Spruiell Eldridge SL, Teetsel JFK, Torres RA, Ulrich CH, Shah VV, Singh D, Zamora MJ, Zamora S, Sater AK.

no number Robert J. Kleberg Jr and Helen C. Kleberg Foundation

             astrocyte activation

	Graphical Abstract
	Figure 1. Focal Impact Injury leads to Edema and Disruption of BBB integrity. Sham tadpoles were anesthetized and transferred temporarily to the injury cradle prior to intraventricular injection with sodium fluorescein (NaF) either 24 h post-injury (h.p.i.) (A) or 48 h.p.i (C). Injured animals were anesthetized and then subjected to focal impact injury at the optic tectum (white arrows). Following either 24 h (B) or 48 h (D) of recovery post-injury, tadpoles were given an intraventricular injection with 10 nL of 1 μg/mL NaF; diffusion of the tracer was visualized using fluorescence microscopy. Arrows in (B,D) show the site of injury. (A–C) White dotted lines indicate the outer borders of the brain. Images are representative of n = 6 animals per treatment group for each time point. (E) * p < 0.05; *** p < 0.001.
	Figure 2. Focal impact injury causes deficits in visually mediated behaviors. NF stage 55 Xenopus laevis tadpoles were evaluated for baseline behavior at 0 h.p.i. in each behavioral assay, then a focal impact injury was administered to the optic tectum (OT) of each animal in the injured group (pink). Sham animals (blue) were anesthetized and moved in and out of the injury cradle after Day 0. (A) Open field testing, C-Start reflex. Open field tests were performed once per day up to 72 h.p.i. following a 0 hpi baseline. The number of C-Start reflexes performed in the injured group was significantly reduced at 24 and 48 h.p.i. (B) Light/Dark box assay. Open field tests were performed once per day up to 72 h.p.i. following a 0 hpi baseline. Light zone time was significantly increased in injured animals at all three timepoints. (C) Visual avoidance behavioral assay. Baseline data were collected at 0 h.p.i., then the injury or sham injury was administered. The test was performed daily to assess the recovery of the visual avoidance behavior. Beginning 48 h.p.i., injured tadpoles display a significant deficit in the avoidance behavior, which they appear to recover over a time course of 7 d.p.i. [Significant p-values from two-way analysis of variance (ANOVA) are shown as * p  <  0.05, ** p  <  0.01, n.s. indicates no significant difference].
	Figure 3. Astrocytes visualized with antibodies against Aldh1L1 via immunofluorescence microscopy. Sections from NF stage 55 N-tubulin-GFP+ Xenopus laevis midbrains of both sham (A–C) and injured (D–F) animals at 3 h post-injury show astrocytes (aldh1L1-positive) in the dorsolateral midbrain and optic tectum (OT). Astrocytes in midbrains 3 h after injury show differences in morphology (B vs. E) and distribution (A vs. D); these differences persist at 24 h post-injury. (G–I), sham; (J–L), injured. (For all images, Alexa Fluor 647 signal was digitally converted to magenta. Sections were also processed to visualize neurons (anti-GFP, green), nuclei (DAPI, blue), and intermediate filaments (anti-Vimentin, red). Sections are 14 µm thick and images selected are representative of all samples examined (n = 6 brains).
	Figure 4. Microglia localize around the ventricle of the injured midbrain. Confocal images of midbrains 3 h after injury; midbrains were isolated from N-tubulin-GFP tadpoles (neurons, green) injected with Ib4-Alexa 647 to label microglia (magenta). Brain were sectioned at 15 µm; nuclei are labeled with DAPI (blue). (A,B) Sham-treated midbrain. (C–E) Midbrain 3 h after injury. (E) Microglia accumulate near the site of injury. (A,C): Scale bar 100 µm. (B,D,E): Scale bar 20 µm. Scale 100 µm. (F–K), confocal images of St. 55 midbrains after 24 h. In the sham brain, microglia are visible both close to the ventricle (G) and toward the periphery of the neuropil (H). In the injured midbrain, microglia form clusters both near the ventricles (J) and in the neuropil near the site of injury (K). (F,I): Scale bar 100 µm. (G,H,J,K): Scale bar 20 µm. Asterisks in (C,I) mark the site of injury (n = 3). * indicates the site of administration for the focal impact injury.
	Figure 5. Evidence of phagocytic astrocytes and microglia following injury. Injured midbrains 24 h post-injury include both astrocytes (D–F) and microglia (J–L) that are positive for N-tubulin-GFP (white arrowheads), as well as for aldh1l1 (astrocytes) or IB4 (microglia); merged fluorescent signal appears white. Magenta (A–F), aldh1l1; Magenta 9 (G–L), IB4; Green, N-Tubulin-GFP; Blue, DAPI. Sham brains (A–C,G–I) contain no more than a few such cells. scale bars, 20 µm.
	Figure 6. Persistence of altered astrocyte morphology and expression of astroglial genes following injury. Visualization of astrocytes (aldh1l1-positive cells, magenta), neurons (N-tubulin-GFP), radial glia (vimentin-positive, red) in sham (A,B) and injured (C–E) 48 h after injury. Amoeboid astrocytes accumulate near the ventricular layer (C,E). Quantitative RT-PCR assays for expression of astroglia-associated genes Aldehyde dehydrogenase 1 family member 1 (aldh1l1) (F), fatty acid-binding protein 7 (fabp7) (G), aquaporin4 (aqp4) (H), vimentin (vim) (I), excitatory amino acid transporter 1 (glast) (J), and nestin (nes) (K). RNA was isolated from sham and injured midbrains at the intervals shown; injured values are normalized to sham-treated controls. Plots show individual data points and a box showing the mean and 95% confidence intervals. n ≥ 6 experiments.
	Figure 7. Expression of genes associated with astrocyte reactivity and neuroprotection. Quantitative RT-PCR assays for expression of the “reactive astrocyte”-associated genes steap4 (A), and timp1 (B), as well as the neuroprotective genes brain-derived neurotrophic factor (bdnf) (C), ubiquitin C-terminal hydrolase-1 (uchl) (D), clusterin (clus) (E), and mesencephalic astrocyte-derived neurotrophic factor (manf) (F). RNA was isolated from sham and injured midbrains at the intervals shown; injured values are normalized to sham-treated controls. Plots show individual data points and a box showing the mean and 95% confidence intervals. n ≥ 6 experiments.
	Figure 8. The microglial response to midbrain injury extends to the forebrain. Confocal images of microglia (IB4-positive cells) in sham (A) and injured (B) forebrains 48 h after injury. Microglia accumulate within the ipsilateral forebrain, at a distance from the injury site at the optic tectum (not pictured). The images are characteristic representations of all samples (n = 3). Brain sections are 50 µm thick. Scale 100 µm. (C–E), Time course of expression of microglia-associated genes following focal impact injury. RNA was isolated from sham and injured midbrains at the intervals shown; injured values are normalized to sham-treated controls. Plots show individual data points and a box showing the mean and 95% confidence intervals. N ≥ 4 experiments.
	Figure 9. Time course of expression of inflammation-associated genes following injury. Quantitative RT-PCR assays for expression of the inflammatory cytokines Interleukin1-β (il1β) (A), Interleukin6 (il6) (B), Tumor Necrosis Factor-α (tnf-α) (C), as well as Nuclear Factor kB (nfkB) (D), matrix metalloproteinase 9 (mmp9) (E), and arginase (arg) (F). RNA was isolated from sham and injured midbrains at the intervals shown; injured values are normalized to sham-treated controls. Plots show individual data points and a box showing the mean and 95% confidence intervals. N ≥ 4 experiments.
	Figure 10. A timeline of response to focal impact injury. The response to focal impact injury begins with the release of inflammatory cytokines, followed by microglial activity, astrocyte reactivity, and edema. Within 48 h, each of these activities has peaked, and all have subsided to near-baseline levels after one week (This image was made using BioRender (Suite 200, Toronto, ON, M5V 2J1)).
	Figure A1. Initial comparisons of focal impact and pressure injury. Midbrain of a st. 48 N-tubulin-GFP tadpole 72 h after focal impact injury. Asterisks mark injured side. (A) Enlarged image of injury site; arrowheads indicate cells that have migrated into the injury area. (B) Entire section shows differences between injured and uninjured sides. Arrowheads show loss of axonal material at the injury site. Red, anti-vimentin (astrocytes and radial glia); Blue, DAPI (nuclei); Green, N-Tubulin-GFP (axons). (C) Quantitative RT-PCR of gene expression in brains from tadpoles subjected to focal impact injury shows expression profile of selected genes at 24, 48, and 72 h post-injury. Genes include markers of reactive astrocytes (timp1, steap4), astrocytes (GLAST, aqp4), radial glia (nestin), and neuroprotective genes (bdnf, clusterin). Open field assays show behavioral alterations in response to focal impact injury (D,E). (D) Comparison of active time in sham vs. injured tadpoles measured during a 10-min interval. Tadpoles were not prescreened for baseline activity. N ≥ 10 tadpoles/ sample. (E) Comparison of pause duration in sham vs. injured tadpoles from the same recordings. N ≥ 10 tadpoles/sample. (F) Quantitative RT-PCR of gene expression in brains subjected to pressure injury at 24, 48, 72, and 120 h post-injury. Genes include markers of astrocytes (vimentin, fabp7, aqp4, xfia2), reactive astrocytes (timp1, steap4), inflammation (cxcl2), and radial glia (nestin). Open Field assays of tadpoles subjected to pressure injury (G,H). (G) Comparison of active time in sham vs. pressure-injured tadpoles measured during a 10-min interval. Again, tadpoles were not prescreened for baseline activity. N = 50 tadpoles/sample. (H) Comparison of pause duration in sham vs. pressure-injured tadpoles from the same recordings. N = 50 tadpoles/sample. (I) Dramatic growth in brain size during the first half of metamorphosis (st. 48–56).

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