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. 2025 Mar 19;8(8):1493–1502. doi: 10.1002/ame2.70014

Impact of different anesthetic protocols during anesthesia for the establishment of a porcine model of acute kidney injury

Axel Guilpin 1,2, Mathieu Magnin 2,3, Axel Aigle 1, Timothée Schuhler 2, Jean‐Yves Ayoub 2, Romain Lac 2, Charlotte Slek 2, Thomas Brichart 1, Abdessalem Hammed 2, Vanessa Louzier 2,3,✉
PMCID: PMC12464876  PMID: 40108868

Abstract

Background

During the establishment of a model of acute kidney injury (AKI) in pigs, we observed a high prevalence of malignant hyperthermia (MH). These complications led us to refine the anesthetic protocol. This publication describes the impact of the choice of anesthetics on the results obtained.

Methods

Pigs were euthanized at the end of the procedure, without recovery from anesthesia. Three anesthetic protocols were used: sevoflurane inhalation (ProtocolA, n = 5), a combination of ketamine, medetomidine and diazepam by intravenous infusion (ProtocolB, n = 5), and a combination of ketamine, diazepam, medetomidine, glucose, and noradrenaline (ProtocolC, n = 5). All pigs received morphine for analgesia. AKI was induced by interrupting renal perfusion for 90 min. MH was diagnosed based on clinical and biological parameters.

Results

All MH pigs belonged to ProtocolA. MH pigs showed significantly higher maximum rectal temperature (p = 0.04), maximum expired carbon dioxide (CO2; p = 0.04), maximum heart rate (HR; p = 0.03), plasma concentration of creatinine and potassium (p < 0.0001). Protocol A pigs had a significantly higher maximum HR (p = 0.01) and hyperkalemia compared to the two other groups (ProtocolB, p = 0.005 and ProtocolC, p < 0.0001). Pigs from ProtocolA had a significantly lower minimum mean arterial pressure (MAP) than ProtocolC group (p = 0.03) and MAP remained below 60 mmHg for longer (p = 0.004). In ProtocolB, minimum glycemia was lower than other groups (p = 0.01).

Conclusion

Sevoflurane use was associated with the occurrence of MH, hemodynamic alterations and changes in plasma concentration of creatinine and potassium. These modifications can have a major impact on the validation of an experimental AKI model.

Keywords: acute kidney injury, anesthesia, ketamine, malignant hyperthermia, sevoflurane

This study details the adaptations required to establish a long‐term anesthetic protocol in pigs. Three protocols were evaluated, with some causing malignant hyperthermia, hypoglycemia, or hemoynamic instability. The final protocol proved effective for developing a porcine AKI model requiring adequate arterial pressure maintenance.

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1. INTRODUCTION

Animal models are essential in biomedical research for preclinical studies on pathological processes and evaluating the safety and efficacy of new treatments. Selection of the appropriate species is based on biological similarities, ensuring the animal's physiological, anatomical, and genetic traits closely resemble those of the target species. 1 , 2 Practical considerations (cost and feasibility) and ethical factors must also be addressed. To minimize animal use, the goal is to reduce inter‐individual variability by using animals with similar characteristics, such as genetics, age, and environment. 1 , 2

In preclinical research, the choice of model is critical. Elements of the model can influence the results obtained. 3 Among the various factors to be considered, anesthesia plays a central role. In addition to ethical concerns, mismanagement of anesthesia can induce stress and lead to results that are difficult to interpret. In addition, the depressant effects of anesthetic agents on the cardiovascular and respiratory systems may lead to the occurrence of complications that may bias the results obtained. 4 , 5 , 6

Pigs are commonly used for acute kidney injury (AKI) models. 7 Indeed, pigs share more similarities with humans in terms of anatomy and physiology than rodents. The renal vascularization of pigs is similar to that of humans. Moreover, repeated blood sampling is possible. 7 The choice of anesthetic protocol is of particular importance in the development of AKI models. The hypotensive effects of numerous anesthetics may induce renal hypoperfusion and ischemic damage, which can affect renal function. 8 , 9 Considering inter‐individual variability, the occurrence of hypotension in a small subset of animals used in a study can lead to the development of heterogeneous kidney failure thus reducing the reproducibility of the model.

Anesthetizing pigs is associated with a multitude of challenges, including predisposition to malignant hyperthermia (MH). 10 , 11 Malignant hyperthermia is a pharmacogenetic disorder defined by a hypermetabolic response characterized by hyperthermia, hypercapnia, exacerbated muscular contraction, and hyperkalemia. 12 This response can be triggered by various stimuli, including pharmacological drugs. 12 Inhalant anesthetics such as halothane, isoflurane, or sevoflurane are the most frequently implicated drugs.

To establish a porcine AKI model, anesthesia was initially performed with sevoflurane inhalation (ProtocolA). However, a higher‐than‐expected incidence of MH necessitated refinement of the anesthetic protocol. Two alternative protocols were developed using ketamine, medetomidine, and diazepam instead of sevoflurane (ProtocolB and ProtocolC, with glucose and noradrenaline added in ProtocolC). This study outlines the evolution of these protocols and reports clinical, hemodynamic, and biochemical observations across the three groups. The focus is not on the AKI model itself, which was already published, 13 but on refining anesthesia and its impact on experimental outcomes.

2. METHODS

This study was not initially planned. Observations made during the development of the intended AKI model led to the collection of various biochemical, hemodynamic, and clinical data related to the anesthesia protocol. The analyses were conducted retrospectively.

2.1. Animals

Fifteen healthy pigs (Youna, Sus scrofa domesticus, 7 males, 8 females), aged 3 months with a median weight of 39 kg (range: 35–45 kg), were used in this study. Following a one‐week acclimatization period in a controlled environment (22 ± 1°C, 65% ± 5% humidity), pigs were fed a protein‐rich soup twice daily, consistent with their prior diet, and had unrestricted water access. Bedding consisted of shavings, with feed scattered to encourage burrowing behavior, supplemented by food hidden in toys. Daily cage maintenance ensured hygiene and comfort, and handlers regularly interacted with the pigs to promote human acclimation.

2.2. Anesthesia

Three anesthetic protocols were sequentially administered. For this reason, there was no randomization. These three anesthetic protocols were termed ProtocolA (n = 5), ProtocolB (n = 5), and ProtocolC (n = 5).

Premedication consisted of tiletamine and zolazepam (Zoletil® 100, Virbac, France) at 6 mg/kg intramuscularly. After intubation, pigs were mechanically ventilated with a tidal volume of 8 mL/kg and a respiratory rate of 15/min, adjusted to maintain end‐tidal carbon dioxide (ETCO2) at 35–45 mmHg (except during MH episodes). Inspired oxygen fraction (FiO2) was set at 50%, supplemented with medical air (at 50%), and adjusted to maintain oxygen saturation (SpO2) > 95%. Saline solution (NaCl) at 0.9% was infused at 5 mL/kg/h. Analgesia was provided with morphine, administered as an initial intravenous (IV) bolus (0.3 mg/kg) followed by a constant infusion (0.3 mg/kg/h, increased to 0.5 mg/kg/h during surgery).

ProtocolA (n = 5): Anesthesia was induced with propofol (Propovet® 10 mg/mL, 20 mL, Zoetis Belgium, Louvain‐la‐Neuve, Belgium) at 4 mg/kg administered IV, and anesthesia was maintained with sevoflurane (1–2.5%; SevoFlo 100%, Zoetis, Belgium) in 50% oxygen–50% air.

ProtocolB (n = 5): Anesthesia was induced with IV ketamine (5 mg/kg), diazepam (0.5 mg/kg), and medetomidine (2.5 μg/kg). Maintenance included ketamine (20 mg/kg/h), diazepam (0.3 mg/kg/h), and medetomidine (2.5 μg/kg/h). During surgery, doses were increased to ketamine (40 mg/kg/h), diazepam (0.45 mg/kg/h), and medetomidine (4 μg/kg/h).

ProtocolC: Identical to Protocol B with the addition of IV glucose 5% and noradrenaline. Glucose 5% was administered when blood glucose was <3 mmol/L, mixed with saline (1/3 glucose, 2/3 NaCl 0.9%) until glycemia reached 3 mmol/L. Noradrenaline was given at 0.05 μg/kg/min if mean arterial pressure (MAP) fell <55 mmHg, adjusted to maintain MAP >60 mmHg to prevent hypoperfusion‐induced AKI. These two adjustments aimed to avoid any hypotension and hypoglycemia.

Anesthesia depth and analgesia effectiveness were assessed every 15 min during surgery and every 30 min during follow‐up.

2.3. Surgical procedures and follow‐up

Using the Seldinger technique, catheters were placed in the right jugular vein and femoral artery for IV anesthetic administration, fluid delivery, arterial blood sampling, and continuous blood pressure monitoring. AKI was induced by bilateral cross‐clamping of renal arteries for 90 min. A 15 cm skin incision and muscle dissection exposed the arteries, which were occluded with laces to induce ischemia. After reperfusion, pigs were monitored for 8 h.

For urine collection, silicone Foley catheters (CH8) were inserted into the ureters. Pigs were placed in dorsal recumbency, and a midline laparotomy from the pubis to the umbilicus exposed the bladder and ureters.

Through anesthesia, several parameters were monitored and regulated, including MAP (mmHg), HR (beats per minute), SpO2 (%), ETCO2 (mmHg), and rectal temperature (°C). These parameters were recorded to the Monitor CARESCAPE B650 (GE Healthcare, Little Chalmont, UK). Rectal temperature was closely managed; in cases of hypothermia, the animal was placed on a heating pad and covered with a thermal blanket. For hyperthermia, cooled fluid packs were applied near the animal (avoiding direct skin contact to prevent burns).

2.4. Euthanasia

Pigs were euthanized with T61 (embutramide, mebezonium, and tetracaine [0.1 mL/kg IV]) under general anesthesia. Euthanasia was confirmed by the absence of pulse waves, apnea, and a drop in expired CO2. Pigs that died spontaneously were under general anesthesia.

2.5. Malignant hyperthermia (MH)

MH was diagnosed if pigs exhibited muscle tremor, rectal temperature >39°C (or an increase >1°C per hour), ETCO2 > 55 mmHg with appropriate controlled ventilation (respiratory frequency at 15 cycles per minute and a tidal volume set at 8 mL/kg), and heart rate > 120 bpm. Concerning MH, pigs were categorized into Yes and No groups.

2.6. Samples collection and biochemical measures

Arterial blood samples were collected in lithium‐heparin tubes for potassium and glycemia analysis using the Stat Profile Prime® Vet analyzer (Nova Medical, USA). Glycemia and acid–base status were recorded every 30 min. Plasma was separated by centrifugation, stored at −80°C, and analyzed for creatine kinase (CK) and creatinine using the Konelab 30i analyzer (Thermo Scientific, France). Urine was collected from each kidney's ureter to measure diuresis and stored at −80°C.

Several parameters were analyzed:

  • T max: highest recorded rectal temperature.

  • ETCO2max: highest recorded end‐tidal CO2.

  • HRmax: highest recorded heart rate.

  • HRmedian: median heart rate over the recording period.

  • MAPmedian: median mean arterial pressure (MAP) during the experiment.

  • MAPmin: lowest recorded MAP.

  • PTU60: percentage of the experiment duration during which MAP was below 60 mmHg.

  • CKmax: highest measured creatine kinase concentration.

  • CKvar: percentage increase in creatine kinase from the start to the end of the experiment.

  • G min: lowest recorded glycemia

2.7. Statistical analysis

Quantitative variables were assessed graphically and presented as medians with interquartile ranges. Fisher's exact test compared MH incidence and mortality across the three protocol groups. Linear mixed models examined associations between time, group, and plasma potassium or creatinine, with time as a fixed effect and ‘animal’ as a random effect, including interactions with anesthesia protocol or MH occurrence. Residuals were checked for homoscedasticity and randomness. Results are reported as regression coefficients with 95% confidence intervals (CI). Kruskal‐Wallis tests with Dunn's post hoc and Mann–Whitney tests for MH‐related groups were used. Spearman correlations assessed AKI biomarkers and hemodynamic parameters. Statistical significance was set at p < 0.05. Analyses were performed using R 4.1.2 with the ‘ggplot2,’ ‘Lme4,’ ‘Lmertest,’ ‘ggpubr,’ and ‘ggstatsplot’ packages.

3. RESULTS

3.1. Missing data

One temperature value (P1) and two ETCO2 values (P1 and P8) were missing due to technical issues.

3.2. Anesthesia and surgery

The median time from premedication to induction was 30 min (20–34). Premedication induced moderate sedation (hypovigilance, lateral recumbency), allowing auricular venous catheter placement in all pigs. Premedication mildly relaxed the jaw and tongue muscles, while maintaining the gag reflex. Surgery time was 3 h 30 min (2 h 50 min–4 h 20 min) and anesthesia time was 13 h 45 min (12 h–14 h 30 min).

3.3. Malignant hyperthermia

3.3.1. Incidence

Three pigs met MH criteria, all in the ProtocolA group (S0). One of them received dantrolene (2 mg/kg IV) to treat MH. The MH incidence was significantly higher in ProtocolA compared to ProtocolB and ProtocolC (p = 0.02). The pig treated with dantrolene was excluded from further analyses.

3.3.2. Diagnosis criteria

Rectal temperature

Maximum rectal temperature (T max) was not significantly different between the three groups of anesthesia (p = 0.69): ProtocolA = 41.0°C (38.7–42.1), ProtocolB = 38.5°C (37.8–39), and ProtocolC = 38.9°C (37.9–39; Figure 1A). T max was significantly higher in the Yes group as compared to the No group (p = 0.04): Yes = 42.1°C (41.6–42.7), and No = 38.5°C (37.4–39.0; Figure 1B). Evolution of rectal temperature is available in Data S1.

FIGURE 1.

FIGURE 1

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Malignant hyperthermia criteria. (A) Maximum rectal temperature (T max) in the three anesthetic related groups. (B) Maximum rectal temperature (T max) in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group). (C) Maximum end tidal carbon dioxide (ETCO2max) in the three anesthetic related groups. (D) Maximum end tidal carbon dioxide (ETCO2max) in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group). (E) Maximum heart rate (HRmax) in the three anesthetic related groups. (F) Maximum heart rate (HRmax) in pigs with malignant hyperthermia (Yes group) and in pigs without malignant hyperthermia (No group).

Respiratory acidosis

Regarding ETCO2max, there were no significant differences between the three groups of anesthesia (p = 0.48): ProtocolA = 97.2 mmHg (66.1–102.5), ProtocolB = 44.7 mmHg (34.1–55.8), and ProtocolC = 47.0 mmHg (45.7–47.8; Figure 1C). ETCO2max was significantly higher in the Yes group as compared to the No group (p = 0.04): Yes = 102.5 mmHg (99.8–105.1), and No = 46.4 mmHg (35–52; Figure 1D). Evolution of ETCO2 is available in Data S2a. A comparison of the area under curve for ETCO2 is available in Data S2b.

Tachycardia

Maximal HR (HRmax) was significantly higher in the ProtocolA group as compared to the ProtocolB (p = 0.02). Other differences were not statically different (Figure 1E): ProtocolA = 187 beats per minutes (bpm) (148–223), ProtocolB = 92 bpm (87–101), and ProtocolC = 93 bpm (92–111). HRmax was significantly higher in the Yes group compared to the No group (p = 0.03), with Yes = 232.5 bpm (223–242), and No = 97 bpm (91–112; Figure 1F). Evolution of heart rate is available in Data S3.

3.3.3. Rhabdomyolysis biomarkers

Plasma potassium

Potassium levels increased significantly faster in the ProtocolA group compared to the ProtocolC group (p < 0.0001), with no significant difference between ProtocolA and ProtocolB (p = 0.31). Mean potassium increase rates were 0.0044 mmol/L/min (95% CI: 0.0037–0.0051) for ProtocolA, 0.0039 mmol/L/min (95% CI: 0.0023–0.0055) for ProtocolB, and 0.0012 mmol/L/min (95% CI: −0.0004 to 0.0028) for ProtocolC (Figure 2A, Data S4a).

FIGURE 2.

FIGURE 2

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Biochemical markers of rhabdomyolysis. (A) Plasma concentration of potassium in the three anesthetic related groups. (B) Plasma concentration of potassium in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group). (C) Percentage of creatine kinase variation (CKvar) between T0 and the end of the experiment in the three anesthetic related groups. (D) Percentage of creatine kinase variation (CKvar) between T0 and the end of the experiment in pigs with malignant hyperthermia (Yes group) and in pigs without malignant hyperthermia (No group). (E) Maximum of creatine kinase (CKmax) between T0 and the end of the experiment. (F) Maximum of creatine kinase (CKmax) in pigs with malignant hyperthermia (Yes group) and in pigs without malignant hyperthermia (No group).

Similarly, potassium levels rose significantly faster in the Yes group compared to the No group (p < 0.0001). The mean rate of increase was 0.0113 mmol/L/min (95% CI: 0.0099–0.0127) for the Yes group and 0.0024 mmol/L/min (95% CI: −0.0932 to 0.0052) for the No group (Figure 2B, Data S4b). Additional potassium evolution data are provided in Data S4c.

Creatine kinase

The CK variation (CKvar) between the beginning and the end of the experiment exhibited no significant differences among the three groups of anesthesia (p = 0.14): ProtocolA = 46.7% (+25.6 to +70.1), ProtocolB = +14.6% (−3.7 to +32.0), and ProtocolC = −15.7% (−20.1 to −7.4; Figure 2C).

CKvar was higher in the Yes group, but this difference was not statistically significant (p = 0.06): Yes = +91.4% (+68.6 to +114.2), and No = −5.5% (−19.0 to +14.6; Figure 2D).

The CK maximal plasma concentration (CKmax) was not significantly different in the three anesthetic groups (p = 0.11): ProtocolA = 2313 UI/mL (1764–2811), ProtocolB = 2395 UI/mL (2155–3252), and ProtocolC = 1329 UI/mL (1169–1792; Figure 2E). There were no significant differences between Yes and No groups for CKmax (Figure 2F, p = 0.17). Evolution of plasma creatine kinase is available in Data S5.

3.4. Hemodynamics

Pigs from the ProtocolA group had a significantly higher median HR (HRmedian) compared to pigs from the ProtocolB (p = 0.02) and the ProtocolC (p = 0.048). There was no significant difference between ProtocolB and ProtocolC (p = 0.60): ProtocolA = 111 bpm (108–124), ProtocolB = 75 (70–86), and ProtocolC = 78 (76–79; Figure 3A). Yes pigs had a higher HRmedian as compared to No group (p = 0.048; Figure 3B).

FIGURE 3.

FIGURE 3

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Hemodynamics. (A) Median heart rate (HRmedian) in the three anesthetic related groups. (B) Median heart rate (HRmedian) in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group). (C) Median of mean arterial pressure (MAPmedian) in the three anesthetic related groups. (D) Median of mean arterial pressure (MAPmedian) in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group). (E) Minimum of mean arterial pressure (MAPmin) in the three anesthetic related groups. (F) Minimum of mean arterial pressure (MAPmin) in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group).

Median MAP (MAPmedian) was significantly lower in ProtocolA as compared to ProtocolC (p = 0.01). The difference was not significant with ProtocolB (p = 0.09): ProtocolA = 54 mmHg (50–56), ProtocolB = 67 mmHg (63–69), and ProtocolC = 71 mmHg (70–78; Figure 3C). Other differences were not significant (Figure 3D).

Pigs from the ProtocolA had a significantly lower minimal MAP (MAPmin) than those from the ProtocolC group (p = 0.048): ProtocolA = 48 mmHg (42–52), ProtocolB = 56 mmHg (52–58), and ProtocolC = 58 mmHg (54–60) (Figure 3E). Other differences were not significant (Figure 3F). Evolution of mean arterial pressure is available in Data S6.

3.5. Acute kidney injury

3.5.1. Prerenal origin

The percentage of time passed under 60 mmHg (PTU60) was significantly higher in the ProtocolA group as compared to the ProtocolC (p = 0.006): ProtocolA = 81% (73–86), ProtocolB = 27% (10–43), and ProtocolC = 5% (0–19). Other differences were not significant (Figure 4A).

FIGURE 4.

FIGURE 4

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Acute Kidney Injury characterization: Prerenal origin and plasma creatinine measurement. (A) Percentage of experiment time passed under 60 mmHg (PTU60) in the three anesthetic related groups. (B) Total diuresis in the three anesthetics related. (C) Plasma concentration of creatinine in the three anesthetic related groups. (D) Plasma concentration of creatinine in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group).

Comparison between Yes and No groups are available in Data S7a.

There was no significant correlation between hypotension and AKI parameters (creatinine, urea, potassium). The results are presented in Data (S7b for table summarizing correlations and S7c‐k for corresponding scatter plot).

3.5.2. Acute kidney injury biomarkers

Evolution of diuresis is available in Data S8a. Total diuresis did not differ between the three anesthetic groups (p = 0.57; Figure 4B) and the two MH groups (Data S8b).

Plasma creatinine increased significantly faster in the ProtocolA group compared to the ProtocolB group (p = 0.003) and the ProtocolC group (p < 0.0001). Mean rates of increase were 0.3257 μmol/L/min (95% CI: 0.2934–0.3578) for ProtocolA, 0.2635 μmol/L/min (95% CI: 0.1908–0.3367) for ProtocolB, and 0.2081 μmol/L/min (95% CI: 0.1369–0.2789) for ProtocolC (Figure 4C, Data S9a).

Similarly, creatinine levels rose significantly faster in the Yes group compared to the No group (p = 0.001; Figure 4D, Data S9b). Additional creatinine evolution data are provided in Data S9c.

3.6. Glycemia

Glycemia values are available in Data S10. Minimal glycemia (G min) was lower in the ProtocolB group compared with ProtocolA group (p = 0.03) and the ProtocolC group (p = 0.03): ProtocolA = 2.6 mmol/L (2.3–3.4), ProtocolB = 1.2 mmol/L (1.1–1.2), and ProtocolC = 0.4 mmol/L (2.4–2.7; Figure 5A). There were no differences in Gmin for MH criteria (Yes group Figure 5B).

FIGURE 5.

FIGURE 5

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Glycemia measurement. (A) Minimum glycemia (G min) in the three anesthetic related groups. (B) Minimum glycemia (G min) in pigs with malignant hyperthermia (Yes group) and pigs without malignant hyperthermia (No group).

3.7. Mortality

Two pigs died spontaneously in the ProtocolA group. These pigs belonged to the MH‐Yes group. Two pigs died spontaneously in the ProtocolB group. Both deaths occurred during an episode of severe hypoglycemia in ProtocolB group. The difference in mortality rate was not statistically significant (p = 0.45).

4. DISCUSSION

We were surprised by the high prevalence of MH observed during our experiments, leading us to refine the anesthetic protocol. This study illustrates how the choice of anesthetic molecules can influence results when setting up an AKI model. Sevoflurane use was associated with MH, while ketamine was not. Notably, MH was linked to severe hyperkalemia, and increased creatinine levels. Sevoflurane also caused hypotension; a complication relevant to AKI models. Ketamine was associated with severe hypoglycemia, treatable with glucose infusions.

Our experiment aimed to develop an AKI model via renal ischemia‐reperfusion, 13 mimicking complications observed in human cardiac surgery. 14 Sevoflurane, initially chosen for its widespread use in human medicine, 15 was replaced with ketamine to mitigate the risk of MH. 12 While ketamine is less common for total intravenous anesthesia in humans 15 —typically performed with propofol—it was selected for its cost‐effectiveness and reduced hypotensive effects compared to both propofol and halogenated gases. 16 , 17 In our study, sevoflurane and, to a lesser extent, ketamine were associated with hypotension, which was effectively managed with noradrenaline in ProtocolC. The anesthetic protocol was adapted from Englehart et al. 17

Analgesia was provided by morphine and ketamine infusion. Doses needed to be increased during surgery due to signs of pain observed in the first animals operated (manifested by contraction of the subcutaneous muscles during the skin incision or by a slight movement of the head when the nasal septum was pinched with forceps). These increases were applied to all animals to ensure comparability between individuals and to limit the influence of anesthetic doses on the severity of AKI. The maximum dose of ketamine was 40 mg/kg/h. This particularly high dose was not administered throughout the entire protocol, only during surgery. High doses of ketamine have also been described in the literature: Englehart et al. reported the use of a dose of 33 mg/kg/h in pigs during the performance of a hemorrhagic shock model. 17 In addition, Boschert et al. recommended repeated bolus administration of ketamine at a dose of 20–30 mg/kg when signs of pain were evident. 18

In this study, sevoflurane was associated with the occurrence of MH, as all three cases were observed in the ProtocolA group, with none in the ketamine‐based protocols. This aligns with existing literature, where halogenated gases, including sevoflurane, are recognized as MH triggers. Historically linked to halothane, all halogenated anesthetics, including sevoflurane, are now understood to pose a similar risk, with episodes of sevoflurane‐induced MH being equally severe. 19 , 20 MH cases during sevoflurane anesthesia have been well documented in both humans and pigs, often with delayed onset, 21 , 22 consistent with the observations in our study. 23 , 24 , 25 , 26 The high incidence (60%) of MH in ProtocolA suggests a possible genetic predisposition in the study animals, which were sourced from the same breeding farm. Genetic profiling could help identify susceptible individuals and reduce the number of animals needed for future experiments.

ETCO2max and T max were higher in the ProtocolA group, although the differences did not reach statistical significance. These increases were primarily driven by the high values recorded in the pigs with MH. Hyperthermia and respiratory acidosis are a classical feature of MH. 12 , 27 Respiratory acidosis and hyperthermia are integral to the pathophysiology of MH, characterized by skeletal muscle hypermetabolism. During MH, massive calcium release within muscle cell cytoplasm triggers sustained muscle contractions, leading to oxygen consumption, elevated CO2 levels, and heat generation. 28 Tachycardia, a classical feature of MH, was observed in the MH‐Yes group as well as in the ProtocolA group, compared to the ProtocolB and ProtocolC groups. 12

In the ProtocolA group, kalemia was elevated compared to the other protocols, likely due to the presence of MH cases. Hyperkalemia is a well‐documented sign of MH, 12 , 29 resulting from sustained muscle contractions that cause ATP depletion, loss of cell membrane integrity, and potassium release into the bloodstream. 28

Unexpectedly, creatine kinase (CK) levels were not significantly higher in the ProtocolA group compared to ProtocolB and ProtocolC, nor in the MH‐Yes group compared to MH‐No. While CK is a recognized marker of MH and rhabdomyolysis, 30 this lack of significance may be explained by the small sample size and hemolysis in some samples. Notably, extremely high CK values were observed in certain non‐MH cases (e.g., PB4 and PB5), likely due to hemolysis, as evidenced by reddish plasma discoloration. Unlike CK, plasma creatinine is thought to remain unaffected by hemolysis. 31

The hypotensive events observed in the ProtocolA group may have multifactorial origins. They could be related to MH and sevoflurane inhalation. Decreases in MAP were commonly noted during MH episodes. 32 The mechanisms underlying hypotension in MH are not fully understood. Instead, they may arise from the release of substances with depressant effects on the heart. 22 , 32 Additionally, hypotension could also be directly associated with the use of sevoflurane, which is known to induce vasodilation and a reduction in cardiac contractility, leading to hypotension. 33 Hypotension could be detrimental to the establishment of an AKI model, as it may exacerbate renal damage on top of that caused by AKI. 34 The MAP difference between sevoflurane and ketamine may also be exacerbated by inotropic positive effect of ketamine.

A more pronounced increase in plasma concentration of creatinine was observed in ProtocolA group. This result was linked to the presence of MH cases in the ProtocolA group. This result could be explained by rhabdomyolysis associated with MH. Indeed, muscular cell lysis leads to the release of myoglobin and other nephrotoxic substances into the bloodstream. 35 Consequently, using sevoflurane for our AKI model may introduce confounding factors and reduce reproducibility.

In our study, ketamine was not associated with MH but was linked to severe hypoglycemia in the ProtocolB group. This effect likely resulted from the high ketamine dose used (40 mg/kg/h) to maintain anesthesia and analgesia. Hypoglycemia induced by ketamine has been reported in rabbits, 36 potentially mediated by opioid and β‐adrenoreceptors, and in the case of overdose in a dog. 37 However, other studies using lower ketamine doses did not observe this effect. 38 The high dosing in our study likely explains the hypoglycemic episodes.

Our study has several limitations. First, the relatively small sample size and the absence of a priori sample size calculation may have reduced the study's statistical power. The evolving nature of the study, initially designed with a single anesthetic protocol, led to the successive implementation of additional protocols in response to observed side effects, without randomization. Second, MH was not confirmed with a definitive diagnostic test, such as the in vitro contracture test, the gold standard for MH diagnosis. Third, the absence of MH in ProtocolB and ProtocolC groups might be due to chance or the lack of genetically susceptible animals in these groups. While all animals came from the same lineage, genetic analyses were not performed to confirm this. Finally, the impact of AKI on the pharmacokinetics of anesthetic agents, such as ketamine, which is renally excreted, was not investigated.

In summary, this study highlights the significant influence of the choice of anesthetic protocol on the results of experimental model development. Plasma creatinine, a common marker of renal failure, was shown to be influenced by the choice of anesthetic. In particular, the use of sevoflurane was associated with MH in some animals, introducing variability that could affect reproducibility. In addition, sevoflurane‐induced hypotension may further affect renal function. Finally, the study highlights the occurrence of severe hypoglycemia associated with high‐dose ketamine administration, a phenomenon rarely documented in literature.

AUTHOR CONTRIBUTIONS

Axel Guilpin: Conceptualization; data curation; formal analysis; investigation; resources; software; visualization; writing – original draft. Mathieu Magnin: Conceptualization; data curation; formal analysis; investigation; methodology; resources; software; supervision; validation; visualization; writing – review and editing. Axel Aigle: Data curation; formal analysis; software; visualization; writing – review and editing. Timothée Schuhler: Investigation; methodology; resources; writing – review and editing. Jean‐Yves Ayoub: Investigation; methodology; resources. Romain Lac: Investigation; resources. Charlotte Slek: Investigation; resources. Thomas Brichart: Funding acquisition. Abdessalem Hammed: Conceptualization; funding acquisition; investigation; methodology; project administration; resources; supervision; validation; visualization; writing – review and editing. Vanessa Louzier: Conceptualization; funding acquisition; investigation; methodology; project administration; resources; supervision; validation; visualization; writing – review and editing.

FUNDING INFORMATION

The authors have reported financial support for this study: BPI (public investment bank) and VetAgro Sup.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ETHICS STATEMENT

This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Ethics Committee of VetAgroSup, Marcy l’Etoile, France (authorization number: 2146), conforming to Directive 2010/63/EU (European Union). This study was performed following the ARRIVE 2.0 guidelines.

Supporting information

Data S1.

AME2-8-1493-s001.docx (2.1MB, docx)

ACKNOWLEDGMENTS

Authors are grateful for the expertise brought by VetAgro Sup Lab center, for all biochemical analysis and the availability of Konelab 30i.

Guilpin A, Magnin M, Aigle A, et al. Impact of different anesthetic protocols during anesthesia for the establishment of a porcine model of acute kidney injury. Anim Models Exp Med. 2025;8:1493‐1502. doi: 10.1002/ame2.70014

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1.

AME2-8-1493-s001.docx (2.1MB, docx)

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

Articles from Animal Models and Experimental Medicine are provided here courtesy of Wiley

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