Post Crush Research Articles

Defining Selective Neuronal Resilience and Identifying Targets of Neuroprotection and Axon Regeneration Using Single-Cell RNA Sequencing: Computational Approaches.

We describe a computational workflow to analyze single-cell RNA-sequencing (scRNA-seq) profiles of axotomized retinal ganglion cells (RGCs)in mice. Our goal is to identify differences in the dynamics of survival among 46 molecularly defined RGC types together with molecular signatures that correlate with these differences. The data consists of scRNA-seq profiles of RGCs collected at six time points following optic nerve crush (ONC) (see companion chapter by Jacobi and Tran). We use a supervised classification-based approach to map injured RGCs to type identities and quantify type-specific differences in survival at 2weeks post crush. As injury-related changes in gene expression confound the inference of type identity in surviving cells, the approach deconvolves type-specific gene signatures from injury responses by using an iterative strategy that leverages measurements along the time course. We use these classifications to compare expression differences between resilient and susceptible subpopulations, identifying potential mediators of resilience. The conceptual framework underlying the method is sufficiently general for analysis of selective vulnerability in other neuronal systems.

Effect of treadmill exercise on pain-related Wnt/β-catenin signaling pathway in dorsal root ganglion neurons at the early phase regeneration of the injured sciatic nerve.

The purpose of this study was to determine whether treadmill walking exercise can improve mechanical allodynia through regulation of Wnt/β-catenin signaling in dorsal root ganglion (DRG) neurons at the early stage of regeneration after sciatic nerve injury (SNI). The experimental rats were divided into seven groups: the normal control, sedentary groups for 3-, 7-, and 14-day post crush (dpc), and exercise group for 3, 7, and 14 dpc. The rats in exercise groups performed treadmill walking exercise at a speed of 8 m/min for 20 min once a day according to experiment duration. For evaluating neuropathic pain-like behavior after SNI, the mechanical allodynia was examined by von Frey apparatus. And the expression levels of pain-related protein were identified in the cytoplasm or nucleus of DRG neurons using Western blot techniques. Mechanical allodynia was significantly ameliorated in the exercise group at 7 and 14 dpc. Treadmill exercise further decreased Wnt3a expression at 3, 7, and 14 dpc compared to in the sedentary group. Also, phosphorylated-low-density lipoprotein receptor 6 was decreased in exercise groups at 3 and 14 dpc. Beta-catenin was significantly decreased in exercise groups at 3 and 14 dpc compared to sedentary groups as well as treadmill exercise decreased translocation of β-catenin towards the nucleus of DRG neurons at 14 dpc. Our findings indicate that treadmill walking exercise may be an important regulator of neuropathic pain after peripheral nerve injury through delayed Wnt/β-catenin signaling pathway in DRG neurons.

Treadmill exercise activates ATF3 and ERK1/2 downstream molecules to facilitate axonal regrowth after sciatic nerve injury.

The purpose of this study was to investigate the effect of treadmill exer-cise on activating transcription factors such as activating transcription factor 3 (ATF3) and extracellular signal-regulated kinase (ERK1/2) sig-naling pathway to facilitate axonal regrowth after sciatic nerve injury (SNI). The experimental rats divided into the normal control (n=10), sedentary groups for 7 (n=10) and 14 days (n=10) post crush, exercise group for 7 (n=10) and 14 days (n=10) post crush (dpc). The rats in ex-ercise groups run on treadmill device at a speed of 8 m/min for 20 min once a day according to exercise duration. In order to evaluate specific regeneration markers and axonal elongation in injured sciatic nerve, we applied immunofluorescence staining and western blot techniques. Treadmill exercise further increased growth-associated protein (GAP-43) expression and axonal regrowth at 7 and 14 dpc than those in sed-entary group. Among mitogen-activated protein kinase downstream molecules, phospho-ERK1/2 (p-ERK1/2) was enhanced by treadmill ex-ercise at only 7 dpc and decreased to basal level 14 days later. But c-Jun N-terminal kinase, c-Jun, and phospho-cyclic adenosine mono-phosphate response element-binding protein showed a tendency to in-crease continuously until 14 dpc by exercise. ATF3 expression in exer-cise group was upregulated at both 7 and 14 dpc compared to the sed-entary group. These results indicate that treadmill exercise had benefi-cial effect on expression of regeneration-related proteins after SNI, suggesting that exercise might be one of various therapeutic strategies for sciatic nerve regeneration.

Human equivalent dose of oral 4-aminopyridine differentiates nerve crush injury from transection injury and improves post-injury function in mice.

4-Aminopyridine (4-AP), an FDA-approved drug for the symptomatic treatment of multiple sclerosis, is used to improve neuromuscular function in patients with diverse demyelinating disorders. We recently demonstrated that local, transdermal or injectable forms of 4-AP improve myelination, nerve conduction velocity, muscle atrophy, and motor function after traumatic peripheral nerve injury in mice. While oral 4-AP is most commonly used in the clinic, it is unknown whether human equivalent oral doses of 4-AP have effects on traumatic peripheral nerve injury differentiation, myelination, muscle atrophy, functional recovery, and post-injury inflammatory processes in animals. Mice with sciatic nerve crush or denervation injury received oral or intraperitoneal 4-AP (10 μg) or vehicle alone and were examined for pharmacokinetics, motor function, muscle mass, intrinsic muscle force, nerve morphological and gene expression profiles. 4-AP showed linear pharmacokinetics and the maximum plasma 4-AP concentrations were proportional to 4-AP dose. Acute single dose of oral 4-AP administration induced a rapid transient improvement in motor function that was different in traumatic peripheral nerve injury with or without nerve continuity, chronic daily oral 4-AP treatment significantly enhanced post crush injury motor function recovery and this effect was associated with improved myelination, muscle mass, and ex vivo muscle force. Polymerase chain reaction array analysis with crushed nerve revealed significant alterations in gene involved in axonal inflammation and regeneration. These findings provide convincing evidence that regardless of the route of administration, 4-AP can acutely differentiate traumatic peripheral nerve injury with or without nerve continuity and can enhance in vivo functional recovery with better preservation of myelin sheaths, muscle mass, and muscle force. The animal experiments were approved by the University Committee on Animal Research (UCAR) at the University of Rochester (UCAR-2009-019) on March 31, 2017.

Regeneration of synapses in the olfactory pathway of locusts after antennal deafferentation.

The olfactory pathway of the locust is capable of fast and precise regeneration on an anatomical level. Following deafferentation of the antenna either of young adult locusts, or of fifth instar nymphs, severed olfactory receptor neurons (ORNs) reinnervate the antennal lobe (AL) and arborize in AL microglomeruli. In the present study we tested whether these regenerated fibers establish functional synapses again. Intracellular recordings from AL projection neurons revealed that the first few odor stimulus evoked postsynaptic responses from regenerated ORNs from day 4-7 post crush on. On average, synaptic connections of regenerated afferents appeared faster in younger locusts operated as fifth instar nymphs than in adults. The proportions of response categories (excitatory vs. inhibitory) changed during regeneration, but were back to normal within 21days. Odor-evoked oscillating extracellular local field potentials (LFP) were recorded in the mushroom body. These responses, absent after antennal nerve crush, reappeared, in a few animals as soon as 4days post crush. Odor-induced oscillation patterns were restored within 7days post crush. Both intra- and extracellular recordings indicate the capability of the locust olfactory system to re-establish synaptic contacts in the antennal lobe after antennal nerve lesion.

Anatomical regeneration and behavioral recovery following crush injury of the trigeminal root in lamprey

In normal larval lamprey, bilateral application of horseradish peroxidase (HRP) to the dorsal part of the anterior oral hood labeled subpopulations of trigeminal components on both sides of the brain; peripherally projecting motoneurons, medullary dorsal cells (sensory), and spinal dorsal cells (sensory), as well as centrally projecting afferents in the trigeminal descending tracts. Following unilateral crush injury of the right trigeminal root, HRP labeling of sensory and motor trigeminal components on the right side gradually increased with increasing recovery time, between 2 weeks and 12 weeks postcrush (PC). Axons of trigeminal motoneurons appeared to exhibit robust regeneration, whereas restoration of projections in the descending trigeminal tract ipsilateral to the injury was incomplete. Control experiments indicated that motor and sensory axons from the intact side of the oral hood did not sprout across the midline to the denervated side. Several results suggested that regenerated trigeminal sensory fibers made synapses with brain neurons that have direct or indirect inputs to reticulospinal (RS) neurons. Following a unilateral crush injury of the right trigeminal root, escape behavior in response to stimulation of the right side of the oral hood gradually returned to normal. Muscle recordings at various recovery times confirmed that anatomical regeneration of trigeminal sensory axons was functional. In addition, at 8 or 12 weeks PC, brief stimulation of the oral hood ipsilateral or contralateral to the crush injury elicited synaptic responses in RS neurons on either side of the brain, similar to that in normal animals. In the lamprey, compensatory mechanisms probably allow recovery of behavioral function despite incomplete regeneration of trigeminal sensory axons within the central nervous system.

Post-translational modification of proteins by arginine and lysine following crush injury and during regeneration of rat sciatic nerves

Following crush injury to rat sciatic nerves, a crude fraction of the 150,000 g supernatant can post-translationally incorporate [3H]Arg and [3H]Lys into endogenous proteins in amounts approximately 10 times uninjured control nerves. These increases occur in the proximal nerve stump within 2 h of injury and 2 weeks later in a distal segment of nerve containing the tips of the regenerating axons. In the present experiments, the endogenous nerve proteins modified by Arg or Lys in these nerve segments have been identified using two-dimensional polyacrylamide gel electrophoresis. The fraction used to assay for protein modification, the void volume of a Sephacryl S-300 column, was found to contain only a few proteins visible by Coomassie blue staining, one of which is likely to be albumin (68 kDa, pI 6.4). While this protein was modified by both Arg and Lys, the majority of label was found in areas not showing Coomassie blue staining. This indicates that of the many potential targets of post-translational arginylation and lysylation, most are proteins of relatively low abundance. A variety of proteins were modified by Arg or Lys alone while others were modified by both Arg and Lys. A high molecular weight protein (175 kDa, pI 9.0) was modified only by Lys and only at 2 h post crush. Of a variety of modified proteins of approximately 17 kDa one (pI 6.3) was modified by both Arg and Lys and at both time points, while another (pI 9.0) was modified at both time points, but only by Lys. The results show that Arg and Lys can be added post-translationally to a large number of low abundance, soluble sciatic nerve proteins, and that some of those proteins are modified only by Arg or Lys while others are modified by both Arg and Lys. Also, the modification of certain proteins appears to be associated specifically with the immediate response of a nerve to injury (e.g. 88 kDa, pI 7.1) while others are associated with the regenerative period (e.g. 56 kDa, pI 7.4).

Dissociation of enhanced ornithine decarboxylase activity and optic nerve regeneration in goldfish

The significance of a previously observed increase in retinal ornithine decarboxylase (ODC, EC 4.1.1.17) following optic nerve crush was investigated in goldfish using a specific irreversible inhibitor of ODC, α-difluoromethylornithine (DFMO). Retinal ODC activity and putrescine levels were reduced 80% and 40%, respectively, by intraperitoneal injection(s) of DFMO. In fish in which the right optic nerve was crushed and DFMO was injected at the time of crush, at 2 days and at 4 days later, retinal ODC levels were maximally decreased after the third injection and returned to normal levels by 14 days post-crush (PC). Retinal tubulin synthesis was examined at 10 days PC. No difference in the post-crush/normal ratio of tubulin synthesis was observed between saline-injected and DFMO-injected groups. Neuritic extension was also examined in retinal explant cultures from 11-day PC retina. The neuritic growth index of DFMO-treated retinas did not differ from that of control retinas. Behavioral studies revealed no difference in the rate of recovery of vision between the two groups. Similar results were obtained in experiments using fish maintained in a 0.1% DFMO solution. These results suggest that the increase in retinal ODC associated with nerve crush does not play a causal role in restoration of function.

Normal and abnormal retinal projections following the crush of one optic nerve in goldfish (Carassius auratus).

Optic nerve regeneration was examined with [3H]proline radioautography in fish that had one nerve crushed. Fibers had not yet grown beyond the crush site at 2 days post-crush (PC) and were at the optic chiasm by 4-5 days PC. By 6 days PC the fibers had reinnervated the rostral pole of the contralateral tectum, the lateral geniculate nucleus and area pretectalis dorsalis and ventralis. Area preopticus, nucleus opticus dorsolateralis and nucleus opticus commissurae posterior were partially reinnervated by 8 days PC. At this time numerous abnormal targets were labeled, including nucleus rotundus, nucleus isthmi, cerebellum, pituitary gland and ipsilateral optic tectum. Optic fibers also entered the posterior, intertectal and horizontal commissures, as well as tractus rotundus, the tectocerebellar, tectobulbar and mesencephalocerebellar tracts. In addition, fibers with the contralateral optic tectum were not restricted to their usual laminae. They were distributed from the superficial edge of the tectum to the ventricle. At 32 days PC only the normal retinal projections were evident, and all of the anomalous projections had disappeared. The anomalous projections may have either retracted or degenerated or become undetectable with radioautography.

Uridine metabolism in the goldfish retina during optic nerve regeneration: whole retina studies.

Accumulation of radioactivity from [3H]uridine in incubations of whole goldfish retinas is increased in the ipsilateral retina during a period of regeneration that follows unilateral optic nerve crush. Brief incubations to investigate the nature of enhanced labeling of the acid-soluble fraction showed a peak uptake 4 days following crush, with a gradual decrease to control levels by 21 days following crush. That nucleoside uptake may not mediate the effect is supported by the observation that the rate of uptake of 5'-deoxyadenosine, a nonmetabolizable nucleoside analog, is the same in post-crush (PC) and normal (N) retinal incubations. Following brief incubations of PC and N retinas with [3H]uridine, there is enhanced labeling in PC retinas relative to N retinas of recovered UMP, UDP, UTP, and uridine nucleotide sugars, whereas recovery of labeled uridine itself is slightly decreased. The results suggest that the increased accumulation of radioactivity in PC retinas following incubation with uridine reflects an increase in the activities of retinal uridine kinase and uridine nucleotide kinases.