Extended Data Fig. 1: Development of a novel model of haemodynamic instability in rodents.

Step 1 (a): We first tested the capacity for an orthostatic challenge to reduce blood pressure in rats 30 days after a T3 spinal cord injury (n = 4). Tilting rats 90° upright did not lead to any reduction in systolic blood pressure (one-way repeated measures ANOVA; F₃ = 0.612; P = 0.62), diastolic blood pressure (one-way repeated measures ANOVA; F₃ = 1.105; P = 0.40), or mean arterial pressure (one-way repeated measures ANOVA; F₃ = 0.915; P = 0.47). Data are mean ± s.e.m. Step 2(b): To confirm that our contusion model disrupted descending control of haemodynamics we used AAV-DJ-hSyn-flex-mGFP-2A-synaptophysin-mRuby injected into the RVLM of TH-Cre rats (n = 4) one month after T3 spinal cord injury. We found a near complete disruption of descending TH^ON sympatho-excitatory axons (paired one-tailed t-test; t = 5.08; P = 0.007) and synapses (paired one-tailed t-test; t = 4.64; P = 0.009) when comparing counts above and below the injury. Bar charts represent the mean with raw data overlaid. Step 3(c): Overview of the time-course used to examine the natural history of haemodynamic instability in uninjured (n = 6) and spinal cord injured animals (n = 6). Confirmation that the lesion site spared minimal white matter (identified using GFAP; mean = 2%). Step 4(d): We implanted a wireless recording system to monitor haemodynamics and sympathetic nerve activity. A blood pressure cannula was inserted into the abdominal aorta and microelectrodes sutured to the sympathetic renal nerve. Step 5(e): Data were recorded 24/7 and automatically uploaded to a server where automated analyses were triggered to quantify blood pressure and sympathetic nerve activity throughout the day and night. Step 6(f): We established the natural history of haemodynamic instability by recording the response to spinal cord injury and automatically detecting outliers (see Methods, ‘Haemodynamic and sympathetic nerve activity monitoring’) for blood pressure and sympathetic nerve activity data for a total of 7 weeks. Step 7(g): Because rats do not exhibit haemodynamic instability in response to an orthostatic challenge (see ‘Step 1(a)’), we developed a servo-controlled negative-pressure approach whereby animals are placed in a chamber and the pressure is dropped and monitored in closed loop (see Methods, ‘Implementation of a simulated orthostatic challenge in rodents and non-human primates’). *P < 0.05; **P < 0.01; ***P < 0.001.

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