Cellular and molecular mechanisms associated with ischemic stroke severity in female mice with chronic kidney disease

Ischemic stroke is highly prevalent in chronic kidney disease (CKD) patients and has been associated with a higher risk of neurological deterioration and in-hospital mortality. To date, little is known about the processes by which CKD worsens ischemic stroke. This work aimed to investigate the cellular and molecular mechanism associated with ischemic stroke severity in an in vivo model of CKD. CKD was induced through right kidney cortical electrocautery in 8-week-old female C57BL/6 J mice followed by left total nephrectomy. Transient middle cerebral artery occlusion (tMCAO) was performed 6 weeks after left nephrectomy. Twenty-four hours after tMCAO, the infarct volumes were significantly wider in CKD than in SHAM mice. CKD mice displayed decreased neuroscore, impaired ability to remain on rotarod device, weaker muscular strength and decreased prehensile score. Apoptosis, neuronal loss, glial cells recruitment and microglia/macrophages M1 signature genes CD32, CD86, IL-1β, IL-6, MCP1 and iNOS were significantly increased within ischemic lesions of CKD mice. This effect was associated with decreased AMP kinase phosphorylation and increased activation of the NFΚB pathway. Pharmacological targeting of AMP kinase activity, which is known to block microglia/macrophages M1 polarization, appears promising to improve stroke recovery in CKD.


Supplementary
Stereotaxic coordinate system does not have a single universally accepted reference point. In the mice the most common reference points are bregma, lambda and the interaural line (IAL) In this study, Bregma was used as the zero reference. A. Stereotaxic atlases coordinate system. Bregma and Lambda are intersections of bone plates on the dorsal skull surface. The diagram indicates the approximate position of the interaural line, but this reference point is not located anatomically. B. Example of stereotaxic regions location using bregma as a zero reference. Commonly, brain structures located between the olfactory bulbs and the bregma (i.e before the zero reference) display positive coordinates. These coordinates represent the distance in millimeter between brain structures (located in the rostral side) and the bregma. This means that the more a structure is close to the olfactory bulbs, the greater is its distance in millimeter from the bregma. Conversely, brain structures located between the bregma and the cerebellum display negative bregma coordinates (they are located behind the zero reference).

Supplementary Figure 2: Stereotaxic regions used to quantify brain ischemic volumes.
A. Coordinates of the 12 stereotaxic sections used to quantify brain ischemic volumes. This schematic representation has been created based on K. Franklin and G. Paxinos stereotaxic atlas (The Mouse Brain in Stereotaxic Coordinates, Academic Press, 2012). B. Examples of a microscope slide stained with cresyl-violet. It is well established that 2 to 6 hours after tMCAO, the histological infarct core is limited to the striatum. Subsequently the core expands Cresyl-violet A.

B. C.
to involve most of the cortical tissue supplied by the middle cerebral artery. This occurs quite rapidly and is complete within 6-12 hours after stroke onset. C. Description of section 6. All the immunohistological analyses were performed on the brain region displaying the coordinates: Bregma 0.00 mm (section 6). In this region, the ischemic core (striatum) is separated from the ischemic penumbra (cortex) by the corpus callosum, a structure that can be easily identified and allows to differentiate the two zones. Correlation between the percent of weight loss (before vs after tMCAO) and brain total infarct volume C. Correlation between mice weight after tMCAO and total infarct volume. Statistical analysis was performed with a non-parametric Spearman correlation test. n = 27 mice (14 SHAM / 13 CKD).

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-Prehensile test: A prehensile test was performed by using a horizontal stainless steel wire 41 (length: 60 cm; diameter: 3 mm) placed 40 cm above a foam pad. The mouse's forepaws were 42 placed onto the wire and the animal was released. The time until the animal fell and the 43 animal's ability to grab the wire with a hind paw were measured. The tested animals were 44 scored as follows: 3 points for holding onto the wire for more than 10 seconds; 2 points for 45 holding on for between 5 and 10 seconds on the wire; 1 point for holding on for between 1 46 and 5 seconds; 0 point for not being able to hang on. An additional point was added if the 47 animal managed to grab the wire with a hind paw.

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-Grip-test: The muscular strength of mice forelimbs was assessed using a grip strength test 49 (Bioseb, Vitrolles, France). The grip strength meter was positioned horizontally and the mice were held by the tail and lowered towards the apparatus. The animals were allowed to grab 51 the metal grid and were then pulled backwards in the horizontal plane. The force applied to 52 the grid just before the animals lost grip was recorded as the peak tension. The muscular 53 strength of mice forelimbs was assessed 5 times per session and the mean of the 5 54 measurements was used for evaluation.

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Immunohistochemical examination of the ischemic area 57 The sections dedicated to immuno-histology were cut with a thickness of 20 µm, i.e. thinner 58 than sections dedicated to cresyl violet staining, in order to facilitate antibodies penetration 59 within brain tissues. Sections were fixed with 4% ice-cold PFA for 5 min at room temperature Briefly, sections of 20-μm were fixed with 4% ice-cold PFA for 5 min at RT and 78 incubated in sodium citrate (1M, pH=6) during 20min at 100°C for antigen retrieval. Section 79 were then quenched in 100 mmol/L glycin in PBS for additional 10 min and permeabilized for 80 1h at RT with 0.3% triton X-100 in PBS containing 1% BSA. Non-specific binding of the