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  Indian J Med Microbiol
 

Figure 1: Strategies for fractionation of optic nerve cells and residual tissue and for targeted deflection resulting in re-innervation. (A–D) A schematic diagram to depict outcome from multiple multi-omics analyses of optic nerve cells and residual tissue. The idea is to obtain clean fractionation of cells and the rest of the tissue environment and perform their multi-omics analyses. (A) The approach of fractionation of isolated retinal ganglion cells (RGCs) from the residual environment and the subsequent multi-omics of both, separately. An ideal approach will be as if a tweezer that could just isolate and pick up RGCs for analysis. As described in the text of the perspective there is no such tweezer or ideal fractionation currently available. However, careful longitudinal sectioning of the optic nerve cylinder and use of a scalpel under microscope to separate fibers to the extent possible, followed by a collagenase treatment may loosen the fibers. After fiber loosening, the cholera toxin B coupled beads binding of RGCs and biochemical approaches to isolate or fractionate bead-bound RGCs will be a step towards this direction. (B) The cross-sectional view of the optic nerve to depict major cell types. A surface marker for each cell type has been shown in grey: myelin-oligodendrocyte glycoprotein (MOG), myelin protein P0 (MPZ), Ionized calcium-binding adaptor molecule 1 (Iba1), α2 isoform of Na(+), K(+) ATPase (α2 NKA) and lipid GM1 for oligodendrocyte, Schwann, microglia, astrocyte and RGC cell types respectively, as indicated. The idea is to fractionate each cell type and subject them to analyses. Three distinct strategies are detailed to complement information: (1) to use conditional adult genetic ablation of different cell types (RGCs, as noted in panel A, astrocytes, microglia, Schwann cells and oligodendrocytes). The analysis of fractionated cells and tissues for each ablation type may inform the changes in omics specially lipidome and metabolome changes. (2) The fractionation of all gross cell types using potential surface marker binding-beads and analyzing them. This will primarily inform non-secretome content of the cells. (3) The analysis of cultured secretome of the isolated cells (as in 2 above). This will inform their secretome albeit in an artificial environment. (C) The fractionated analysis of RGCs and residual environment in P0–P5 (plasticity permissive) and higher time points (plasticity non-permissive). (D) The outcome of multi-omics is likely to inform comprehensively about intrinsic and extrinsic factors, plasticity breaks and about potential deflectors to test in animal models of regeneration and re-innervation to restore functional vision. (E, F) The targeted controlled deflection for re-innervation. The idea is to enable targeted deflection of the long distance regenerated RGCs for re-innervation. (E) The depiction of the need for targeted deflection, while some long distance regenerated neurons may travel a straight path, others, may need to be deflected towards ipsilateral (or contralateral as it may be) side. A convoluted arrow represents the deflectors. (F) The depiction of deflection using laser spots generating heat (optical guidance) or using fine-tuned concentration of specific lysolipids such as lysophospatidic acid (LPA).

Figure 1: Strategies for fractionation of optic nerve cells and residual tissue and for targeted deflection resulting in re-innervation.
(A–D) A schematic diagram to depict outcome from multiple multi-omics analyses of optic nerve cells and residual tissue. The idea is to obtain clean fractionation of cells and the rest of the tissue environment and perform their multi-omics analyses. (A) The approach of fractionation of isolated retinal ganglion cells (RGCs) from the residual environment and the subsequent multi-omics of both, separately. An ideal approach will be as if a tweezer that could just isolate and pick up RGCs for analysis. As described in the text of the perspective there is no such tweezer or ideal fractionation currently available. However, careful longitudinal sectioning of the optic nerve cylinder and use of a scalpel under microscope to separate fibers to the extent possible, followed by a collagenase treatment may loosen the fibers. After fiber loosening, the cholera toxin B coupled beads binding of RGCs and biochemical approaches to isolate or fractionate bead-bound RGCs will be a step towards this direction. (B) The cross-sectional view of the optic nerve to depict major cell types. A surface marker for each cell type has been shown in grey: myelin-oligodendrocyte glycoprotein (MOG), myelin protein P0 (MPZ), Ionized calcium-binding adaptor molecule 1 (Iba1), α2 isoform of Na(<sup>+</sup>), K(<sup>+</sup>) ATPase (α2 NKA) and lipid GM1 for oligodendrocyte, Schwann, microglia, astrocyte and RGC cell types respectively, as indicated. The idea is to fractionate each cell type and subject them to analyses. Three distinct strategies are detailed to complement information: (1) to use conditional adult genetic ablation of different cell types (RGCs, as noted in panel A, astrocytes, microglia, Schwann cells and oligodendrocytes). The analysis of fractionated cells and tissues for each ablation type may inform the changes in omics specially lipidome and metabolome changes. (2) The fractionation of all gross cell types using potential surface marker binding-beads and analyzing them. This will primarily inform non-secretome content of the cells. (3) The analysis of cultured secretome of the isolated cells (as in 2 above). This will inform their secretome albeit in an artificial environment. (C) The fractionated analysis of RGCs and residual environment in P0–P5 (plasticity permissive) and higher time points (plasticity non-permissive). (D) The outcome of multi-omics is likely to inform comprehensively about intrinsic and extrinsic factors, plasticity breaks and about potential deflectors to test in animal models of regeneration and re-innervation to restore functional vision. (E, F) The targeted controlled deflection for re-innervation. The idea is to enable targeted deflection of the long distance regenerated RGCs for re-innervation. (E) The depiction of the need for targeted deflection, while some long distance regenerated neurons may travel a straight path, others, may need to be deflected towards ipsilateral (or contralateral as it may be) side. A convoluted arrow represents the deflectors. (F) The depiction of deflection using laser spots generating heat (optical guidance) or using fine-tuned concentration of specific lysolipids such as lysophospatidic acid (LPA).