Kelly Matsudaira Yee

Regenerative Growth of Corticospinal Tract Axons via the Ventral Column after Spinal Cord Injury in Mice

Studies that have assessed regeneration of corticospinal tract (CST) axons in mice after genetic modifications or other treatments have tacitly assumed that there is little if any regeneration of CST axons in normal mice in the absence of some intervention. Here, we document a previously unrecognized capability for regenerative growth of CST axons in normal mice that involves growth past the lesion via the ventral column. Mice received dorsal hemisection injuries at thoracic level 6–7, which completely transect descending CST axons in the dorsal and dorsolateral column. Corticospinal projections were traced by injecting biotinylated dextran amine (BDA) into the sensorimotor cortex of one hemisphere either at the time of the injury or 4 weeks after injury, and mice were killed at 20–23 or 46 d after injury. At 20–23 d after injury, BDA-labeled CST axons did not extend past the lesion except in one animal. By 46 d after injury, however, a novel population of BDA-labeled CST axons could be seen extending from the gray matter rostral to the injury into the ventral column, past the lesion, and then back into the gray matter caudal to the injury in which they formed elaborate terminal arbors. The number of axons with this highly unusual trajectory was small (∼1% of the total number of labeled CST axons rostral to the injury). The BDA-labeled axons in the ventral column were on the same side as the main tract and thus are not spared ventral CST axons (which would be contralateral to the main tract). These results indicate that normal mice have a capacity for CST regeneration that has not been appreciated previously, which has important implications in studying the effect of genetic or pharmacological manipulations on CST regeneration in mice.

Response to: Kim et al., "axon regeneration in young adult mice lacking Nogo-A/B." Neuron 38, 187-199.

Department of Neurosciences, University of California at San Diego, La Jolla, CA 92093, USA*Correspondence: osteward@uci.eduDOI 10.1016/j.neuron.2007.04.004IntroductionNogohasbeenproposedtobeamajormyelin-derived inhibitor of axon re-generation in the mammalian centralnervous system (CNS). Three studiestested this hypothesis by assessingregeneration in various Nogo-deficientmice following spinal cord injury (Kimet al., 2003; Simonen et al., 2003;Zhengetal.,2003),yetdifferentregen-erative responses were reported. Instudies of a Nogo gene trap mutant,Kim et al. (2003) reported dramaticallyenhanced sprouting above the lesion,reflected by the presence of CSTaxons in ectopic locations (the lateralfuniculus ipsilateral to the injection) inall (12/12) Nogo knockout mice. Evi-dence of striking long-distance regen-eration was the presence of ectopicBDA-labeled axons more than 5 mmcaudal to the injury in 7/11 Nogoknockout mice that received spinalcord injuries at 7.5–9 weeks of age.Three mice that underwent surgery at11–14 weeks of age did not exhibit ev-idence of long-distance regeneration.The labeled axons in caudal segmentsseemed to be especially compellingevidence of robust but disorderly re-generationoftheCSTbecauselabeledaxons were present bilaterally in thelateral funiculus (an ectopic location).In contrast, Simonen et al. (2003)reported increased sprouting nearthe lesion site in a Nogo-A-targetedknockout(inwhichNogo-Bexpressionwas upregulated) and evidence foren-hanced regeneration below the lesionin a subset (4/16) of mutants, althoughthere was no statistically significantdifferencebetweenknockoutandcon-trols. Our group found no statisticallysignificant difference between Nogo-A,B(orNogo-A,B,C)mutantsandwild-type controls in the extent of CSTsprouting above the lesion or regener-ation past the lesion (Zheng et al.,2003). One enigmatic finding was thatectopic axons similar to those re-ported by Kim et al. were observed inone control mouse (see Figure S2 inthe Supplemental Data of Zhenget al., 2003).Possible reasons for the discrepantresults have been discussed (Woolf,2003), but there has been no satisfac-tory explanation of the apparently ro-bust regeneration in one study andmodest or no enhanced regenerationin the others. Accordingly, we set outto perform a full replication of the ex-periments in the knockout line usedin our study (Zheng et al., 2003) andthe gene trap line used by Kim et al.(2003), which were reported to exhibitstriking regeneration. In the course ofour studies, we discovered that axonsin the white matter stain artifactuallywhenBDAleaksintothecerebrospinalfluid (CSF) in the cerebral ventricle,producing a pattern of ectopic labeledaxons that is remarkably similar towhat was interpreted as massivesprouting/regeneration by Kim et al.Inthisreport,wedocumenttheartifac-tual labeling, show that it occurs asa result of BDA entering the CSF, anddemonstrate that the artifact may beavoided by delaying the tract tracinginjections for 4 weeks after spinalcord lesions.ResultsIn all three studies of Nogo knockoutmice, regeneration was assessed fol-lowing transection of the dorsal halfof the spinal cord (a dorsal hemi-section) atT8, which in mice interruptsdescending CST axons while sparingwhite matter and gray matter in ventralportions of the spinal cord. Possibleregeneration of cortico-spinal tract(CST) axons was then assessed byinjecting biotinylated dextran amine(BDA) into the sensorimotor cortex.Importantly, all three studies used asimilar, short-term protocol: the corti-cal BDA injection was made in thesame operative procedure as the spi-nal cord lesion, and regeneration wasassessed 2–3 weeks later.In our replication experiment, weusedthesameprotocoltoassesspos-sibleCSTregenerationinhomozygousmutant and control (wild-type and het-erozygotes). BDA was injected usingthe coordinates described in Zhenget al. (2003) (Kim et al. [2003] did notreport their coordinates). One smallprocedural difference that turned outto be important was that, in our previ-ous study (Zheng et al., 2003), BDAwas injected at a depth of 0.5 mm inthe cortex, whereas in the repeatexperiment we lowered the syringebelow 0.5 mm and then withdrew tothe final location. This was done toachieve greater accuracy becausethe cortex is frequently displacedslightlyasthemicrosyringe islowered.As discussed below, this had theunin-tended consequence that the syringesometimes penetrated the ventricle.In intact mice (that is, without spinalcord injuries), injections of BDA intothe sensorimotor cortex producerobust labeling of dorsal main CST inthe ventral part of the dorsal columnNeuron 54, April 19, 2007 a2007 Elsevier Inc. 191

Long-Distance Migration and Colonization of Transplanted Neural Stem Cells

A recent study reported remarkable survival and exuberant axon outgrowth from transplants of neural stem cells (NSCs) in a fibrin matrix with growth factors that were grafted into a complete spinal cord transection site in rats (Lu et al., 2012). As part of the NIH-supported replication project (Facilities of Research Excellence-Spinal Cord Injury), we repeated key parts of that study. Because this is a surgical intervention that may depend on skills that require extensive experience, we felt that the goals of the replication would be best served if the same surgeon performed the lesion surgeries and transplants.

Salmon fibrin treatment of spinal cord injury promotes functional recovery and density of serotonergic innervation.

The neural degeneration caused by spinal cord injury leaves a cavity at the injury site that greatly inhibits repair. One approach to promoting repair is to fill the cavity with a scaffold to limit further damage and encourage regrowth. Injectable materials are advantageous scaffolds because they can be placed as a liquid in the lesion site then form a solid in vivo that precisely matches the contours of the lesion. Fibrin is one type of injectable scaffold, but risk of infection from blood borne pathogens has limited its use. We investigated the potential utility of salmon fibrin as an injectable scaffold to treat spinal cord injury since it lacks mammalian infectious agents and encourages greater neuronal extension in vitro than mammalian fibrin or Matrigel®, another injectable material. Female rats received a T9 dorsal hemisection injury and were treated with either salmon or human fibrin at the time of injury while a third group served as untreated controls. Locomotor function was assessed using the BBB scale, bladder function was analyzed by measuring residual urine, and sensory responses were tested by mechanical stimulation (von Frey hairs). Histological analyses quantified the glial scar, lesion volume, and serotonergic fiber density. Rats that received salmon fibrin exhibited significantly improved recovery of both locomotor and bladder function and a greater density of serotonergic innervation caudal to the lesion site without exacerbation of pain. Rats treated with salmon fibrin also exhibited less autophagia than those treated with human fibrin, potentially pointing to amelioration of sensory dysfunction. Glial scar formation and lesion size did not differ significantly among groups. The pattern and timing of salmon fibrins effects suggest that it acts on neuronal populations but not by stimulating long tract regeneration. Salmon fibrin clearly has properties distinct from those of mammalian fibrin and is a beneficial injectable scaffold for treatment of spinal cord injury.

Characterization of Ectopic Colonies That Form in Widespread Areas of the Nervous System with Neural Stem Cell Transplants into the Site of a Severe Spinal Cord Injury

We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies.

Delayed degradation and impaired dendritic delivery of intron-lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc transgenic mice

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized Arc mRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3′UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3′UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3′UTR.