John H. Brock

Long-Distance Growth and Connectivity of Neural Stem Cells after Severe Spinal Cord Injury

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury.

Although axonal regeneration after CNS injury is limited, partial injury is frequently accompanied by extensive functional recovery. To investigate mechanisms underlying spontaneous recovery after incomplete spinal cord injury, we administered C7 spinal cord hemisections to adult rhesus monkeys and analyzed behavioral, electrophysiological and anatomical adaptations. We found marked spontaneous plasticity of corticospinal projections, with reconstitution of fully 60% of pre-lesion axon density arising from sprouting of spinal cord midline-crossing axons. This extensive anatomical recovery was associated with improvement in coordinated muscle recruitment, hand function and locomotion. These findings identify what may be the most extensive natural recovery of mammalian axonal projections after nervous system injury observed to date, highlighting an important role for primate models in translational disease research.

Long-Distance Axonal Growth from Human Induced Pluripotent Stem Cells after Spinal Cord Injury

UNLABELLED Human induced pluripotent stem cells (iPSCs) from a healthy 86-year-old male were differentiated into neural stem cells and grafted into adult immunodeficient rats after spinal cord injury. Three months after C5 lateral hemisections, iPSCs survived and differentiated into neurons and glia and extended tens of thousands of axons from the lesion site over virtually the entire length of the rat CNS. These iPSC-derived axons extended through adult white matter of the injured spinal cord, frequently penetrating gray matter and forming synapses with rat neurons. In turn, host supraspinal motor axons penetrated human iPSC grafts and formed synapses. These findings indicate that intrinsic neuronal mechanisms readily overcome the inhibitory milieu of the adult injured spinal cord to extend many axons over very long distances; these capabilities persist even in neurons reprogrammed from very aged human cells. VIDEO ABSTRACT

Extensive Spinal Decussation and Bilateral Termination of Cervical Corticospinal Projections in Rhesus Monkeys

To examine neuroanatomical mechanisms underlying fine motor control of the primate hand, adult rhesus monkeys underwent injections of biotinylated dextran amine (BDA) into the right motor cortex. Spinal axonal anatomy was examined using detailed serial‐section reconstruction and modified stereological quantification. Eighty‐seven percent of corticospinal tract (CST) axons decussated in the medullary pyramids and descended through the contralateral dorsolateral tract of the spinal cord. Eleven percent of CST axons projected through the dorsolateral CST ipsilateral to the hemisphere of origin, and 2% of axons projected through the ipsilateral ventromedial CST. Notably, corticospinal axons decussated extensively across the spinal cord midline. Remarkably, nearly 2‐fold more CST axons decussated across the cervical spinal cord midline (≈12,000 axons) than were labeled in all descending components of the CST (≈6,700 axons). These findings suggest that CST axons extend multiple segmental collaterals. Furthermore, serial‐section reconstructions revealed that individual axons descending in either the ipsilateral or contralateral dorsolateral CST can: 1) terminate in the gray matter ipsilateral to the hemisphere of origin; 2) terminate in the gray matter contralateral to the hemisphere of origin; or 3) branch in the spinal cord and terminate on both sides of the spinal cord. These results reveal a previously unappreciated degree of bilaterality and complexity of corticospinal projections in the primate spinal cord. This bilaterality is more extensive than that of the rat CST, and may resemble human CST organization. Thus, augmentation of sprouting of these extensive bilateral CST projections may provide a novel target for enhancing recovery after spinal cord injury. J. Comp. Neurol. 513:151–163, 2009.

Local and Remote Growth Factor Effects after Primate Spinal Cord Injury

Primate models of spinal cord injury differ from rodent models in several respects, including the relative size and functional neuroanatomy of spinal projections. Fundamental differences in scale raise the possibility that retrograde injury signals, and treatments applied at the level of the spinal cord that exhibit efficacy in rodents, may fail to influence neurons at the far greater distances of primate systems. Thus, we examined both local and remote neuronal responses to neurotrophic factor-secreting cell grafts placed within sites of right C7 hemisection lesions in the rhesus macaque. Six months after gene delivery of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) into C7 lesion sites, we found both local effects of growth factors on axonal growth, and remote effects of growth factors reflected in significant reductions in axotomy-induced atrophy of large pyramidal neurons within the primary motor cortex. Additional examination in a rodent model suggested that BDNF, rather than NT-3, mediated remote protection of corticospinal neurons in the brain. Thus, injured neural systems retain the ability to respond to growth signals over the extended distances of the primate CNS, promoting local axonal growth and preventing lesion-induced neuronal degeneration at a distance. Remote cortical effects of spinally administered growth factors could “prime” the neuron to respond to experimental therapies that promote axonal plasticity or regeneration.

Animal Models of Neurologic Disorders: A Nonhuman Primate Model of Spinal Cord Injury

Primates are an important and unique animal resource. We have developed a nonhuman primate model of spinal cord injury (SCI) to expand our knowledge of normal primate motor function, to assess the impact of disease and injury on sensory and motor function, and to test candidate therapies before they are applied to human patients. The lesion model consists of a lateral spinal cord hemisection at the C7 spinal level with subsequent examination of behavioral, electrophysiological, and anatomical outcomes. Results to date have revealed significant neuroanatomical and functional differences between rodents and primates that impact the development of candidate therapies. Moreover, these findings suggest the importance of testing some therapeutic approaches in nonhuman primates prior to the use of invasive approaches in human clinical trials. Our primate model is intended to: 1) lend greater positive predictive value to human translatable therapies, 2) develop appropriate methods for human translation, 3) lead to basic discoveries that might not be identified in rodent models and are relevant to human translation, and 4) identify new avenues of basic research to “reverse-translate” important questions back to rodent models.

Methods for functional assessment after C7 spinal cord hemisection in the rhesus monkey

Background. Reliable outcome measures are essential for preclinical modeling of spinal cord injury (SCI) in primates. Measures need to be sensitive to both increases and decreases in function in order to demonstrate potential positive or negative effects of therapeutics. Objectives. To develop behavioral tests and analyses to assess recovery of function after SCI in the nonhuman primate. Methods. In all, 24 male rhesus macaques were subjected to complete C7 lateral hemisection. The authors scored recovery of function in an open field and during hand tasks in a restraining chair. In addition, EMG analyses were performed in the open field, during hand tasks, and while animals walked on a treadmill. Both control and treated monkeys that received candidate therapeutics were included in this report to determine whether the behavioral assays were capable of detecting changes in function over a wide range of outcomes. Results. The behavioral assays are shown to be sensitive to detecting a wide range of motor functional outcomes after cervical hemisection in the nonhuman primate. Population curves on recovery of function were similar across the different tasks; in general, the population recovers to about 50% of baseline performance on measures of forelimb function. Conclusions. The behavioral outcome measures that the authors developed in this preclinical nonhuman primate model of SCI can detect a broad range of motor recovery. A set of behavioral assays is an essential component of a model that will be used to test efficacies of translational candidate therapies for SCI.

Neural Stem Cell Dissemination after Grafting to CNS Injury Sites

Steward and colleagues report that implants of E14 rat spinal-cord-derived multipotent neural progenitor cells are associated with ectopic deposits of cells, occasionally at long distances from a T3 spinal cord lesion and grafting site. Of 20 grafted rats, half showed ectopic cell deposits. One rat had a deposit of cells in the 4th ventricle, from which relatively few axons extended into the tegmentum. Half of animals had cells within six spinal segments of the lesion site, and of these, three had deposits more distantly.

Distribution and injury-induced plasticity of cadherins in relationship to identified synaptic circuitry in adult rat spinal cord

Cadherins are synaptically enriched cell adhesion and signaling molecules. In brain, they function in axon targeting and synaptic plasticity. In adult spinal cord, their localization, synaptic affiliation, and role in injury-related plasticity are mostly unexplored. Here, we demonstrate in adult rat dorsal horn that E- and N-cadherin display unique patterns of localization to functionally distinct types of synapses of intrinsic and primary afferent origin. Within the nociceptive afferent pathway to lamina II, nonpeptidergic C-fiber synapses in the deeper half of lamina II (IIi) contain E-cadherin but mostly lack N-cadherin, whereas the majority of the peptidergic C-fiber synapses in the outer half of lamina II (IIo) contain N-cadherin but lack E-cadherin. Approximately one-half of the Aβ-fiber terminations in lamina III contain N-cadherin; none contain E-cadherin. Strikingly, the distribution and levels of these cadherins are differentially affected by sciatic nerve axotomy, a model of neuropathic pain in which degenerative and regenerative structural plasticity has been implicated. Within the first 7 d after axotomy, E-cadherin is rapidly and completely lost from the dorsal horn synapses with which it is affiliated, whereas N-cadherin localization and levels are unchanged; such patterns persist through 28 d postlesion. The loss of E-cadherin thus occurs before the onset of mechanical hyperalgesia (∼10-21 d postlesion), as reported previously. Together, the synaptic specificity displayed by these cadherins, coupled with their differential response to injury, suggests that they may proactively contribute to the maintenance of some, and incipient dismantling of other, synaptic circuits in response to nerve injury. Speculatively, such changes may ultimately contribute to subsequently emerging abnormalities in pain perception.

Neuropathic pain- and glial derived neurotrophic factor-associated regulation of cadherins in spinal circuits of the dorsal horn

&NA; Neuropathic pain is associated with reorganization of spinal synaptic circuits, implying that adhesion proteins that normally build and modify synapses must be involved. The adhesion proteins E‐ and N‐cadherin delineate different synapses furnished by nociceptive primary afferents, but dynamic aspects of cadherin localization in relationship to onset, maintenance or reversibility of neuropathic pain are uncharacterized. Here, we find very different responses of these cadherins to L5 spinal nerve transection (SNT)‐induced mechanical allodynia and to intrathecal glial derived neurotrophic factor (GDNF), which has potent analgesic effects in this pain model. In L5, E‐cadherin is rapidly eliminated in patches within lamina IIi contemporaneously with the onset of mechanical allodynia. Intrathecal GDNF in conjunction with, or at 7 days after, L5 SNT prevents or reverses both the loss of E‐cadherin and abnormal pain sensation. In contrast, N‐cadherin undergoes a delayed and transient increase uniformly across lamina I–II that is insensitive to GDNF. Some N‐cadherin‐labeled profiles codistribute with GAP‐43, suggesting a role in axon sprouting. Patterns of immunolabeling for GDNF receptor components GFRα1, NCAM, and RET after L5 SNT suggest that GFRα1 and NCAM are the principal receptors operative in this model. In addition, GFRα1 codistributes with E‐cadherin, but not N‐cadherin, profiles. Together, these data indicate strikingly divergent patterns of temporal and molecular regulation of different cadherins at distinct nociceptive circuits in response to spinal nerve injury, suggesting that the two cadherins and the circuits with which they are affiliated participate in different aspects of synaptic and circuit reorganization associated with neuropathic pain. Two synaptic cadherins in rat spinal dorsal horn are differentially regulated by L5 spinal nerve lesion, which causes neuropathic pain, and by glial derived neurotrophic factor (GDNF), a potent analgesic.