
==== Front
Brain Spine
Brain Spine
Brain & Spine
2772-5294
Elsevier

S2772-5294(24)00089-4
10.1016/j.bas.2024.102833
102833
Special Issue Section: ICP 2022
Development of a piglet model for cerebrovascular autoregulation assessment with altered PaCO2
Dietvorst Sofie sofie.dietvorst@alderhey.nhs.uk
ab⁎
Desloovere Veerle cd
Meyfroidt Geert de
Depreitere Bart ab
a Department of Neurosurgery, University Hospitals Leuven, Belgium
b Research Group Experimental Neurosurgery and Neuroanatomy, KULeuven, Belgium
c Department of Anesthesiology, University Hospitals Leuven, Belgium
d Laboratory of Intensive Care Medicine, KULeuven, Belgium
e Department of Intensive Care Medicine, University Hospitals Leuven, Belgium
⁎ Corresponding author. Department of Neurosurgery, University Hospitals Leuven, Belgium. sofie.dietvorst@alderhey.nhs.uk
07 5 2024
2024
07 5 2024
4 10283324 5 2023
1 4 2024
6 5 2024
© 2024 Published by Elsevier B.V. on behalf of EUROSPINE, the Spine Society of Europe, EANS, the European Association of Neurosurgical Societies.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction

Cerebrovascular autoregulation (CA) capacity can be impaired in the aftermath of acute brain injuries. Altered physiological states, such as hypo- and hypercapnia, affect CA. Although these effects have been demonstrated in several animal experiments, the exact effect of PaCO₂ on the plateau of cerebral blood flow (CBF) across the spectrum of arterial blood pressures has not been fully disclosed.

Research question

The aim was to explore pial vasodynamics in response to changing PaCO₂ in a porcine cranial window model, as preparation for an experimental setup in which the CBF plateau position is investigated under different PaCO₂ conditions.

Material and methods

Five piglets were brought under anesthesia, intubated, ventilated and instrumented with a cranial window through which pial arteriolar diameters could be microscopically observed. By changing ventilation to either hyper- or hypoventilation we were able to investigate a range of PaCO2 from 25 till 90 mmHg.

Results

Altering the respiratory rate to manipulate PaCO₂ by ventilation appeared to be feasible and reliable.

Discussion and conclusion

We found that ETCO₂ reliably represents PaCO₂ in our model. Pial arteriolar diameter changes followed the direction of PaCO₂ changes, but the effect of PaCO₂ on the diameters was not linear. Only in the hypercapnia setting did we observe a clear and consistent vasodilation of the pial arterioles.

Highlights

• In our exploratory study we investigated the direct influence of PaCO2 on cerebrovascular resistance in pial arterioles.

• The effect of PaCO2 on the pial diameters is not linear; it is not the value but the direction of change of PaCO2 that alters the cerebrovascular resistance.

Keywords

Cerebrovascular autoregulation
Cerebral blood flow
Respiratory acidosis/alkalosis
Handling editor: Dr W Peul
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pmc1 Introduction

Carbon dioxide (CO2) is a potent vasodilator in cerebral hemodynamics. However, not CO2 itself, but the change of extracellular pH in the interstitial fluid secondary to CO2, has been shown to be the main driver affecting cerebrovascular resistance (CVR). It was proven that perivascular pH is inversely related to pial arterial diameter (Muizelaar et al., 1988; Madden, 1993; Yoon et al., 2012). This was confirmed in experiments with tromethamine (THAM), an alkaline buffer that does not affect CO2, but does lower intracranial pressure (ICP) in acute neurological injury (Zeiler et al., 2014; Muizelaar et al., 1991; Wolf et al., 1993). CO2 can have an effect on CVR during the transient shift in interstitial pH. This effect lessens once metabolic compensation for the pH change caused by CO2 fluctuations has occurred, which has been shown to take about 12–24 h (Berend et al., 2014; Brian, 1998). However, because CO2 is lipophilic and can cross the blood-brain barrier (BBB), whereas loaded H+ ions cannot, respiratory acidosis has a greater influence on CVR than metabolic acidosis. As a result, arterial carbon dioxide partial pressure (PaCO2) is the main determinant of interstitial pH(Muizelaar et al., 1988; Kontos et al., 1977). Several feedback loops counteract elevated PaCO2 and its effects. Due to vasodilation, the subsequent increase in cerebral blood flow (CBF) leads to a washout of CO2. Further, the central respiratory chemoreflex (situated in the ventral medulla oblongata) responds to increases of PaCO2 by increasing ventilation frequency and/or depth. Conversely, the peripheral respiratory chemoreflex initiated by the carotid and aortic sinuses acts upon decreases in PaO2 which occurs when it drops below 50 mmHg (Ogoh, 2019; Schaeffer and Iadecola, 2021). Partial pressures of CO₂ in the arterial blood and in the cerebrospinal fluid measured in the intracranial cisterns have similar values (Ogoh, 2019). However, they are not similar to those in lumbar cerebrospinal fluid since the response to CO2 changes here is delayed due to distance (Andrews et al., 1994). Changing PaCO2 also influences the Hemoglobin (Hb) dissociation curve through the change in arterial pH. This curve depicts the relation between PaO2 (in mmHg) and Oxyhemoglobin (saturation in %). Decreasing pH shifts the curve to the right, so the Hb affinity for O2 decreases. This helps deliver O2 to tissues and is called the Bohr effect (Benner et al., 2022; Bö et al., 2020).

In spite of the mentioned experimental physiological knowledge, the exact effect of PaCO₂ on the height and width of the plateau of CBF across the spectrum of arterial blood pressures (ABP) in large animals nor humans has neither been fully disclosed nor quantified. In particular, Gelb et al. published a review of the available research on the impact of hypo- and hypercapnia on cerebrovascular autoregulation (CA), where they concluded that several hypotheses exist for the relation of hypocapnia and the upper limit of autoregulation (Meng and Gelb, 2015).

Our lab(Klein et al., 2019) has been able to revisit the autoregulation curve, first introduced by Lassen, by applying mechanically induced blood pressure changes in a modified porcine cranial window model which allowed to record both changes in pial arteriolar diameter and red blood cell velocity (Klein et al., 2019; Lassen, 1959). Pigs, as opposed to other animal models, have a brain morphology and physiology (including BBB and CA) and a cardiopulmonary physiology close to humans (Duhaime, 2006). We want to investigate what the direct influence of different levels of PaCO₂ is on pial diameters, to confirm if hypercapnia induces vasodilation and hypocapnia induces vasoconstriction of pial arterioles in our model.

2 Materials and methods

2.1 Ethical considerations

All animal care and procedures were approved by the Ethics Committee Animal Research Center, KULeuven (Ethical Approval: P107-2019) in compliance with the Belgian Royal Decree (May 29, 2013) and European Directive 2010/63/EU on the protection for animals used for scientific purposes. All animal procedures were conducted under veterinarian supervision according to the guidelines imposed by the Ethical Committee.

2.2 Experimental setup

To alter PaCO2 in sedated and ventilated animals, there are two possibilities. The first is to blend CO2 in the inhaled mixture of air and O2. This is probably the easiest method and does not influence the ventilation settings. The second is adjusting the ventilator to hyper- or hypoventilation and hereby influencing the level of PaCO2. However, this might also influence the intrathoracic pressure and thus alters also the basic cardiopulmonary physiology (Stocchetti et al., 2005). We decided to alter ventilation, since this seems the most comparable to human data. Hyper- and hypoventilation are not only common in diseases but are also used as a therapy. We kept the tidal volume (TV) constant but changed the respiration rate (RR), since it has been shown that mechanical hyperventilation with low RR and large TV generally reduces the blood flow to tissues. By increasing RR with a constant TV, there are no changes in cardiac output (CO) expected, which is important in our model where we want to strive for a normal physiological setting (Stocchetti et al., 2005; KARLSSON et al., 1994).

For this exploratory study, PaCO2 was manipulated from the lowest achievable PaCO2 possible to the highest achievable or vice versa, where the animals remained hemodynamically stable. Experiments were based on the porcine cranial window model described by Klein et al. (2019) Five 6-week-old male piglets (domestic swine, Zootechnical Center KU Leuven) were brought under general anesthesia by intravenous propofol, pancuronium, midazolam and fentanyl, intubated and ventilated. An arterial line was placed in the left femoral artery for continuous ABP monitoring, placed 5 cm above the diaphragm. Subsequently, we made two small cranial burr holes posterior to the coronal suture, on the right side, one for ICP with brain tissue oxygen monitoring (PbO2) (Neurovent-PTO, Raumedic AG, Muenchenberg Germany) and laser Doppler flow (LDF) (Moor VMS-LDF1 with VP14–CBF probe, Moor Instruments, Devon UK) monitoring. Anterior to the coronal suture, a round craniotomy was performed for placement of the cranial window, which was cemented to the skull.

2.3 Monitoring

ABP, ICP, PbO2, brain temperature, LDF and heart rate (3 lead ECG and arterial pulse wave analysis) were continuously monitored (Philips Intellivue monitor, Philips Medical Systems, the Netherlands). Cerebral perfusion pressure (CPP) is calculated as ABP minus ICP. Blood oxygen level was measured with a pulse oximeter and kept above 95%. Inspired and expired concentrations of CO₂ and O₂ were measured with a gas analyzer (Phillips M1026B, Philips Medical Systems, The Netherlands). Rectal temperature was kept at normothermia of 38–39 °C by warming mattress and blankets. The ICM + software (Cambridge University, Cambridge, United Kingdom, https://icmplus.neurosurg.cam.ac.uk/) was used to integrate and record all the monitoring data. ABP, ICP and LDF signals were sampled real-time at 250Hz.

2.4 Cranial window in vivo imaging

Pial arterioles were observed through the cranial window using an epifluorescence microscope (SMZ18 with P2-SHR Plan Apo 1x, Nikon), illuminated with a solid-state light engine (SOLA SM2, Lumencor), and captured with a high-speed digital CMOS camera (Orca Flash 4.0 V2, Hamamatsu) controlled by NIS-Elements software (Nikon). A green fluorescent filter (P2-EFL GFP-B Filter Cube 470–535 nm, Nikon) was used. Images were acquired at 200 frames per second and digitally stored for offline analysis. For this study, we randomly selected up to 5 arterioles per cranial window experiment for further analysis, with baseline diameter ranging from 30 to 250 μm.

2.5 Ventilation

Ventilation was accomplished with a volume-controlled ventilator (Cato® Dräger, Lübeck, Germany) with the following settings: tidal volume (TV) of 10 ml/kg, PEEP 5, I/E ½, peak pressure 30cmH2O and respiratory rate (RR) of 20–26/min adjusted to maintain an end-tidal carbon dioxide (ETCO2) tension of 40 mmHg, verified by arterial blood gas sampling. We explored the PaCO2 continuum to assess the effect of PaCO2 on CVR. Hypocapnia was induced in steps of 5 mmHg by increasing the RR until the lowest obtainable value of PaCO2. Hypercapnia was achieved in steps of 10 mmHg by slowly reducing the RR and increasing dead space ventilation by means of a swivel distal to the ventilation tubes, to arrive at the highest obtainable PaCO2 value. Previous experiments have shown that hyperventilation is more delicate to achieve, therefore smaller steps of decreasing PaCO2 are used (Van Hulst et al., 2002; Van Hulst et al., 2004). Each level of ETCO2 lasted for approximately 15 min and was checked by arterial blood gas when the level of ETCO2 was stable for 10 min. We did not want a continuous increase or decrease of ETCO2 since we wanted to give the animals time to recuperate and to establish a steady interstitial pH. In this short time span, we expect no interference of metabolic compensation. Two animals were first brought to hypercapnia and then slowly brought to hypocapnia. Three animals were first brought to hypocapnia and then brought to hypercapnia.

2.6 Statistical analysis

Analysis was performed using Excel (Microsoft, Washington, US) and R statistical software (R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/). We used the following packages: tidyverse, lubridate, ggplot2, segmented, stringr, imputeTS, ggpubr.

2.7 Goal

We want to develop a solid piglet model to measure CA in a state of hyper- or hypocapnia. First, we want to confirm that this is possible solely by altering ventilation. Second, we want to know if PaCO2 and ETCO2 can be used as interchangeable parameters. Third, we want to investigate how PaCO2 alters CVR of the arteriolar diameters, but also what the impact is on ICP, PbO2 and LDF.

3 Results

We were able to bring PaCO2 by hyperventilation to values as low as 25 mmHg (corresponding to a maximal RR of 51, though there are big differences between animals). Obtaining even lower PaCO2 seemed impossible by further increasing RR, since the pigs became hemodynamically unstable with rapid changes in ABP along with tachycardia. They also started shivering, which led to a rise in PaCO2 again. By hypoventilation combined with the use of a swivel, we were able to build up PaCO2 as high as 90 mmHg (corresponding to a minimal RR of 14). 90 mmHg was the highest PaCO2 measurable by our gas analyzer. When comparing the values of ETCO2 with the values of PaCO2, obtained by sampling arterial blood gases after 10 min of stabilization at the level of ETCO2, no significant difference was found (two-tailed paired t-test with equal variance, p = 0.93).

In two out of five animals we first manipulated PaCO2 to hyper- and then hypocapnia. The quality of the microscopic imaging in one of these animals turned out to be uninterpretable. In three out of five animals we manipulated first to hypo- and then to hypercapnia. Through the alteration of ventilation and PaCO2, we did not see any significant changes in ABP (Pearson correlation ETCO2 and ABP; R = 0.19). The direction of PaCO2 change drove the change in pial arteriolar diameter, as displayed in Fig. 1, Fig. 2. When using the Granger causality test, there was a significant causal relationship between ETCO2 and both diameters in the hyper-then hypocapnia experiment, depicted in Fig. 1 (p < 0.05). There was no significant causal relationship in the other experiments between ETCO2 and any of the measured diameters, all of which were hypo-then hyperventilation experiments.Fig. 1 The level of ETCO2 is depicted over time for one animal, from normocapnia till hypercapnia (maximum 85 mmHg in this experiment), after which ETCO2 is decreased to 25 mmHg. Both pial arterioles (bv1 and bv2, resp. 239 μm and 71 μm when PaCO2 was 40 mmHg) dilate with increasing ETCO2, however when RR is slightly increased there is a rapid decline of ETCO2, and an even more rapid decline in diameter. By lowering ETCO2, the diameter at ETCO2 60 mmHg is smaller compared to the starting point of ETCO2 at 40 mmHg. This experiment took place over a period of 3 h, which prevented full metabolic compensation.

Fig. 1

Fig. 2 The level of ETCO2 is depicted over time for one animal, the three experiments in which hypocapnia was obtained first followed by hypercapnia look comparable. There is no change in diameter of the pial arterioles when decreasing ETCO2 till 25 mmHg, independent on the baseline size of the blood vessel. When increasing ETCO2 there is a rapid increase in diameter. Starting diameters at PaCO2 of 40 mmHg in this experiment where 229 μm for bv1, 77 μm for bv2 and 152 μm for bv3.

Fig. 2

Correlation coefficients between PaCO2 on the one hand and PbO2, ICP and LDF on the other hand were R = 0.31 (PbO2), R = 0.46 (ICP) and R = 0.58 (LDF). In the higher ranges of ETCO2, there seems to be an increase in PbO2 and ICP. Since these experiments were limited in numbers as exploratory research, we cannot draw any statistical conclusion (Fig. 3).Fig. 3 3a depicts ETCO2 evolution over time in the four experiments. 3b depicts the absolute values of ICP, PbO2 and LDF in the same time scale, however LDF is a relative number as it has no absolute numeric. We can see the relation of these values with ETCO2, how they increase with hypercapnia and decrease with hypocapnia. Especially PbO2 had very different absolute values at start (PaCO2 of 40 mmHg), but there is a change seen in every experiment directly after adjusting ETCO2.

Fig. 3

4 Discussion

In the current study in which pial arteriolar diameters were studied through a cranial window in piglets while changing PaCO2 by ventilation settings, diameter changes followed the direction of PaCO2 changes, but the effect of PaCO2 on the diameters was not linear. It seems that rather the direction of change is the main driver of change in CBF. The biggest effects were seen in experiment 2, which is the only experiment where we first went to hypercapnia and then hypocapnia. This is the only experiment where a causality was proven between ETCO₂ and diameter by the Granger causality test, possibly since this is the only experiment where a clear decrease in diameter is proven. In the other experiments, we did not see a clear decrease in diameter when decreasing from 40 till 25 mmHg of PaCO₂, which is different than previous experiments have shown (Muizelaar et al., 1988; Brian, 1998; Kontos et al., 1977). The effect of different PaCO2 levels on pial vasoreactivity in response to ABP changes (i.e. CA) has been explored in small animals, but is still totally unclear in humans and large animals, and the current experiments are a preparation for such investigation in our piglet cranial window model (Klein et al., 2019). Based on the current study, it was decided to investigate vasoreactivity to ABP changes at PaCO2 levels of 25 mmHg and 60 mmHg. 25 mmHg was the lowest achievable level of PaCO2 with the animal remaining hemodynamically stable. 60 mmHg was chosen as the level of hypercapnia, since there was a clear arteriolar vasodilation at this level, while not being an extremely high level of PaCO2 far outside the normal range.

The porcine cranial window model enables visualization of the pial arterioles, which constitute 21% of arterial resistance (Iadecola, 2017). The method however does not visualize the entire cerebrovascular tree. At present, no methods have been described that depict diameters of penetrating arterioles in real time with sufficient temporal and spatial resolution. Upstream, large arteries can be visualized with transcranial Doppler. Attempts were made to add transcranial Doppler measurements to the current set up, but with the instrumentation already used the piglet head did not allow for reliably securing the Doppler probe. The piglet head also proved too small for reliable application of Near Infrared Spectroscopy probes (NIRS). An estimation of cerebral blood flow was obtained by LDF. LDF is not often clinically used since it is very prone to displacement, but in a controlled laboratory setting it is easily applicable.

PaCO2 is usually measured on arterial blood bases. In the present study, we found a strong and significant association between ETCO2 and PaCO2, and ETCO2 is more practical to monitor in this complex setup. As stated, the actual driver of arteriolar diameter change is the interstitial pH, but its main determinant in the short term and in the present study is PaCO2. Ideally, interstitial pH is included in the monitoring. Microdialysis is unpractical to add to the current model, since it takes too much time to collect readings from the pump that operates at a specific pace, and this is too slow to follow our adjustment of RR. Alternative pH probes were too bulky to apply on the small piglet brain. The Paratrend probe (initially produced by Diametrics Medical Ltd, UK), which can monitor brain interstitial pH and PaCO2, is not available anymore. By modifying PaCO2, indirectly we also modify PaO2. On arterial blood gas, we made sure that the value of PaO2 was always kept between 180 and 200 mmHg. As discussed in the introduction, due to the Bohr effect the decreasing pH increases the delivery of O2 to tissues. Increasing pH shifts the curve to the left and withholds O2 to separate from the Hb and leads to a local increase in CO2 to counteract the increase in pH(Benner et al., 2022; Bö et al., 2020; Stocchetti et al., 2005; Patel et al., 2022)

Finally, we did not observe any change in ABP secondary to changing PaCO2 and pH. This means that, in contrast to cerebral arterioles, the susceptibility of the systemic circulation to changes in PaCO2 is minimal (Frö et al., 2018; Caldwell et al., 2021). This is important for our next experimental setup, where we want to investigate the effect of altering ABP to investigate CA in a state of hyper- or hypoventilation. If the level of PaCO2 already had a clear influence on ABP it would blur our methods and results, since the influence of changing ABP is the main variable that we want to explore during the experiment. It will be of paramount importance to maintain a very stable level of PaCO2 during the experiment, since small alterations could have an effect on CVR and may lead to a wrongful interpretation of our results.

5 Conclusion

Altering the RR to manipulate PaCO2 by ventilation appeared to be feasible and reliable. We found that ETCO2 reliably represents PaCO2 to be used in our piglet cranial window model. Pial arteriolar diameter changes followed the direction of PaCO2 changes, but the effect of PaCO2 on the diameters was not linear.

Author contribution statement

SD, GM and BD designed the study concept. SD developed the protocol and performed all the experiments. VDS contributed to the experimental work and optimized the anesthesiology setup. SD analyzed the data and wrote the manuscript. GM and BD supervised the project.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Sincere thanks to Stéphanie De Vleeschauwer, who works as a veterinarian for the large animal facility of KULeuven.
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