Fig 2: Hydrogel-evoked FBRs vary and exhibit cellular features of CNS wound response.a Schematic and chemical structures of synthetic hydrogels used as tools to study CNS FBR. b Experimental model of in vivo injections into caudate putamen (CP) of mouse forebrain. c, d Survey and detail images of stromal and inflammatory cells (CD13), astrocytes (GFAP), and neurons (NeuN) at 1 week after injections of PBS, hydrogels, or L-NIO-induced stroke. e–g Quantification of total immunohistochemical staining. e Total CD13 in CP. not significant (NS), ***P < 0.0001 versus PBS injection, one-way ANOVA with Bonferroni. f Total CD13 in either non-neural or neural tissue compartments. NS or ***P < 0.0001 versus PBS injection, two-way ANOVA with Bonferroni. g Total GFAP in CP. NS or ***P < 0.0001 versus DCHMO, one-way ANOVA with Bonferroni. All graphs are mean ± s.e.m with individual data points superimposed showing n = 10, 10, 12, and 4 mice per group for PBS, DCHMO, DCHK, and L-NIO, respectively. AU arbitrary units, G hydrogel, IC internal capsule.

Fig 3: Immunohistochemical staining of retinal cryosections to label microglial cells (TMEM119, antibody by Synaptic Systems, red), ganglion cells (NeuN, green), and astrocytes (GFAP, green) in retinas of young and old mice, as indicated. Only inner layers of the retinas are shown. White arrowheads point to TMEM119-positive retinal ganglion cells. Scale bar: 50 µm.

Fig 4: Phosphorylated MLKL, a marker of necroptosis is reduced in RIP knockout animals. a. Exemplary staining of pMLKL and NeuN (upper panel) and a pMLKL positive neuron at higher magnification (lower panel) in the rim of a traumatic contusion in a wild type mouse. b. pMLKL is significantly reduced in a RIPK3 deficient mouse. c and d p-MLKL signal intensity was significantly increased after TBI in wild type animals (white bars) compared to sham animals indicating significant presence of necroptosis three months after TBI; this increase was significantly blunted in RIPK1 (c) as well as in RIPK3 d mice where there was no difference between TBI and sham animals. Data are presented as mean ± SD; n = 9–10 for RIPK1, n = 8–9 for RIPK3. Two-way RM ANOVA with Tukey’s multiple comparisons test was used. ***p < 0.001, n.s. indicates no significant statistical difference between groups

Fig 5: Validation of CaMKIIa and CaMKIIß antibodies for immunofluorescence in the spinal dorsal horn. a CaMKIIa (green) and NeuN (magenta) immunofluorescence in the superficial dorsal horn of wild-type and CaMKIIa-/- mice. CaMKIIa immunolabeling was essentially absent in CaMKIIa-deficient mice. b CaMKIIß (green) and NeuN (magenta) immunofluorescence in the superficial dorsal horn of wild-type and CaMKIIß-/- mice. CaMKIIß immunolabeling was essentially absent from CaMKIIß-/- mice, apart from a weak staining of myelin. Dashed lines in a and b indicate the dorsal border of lamina I. Micrographs are single optical sections obtained using a 63×/1.4 objective. Scale bar, 10 µm, valid for a and b. c Example of an Iba1+ microglial cell in lamina II of the rat spinal cord devoid of both CaMKIIa and CaMKIIß immunoreactivity. Bottom panels are pseudocolored to better visualize weak immunolabeling. Arrowheads indicate the microglial cell body. Micrographs are singe optical sections obtained with a 63×/1.4 objective. Scale bar, 10 µm