Jed S. Shumsky

Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations

Bone marrow stromal cells (MSC) are non-hematopoietic support cells that can be easily derived from bone marrow aspirates. Human MSC are clinically attractive because they can be expanded to large numbers in culture and reintroduced into patients as autografts or allografts. We grafted human MSC derived from aspirates of four different donors into a subtotal cervical hemisection in adult female rats and found that cells integrated well into the injury site, with little migration away from the graft. Immunocytochemical analysis demonstrated robust axonal growth through the grafts of animals treated with MSC, suggesting that MSC support axonal growth after spinal cord injury (SCI). However, the amount of axon growth through the graft site varied considerably between groups of animals treated with different MSC lots, suggesting that efficacy may be donor-dependent. Similarly, a battery of behavioral tests showed partial recovery in some treatment groups but not others. Using ELISA, we found variations in secretion patterns of selected growth factors and cytokines between different MSC lots. In a dorsal root ganglion explant culture system, we tested efficacy of conditioned medium from three donors and found that average axon lengths increased for all groups compared to control. These results suggest that human MSC produce factors important for mediating axon outgrowth and recovery after SCI but that MSC lots from different donors vary considerably. To qualify MSC lots for future clinical application, such notable differences in donor or lot-lot efficacy highlight the need for establishing adequate characterization, including the development of relevant efficacy assays.

Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration

Ex vivo gene therapy, utilizing modified fibroblasts that deliver BDNF or NT-3 to the acutely injured spinal cord, has been shown to elicit regeneration and recovery of function in the adult rat. Delayed grafting into the injured spinal cord is of great clinical interest as a model for treatment of chronic injury but may pose additional obstacles that are not present after acute injury, such as the need to remove an established scar, increased retrograde cell loss and/or atrophy, and diminished capacity for regeneration by neurons which may be doubly injured. The purpose of the present study was to determine if delayed grafting of neurotrophin secreting fibroblasts would have anatomical effects similar to those seen in acute grafting models. We grafted a mixture of BDNF and NT-3 producing fibroblasts or control fibroblasts into a complete unilateral cervical hemisection after a 6-week delay. Fourteen weeks after delayed grafting we found that both the neurotrophin secreting fibroblasts and control fibroblasts survived, but that only the neurotrophin secreting grafts provided a permissive environment for host axon growth, as indicated by immunostaining for RT-97, a marker for axonal neurofilaments, GAP-43, a marker for elongating axons, CGRP, a marker for dorsal root axons, and 5-HT, a marker for raphe spinal axons, within the graft. Anterograde tracing of the uninjured vestibulospinal tract showed growth into neurotrophin producing transplants but not into control grafts, while anterograde tracing of the axotomized rubrospinal tract showed a small number of regenerating axons within the genetically modified grafts, but none in control grafts. The neurotrophin expressing grafts, but not the control grafts, significantly reduced retrograde degeneration and atrophy in the injured red nucleus. Grafts of BDNF + NT-3 expressing fibroblasts delayed 6 weeks after injury therefore elicit growth from intact segmental and descending spinal tracts, stimulate modest regenerative growth by rubrospinal axons, and partially rescue axotomized supraspinal neurons and protect them from atrophy. The regeneration of rubrospinal axons into delayed transplants was much less than has been observed when similar transplants were placed acutely into a lateral funiculus or, after a 4-week delay, into a hemisection lesion. This suggests that the regenerative capacity of chronically injured red nucleus neurons was markedly diminished. The increased GAP43 reactivity in the corticospinal tracts ipsilaterally and contralaterally to the combination grafts suggests that these axons remain responsive to the neurotrophins, that the neurotrophins may stimulate both regenerative and sprouting responses, and that the grafted cells continue to secrete the neurotrophins.

Transplantation of Neuronal and Glial Restricted Precursors into Contused Spinal Cord Improves Bladder and Motor Functions, Decreases Thermal Hypersensitivity, and Modifies Intraspinal Circuitry

Transplanting neuronal and glial restricted precursors (NRP/GRP) into a midthoracic injury 9 d after contusion improved bladder and motor function, diminished thermal hypersensitivity, and modified lumbosacral circuitry compared with operated controls (OP-controls). Histological analysis showed that NRP/GRP survived, filled the lesion site, differentiated into neurons and glia, and migrated selectively. Volume of spinal cord spared was increased in NRP/GRP recipients, suggesting local protection. Bladder areflexia developed in both operated groups, but NRP/GRP recipients exhibited an accelerated recovery, with decreased micturition pressure and fewer episodes of detrusor hyperreflexia. Because noradrenergic receptors proliferate after spinal injury and descending noradrenergic pathways contribute to regulation of bladder control, we examined the effects of administering an α-1A-adrenergic antagonist, Tamsulosin, on urodynamics. This improved all cystometric parameters in both operated groups, and micturition pressure in NRP/GRP rats recovered to normal levels. Both operated groups initially showed increased sensitivity to a thermal stimulus applied to the tail; the NRP/GRP rats showed significant improvement over time. NRP/GRP grafts also produced greater recovery of hindlimb function in several tests, although both groups showed persistent and similar deficits in locomotion on a grid. Because bladder, hindlimb, and tail sensory and motor functions are organized through lumbosacral cord, we examined descending and primary afferent projections at L6-S1. The density of serotonergic, noradrenergic, and corticotrophin releasing factor-positive fibers increased in the NRP/GRP group compared with OP-controls, suggesting some sparing and/or sprouting of these modulatory pathways. Immunocytochemical staining density of dorsal root axons in the dorsal horn increased in the OP-controls but appeared normal in the NRP/GRP group. Synaptophysin immunoreactivity in the lumbosacral dorsal horn was similar among groups, consistent with restoration of synaptic density in both groups of operated animals but by different pathways. We suggest that local protection provided by NRP/GRP resulted in increased sparing/sprouting of descending pathways, which prevented sprouting by dorsal root axons, and that this modification in lumbosacral circuitry contributes to the recovery of function.

Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT-3 into a spinal cord injury site is associated with limited recovery of function

Delivery of neurotrophic factors in acute models of spinal cord injury in adult rats can rescue axotomized neurons, promote axonal growth, and partially restore function. The extent to which repair and recovery of function can be achieved after chronic injury has received less attention. In the companion paper we show that transplanting fibroblasts genetically modified to produce neurotrophic factors into chronic (6-week) hemisection injuries results in sprouting, partial neuroprotection, but only limited regeneration. Here we describe functional consequences of this treatment using a series of behavioral tests. Adult rats received a complete unilateral C3/C4 hemisection and recovery from the injury was assessed over 5 weeks. At 6 weeks postoperative, the experimental group received grafts of a combination of fibroblasts modified to secrete BDNF or NT-3. The operated control groups received grafts of either gelfoam or gelfoam with fibroblasts expressing GFP into the lesion site. Behavioral recovery in the three groups was assessed over the next 10 weeks. Severe deficits with no recovery in any of the groups were observed in several tests (BBB, limb preference, narrow beam, horizontal rope test) that measure primarily motor function. Recovery was observed in the grid test, a measure of sensorimotor function, and the von Frey test, a measure of response to mechanical stimulation, but there were no differences between the operated control or experimental groups. Both groups also showed recovery from heat-induced hyperalgesia, with the experimental group exhibiting greater recovery than the operated control groups. In this test, delivery of neurotrophic factors from transplanted fibroblasts does not worsen responses to nociceptive stimuli and in fact appears to reduce hypersensitivity. Our data also demonstrate that additional damage to the spinal cord upon placement of a graft further compromises behavioral recovery for locomotor and postural function. Additional therapeutic interventions will be necessary to provide greater levels of recovery after chronic injuries.

Transplants of fibroblasts expressing BDNF and NT-3 promote recovery of bladder and hindlimb function following spinal contusion injury in rats

We examined whether fibroblasts, genetically modified to express BDNF and NT-3 (Fb-BDNF/NT3) and transplanted into a thoracic spinal injury site, would enhance recovery of bladder function and whether this treatment would be associated with reorganization of lumbosacral spinal circuits implicated in bladder function. Rats received modified-moderate contusion injuries at T8/9, and 9 days later, Fb-BDNF/NT3 or unmodified fibroblasts (OP-controls) were delivered into the cord. Fb-BDNF/NT3 rats recovered from areflexic bladder earlier, showed decreased micturition pressure and fewer episodes of detrusor hyperreflexia, compared to OP-controls. There were also improvements in hindlimb function in the Fb-BDNF/NT3 group although locomotion on a more challenging substrate (grid) and tail withdrawal latency in response to a thermal stimulus showed persisting deficits, little recovery, and no differences between the groups. Immunocytochemistry at L6-S1 revealed changes in density of afferent and descending projections to L6-S1 cord. The density of small dorsal root axons increased in the superficial layers of the dorsal horn in OP-controls but not in Fb-BDNF/NT3, suggesting sprouting of primary afferents following injury that was inhibited by Fb-BDNF/NT-3. In contrast, the trophic factor secreting transplants stimulated sprouting and/or sparing of descending modulatory pathways projecting to the lumbosacral spinal cord. No differences in synaptophysin immunoreactivity were seen in the dorsal horn which suggested that synaptic density was similar but achieved by sprouting of different systems in the two operated groups. Fb-BDNF/NT3 transplanted into injured spinal cord thus improved both bladder and hindlimb function, and this was associated with reorganization of spinal circuitry.

Prenatal cocaine exposure produces consistent developmental alterations in dopamine-rich regions of the cerebral cortex

Administration of cocaine to pregnant rabbits produces robust and long-lasting anatomical alterations in the dopamine-rich anterior cingulate cortex of offspring. These effects include increased length and decreased bundling of layer III and V pyramidal neuron dendrites, increases in parvalbumin expression in the dendrites of interneurons, and increases in detectable GABAergic neurons. We have now examined multiple cortical regions with varying degrees of catecholaminergic innervation to investigate regional variations in the ability of prenatal cocaine exposure to elicit these permanent changes. All regions containing a high density of tyrosine hydroxylase-immunoreactive fibers, indicative of prominent dopaminergic input, exhibited alterations in GABA and parvalbumin expression by interneurons and microtubule-associated protein-2 labeling of apical dendrites of pyramidal neurons. These regions included the medial prefrontal, entorhinal, and piriform cortices. In contrast, primary somatosensory, auditory and motor cortices exhibited little tyrosine hydroxylase staining and no measurable cocaine-induced changes in cortical structure. From these data we suggest that the presence of dopaminergic afferents contributes to the marked specificity of the altered development of excitatory pyramidal neurons and inhibitory interneurons induced by low dose i.v. administration of cocaine in utero.

Ex vivo MR determined apparent diffusion coefficients correlate with motor recovery mediated by intraspinal transplants of fibroblasts genetically modified to express BDNF.

The purpose of this study was to determine whether apparent diffusion coefficients (ADCs) in ex vivo spinal cord white matter, calculated from diffusion weighted MR (DWI) images, correlate with axonal growth and behavioral recovery following subtotal hemisection and transplantation of fibroblasts genetically modified to express brain derived neurotrophic factor (BDNF). These genetically modified fibroblasts have been shown to promote axonal growth, diminish retrograde degenerative changes in axotomized Red nucleus neurons, and are associated with behavioral recovery. Since changes in ADC appear to reflect damage to axons and myelin sheaths, which conventional MR techniques do not identify, partial repair mediated by BDNF-secreting fibroblasts should be detected with ADC measures. Accordingly, we transplanted unmodified fibroblasts (Fb-UM) or fibroblasts modified to secrete BDNF (Fb-BDNF) into cervical subtotal hemisection cavities in adult rats. Rats with Fb-BDNF transplants showed significantly greater behavioral recovery over 12 weeks, as measured by tests of forelimb exploration and open field locomotor activity. Lesion sizes and transplant survival did not differ between the two groups, but immunocytochemical examination showed substantial growth of axons into the Fb-BDNF grafts and little growth into the Fb-UM grafts. Fixed spinal cords were imaged in a 9.4-T magnet. ADCs perpendicular (tADC) and parallel (lADC) to the long axis of the cord were measured in the dorsal lateral white matter, rostral and caudal to the transplant. tADC values and anisotropy index (AI = tADC/lADC) were elevated in both transplant types, indicating white matter damage, but were closer to normal in rats with Fb-BDNF, consistent with known neuroprotection and axonal growth elicited by BDNF. Closer to normal tADC and AI values correlated with improved behavioral recovery. These findings suggest that high-resolution imaging with measurement of tADC and lADC can provide a measure of functionally significant repair that may otherwise go undetected with conventional MR techniques.

Differential Sensitivity to Psychostimulants Across Prefrontal Cognitive Tasks: Differential Involvement of Noradrenergic α1- and α2-Receptors

BACKGROUND Psychostimulants improve a variety of cognitive and behavioral processes in patients with attention-deficit/hyperactivity disorder (ADHD). Limited observations suggest a potentially different dose-sensitivity of prefrontal cortex (PFC)-dependent function (narrow inverted-U-shaped dose-response curves) versus classroom/overt behavior (broad inverted U) in children with ADHD. Recent work in rodents demonstrates that methylphenidate (MPH; Ritalin) elicits a narrow inverted-U-shaped improvement in performance in PFC-dependent tests of working memory. The current studies first tested the hypothesis that PFC-dependent tasks, in general, display narrow dose sensitivity to the beneficial actions of MPH. METHODS The effects of varying doses of MPH were examined on performance of rats in two tests of PFC-dependent cognition, sustained attention and attentional set shifting. Additionally, the effect of pretreatment with the α₁-antagonist prazosin (.5 mg/kg) on MPH-induced improvement in sustained attention was examined. RESULTS MPH produced a broad inverted-U-shaped facilitation of sustained attention and attentional set shifting. Prior research indicates α₁-receptors impair, whereas α₂-receptors improve, working memory. In contrast, attentional set shifting is improved with α₁-receptor activation, whereas α₂-receptors exert minimal effects in this task. Given the similar dose sensitivity of sustained attention and attentional set-shifting tasks, additional studies examined whether α₁-receptors promote sustained attention, similar to attentional set shifting. In these studies, MPH-induced improvement in sustained attention was abolished by α₁-receptor blockade. CONCLUSIONS PFC-dependent processes display differential sensitivity to the cognition-enhancing actions of psychostimulants that are linked to the differential involvement of α₁- versus α₂-receptors in these processes. These observations have significant preclinical and clinical implications.

Combined Effects of Neurotrophin Secreting Transplants, Exercise, and Serotonergic Drug Challenge Improve Function in Spinal Rats

Objectives. To determine the effects of neurotrophin-secreting transplants combined with exercise and serotonergic drug challenges on recovery of hindlimb function in rats with midthoracic spinal cord transection injuries. Methods. Spinalized animals received transplants of fibroblasts genetically modified to express brain-derived neurotrophic factor and neurotrophin-3 and daily cycling exercise. Hindlimb movement in an open-field test (BBB) was scored weekly. Serotonin agonists were used monthly to further stimulate motor function. Axonal growth was quantified in the transplant and at L5 using immunocytochemical markers. Weights of hindlimb muscles were used to assess muscle atrophy. Results. Neurotrophin-secreting transplants stimulated axonal growth, and cycling prevented muscle atrophy, but individual treatments did not improve motor scores. Combined treatments resulted in improvements in motor function. Serotonergic agonists further improved function in all groups, and transplant groups with exercise achieved weight-supporting levels following drug treatment. Conclusion. Combined treatments, but not individual treatments, improved hindlimb function.

Aspiration of a cervical spinal contusion injury in preparation for delayed peripheral nerve grafting does not impair forelimb behavior or axon regeneration.

A peripheral nerve graft model was used to examine axonal growth after a unilateral cervical (C) contusion injury in adult rats and to determine if manipulation of an injury site prior to transplantation affects spontaneous behavioral recovery. After a short delay (7 d) the epicenter of a C4 contusion was exposed and aspirated without harming the cavity walls followed by apposition with one end of a pre-degenerated tibial nerve to the rostral cavity wall. After a longer delay (28 d) the aspirated cavity was treated with GDNF to promote regeneration by chronically injured neurons. In both groups forelimb and hindlimb locomotor scores decreased significantly 2 d after lesion site manipulation, but by 7 d, the forelimb score was not different from the pre-manipulation score. There was no significant difference in grid walking or grip strength scores for the affected forelimb in either group 7 d after contusion vs. 7 d after manipulation. Over 1500 brain stem and propriospinal neurons grew axons into the graft with either delay. These results demonstrate that a contusion injury site can be manipulated prior to transplantation without causing long-lasting forelimb or hindlimb behavioral deficits and that peripheral nerve grafts support axonal growth after acute or chronic contusion injury.