Özugur S et al. (2022), Transcardial injection and vascular distributio...

XB-ART-58928

STAR Protoc 2022 Mar 15;32:101250. doi: 10.1016/j.xpro.2022.101250.

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Transcardial injection and vascular distribution of microalgae in Xenopus laevis as means to supply the brain with photosynthetic oxygen.

Özugur S , Chávez MN , Sanchez-Gonzalez R , Kunz L , Nickelsen J , Straka H .

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Oxygen in vertebrates is generally provided through respiratory organs and blood vessels. This protocol describes transcardial injection, vascular distribution, and accumulation of phototrophic microalgae in the brain of Xenopus laevis tadpoles. Following tissue isolation, oxygen dynamics and neuronal activity are recorded in semi-intact whole-head preparations. Illumination of such microalgae-filled preparations triggers the photosynthetic production of oxygen in the brain that, under hypoxic conditions, rescues neuronal activity. This technology is potentially able to ameliorate consequences of hypoxia under pathological conditions. For complete details on the use and execution of this protocol, please refer to Özugur et al. (2021).

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Species referenced: Xenopus laevis

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	Figure 1. Eukaryotic green algae (C. reinhardtii) and prokaryotic cyanobacteria (Synechocystis 6803) produce oxygen upon illumination(A and B) Microphotographs depicting an overview and higher magnification (insets) of C. reinhardtii (A) and Synechocystis 6803 (B).(C) Setup for measurements of photosynthetic O2 production upon illumination in isolated suspensions of microorganisms.(A–C) adapted from and Özugur (2021) and Özugur et al. (2021), with permission under the CC BY-NC-ND license agreement.
	Figure 2. Setup for transcardial injections of algae suspensions into the vascular system of Xenopus laevis tadpolesThe spatial arrangement of the binocular, micromanipulator, light source and chamber, holding the animals during the injection have been optimized for visibility and accessibility of the tissue for the injection.
	Figure 3. Transcardial injection of algae suspensions completely fill blood vessels with phototrophic microorganisms(A) Ventral view of the exposed heart of a stage 53 Xenopus tadpole depicting the glass capillary injecting a C. reinhardtii suspension visualized by the gradual filling of blood vessels with the microorganisms over a period of 30 min, illustrated as four sequential images each separated by ∼10 min (from left to right); note the progressively augmenting green colorization of the tissue.(B–F) Overview (B–D) and higher magnification images (E and F) depicting a dorsal, ventral and lateral view of a stage 53 Xenopus tadpole after completion of the injection of cyanobacteria (Synechocystis 6803); note that the cyanobacteria have invaded the entire vascular system, likely filling even the smallest blood vessels in the body (E) and tail (F). Calibration bars in (B and E) apply to (C, D, and F), respectively.Adapted from Özugur (2021) with permission under the CC BY-NC-ND license agreement.
	Figure 4. Photosynthetic microorganisms in brain blood vessels(A) Confocal reconstruction of a Xenopus laevis whole brain illustrating isolectin-labeled endothelial walls of blood vessels (magenta) and chlorophyll autofluorescence of transcardially injected Synechocystis 6803 (green).(B–E) Confocal reconstruction of hindbrain tissue, depicting intravascular accumulations of C. reinhardtii (B and D) and Synechocystis 6803 (C and E); phototrophic microorganisms in green due to chlorophyll autofluorescence and isolectin-stained endothelial walls of blood vessels in magenta.Te, telencephalon; Di, diencephalon; OT, optic tectum; Cb, cerebellum; Hb, hindbrain. Adapted from Özugur et al. (2021) with permission under the CC BY-NC-ND license agreement.
	Figure 5. Recording of neuronal activity and O2 production in semi-intact preparations of Xenopus tadpoles(A) Schematic depicting the individual components required to maintain viable semi-intact preparations in a recording chamber; frog Ringer solution is hydrostatically supplied from an elevated storage vessel through a tube system that allows an adjustment of the temperature (ice box) and the O2 level (by aeration with N2 or carbogen in an antechamber); recording electrodes for neuronal activity and O2 are positioned with micromanipulators.(B) Images of the antechamber and the recording chamber.(C) Photograph, depicting a semi-intact head preparation and the placements of the electrodes for the recording of O2 concentration dynamics in the IVth ventricle (blue electrode) and of multiunit spike discharge from the superior oblique motor nerve (pink electrode). OT, optic tectum; Te, telencephalon. (C), adapted from Özugur et al. (2021) with permission under the CC BY-NC-ND license agreement.

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