatic increases in the levels of GFAP mRNA in the hippocampus ipsilateral to the EC lesions. The increases were biphasic, with a large peak in mRNA levels at 1-2 days postlesion (about 10-fold greater than control) and a second peak at 6-8 days. In most animals, the increases were predominantly ipsilateral to the lesion. However, in some animals, there were also large increases on the contralateral side. In situ hybridization experiments revealed two different spatial patterns of increased gene expression, one in which the increases in GFAP mRNA occurred bilaterally and one in which increases were restricted primarily to the hippocampus ipsilateral to the lesion. Immunochemical measures revealed that GFA protein levels increased gradually in the hippocampus ipsilateral to the lesion, reaching a peak at about 2-fold higher than control at 4 days postlesion, and then remained near this level until at least 10 days postlesion. In the contralateral hippocampus, GFA protein levels were increased to about the same extent as on the ipsilateral side at 1, 2, and 4 days postlesion, but then began to decline, returning to near control levels by 8 days. Increases in immunostaining occurred with about the same time course as the increases in GFA protein levels as measured immunochemically. These results define the temporal relationship between increases in GFAP mRNA and increases in GFA protein, providing new insights into the regulation of gene expression in reactive astrocytes.

Progressive entorhinal cortex lesions accelerate hippocampal sprouting and spare spatial memory in rats.

Accelerating hippocampal sprouting by making unilateral progressive lesions of the entorhinal cortex spared the spatial memory of rats tested for retention of a learned alternation task. Subsequent transection of the sprouted crossed temporodentate pathway (CTD), as well as a simultaneous CTD transection and progressive entorhinal lesion, produced a persistent deficit on the memory task. These results suggest that CTD sprouting, which is homologous to the original perforant path input to the dentate gyrus of the hippocampus, is behaviorally significant and can ameliorate at least some of the memory deficits associated with hippocampal deafferentation.

c-Jun expression in adult rat dorsal root ganglion neurons: differential response after central or peripheral axotomy.

The response of the mature central nervous system (CNS) to injury differs significantly from the response of the peripheral nervous system (PNS). Axotomized PNS neurons generally regenerate following injury, while CNS neurons do not. The mechanisms that are responsible for these differences are not completely known, but both intrinsic neuronal and extrinsic environmental influences are likely to contribute to regenerative success or failure. One intrinsic factor that may contribute to successful axonal regeneration is the induction of specific genes in the injured neurons. In the present study, we have evaluated the hypothesis that expression of the immediate early gene c-jun is involved in a successful regenerative response. We have compared c-Jun expression in dorsal root ganglion (DRG) neurons following central or peripheral axotomy. We prepared animals that received either a sciatic nerve (peripheral) lesion or a dorsal rhizotomy in combination with spinal cord hemisection (central lesion). In a third group of animals, several dorsal roots were placed into the hemisection site along with a fetal spinal cord transplant. This intervention has been demonstrated to promote regrowth of severed axons and provides a model to examine DRG neurons during regenerative growth after central lesion. Our results indicated that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but following a central axotomy, only 18% of the neurons expressed c-Jun. Following dorsal rhizotomy and transplantation, however, c-Jun expression was upregulated dramatically; under those experimental conditions, 63% of the DRG neurons were c-Jun-positive. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions.

Direct digital imaging with and without niobium filtration for detection of density differences beneath steel orthodontic bands.

An in vitro investigation was carried out to determine the efficacy of the RVG 32000 (Trophy Radiologie, Vincennes, France) in detecting subtle density variations in a standard aluminum test object through steel orthodontic bands. The density variations were of the same magnitude as those found when dental caries develops beneath bands during orthodontic therapy. The procedure was carried out with both standard aluminum filtration and added niobium filtration. This study revealed the imaging system to have a wide recording latitude with no significant differences in the diagnostic decisions being made between with entrance doses ranging from 189-517 microGy without niobium, and 169-267 microGy with added niobium. No significant difference was found between the diagnostic yield of images made with and without added niobium filtration. The accuracy was 89% with added niobium and 90% without added niobium. Specificity was 99% for both filtration conditions. It was generally possible to detect defects as small as 0.2-0.3 mm in 7 mm of aluminum through 0.26 mm steel orthodontic band material. It is concluded that the RVG 32000 has a wide recording latitude which permits detection of small density changes beneath orthodontic band material. The addition of niobium filtration did not interfere with this diagnostic task.

Transplants and neurotrophic factors prevent atrophy of mature CNS neurons after spinal cord injury.

Axotomy of mature rubrospinal neurons leads to a substantial atrophy of these neurons within days after surgery. In addition, these neurons do not successfully regenerate following axotomy. The relationship of atrophy to regenerative failure is not clear, and the signals which regulate these events have not been identified. However, it is possible that the atrophy of these neurons plays a role in preventing regeneration. In the present study, we evaluated the hypothesis that interventions which have been shown to promote growth of axotomized CNS neurons are also capable of reversing the axotomy-induced atrophy. To test this hypothesis, adults rats received thoracic spinal cord hemisection alone or in combination with transplants of fetal spinal cord tissue and/or neurotrophic factor support. Our data indicate that application of either transplants or neurotrophic factors partially reverse the axotomy-induced atrophy in rubrospinal neurons, but that both interventions together reverse the atrophy completely. These results suggest that the same pathways that are activated to enhance growth of rubrospinal neurons after axotomy may also be involved in the maintenance of cell morphology.

Fetal spinal cord transplants and exogenous neurotrophic support enhance c-Jun expression in mature axotomized neurons after spinal cord injury.

The responses of the central (CNS) and peripheral (PNS) nervous system to axotomy differ in a number of ways; these differences can be observed in both the cell body responses to injury and in the extent of regeneration that occurs in each system. The cell body responses to injury in the PNS involves the upregulation of genes that are not upregulated following comparable injuries to CNS neurons. The expression of particular genes following injury may be essential for regeneration to occur. In the present study, we have evaluated the hypothesis that expression of the inducible transcription factor c-Jun is associated with regrowth of axotomized CNS neurons. In these experiments, we compared c-Jun expression in axotomized brainstem neurons after thoracic spinal cord hemisection alone (a condition in which no regrowth occurs) and in groups of animals where hemisections were combined with treatments such as transplants of fetal spinal cord tissue and/or application of neurotrophic factors to the lesion site. The latter conditions enhance the capacity of the CNS for regrowth. We have demonstrated that hemisections alone do not upregulate expression of c-Jun, indicating that this particular cell body response is not a direct result of axotomy. However, c-Jun expression is upregulated in animals that received application of transplants and neurotrophins. Because these interventions also promote sprouting and regrowth of CNS axons after spinal cord lesions, we suggest that transplants and exogenous neurotrophic factor application activate a cell body response consistent with a role for c-Jun in axonal growth.