Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration.

Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn2+) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn2+ accumulation in amacrine cell processes involves the Zn2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn2+ chelation extends for several days after nerve injury. These results show that retinal Zn2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

Robust Axonal Regeneration Occurs in the Injured CAST/Ei Mouse CNS.

Axon regeneration in the CNS requires reactivating injured neurons' intrinsic growth state and enabling growth in an inhibitory environment. Using an inbred mouse neuronal phenotypic screen, we find that CAST/Ei mouse adult dorsal root ganglion neurons extend axons more on CNS myelin than the other eight strains tested, especially when pre-injured. Injury-primed CAST/Ei neurons also regenerate markedly in the spinal cord and optic nerve more than those from C57BL/6 mice and show greater sprouting following ischemic stroke. Heritability estimates indicate that extended growth in CAST/Ei neurons on myelin is genetically determined, and two whole-genome expression screens yield the Activin transcript Inhba as most correlated with this ability. Inhibition of Activin signaling in CAST/Ei mice diminishes their CNS regenerative capacity, whereas its activation in C57BL/6 animals boosts regeneration. This screen demonstrates that mammalian CNS regeneration can occur and reveals a molecular pathway that contributes to this ability.

Long-distance axon regeneration in the mature optic nerve: contributions of oncomodulin, cAMP, and pten gene deletion.

The inability of retinal ganglion cells (RGCs) to regenerate damaged axons through the optic nerve has dire consequences for victims of traumatic nerve injury and certain neurodegenerative diseases. Several strategies have been shown to induce appreciable regeneration in vivo, but the regrowth of axons through the entire optic nerve and on into the brain remains a major challenge. We show here that the induction of a controlled inflammatory response in the eye, when combined with elevation of intracellular cAMP and deletion of the gene encoding pten (phosphatase and tensin homolog), enables RGCs to regenerate axons the full length of the optic nerve in mature mice; approximately half of these axons cross the chiasm, and a rare subset (∼1%) manages to enter the thalamus. Consistent with our previous findings, the axon-promoting effects of inflammation were shown to require the macrophage-derived growth factor Oncomodulin (Ocm). Elevation of cAMP increased the ability of Ocm to bind to its receptors in the inner retina and augmented inflammation-induced regeneration twofold. Inflammation combined with elevated cAMP and PTEN deletion increased activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways and augmented regeneration ∼10-fold over the level induced by either pten deletion or Zymosan alone. Thus, treatments that synergistically alter the intrinsic growth state of RGCs produce unprecedented levels of axon regeneration in the optic nerve, a CNS pathway long believed to be incapable of supporting such growth.

Spatiotemporal quantification of tumor necrosis factor-alpha and interleukin-10 after crush injury in rat sciatic nerve utilizing immunohistochemistry.

The purpose of this study was to investigate quantitatively the longitudinal temporal, spatial changes of the tumor necrosis factor-alpha (TNF) and interleukin-10 (IL-10) immunopositive cells during Wallerian degeneration and the following regeneration after crush injury in rat sciatic nerve using immunohistochemistry and enzyme linked immunosorbent assay (ELISA). The number of TNF-immunopositive cells reached its peak and increased significantly in all the segments distal to the crush site 3 days after injury. On Day 7, TNF-immunopositive cells decreased in all the segments distal to the crush site, and a significant decrease was observed 14 days after injury. From Day 21 to Day 56, there were no significant differences in the numbers of TNF-immunopositive cells. The average size of TNF immunopositive cells became significantly larger with degeneration. The number of IL-10-immunopositive cells decreases significantly 1 day after crush injury. IL-10-immunopositive cells increased on Day 3, returning to control levels. Seven days after injury, a significant increase in the number of IL-10-immunopositive cells was observed. There was also no significant difference in the number of IL-10-immunopositive cells beyond Day 14 except for a part of distal segments. The number of IL-10-immunopositive cells showed no significant differences in all the segments on Day 56. The protein levels of IL-10 measured by ELISA were similar to the result of immunohistochemistry. These results suggest that the significant change in IL-10 occurred prior to the significant change in TNF and that IL-10 may be the key to the change in TNF.

Different expressions of BDNF, NT3, and NT4 in muscle and nerve after various types of peripheral nerve injuries.

The changes in the expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) in the rat neuromuscular system as a result of three different types of sciatic nerve injuries have been evaluated. The changes in mRNA and protein levels for BDNF, NT-3, and NT-4 in the soleus muscle and sciatic nerve were assessed 4-28 days after sciatic nerve transection (neurotmesis), sciatic nerve crush (axonotmesis), and mild acute compression (neurapraxia). BDNF mRNA levels increased dramatically with nerve transection in the soleus muscle and the sciatic nerve 7-14 days after injury, whereas the changes were low in other types of injury. The changes of protein levels for BDNF were also similar. The mRNA and the protein levels of NT-3 in the soleus muscle did not show any significant difference. The mRNA for NT-4 in the soleus muscle decreased from 4 to 14 days after sciatic nerve transection, and the protein level was also minimum 14 days after sciatic nerve transection. Our results indicate that the neurotrophic factors in the neuromuscular system could play a role in differentiating peripheral nerve injury.

Spatiotemporal quantification of recruit and resident macrophages after crush nerve injury utilizing immunohistochemistry.

The purpose of this study was to investigate quantitatively the temporal and spatial regulation and the morphological changes of the recruit and resident macrophages in the sciatic nerve during Wallerian degeneration and the following regeneration using immunohistochemistry. Sciatic nerves in Sprague-Dawley (SD) rats were examined after nerve crush. The rats were anesthetized with 100 mg of ketamine and 20 mg of xylazine in a dose of 1 ml/kg by intraperitoneal injection. Anti-ED-1 antibody was used to detect phagocytic macrophage and anti-OX-6 antibody was used to detect MHC class II cells. Few ED-1-immunopositive cells were seen within the normal sciatic nerve. After crush injury the number and the size of ED-1-immunopositive cells started to increase in all the segments distal to the crush site 3 days after injury and the number and size reached its peak on day 14 when the population of macrophage was 150 times higher in all the segments compared to controls. However, the number of ED-1-immunopositive cells and the size of the cells remains significantly high even after day 56 when functional recovery and axonal regeneration were complete. OX-6-immunopositive cells were observed within the control sciatic nerves. The number decreases significantly 3 days after injury in all the segments distal to the crush site but showed no significant difference thereafter. There were also no significant differences in the cell areas. ED-1-immunopositive phagocytic macrophages show significant differences temporally in both the cell number and the size even after axonal regeneration.

The differential expression patterns of messenger RNAs encoding Nogo-A and Nogo-receptor in the rat central nervous system.

Nogo-A and Nogo-receptor have been considered to play pivotal roles in controlling axonal regeneration and neuronal plasticity. We investigated the total distribution of Nogo-A and Nogo-receptor mRNAs in the adult rat central nervous system using in situ hybridization histochemistry. Nogo-A is abundantly expressed in both neurons and oligodendrocytes throughout the central nervous system. Interestingly, we could not find any neuron which lacks Nogo-A mRNA expression, indicating that Nogo-A mRNA is universally expressed in all neurons. In contrast, Nogo-R mRNA expression was very restricted. Nogo-R mRNA was expressed in the olfactory bulb, hippocampus, tentia tecta, some amygdala nuclei, cerebral cortex, some thalamic nuclei, medial habenular, whereas we could not detect it in the other regions. Interestingly, we did not detect Nogo-R mRNA in monoaminergic neurons, which are known to have high regenerative capacity, in the substantia nigra, ventral tegmental area, locus caeruleus, and raphe nuclei. In addition, although neurons in the reticular thalamus and cerebellar nuclei are also known to show high capacity for regeneration, Nogo-R mRNA was not detected there. These data indicate that Nogo-A and Nogo-R mRNAs were differentially expressed in the central nervous system, and suggest that the lack of Nogo-R expression in a given neuron might be necessary to keep its high regenerative capacity.

A mild acute compression induces neurapraxia in rat sciatic nerve.

The pressure that induces neurapraxia in rat remains unrevealed. To determine the appropriate force to induce neurapraxia, two types of clips were applied to the sciatic nerve and were evaluated with functional, electrophysiological, and histological examinations. With a compression of 60 g/mm2, walking track analysis showed complete sciatic nerve paralysis one day postoperatively, but became normal in 14 days. Electrophysiologically, complete conduction block occurred one day post operatively, whereas the motor conduction velocity (MCV) below the compression site remained normal. Histologically, only limited signs of Wallerian degeneration were seen. The model in this study exhibited the features of neurapraxia.

The recovery of blood-nerve barrier in crush nerve injury--a quantitative analysis utilizing immunohistochemistry.

The purpose of this study is to reveal whether the application of immunohistochemical examinations to the peripheral nervous system (PNS) can be a reliable method for the quantitative analysis of the blood-nerve barrier (BNB) and the relationship between restoration of BNB and nerve regeneration. Sciatic nerves in rats were examined after nerve crush. Immunohistochemical staining with anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells and anti-endothelial barrier antigen (anti-EBA) for the detection of barrier-type endothelial cells were used. Neurofilament for staining axons was also performed. A quantitative analysis of the BNB was assessed using the ratio of EBA positive cells and RECA-1 positive cells. The ratio of EBA/RECA-1 decreased significantly 3 days postoperatively and reached its lowest level at day 7 in the segment 5 mm proximal and the entire distal stump. The ratio gradually recovered from the proximal and the regeneration of axons started a week earlier than BNB. The ratio of EBA/RECA-1 applied to the PNS can be a reliable method for the quantitative analysis of BNB. In crush injuries, the breakdown of BNB occurred simultaneously in the segment 5 mm proximal and the entire distal stump; restoration began from the proximal to distal and followed a week later to nerve regeneration.