We have studied the time course of regeneration after giving a crush injury to the adult zebrafish spinal cord. To characterize the injury, we have documented the basic cellular events during regeneration at different time points after injury by using H&E, luxol fast blue, and cresyl violet (Fig. 1), trichrome (Fig. 2) staining, as well as ultrastructural analysis by TEM (Figs.1 and 2). Immediately after injury, there is a loss of the blood-brain barrier, which results in infiltration of blood cells to the injury site, disruption of ependyma, and a substantial loss of tissue, both in white and grey matter (Figs. 1B and 2A). Infiltrating blood cells include macrophages as evidenced by their phagocytic nature (Fig. 2D–G), polymorphonuclear neutrophils (Fig. 2H), monocytes with a horse-shoe-shaped nucleus (Fig. 2I), and red blood cells (RBC; Figs. 1C, 2B), all of which can be seen in 3-day post-injury (dpi) cord. These macrophages are most likely blood-derived since they are located in the close vicinity of the blood vessel (Fig. 2F). A substantially higher number of macrophage infiltrations can be demonstrated at the injured site in 3dpi cord and many macrophages are present around a dying neuron (Fig. 2F), which have a characteristic electron-dense fragmented body in nucleus and retracted cytoplasm (Naganska and Matyja, 2001). At the same time point, damaged axons are shown about to be engulfed by macrophages (Fig. 2E), whereas 10dpi cord shows fewer macrophages at the site of injury (Fig. 2G), although lipophilic inclusions, vacuoles, and some digested residues of myelin debris that were engulfed earlier are present inside the macrophage. Presence of RBC can be predominantly seen close to the injury epicenter in 1-, 3-, and 5dpi cord (Fig. 2A–C) and afterwards the infiltration of RBC decreased considerably (data not shown). Both 1dpi and 3dpi cord showed damaged tissue in grey matter, as disruption of ependyma and compression of the cord is evident (Figs. 1B,C,K, 2A,B). Tissue edema can be observed as a consequence of injury in 3- to 7dpi cord and is reduced afterwards. Loss of white matter (Figs. 1B,C,K, 2A,B), can also be confirmed in injured cord both by luxol fast blue staining (Fig. 1K) and by ultrastructural analysis, which revealed demyelination of axons (Fig. 1O,P) when compared to normal uninjured cord (Fig. 1N). There are accumulations of some cells around the cut ends of ependyma as early as 3dpi, which replace the initial loss of tissue. Cells around the ependymal canal begin to migrate and initiate ependymal sealing at the same time (Fig. 1C, C.1). Accumulation of cells around the injury site continues to occur at 5dpi and 7dpi (Fig. 1D and E, respectively). Progressive ependymal sealing, which is an early regenerative event, can be observed at around 5dpi (Fig. 1D, D.1) when ependymal bulb formation involving ependyma and the ependymal canal is obvious. As both ends of the ependymal bulb join, there is continuation of the ependymal canal, which forms a cavity-like structure in the grey matter of 7dpi (Fig. 1E) and 10dpi cord (Fig. 1F). It is worth mentioning that the size of the cavity-like structure observed at different levels in subsequent sections (data not shown) of 7dpi cord is quite large (1E). The size reduces later at day 10 (Fig. 1F) and further regression can be seen after 15dpi (Fig. 1G and G.1) and at 1 month of injury (Fig. 1H, L) as regeneration progresses. These cavity-like structures are seen only in the grey matter lined by ependymal cells and may not be equivalent to what we see in mammalian SCI, which is a fluid-filled astrocyte-lined cavity present in white matter (Balentine, 1978a, b; Fitch et al., 1999; Renault-Mihara et al., 2008). We also observed newly formed neuronal cell bodies at the injury epicenter, which are small in size at 10dpi cord (Fig. 1F.1) and identity of these cells is further confirmed by using neuronal markers like Hu along with luxol fast blue/cresyl violet staining (see Fig. 6H–K). Both grey matter and white matter regeneration has occurred by day 15 post-injured cord. A regenerated ependyma, subependyma with newly formed neurons in the injury epicenter, and regenerating axons in white matter (Fig. 1G and G.1) can be observed, although white matter volume has not regained the original volume that was present before injury. We followed the regeneration time course for a much longer time period, up to 1 month (Fig. 1H and H.1, I) and 1.5 months post-injury (data not shown). Complete regeneration of ependyma is observed at 1 month post-injured cord with reappearance of lost neurons in the subependyma near the injury epicenter (Fig. 1H and H.1, L and L.1) and axons (Fig. 1L and L.1). Regeneration of white matter took place without formation of a cavity, but even at 1 and 1.5 month(s) after injury, full white matter volume was not regained.
Figure 2. Trichrome staining (A-C) and ultrastructural analysis (D-I) of injured zebrafish spinal cord. A: 1dpi cord shows infiltration of large number of RBC (↓) both at the injury epicenter (double arrowheads) and also towards the normal part of the cord. B: A 3dpi cord showing less number of infiltrating R.B.C. C: A 5dpi cord shows presence of very few blood cells and the appearance of ependymal bulb (epb) at the injury epicenter. A.1, B.1, C.1: Higher magnification representation of A, B, and C, respectively, showing the presence of blood cells at the injury epicenter. D–G: Presence of macrophages (ma) at the injury epicenter in 3dpi (D–F) and 10dpi (G) cord. D, E: Macrophage (ma) with a typical phagocytic process (↑) and in close proximity to a demyelinating axon (dax), respectively. F: A dying neuron (n) with condensed nuclear matter surrounded by infiltrated macrophages (ma) and a blood vessel (bv) nearby. G: Macrophage (ma) with many lipophillic vacuoles (▸) and phagocytosed myelin debris (thick arrow). H, I: The presence of neutrophils (neu) and monocytes (mon), respectively, in 3dpi cord. Scale bar = 250 μm (A–C), 50 μm (A.1–C.1), 5 μm (D, F), 2 μm (H, I), 1 μm (E, G).
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TEM analysis showed presence of oligodendrocytes and myelinated axons with compact myelin sheath and sparsely distributed neurofilament in the axonal body in uninjured cord (Fig. 1M,N). In 3dpi cord, the axons are denuded and typical features of these demyelinated axons are the presence of denser neurofilament within the axonal body (Fig. 1O,P), whereas myelin sheaths are either less compact with irregular shapes or are disintegrating. We observed the presence of Schwann cells around the injured demyelinated axons in 10dpi cord (Fig. 1R), although no Schwann cells are observed in uninjured cord and Schwann cells are seen dividing (Fig. 1Q), which would probably start remyelination (Fig. 1R). Later on, some of these cells are wrapping the axons through basal membrane, suggesting that these cells are indeed involved in the remyelination process after injury (Fig. 1S and T). In regenerated cord 1 month post-injury, the presence of remyelinated axons with thinner myelin sheaths at the injury epicenter can be seen (Fig. 1S,T) when compared to uninjured cord (Fig. 1N).