David P. Crockett

Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine.

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.

Differentiation of the midbrain dopaminergic pathways during mouse development

Dopaminergic (DA) neurons in the substantia nigra (SN) and ventral tegmental area (VTA) of the midbrain project to the dorsolateral caudate/putamen and to the ventromedially located nucleus accumbens, respectively, establishing the mesostriatal and the mesolimbic pathways. Disruptions in this system have been implicated in Parkinsons disease, drug addiction, schizophrenia, and attention deficit hyperactivity disorder. However, progress in our understanding has been hindered by a lack of knowledge of how these pathways develop. In this study, different retrograde tracers, placed into the dorsolateral caudate/putamen and the nucleus accumbens, were used to analyze the development of the dopaminergic pathways. In embryonic day 15 mouse embryos, both SN and VTA neurons, as well as their fibers, were doubly labeled by striatal injections into the dorsolateral and ventromedial striatum. However, by birth, the SN DA neurons were labeled exclusively by DiA placed in the dorsolateral striatum, and the VTA DA neurons were labeled only by DiI injected into the ventromedial striatum. These data suggest that initial projections from midbrain DA neurons target nonspecifically to both the dorsolateral striatum and the nucleus accumbens. Later during development, the separate mesostriatal and mesolimbic pathways differentiate through the selective elimination of mistargeted collaterals. J. Comp. Neurol. 476:301–311, 2004.

Mistargeting hippocampal axons by expression of a truncated Eph receptor

Topographic mapping of axon terminals is a general principle of neural architecture that underlies the interconnections among many neural structures. The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been implicated in the formation of topographic projection maps. We show that multiple Eph receptors and ligands are expressed in the hippocampus and its major subcortical projection target, the lateral septum, and that expression of a truncated Eph receptor in the mouse brain results in a pronounced alteration of the hippocamposeptal topographic map. Our observations provide strong support for a critical role of Eph family guidance factors in regulating ontogeny of hippocampal projections.

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair. Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20. We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Number of oligodendrocyte progenitors recruited to the lesioned spinal cord is modulated by the levels of the cell cycle regulatory protein p27Kip-1

Remyelination is a critical step for recovery of function after demyelination and defines the ability to generate new myelin. This repair process is dependent on the presence of resident oligodendrocyte progenitors (OLPs) that have been shown to remyelinate axons after demyelination. We have previously shown that the levels of the cell cycle inhibitor p27Kip‐1 modulate the number of neonatal cortical OLPs. We now asked whether this cell cycle molecule plays also a role in regulating the number of adult OLP in the spinal cord after demyelination induced by lysolecithin injection. The proliferative response of OLP in the spinal cord of injected wild‐type (wt) and p27Kip‐1 null mice was evaluated 3 days after lesion. In vivo labeling with bromodeoxyuridine (BrdU) was used to identify cells in S phase. Double immunofluorescence for the OLP marker NG2, and for BrdU was used to count the number of proliferating progenitors. Consistent with a role of p27Kip‐1 in regulating the number of adult OLP in the injured spinal cord, a larger number of proliferating OLPs was observed in p27Kip‐1null mice compared with wild‐type controls. These cells were able to differentiate as assessed by the presence of MBP+ cells in the spinal cord 14 days after injury. We conclude that the cellular levels of the cell cycle inhibitor p27Kip‐1 modulate the repair response of OLPs to injury in the adult spinal cord.

Na+/H+ exchanger inhibition modifies dopamine neurotransmission during normal and metabolic stress conditions

Na+/H+ exchanger (NHE) proteins are involved in intracellular pH and volume regulation and may indirectly influence neurotransmission. The abundant NHE isoform 1 (NHE1) has also been linked to brain cell damage during metabolic stress. It is not known, however, whether NHE1 or other NHE isoforms play a role in striatal dopamine (DA) neurotransmission under normal or metabolic stress conditions. Our study tested the hypothesis that NHE inhibition with cariporide mesilate (HOE‐642) modifies striatal DA overflow and DAergic terminal damage in mice caused by the mitochondrial inhibitor malonate. We also explored the expression of NHE1–5 in the striatum and substantia nigra. Reverse microdialysis of HOE‐642 elicited a transient elevation followed by a reduction in DA overflow accompanied by a decline in striatal DA content. HOE‐642 pre‐treatment diminished the malonate‐induced DA overflow without reducing the intensity of the metabolic stress or subsequent DAergic axonal damage. Although NHE isoforms 1–5 are expressed in the striatum and midbrain, NHE1 protein was not co‐located on nigrostriatal DAergic neurons. The absence of NHE1 co‐location on DAergic neurons suggests that the effects of HOE‐642 on striatal DA overflow are either mediated via NHE1 located on other cell types or that HOE‐642 is acting through multiple NHE isoforms.

Neurotrophin receptor (p75) in the trigeminal thalamus of the rat: Development, response to injury, transient vibrissa-related patterning, and retrograde transport

We report on the transient, patterned expression of p75 in the ventrobasal (VB) thalamus, the major thalamic relay for somatosensation. We immunostained the brains of developing rats ranging in age from embryonic day (E) 14.5 to postnatal day (PD) 15 with an antibody against p75. To compare p75 expression with the developing synaptic organization within VB, we also immunolocalized the synaptic‐vesicle‐associated protein, synaptophysin (SYN), on alternate sections. p75‐immunoreactivity (IR) was dense and uniform in the ventroposterior medial nucleus (VPM) in the late embryonic and early postnatal periods (E 16.5 to PD 3). In contrast, from PD 4–10, p75‐IR in the VPM was patterned, reminiscent of cytochrome‐oxidase‐stained barreloids, a characteristic feature of the VB in rodents. By PD 14, p75‐IR in the VPM was no longer detectable. The ventroposterior lateral nucleus (VPL), in contrast, exhibited no p75‐IR. No p75‐IR was detected in the ventroposterior lateral nucleus (VPL) at any developmental stage in which VPM could be distinguished from VPL. Light, but clearly patterned SYN‐IR, first detectable on PD 2–3, increased in intensity in both VPL and VPM through PD 15. Sectioning the infraorbital nerve on PD 0 resulted in blurred patterns of p75‐ and SYN‐IR within VPM in PD 7–9 rat pups. Removing large portions of the somatosensory cortex on PD 0 resulted in subsequent greatly reduced p75‐ and SYN‐IR within VB. To specify the source of the p75‐IR terminals, we stereotaxically injected into the VPM of PD 4–5 rats a monoclonal antibody to p75. One to 2 days later, IR of retrogradely transported p75 antibodies could be traced within axons and cell bodies of neurons associated with the trigeminothalamic pathway through the caudal diencephalon and mesencephalon; labelling was confined to the contralateral trigeminal principal sensory nucleus. The observed, transiently patterned p75‐IR in VPM the early postpartum period suggests a role for p75 in synaptogenesis and pattern formation. Anat Rec 259:446–460, 2000.

Genetic and pharmacological intervention of the p75NTR pathway alters morphological and behavioural recovery following traumatic brain injury in mice.

Abstract Primary objective: Neurotrophin levels are elevated after TBI, yet there is minimal regeneration. It was hypothesized that the pro-neurotrophin/p75NTR pathway is induced more than the mature neurotrophin/Trk pathway and that interfering with p75 signalling improves recovery following TBI. Research design: Lateral Fluid Percussion (LFP) injury was performed on wildtype and p75 mutant mice. In addition, TrkB agonist 7,8 Dihydroxyflavone or p75 antagonist TAT-Pep5 were tested. Western blot and immunohistochemistry revealed biochemical and cellular changes. Morris Water Maze and Rotarod tests demonstrated cognitive and vestibulomotor function. Main outcomes and results: p75 was up-regulated and TrkB was down-regulated 1 day post-LFP. p75 mutant mice as well as mice treated with the p75 antagonist or the TrkB agonist exhibited reduced neuronal death and degeneration and less astrocytosis. The cells undergoing apoptosis appear to be neurons rather than glia. There was improved motor function and spatial learning in p75 mutant mice and mice treated with the p75 antagonist. Conclusions: Many of the pathological and behavioural consequences of TBI might be due to activation of the pro-neurotrophin/p75 toxic pathway overriding the protective mechanisms of the mature neurotrophin/Trk pathway. Targeting p75 can be a novel strategy to counteract the damaging effects of TBI.

Distribution of the low-affinity neurotrophin receptor (p75) in the developing trigeminal brainstem complex in the rat

The low‐affinity neurotrophin receptor (p75) binds all members of the neurotrophin family. In the rat, during the first week postpartum, dense p75‐immunoreactivity (IR) is present throughout all components of the trigeminal brainstem complex (TBC), largely associated with primary sensory afferents. Within subnucleus caudalis (SpC) of the TBC, intense p75‐IR is present in all laminae at birth. During the second and third postnatal weeks, p75‐IR in SpC gradually fades within the deeper laminae, becoming generally restricted in the adult to laminae I and II. Similar declines in p75‐IR intensity occur in the subnucleus oralis (SpO); in the SpO in the adult, p75‐IR is confined to the dorsalmost portion of SpO. In subnucleus interpolaris, an emerging, vibrissa‐related pattern of p75‐IR is detectable on PD0 (first 24 hr postpartum), which becomes fully differentiated during PD4–PD7. However, this pattern gradually disappears during the third postnatal week. Ventrally in the nucleus principalis (PrV), a pattern of p75‐IR that mirrors the topographical arrangement of the vibrissae is detectable by PD0–PD1, is fully differentiated by the end of the first postnatal week, and persists into adulthood. Perinatal unilateral sectioning of the infraorbital nerve on PD0–PD1, but not as late as PD4, disrupts p75‐IR patterning in the adult PrV.

Acute adaptive responses of central sensorimotor neurons after spinal cord injury

Spinal cord injury (SCI) can be a lifelong, devastating condition for both the patient and the caregiver, with a daunting incidence rate. Still, there are only limited available therapies and the effectiveness of precise regeneration within the central nervous system is minimal throughout postnatal life. Recently, improved regeneration after SCI was seen by manipulating a pathway in sensorimotor neocortices that is involved in phosphorylation of an RNA binding protein (RBP) required for mRNA translation, the Eukaryotic translation initiation factor 4E (eIF4E). Our data identifies rapid molecular alterations of eIF4E in the sensorimotor neocortices 1 and 3 days after a lateral hemisection SCI, used as a model for Brown-Séquard syndrome. The function of an RBP depends on both its distribution sites within the cell and its phosphorylation states. Indeed, we found both to be affected after SCI. There was a distinct subcellular redistribution of eIF4E and phosphorylated-eIF4E was reduced, indicating that the eIF4E’s translation was disrupted. Upon identification and analysis of the mRNA cargo of eIF4E in uninjured sensorimotor neocortices, we found that eIF4E binds both Importin-13 (Ipo13) and Parvalbumin (Pv) mRNAs, indicating a role in their translation. Remarkably, eIF4E’s interaction with both Ipo13 and Pv mRNAs was disrupted 1 and 3 days after SCI, despite preservation of total Ipo13 and Pv mRNA levels. Finally, we detected a selective loss of expression of both IPO13 and PV proteins in projection neurons of sensorimotor neocortices, as well as their disrupted dendritic polarity. Since IPO13 is predominantly expressed in neocortical projection neurons and PV in a subset of neocortical interneurons, these data suggest a strong acute effect of SCI on neocortical microcircuitry. Taken together, these data indicate that neocortical eIF4E and a subset of mRNAs may be rapidly recruited to translational machinery after SCI to promote adaptive regeneration response of sensorimotor neurons.