Differential and prolonged expression of Fos-lacZ and Jun-lacZ in neurons, glia, and muscle following sciatic nerve damage.

Fos-lacZ and Jun-lacZ transgenic mice were used to assess the involvement of immediate-early genes in the axotomy-transcription coupling pathway triggered by sciatic nerve injury in neonates and adults. Nerve transection transiently induced Fos-lacZ in degenerating (neonatal) and regenerating (adult) motor, but not sensory, neurons. In contrast, Jun-lacZ was persistently up-regulated in both axotomized motor and sensory neurons in neonates and adults. Thus, expression of these genes did not predict neuronal death or survival. As Jun-lacZ was induced in some undamaged sensory neurons, this gene can be regulated by direct (axotomy) and indirect (transcellular) mechanisms. Indirect mechanisms also mediate expression of both genes in denervated muscle, Schwann cells in the distal and proximal stumps, and satellite cells in the DRG following axotomy. Thus, either these genes may regulate distinct sets of target genes in different cell types or they may subserve a single mechanism that is common to many cell types.

Regenerating motor neurons express Nna1, a novel ATP/GTP-binding protein related to zinc carboxypeptidases.

To identify genes involved in axon regeneration, differential screening was applied to RNA isolated from spinal cord of mice subjected to sciatic nerve transection or crush injury. A 4-kb transcript, termed nna1, was identified that was rapidly induced in affected motor neurons in both paradigms. The levels of nna1 transcript levels declined in motor neurons within 1-2 weeks after nerve crush, coincident with target reinnervation. If reinnervation was blocked by nerve cut and ligation, nna1 was continuously expressed in motor neurons. In addition, in situ analysis of developing embryonic nervous tissue showed nna1 was highly expressed in differentiating neurons, but not proliferating populations. Nna1 is predicted to be a zinc carboxypeptidase that contains nuclear localization signals and an ATP/GTP binding motif. Cultured neurons transfected with green fluorescent protein (GFP)-nna1 expressed GFP-Nna1 in cytoplasmic and nuclear compartments. Thus, Nna1 may contribute to nuclear signaling events in differentiating and regenerating neurons.

Expression patterns of the hepatic leukemia factor gene in the nervous system of developing and adult mice.

Hepatic leukemia factor (HLF) is a bZIP transcription factor related to the CES-2 protein, which controls apoptosis of the NSM serotoninergic neurons in Caenorhabditis elegans. Ectopic expression of HLF as an E2A-HLF fusion protein in t(17;19)-positive human pro-B cell acute lymphoblastic leukemias is believed to promote malignancy by interfering with apoptosis. While HLF has been linked to malignancies of the lymphoid system, it is not normally expressed in these cells. Rather, HLF transcripts are detected in the liver, kidney, lung and adult nervous system by Northern blotting. Despite the links to cell death, little is known of the distribution or function of HLF in the adult and developing mammalian nervous system. Therefore, we cloned mouse Hlf and studied its expression by in situ hybridization. During embryonic brain development, Hlf expression was restricted to the anterior pituitary and meninges. By early postnatal life, Hlf was highly expressed in somatosensory cortex, thalamic nuclei, and structures arising from ectodermal placodes. Subsequently, Hlf expression increased in the central nervous system and was found throughout the brain by adulthood. In the developing pituitary gland, Hlf was highly expressed in the rostral tip of the anterior lobe. This pattern is similar to that of Tef, an Hlf-related bZIP protein. However, while Tef is expressed in the anterior pituitary of the adult mouse, Hlf was detected in both the anterior and posterior pituitary. Hlf expression was not associated with cells undergoing programmed cell death in the nervous system. Hlf expression increased markedly with synaptogenesis and was coincident with barrel formation revealed by cytochrome oxidase staining. Together, these data suggest that Hlf plays a role in the function of differentiated neurons in the adult nervous system rather than programmed cell death.

Atm expression patterns suggest a contribution from the peripheral nervous system to the phenotype of ataxia-telangiectasia.

Ataxia-telangiectasia is a human autosomal recessive disease characterized by neurodegeneration, cancer predisposition and sensitivity to ionizing radiation. One of the earliest features of this disease is ataxia, which is thought to be attributable to a progressive cerebellar degeneration associated with a disruption of Purkinje cell cytoarchitecture and positioning. To investigate the neuropathology of ataxia-telangiectasia, we used in situ hybridization to map Atm (the gene mutated in ataxia-telangiectasia) expression during mouse development. Atm expression was highest in the embryonic mouse nervous system, where it was predominantly associated with regions undergoing mitosis. During the period of Purkinje cell neurogenesis, Atm was highly expressed in the area containing Purkinje cell precursors (the ventricular zone of the fourth ventricle). However, in the postnatal cerebellum, Atm expression in Purkinje cells was very low, while expression in proliferating granule neurons was high. The only region of the adult nervous system that exhibited elevated Atm expression were the postmitotic sensory neurons of the dorsal root ganglia. The data suggest an early developmental requirement for ATM in the cerebellum, and other regions of the central nervous system, and a potential contribution of the dorsal root ganglia/sensory input pathway to the ataxic phenotype of ataxia-telangiectasia.

The Ca2+ binding protein, frequenin is a nervous system-specific protein in mouse preferentially localized in neurites.

Frequenin is a Ca2+-binding protein that has been implicated in the regulation of neurotransmitter release at the neuromuscular junction [15,16]. However, its cellular and subcellular localization in brain have not been determined. Therefore, we cloned mouse frequenin (Mfreq) and investigated its expression both in vivo and in vitro. The amino acid sequence of Mfreq is homologous to that of frequenins from other species. Northern and Western blot analyses indicated that the Mfreq mRNA is a single species of 4.2 kb, and that the protein has a mass of 24 kDa protein on SDS gel, respectively. Expression of Mfreq is nervous system specific. However, Mfreq mRNA and protein are widely distributed in the brain, spinal cord, and dorsal root ganglia. Mfreq is expressed in early embryonic brain and the levels of Mfreq remain high throughout development. In situ hybridization and immunocytochemistry demonstrated that Mfreq is expressed primarily in neurons and presumptive astrocytes. The Mfreq protein was preferentially localized in neurites (dendrites and axons). Double immunofluorescence microscopy established that Mfreq was co-localized with the dendritic marker, MAP-2 and the synapse marker, SV2 in cultured hippocampal neurons. The distribution and subcellular localization of Mfreq may help understand its cellular function.

Inflammatory leukocytic recruitment and diffuse neuronal degeneration are separate pathological processes resulting from traumatic brain injury.

The present study characterized whether inflammatory leukocytic infiltration is temporally and regionally correlated with neuronal degeneration and/or blood brain barrier (BBB) breakdown resulting from traumatic brain injury. Adult rats were sacrificed at 5 min, 2, 4, 12, 24, and 72 hr after lateral fluid percussion brain injury. BBB breakdown, neuronal degeneration and leukocyte infiltration were assessed using immunocytochemistry, silver impregnation and toluidine blue and eosin staining. BBB breakdown and neuronal degeneration occurred concomitantly in injured cortex, hippocampus, and along the dorsolateral quadrant of the diencephalon. However, neuronal degeneration within deep diencephalic structures transpired in the absence of IgG extravasation. Neutrophils were observed only in regions exhibiting BBB damage and were first apparent in injured cortex and hippocampus between 2-12 hr posttrauma lining the vasculature and filling subarachnoid/subdural spaces. Neutrophils then migrated from damaged vasculature into traumatized cortical and hippocampal parenchyma by 24 hr after lateral fluid percussion injury. Macrophages were also observed within cortical parenchyma at 24 hr and completely filled the cortical lesion site by 72 hr after injury. Macrophages were not as abundant throughout hippocampal parenchyma and were found only in hippocampal regions exhibiting focal hemorrhage at 72 hr. Finally, neutrophils did not migrate to deep diencephalic structures that showed no BBB damage despite extensive neuronal degeneration. Indeed, lateral fluid percussion elicits inflammatory leukocytic recruitment only in regions experiencing concomitant BBB damage and neuronal degeneration. In summary, inflammatory leukocytic recruitment and diffuse neuronal degeneration are separate pathological processes resulting from traumatic brain injury.

Fetal hippocampal transplants attenuate CA3 pyramidal cell death resulting from fluid percussion brain injury in the rat.

Transplantation of fetal neural tissue has been demonstrated to prevent neuronal loss in a number of CNS injury models including spinal cord contusion. However, no studies have examined the neuroprotective role of fetal transplants in models of traumatic brain injury. The present study examined the ability of fetal neural grafts to attenuate neuronal loss resulting from lateral fluid percussion (FP) brain injury in the rat. Lateral FP in the rat elicits a focal contusion within the parietal/temporal cortex and induces cell death in a subset of hippocampal CA3 pyramidal neurons. To examine potential neuroprotective effects of fetal neural grafts, either E16 fetal hippocampus, E16 fetal cortex, or sterile lactated Ringers was stereotaxically transplanted directly into contused cortex 2 days after FP brain injury. The effects of fetal transplants upon adjacent injured hippocampal CA3 regions were then assessed at 4 weeks after grafting utilizing quantitative image analysis. Both fetal cortex and hippocampal grafts survived within contused cortex. Fetal hippocampal grafts significantly attenuated CA3 cell death resulting from lateral fluid percussion, while fetal cortical transplants induced a small, but nonsignificant, amelioration of CA3 pyramidal loss. Thus, neuroprotection by fetal grafts appeared to be tissue specific with hippocampal, but not cortical, fetal transplants significantly reducing posttraumatic CA3 loss. In summary, fetal neural transplantation can ameliorate hippocampal cell death following experimental brain injury.

A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.

The autosomal recessive mouse mutation reeler leads to impaired motor coordination, tremors and ataxia. Neurons in affected mice fail to reach their correct locations in the developing brain, disrupting the organization of the cerebellar and cerebral cortices and other laminated regions. Here we use a previously characterized reeler allele (rl(tg)) to close a gene, reelin, deleted in two reeler alleles. Normal but not mutant mice express reelin in embryonic and postnatal neurons during periods of neuronal migration. The encoded protein resembles extracellular matrix proteins involved in cell adhesion. The reeler phenotype thus seems to reflect a failure of early events associated with brain lamination which are normally controlled by reelin.

A model of parasagittal controlled cortical impact in the mouse: cognitive and histopathologic effects.

Controlled cortical impact (CCI), using a pneumatically driven impactor to produce traumatic brain injury, has been characterized previously in both the ferret and in the rat. In the present study, we applied this technique to establish and characterize the CCI model of brain injury in another species, the mouse, evaluating cognitive and histopathologic outcome. In anesthetized (sodium pentobarbital, 65 mg/kg) male C57BL mice, we performed sham treatment (no injury, n = 12) or CCI injury (n = 12) at a velocity of 5.7-6.2 m/sec and depth of 1 mm, using a 3-mm diameter rounded-tip impounder, positioned over the left parietotemporal cortex (parasagittal). At this level of injury, we observed highly significant deficits in memory retention of a Morris water maze task 2 days following injury (p < 0.001). Postmortem histopathologic analysis performed at 48 h following injury revealed substantial cortical tissue loss in the region of impact and selective hippocampal neuronal cell loss in the CA2, CA3, and CA3c regions, using Nissl staining. Analysis of degenerating neurons using modified Gallyas silver staining techniques demonstrated consistent ipsilateral injury of neurons in the cortex adjacent to the impact site and in the dentate gyrus of the ipsilateral hippocampus. Bilateral degeneration was observed at the gray matter-white matter interface along the corpus callosum. Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed extensive reactive gliosis appearing diffusely through the bilateral cortices, hippocampi, and thalami at 48 h postinjury. Breakdown of the blood-brain barrier was demonstrated with antimouse IgG immunohistochemistry, revealing extravasation of endogenous IgG throughout the ipsilateral cortex, hippocampus, and thalamus. These results suggest that this new model of parasagittal CCI in the mouse mimics a number of well-established sequelae observed in previously characterized brain injury models using other rodent species. This mouse model may be a particularly useful experimental tool for comparing behavioral and histopathologic characteristics of traumatic brain injury in wild-type and genetically altered mice.

Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death.

In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.

Development of prolonged focal cerebral edema and regional cation changes following experimental brain injury in the rat.

The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid-percussion brain injury. Animals receiving fluid-percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid-percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p less than 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p less than 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p less than 0.05) and injured hippocampus (10 min post injury, p less than 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p less than 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24-h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid-percussion brain injury and that these changes may persist for a prolonged period after brain injury.