Section 2
Methods
The Program 07 salvage protocol as executed on subject MU-01, transcribed from the pre-registered document (KITC-VET-07, v4.2) with deviations noted in Section 2.7 of the supplementary appendix. All timestamps derive from a single synchronised theatre clock (network time protocol, drift under 40 ms) shared by the perfusion pump, the electroencephalograph, the ventilator and the video record, so that every value reported here is co-registered to the same time base.
2.1 Subject and certification
Subject MU-01 was a 9-year-old intact male Shiba Inu, 11.4 kg, with no prior neurological history. Death was certified independently by two attending veterinarians on the basis of asystole on continuous three-lead ECG for greater than 5 minutes, absent brainstem reflexes, and absent spontaneous respiration. Certification preceded any Program 07 intervention and was not performed by any member of the research team.
2.2 Consent and governance
Enrolment followed pre-existing written owner consent under the institute's post-mortem veterinary research programme (KITC-IRB-2024-118). An independent monitor was present in theatre throughout. The protocol permitted immediate withdrawal at the monitor's sole discretion at any stage, including after return of circulation.
2.3 Access and washout
Bilateral femoral arterial and venous access was established by open cut-down at T+00:07. Circulating blood volume was displaced with 4.2 L of KRP-7 at 6 °C over 6 minutes 20 seconds until venous effluent haematocrit fell below 3 %. Flow was non-pulsatile and held at 45 mL·kg⁻¹·min⁻¹.
2.4 Hypothermic hold
Core oesophageal temperature reached the target of 18.2 °C at T+00:24 and was held to ±0.3 °C. Estimated cerebral metabolic rate at target was 11 % of normothermic baseline. Total hold duration was 17 minutes; no oxygen was delivered during the hold.
2.5 Graded reoxygenation
Oxygen tension was raised in nine pre-specified steps (Table 2) with simultaneous rewarming at 0.7 °C per 10 minutes. Advance to each step required frontal near-infrared saturation to be stable for 120 seconds and lactate flux to be non-increasing. The schedule was designed to prevent the oxidative burst that dominates injury in flash-reperfusion controls.
2.6 Monitoring and endpoints
Continuous 8-channel EEG, bilateral near-infrared spectroscopy, invasive arterial pressure, serial arterial blood gas at 5-minute intervals, and serum neurofilament light chain at 0, 6, 24, 72 and 168 hours. The pre-registered primary endpoint was return of coordinated cortical activity, defined as inter-hemispheric coherence above 0.4 in the 4 to 12 Hz band sustained for 60 seconds. Secondary endpoints, in hierarchical testing order, were return of spontaneous circulation without inotropic support, quantitative EEG spectral power at day 6, handler voice discrimination at day 9, and survival to 180 days.
2.7 Perfusate manufacture and quality control
KRP-7 was compounded under ISO 14644-1 Class 5 conditions from United States Pharmacopeia grade constituents, sterile-filtered at 0.22 µm, and released against endotoxin below 0.25 EU·mL⁻¹ by kinetic chromogenic assay. Each lot was verified for osmolality (298 to 306 mOsm·kg⁻¹), pH at 6 °C (7.58 to 7.64), and KI-118 concentration by reverse-phase high performance liquid chromatography with a coefficient of variation under 3 percent. Lot KRP7-2601-B was used throughout the case; retained samples are archived at minus 80 °C for independent assay.
2.8 Circuit configuration
The circuit comprised a centrifugal pump, a polymethylpentene hollow-fibre oxygenator with an integrated heat exchanger rated to 3.5 kW, a 40 µm arterial line filter, an in-line haemoconcentrator, and dual independent thermistors at the arterial outflow and the oesophagus. Priming volume was 620 mL. Gas blending used calibrated mass flow controllers so that fraction of inspired oxygen could be commanded in increments of 1 percent, which is the resolution required to execute the nine-step schedule with the reported fidelity. A redundant pump head and a second oxygenator were primed in parallel and never required.
2.9 Tissue controls and blinding
Cortical, hippocampal, renal and hepatic needle biopsies were taken at fixed intervals from six flash-reperfusion control animals and from four normothermic ischaemia references, processed to paraffin and to glutaraldehyde-fixed resin for electron microscopy, and coded before analysis. Two neuropathologists scored spectrin breakdown, cytochrome c translocation, terminal deoxynucleotidyl transferase labelling and mitochondrial cristae integrity independently while blinded to arm and to time point, with disagreements adjudicated by a third reader. Inter-rater agreement on the primary histological marker was 0.91 by Cohen kappa.
2.10 Protocol deviations
Three deviations were recorded and reported to the monitor in real time. Step 4 of the reoxygenation schedule was held for an additional 4 minutes 10 seconds after arterial lactate rose by 0.4 mmol·L⁻¹. Step 7 was held for 2 minutes 30 seconds for the same reason. The 72-hour neurofilament sample was drawn 41 minutes late owing to venous access difficulty. No deviation altered a pre-registered endpoint definition, and none was judged by the monitor to affect interpretability.
Table 1
KRP-7 perfusate composition
| Component | Concentration | Role |
|---|---|---|
| Sodium chloride | 118 mmol·L⁻¹ | Osmotic base |
| Potassium chloride | 3.6 mmol·L⁻¹ | Deliberately sub-physiological |
| THAM buffer | 22 mmol·L⁻¹ | pH stability at low temperature |
| Cyclosporin-A analogue KI-118 | 1.4 µmol·L⁻¹ | mPTP inhibition |
| Sodium thiosulfate (H₂S donor) | 0.9 mmol·L⁻¹ | Metabolic suppression |
| Calpain antagonist SNJ-1945 | 6 mg·kg⁻¹ | Proteolysis arrest |
| Hydroxyethyl starch 130/0.4 | 40 g·L⁻¹ | Colloid oncotic support |
| Glutathione + ascorbate | 3.0 / 1.2 mmol·L⁻¹ | Radical scavenging |
Table 2
Nine-step reoxygenation schedule
| Step | Elapsed (h:mm) | FiO₂ (%) | Flow (mL·kg⁻¹·min⁻¹) |
|---|---|---|---|
| 1 | 0:00 | 21 | 45 |
| 2 | 0:08 | 26 | 45 |
| 3 | 0:18 | 32 | 50 |
| 4 | 0:29 | 38 | 55 |
| 5 | 0:41 | 46 | 60 |
| 6 | 0:54 | 55 | 65 |
| 7 | 1:08 | 66 | 70 |
| 8 | 1:22 | 78 | 72 |
| 9 | 1:36 | 92 | 75 |