Dendritic shrinkage after injury: a cellular killer or a necessity for axonal regeneration?

Dendrites form an essential component of the neuronal circuit have been largely overlooked in regenerative research. Nevertheless, subtle changes in the dendritic arbors of neurons are one of the first stages of various neurodegenerative diseases, leading to dysfunctional neuronal networks and ultimately cellular death. Maintaining dendrites is therefore considered an essential neuroprotective strategy. This mini-review aims to discuss an intriguing hypothesis, which postulates that dendritic shrinkage is an important stimulant to boost axonal regeneration, and thus that preserving dendrites might not be the ideal therapeutic method to regain a full functional network upon central nervous system damage. Indeed, our study in zebrafish, a versatile animal model with robust regenerative capacity recently unraveled that dendritic retraction is evoked prior to axonal regrowth after optic nerve injury. Strikingly, inhibiting dendritic pruning upon damage perturbed axonal regeneration. This constraining effect of dendrites on axonal regrowth has sporadically been proposed in literature, as summarized in this short narrative. In addition, the review discusses a plausible underlying mechanism for the observed antagonistic axon-dendrite interplay, which is based on energy restriction inside neurons. Axonal injury indeed leads to a high local energy demand in which efficient axonal energy supply is fundamental to ensure regrowth. At the same time, axonal lesion is known to induce mitochondrial depolarization, causing energy depletion in the axonal compartment of damaged neurons. Mitochondria, however, become mostly stationary after development, which has been proposed as a potential underlying reason for the low regenerative capacity of adult mammals. Per contra, upon reduced neuronal activity, mitochondrial mobility enhances. In this view, dendritic shrinkage after axonal injury in zebrafish could result in less synaptic input and hence, a release of mitochondria within the soma-dendrite compartment that then translocate to the axonal growth cone to stimulate axonal regeneration. If this hypothesis proofs to be correct, i.e. dendritic remodeling serving as fuel for axonal regeneration, we envision a major shift in the research focus within the neuroregenerative field and in the potential uncovering of various novel therapeutic targets.     

Functional outcome after diffuse axonal injury in childhood traumatic brain injury

We investigated the functional prognosis after traumatic diffuse axonal injury in children. We evaluated the status of the acute stage, as well as the functional independence measure (FIM) and intelligence quotient (IQ) at 4, 12, and 24 months after the injury. Physical disabilities persisted in all but 1 case, but 5 cases could walk by themselves after 1 year. IQ at 2 years after the injury was 64 in one case, but between 81 and 100 in others. Concerning the higher cortical function, all cases showed memory disturbance. None developed epilepsy. All cases showed abnormalities on cerebral MRI. Five of the 7 cases showed EEG abnormalities. As to the course of recovery scaled by FIM and IQ, marked improvement was seen in 4 cases during the first 4 months, 3 cases during the first 1 year. After 1 year, the degree of improvement became slower in all. All cases showed learning disability at school.     

Distribution of neurofilament antigens after axonal injury

Phosphorylated and nonphosphorylated epitopes of neurofilament (NF) proteins are distributed in different regions of individual neurons. Immunocytochemical methods, with monoclonal antibodies directed against phosphorylated and nonphosphorylated NF, demonstrated nonphosphorylated NF in perikarya and proximal axonal segments of neurons in dorsal root ganglia, while phosphorylated NF proteins were present in axons of these cells. The distribution of these epitopes of NF were examined at various times following injury of axons in the rat sciatic nerve. Between one and 21 days after crush of the proximal nerve, phosphorylated NF were present in neuronal perikarya. We have compared patterns of perikaryal immunoreactivity at one time point (three weeks) following a more distal crush or complete transection of the sciatic nerve. At this time period, following transection/ligation, phosphorylated NF immunoreactivity was not present in perikarya, but abnormal staining was observed after nerve crush. These altered distributions of phosphorylated epitopes of NF are of interest because several recent reports have indicated that similar, but not identical, abnormal staining patterns occur in human neurological diseases, including Alzheimer's disease and Parkinson's disease. In accord with previous studies, this investigation indicates that one response of neurons to injury, or to disease, is an abnormal distribution of phosphorylated epitopes of NF proteins.     

Modeling axonal injury in vitro: injury and regeneration following acute neuritic trauma

Traumatic injury to axons was modeled in vitro using sympathetic principal neurons from the rat superior cervical ganglion. Neurons were grown as a pure culture on collagen in parallel tracks, with cell somata confined to the center, and neurites occupying the periphery of the culture dish. Growing as fascicles on tracks, the neurites demonstrated periodic varicosities. Neuritic transection was reliably and reproducibly achieved with a motor driven rubber impactor injury device. During a period lasting at least 1 h, dieback involving the proximal neurites averaged 105 +/- 10 microm. This was followed by neurite regeneration, with the injured segment being traversed within 36 h at an average rate of regeneration of 595 +/- 15 microm/day. The distal neurite segments showed degenerative changes within 1 h following transection, with initial receding of neurites progressing to vacuolation, beading, blebbing, and eventual detachment from the underlying matrix. This in vitro model of axonal injury allows neuritic injury to be studied at the cellular and molecular levels, and also provides a unique opportunity to test potential neuromodulatory and neuroprotective strategies.     

Proximal axonal changes after peripheral nerve injury in man

Peripheral nerve injury leads to changes in the proximal axon. Traumatic nerve injuries in humans were investigated to characterize such electrophysiological changes. Mixed nerve conduction studies (MNCS) and motor conduction studies (MCS) were performed proximal to the injury. Control values were obtained from the uninjured limb. Median (n = 24) and ulnar (n = 35) nerve injuries were studied. The injured nerves had significant mixed nerve action potential (MNAP) amplitude reductions (median: P < 0.0001; ulnar: P < 0.0001). The majority of the MNAP amplitude reductions were severe and early. There was slowing in the mixed nerve conduction velocity (MNCV) (median: P = 0.09; ulnar: P = 0.04) and motor conduction velocity (MCV) (median: P = 0.046; ulnar: P = 0.005). Axonal loss appears to play a significant role in producing the MNCS changes observed, and its early occurrence is noteworthy. Proximal MCV reduction could be secondary to the effects of injury as well as collateral sprouting of uninjured axons. Proximal axonal changes may have an impact on recovery.     

Spontaneous axonal regeneration in rodent spinal cord after ischemic injury

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.     

Spontaneous axonal regeneration after optic nerve injury in adult rat

Optic nerves of adult rats were crushed 2 mm behind the eye to examine the ability of retinal ganglion cells (RGCs) to regenerate their axons. Some animals were treated with the immunophilin ligands FK 506 or GPI 1046 for up to 4 weeks. After 10 days to 16 months, regenerating RGC axons were visualized using anterograde tracing and/or electron microscopy. A small proportion of RGC axons regenerated across the lesion site and grew very slowly along the entire optic nerve. Immunophilin ligands had no obvious effect. The regenerating axons were about 0.2 microm in diameter, and usually in clusters surrounded by astrocyte processes. Thus, some CNS axons can spontaneously regenerate long distances within degenerate white matter and this slow regeneration is not accelerated by immunophilin ligands.     

RhoA-inhibiting NSAIDs promote axonal myelination after spinal cord injury

Nonsteroidal anti-inflammatory drugs (NSAIDs) are extensively used to relieve pain and inflammation in humans via cyclooxygenase inhibition. Our recent research suggests that certain NSAIDs including ibuprofen suppress intracellular RhoA signal and improve significant axonal growth and functional recovery following axonal injury in the CNS. Several NSAIDs have been shown to reduce generation of amyloid-beta42 peptide via inactivation of RhoA signal, supporting potent RhoA-repressing function of selected NSAIDs. In this report, we demonstrate that RhoA-inhibiting NSAIDs ibuprofen and indomethacin dramatically reduce cell death of oligodendrocytes in cultures or along the white matter tracts in rats with a spinal cord injury. More importantly, we demonstrate that treatments with the RhoA-inhibiting NSAIDs significantly increase axonal myelination along the white matter tracts following a traumatic contusion spinal cord injury. In contrast, non-RhoA-inhibiting NSAID naproxen does not have such an effect. Thus, our results suggest that RhoA inactivation with certain NSAIDs benefits recovery of injured CNS axons not only by promoting axonal elongation, but by enhancing glial survival and axonal myelination along the disrupted axonal tracts. This study, together with previous reports, supports that RhoA signal is an important therapeutic target for promoting recovery of injured CNS and that RhoA-inhibiting NSAIDs provide great therapeutic potential for CNS axonal injuries in adult mammals.     

Rho-kinase inhibition enhances axonal regeneration after peripheral nerve injury

In injured adult neurons, the process of axonal regrowth and reestablishment of the neuronal function have to be activated. We assessed in this study whether RhoA, a key regulator of neurite elongation, is activated after injury to the peripheral nervous system. RhoA is activated in motoneurons but not in Schwann cells after mouse sciatic nerve injury. To examine whether the activation of RhoA and its effector, Rho-kinase, retards axon regeneration of injured motoneurons, we employed a Rho-kinase inhibitor, fasudil. Amplitudes of distally evoked compound muscle action potentials are increased significantly faster after axonal injury in mice treated with fasudil compared with controls. Histological analysis shows that fasudil treatment increases the number of regenerating axons with large diameter, suggesting that axon maturation is facilitated by Rho-kinase inhibition. In addition, fasudil does not suppress the myelination of regenerating axons. These findings suggest that RhoA/Rho-kinase may be a practical molecular target to enhance axonal regeneration in human peripheral neuropathies.     

弥漫性轴索损伤后海马生长抑素样神经元的变化

目的 :弥漫性轴索损伤 (DAI)能导致伤后认知障碍。本实验通过建立实验性 DAI动物模型 ,以了解在 DAI伤后与认知功能关系密切海马生长抑素 (Ss)样神经元的变化。方法 :采用 Marmarou打击装置建立 DAI动物模型 ,免疫组织化学染色以显示海马 Ss样神经元。结果 :1海马 Ss样神经元在重伤组、轻伤组、对照组有显著差异 (P<0 .0 1)。 2损伤后二周组神经元减少与一周组比较有显著差异 (P<0 .0 1或 P<0 .0 5 )。结论 :1DAI后海马 Ss样神经元的减少可能是伤后认知障碍 ,甚至是 DAI后植物生存的主要病理改变之一。 2伤后的迟发性细胞死亡在该种神经元的减少中起着重要作用。     

弥漫性轴索损伤

弥漫性轴索损伤(DAI)属于闭合性原发弥漫性脑损伤。是由于头部成角、加(减)速运动或旋转性暴力出现弥漫性轴索扭曲、肿胀、断裂及皮髓质交界区穿行血管中断所致。好发于皮髓质交界区、胼胝体、尾状核、丘脑、内囊及中脑被盖的背外侧。其病理变化包括:(1)广泛性轴索损害,累及大脑、脑干和小脑的白质和大脑深部核质,包括中线旁皮质下白质、胼胝体、穹窿柱、内囊、基底节及丘脑、齿状核背侧小脑叶、皮质脊髓束、内侧丘脑系、内侧纵束等。(2)胼胝体局限性出血灶。(3)上脑干背外     

Retrograde axonal transport in rat sciatic nerve after nerve crush injury

We investigated the quantitative alterations in retrograde transport of proteins following a nerve crush injury using the 3H N-succinimidyl propionate (3H NSP) method in rat sciatic nerve. After subepineurial injection of 3H NSP into the nerve the amount of radioactively labeled proteins accumulating in the cell bodies of the motor and sensory neurons was determined 1 day or 7 days later in nerves which had been crushed distal to the injection site 1, 3, 5, 7, or 33 days prior to 3H NSP labeling. One day accumulation in the DRG and spinal cord was not altered by nerve crush. Seven day accumulation in the DRG was initially slightly increased, then fell to 73% of control by 7 days, remaining reduced 33 days after crush. Seven day accumulation in the spinal cord was reduced to 25% of control 1 day after crush and remained at that low level except for 5 days post-crush when a normal amount of labeled protein was transported to the spinal cord. The time course of these changes suggests that quantitative alterations in retrograde transport may be involved in the long-term trophic interactions between the cell body and periphery, but are too slow to account for the earliest perikaryal responses to injury. In addition, the difference between the alterations of retrograde transport in motor and sensory neurons may reflect fundamental differences in the composition of retrograde transport in those different systems.     

大鼠弥漫性轴突损伤后神经丝68蛋白水平减少

目的：通过检测大鼠弥漫性轴突损伤（DAI）后神经丝蛋白68（NF68）水平，探讨弥漫性轴突损伤的病理机制。方法：用一种对大鼠头颅旋转装置致伤引发大鼠DAI。Sprague-Dawley（SD）大鼠18只随机分成3组（30min,24,72h组），每组6只，在不同时间点取脑白质、海马、胼胝体和脑干标本进行NF68印记杂交（Western blot)，检查神经丝蛋白68 水平。结果：Western blot看到对照动物NF68水平没有变化，而损伤动物明显NF68的丢失，特别是脑干。NF68是蛋白质水平减少在损伤后30min即出现直到损伤后72h。在NF68丢失的同时伴有31、42．7和52Kda低分子量蛋白条带的出现，在24h最明显。结论：NF68降解是由病理性钙蛋白酶激活引起的。损伤后24h内是最佳治疗时机。     

Role of myelin-associated inhibitors in axonal repair after spinal cord injury

Myelin-associated inhibitors of axon growth, including Nogo, MAG and OMgp, have been the subject of intense research. A myriad of experimental approaches have been applied to investigate the potential of targeting these molecules to promote axonal repair after spinal cord injury. However, there are still conflicting results on their role in axon regeneration and therefore a lack of a cohesive mechanism on how these molecules can be targeted to promote axon repair. One major reason may be the lack of a clear definition of axon regeneration in the first place. Nevertheless, recent data from genetic studies in mice indicate that the roles of these molecules in CNS axon repair may be more intricate than previously envisioned.     

Glial response to axonal injury: in vitro manifestation and implication for regeneration

Crushed fish optic axons readily regenerate, while similarly injured rat optic axons do not; the reasons for the differences in regeneration ability may lie in differences in the environment of the axons. We have cultured glial cells from previously crushed optic nerves of fish and rat to determine whether a relationship exists between the ability to regenerate and the nature of the responses of the associated nonneuronal cells to injury. The glial cells were examined using indirect immunofluorescence with antibodies to known glial markers. In the rat cultures, mature GalC oligodendrocytes, which are known to be nonpermissive for axonal growth, were abundant. In contrast, in the fish cultures mature oligodendrocytes were rare, but A2B5 positive cells were abundant. The high number of A2B5 positive cells in the fish may suggest a high number of immature cells. This interpretation, however, should wait until evidence for glial cell lineage of the fish is available. Additional indication is provided also in the present study that the number of mature oligodendrocytes in the fish is regulated by elements external to the nerve. This study thus demonstrates an important difference between rat and fish optic nerves in the response of glial cells to the optic nerve injury.     

Ischemic axonal injury and its recovery after focal cerebral ischemia

After focal cerebral infarction by occluding the middle cerebral artery (MCA) of the rat, the neuronal death occurred in the ipsilateral thalamic neurons, because axons of the thalamic neurons were injured by infarction and retrograde degeneration occurred in the thalamic neurons. However, cortical neurons adjacent to the infarction survived despite their axons injured by ischemia. We employed immunohistochemical staining for 200 kilodalton (kD) neurofilament (NF), in order to study those responses of cortical and thalamic neurons against axonal injury caused by focal cerebral infarction. In the sham operated rats the immunoreactivity to the anti-200 kD NF antibody was only detected in the axon but not in the cell bodies and dendrites. At 3 days after MCA occlusion, axonal swelling proximal to the site of ischemic injury was found in the caudoputamen and internal capsule of the ipsilateral side. At 7 days after occlusion, cell bodies and dendrites of the neurons in the ipsilateral cortex and thalamus were strongly stained with anti-NF antibodies. At 2 weeks after occlusion these responses disappeared in the cortex, but lasted in the thalamus. These phenomena are caused by stasis of the slow axonal transport, because the NF is transported by slow axonal transport. In the cortical neurons impairment of slow axonal transport recovered in the early phase after injury, but in the thalamic neurons the impairment prolonged up to 3 weeks after occlusion. The early recovery of axonal transport from ischemia seemed to be essential for survival of neurons after ischemic axonal injury.     

Exercise training and axonal regeneration after sciatic nerve injury.

In the present study, we aimed to investigate the relationship between exercise training and peripheral nerve regeneration after crush injury. For this purpose, HRP neurohistochemistry and modified Pal-Weigert methods were used to assess the axonal regeneration. In the 2nd and 3rd regeneration week groups, myelin debris was observed, and there was no significant difference between exercise trained and sedentary groups. In the 4th regeneration week group, it was seen that myelin debris was removed, and some myelinated fibers were observed in the exercise trained group. On the other hand, there was no myelinated fiber in the sedentary group, and there was a significant difference between exercise trained and sedentary groups. Consequently, we think that exercise is effective in the 4th regeneration week.     

A predominantly glial origin of axonal ribosomes after nerve injury

Axonal mRNA transport and local protein synthesis are crucial for peripheral axon regeneration. To date, it remains unclear how ribosomes localize to axons. They may be co‐transported with mRNAs or, as suggested by recent studies, transferred from Schwann cells (SC). Here, we generated transgenic “RiboTracker” mice expressing tdTomato‐tagged ribosomal protein L4 in specific cell types when crossed with Cre lines. Two neuronal RiboTracker‐Cre lines displayed extremely low levels of axonal L4‐tdTomato‐positive ribosomes. In contrast, two glial RiboTracker‐Cre lines revealed tagged ribosomes in sciatic nerve (SN) axons with increasing amounts after injury. Furthermore, non‐RiboTracker dorsal root ganglia co‐cultured with L4‐tdTomato‐expressing SCs displayed tagged ribosomes in axons. These data provide unequivocal evidence that SN axons receive ribosomes from SCs upon injury and indicate that glial cells are the main source of axonal ribosomes.