Transcardiac Perfusion in Mice: Comparing Heart-Beating and Non-Beating Conditions for Brain Histological and Electrophysiological Analyses

Transcardiac Perfusion in Mice: Comparing Heart-Beating and Non-Beating Conditions for Brain Histological and Electrophysiological Analyses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transcardiac Perfusion in Mice: Comparing Heart-Beating and Non-Beating Conditions for Brain Histological and Electrophysiological Analyses NADEGE SARRAZIN, Delphine Roussel, Joanna Droesbeke, Charlotte Deleuze, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8544231/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract In vivo and ex vivo animal model experiments are essential for understanding the mechanisms underlying neurological diseases. These investigations bridge the gap between organism behavior and cellular pathways, providing a holistic view of brain function. A key factor is the quality of tissue, which depends on the efficacy of the perfusion process. Rapid and uniform delivery of fixative or physiological solutions is crucial to preserve tissue integrity, yet the effectiveness of perfusion under non-beating heart conditions remains debated. Our study aimed to optimize perfusion conditions for quality brain tissue samples, complying with ethical standards. We tested both beating heart conditions with xylazine/ketamine and non-beating heart conditions with xylazine/pentobarbital, with perfusion following cardiorespiratory arrest, occurring within 5 minutes in our conditions. Electroencephalography and electrocardiography measurements following pentobarbital injection demonstrated a rapid cessation of brain activity coinciding with the onset of irregular, non-sinusoidal cardiac rhythms. Death was confirmed at the time of thoracotomy, performed after cardiorespiratory arrest, which consistently occurred within a 5 minutes window post-injection. Semi-quantitative histology and neurotransmitter immunohistochemistry showed no significant differences between the 2 conditions. Electron microscopy confirmed good tissue quality in both, with similar results in functional studies using electrophysiological approaches. This study demonstrates that post-mortem transcardiac perfusion in mice, performed under conditions where perfusion is carried out very quickly, reliably yields high-quality brain samples for histology, cytology, and electrophysiology. Our findings help address controversies regarding perfusion efficacy and highlight the need to reconsider euthanasia practices to ensure sample quality while minimizing impact on animals and researchers. Biological sciences/Biological techniques/Experimental organisms/Model vertebrates/Mouse Biological sciences/Biological techniques/Biological models/Neurological models transcardiac perfusion electrophysiology histology animal welfare ethics euthanasia methods mice 3R wellbeing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION In neuroscience research, TransCardiac Perfusion (TCP) is a widely used method to preserve brain tissue integrity. This technique delivers fixative or physiological solutions uniformly through the cerebral vascular system. While direct immersion treatment works for small samples, it is less reliable for larger ones due to inconsistent penetration, affecting tissue viability and morphology. Despite the risk of irreversible brain damage with delayed perfusion, many researchers argue that TCP is feasible post-mortem. Training staff in TCP presents significant challenges, requiring advanced skills and precision, while considering ethical and scientific standards under the 3Rs principles. This study, based on internal discussions among stakeholders, addresses specific questions related to TCP. Pain-free methods The brain harvesting process requires careful oversight to ensure it is painless. The alkaline pH of pentobarbital can lead to peritoneal irritation and pain in mice highlighting the need for refinements 1 . Peritoneal local anesthesia reduces pain 2 , but it has limitations, offering no overall relaxation or sedation. In contrast, xylazine offers both rapid sedation and analgesic effects 3 . This enhancement quickly calms the animal, reducing distress for both the animal and the operator. This study aims to evaluate the physiological effects of administering xylazine prior to a pentobarbital overdose on heart and brain activity in mice, measured through Electrocardiogram (ECG) and Electroencephalogram (EEG) recordings. To ensure perfusion with a beating heart, deep anesthesia is achieved using a combination of xylazine and ketamine. This approach aims to determine the ideal timing to ensure the animal is fully dead before perfusion begins, allowing the tissue processing to be adapted to the specific requirements of the sample. It also upholds ethical and welfare standards. Challenges associated with lived and fixed tissue. The brain sample must be carefully adapted to analysis to be performed. Tissue fixation must maintain the structure of tissues and cells as closely as possible to their natural state. Using the appropriate fixative solution is critical because it is perfused through the cerebral vasculature and diffuses into the surrounding tissue and therefore, directly affecting preservation. It is formaldehyde's ability to diffuse rapidly in tissues to preserve cellular proteins that makes it invaluable a general-purpose fixative in histology 4 . Fixation for ultrastructural studies is more delicate and too long perfusion time could cause structural changes in the synapses 5 . Fixation is achieved through a succession of treatments. First, glutaraldehyde, a fixative that reacts with proteins, preserves tissue structure. Osmium tetroxide and uranyl acetate are then applied to stabilize membrane lipids and enhance contrast, respectively 6 . Live tissues are used for functional studies using electrophysiology and imaging approaches. Brain slices provide important information on membrane excitability, firing profile, synaptic activity and plasticity, essential for understanding neuronal networks. Good tissue viability is crucial for successful electrophysiological recordings. However, this can be difficult to obtain, particularly in adult mice that are more susceptible to anoxia. To address this issue, TCP can be used to rapidly perfuse the brain with ice-cold artificial cerebrospinal fluid (aCSF), preserving tissue viability, integrity, and functionality, and improving the quality of electrophysiological recordings from brain slices 7 . In this study (Fig. 1), we aimed to (1) determinate the most suitable euthanasia method and timing corresponding to various states of cardiac and brain activity until death, and (2) evaluate the effectiveness of TCP under Non-Beating Heart (NBH) and Beating Heart (BH) conditions. This evaluation was based on histological analysis of fixed tissues and the assessment of live tissue functionality using electrophysiological techniques. RESULTS Euthanasia by overdose of pentobarbital versus xylazine/Ketamine We aimed to understand the stages leading to death during the TCP-specific euthanasia protocol by performing EEG and ECG recordings, ensuring the timeline closely aligns with the animal's death to minimize tissue damage. In NBH condition, mice received a xylazine administration followed by pentobarbital overdose or in BH condition a xylazine-ketamine overdose (Fig 2a). Using Fast Fourier Transform (FFT), EEG were visualized in a color-coded format, revealing a rapid onset of deep blue hues indicative of suppressed brain activity and reduced neuronal excitability. Quantitative analysis showed that in the NBH group (n= 15), the mean duration of EEG activity after injection was 133.6 ± 47.49 seconds, whereas in the BH group (n= 5), it averaged 410 ± 91.95 seconds (Fig 2b). This difference was statistically significant (P<0.0001), indicating that pentobarbital, at our concentration induces a faster and more consistent cessation of brain activity compared to xylazine/ketamine overdose. Heart rate monitoring revealed a significant decrease following xylazine administration, consistent with its expected sedative and bradycardic effects. Cardiac arrest occurred rapidly, with fibrillation persisting for extended durations. Analysis of RR intervals showed disruption within the first 100 seconds after supine positioning (Fig 2c), and residual ECG activity persisted beyond 200 seconds, reflecting isolated cardiomyocyte contractions. This loss of coordinated pump function led to ineffective circulation, evident from distinct color changes in the heart, with darker atria. These results suggest that performing post-mortem TCP within five minutes of pentobarbital administration ensures the animal is deceased, with a non-beating heart. Influence of Perfusion Conditions on Histological Samples Firstly, we independently evaluated samples using a scoring system that assessed various criteria: overall staining, border effects, presence of dark neurons, vacuolization, and periventricular space (Fig 3a). No significant difference was observed for the NBH and NH group (P=565). Additionally, we tested the effects of heparin usage and compared samples from mice perfused with powdered PFA, no discernible structural differences were observed (Fig 3b). Secondly, we performed immunohistochemistry for Iba1, GFAP in cortex (Fig 3c), and VGluT1 in the cortex and cerebellum (Fig 3d), which showed no discernible difference between perfusion methods, confirming the consistency of our findings across different analytical techniques to ensure that the nature of perfusion had no effect. Consistent with the results obtained in histology, the samples (6NBH, 6BH) observed in electron microscopy show no difference between groups at least in the CA1 hippocampus (Fig 4). The ultrastructural integrity of the cells is maintained in both NBH and BH samples (Fig 5). Brain sections of post-mortem samples (Fig 4b, d) revealed a diversity of cell types with resolution and quality comparable to those observed in ante-mortem samples (Fig 4a, c). These included microglia, endothelial cells, oligodendrocytes, and neurons, each displaying well-defined cellular organelles such as the nucleus, mitochondria, and endoplasmic reticulum, all observed at high resolution (Fig 5a-f). We looked for synapses in the CA1 stratum radiatum region of the hippocampus because of the homogeneity of the synapses (mainly glutamatergic). The two components of synapses were observed: presynaptic boutons with numerous vesicles of homogenous size (20 to 30 nm in diameter) and post synaptic element showing a more or less thick, electron-dense zone in front of the presynaptic element (Fig 5c). Excellent visibility of the different organelles was achieved in cells: the nuclear field with it’s double layered envelope interrupted from place to place by a nuclear pore (not shown), cisternae of endoplasmic reticulum often associated with rows of ribosomes, Golgi apparatus stacks with budding vesicles, microtubules and neurofilaments (Fig 5b). There was no difference in the number of lysosomes per neuron both in NBH and BH samples. Also in both cases, the mitochondria were the same showing well-preserved cristae. With this absence of difference in number and morphology, we decided to stay at this qualitative level. Functional impact of perfusion conditions on electrophysiological properties of neurons. We investigated whether NBH TCP were associated with any functional changes in neuronal activity, using electrophysiological approaches. First, basal synaptic transmission and short-term plasticity were assessed at the CA3-CA1 hippocampal synapse using Multielectrode array (MEA) technique (Fig 6a1). The input-output (I/O) relationships between evoked fPSP slope and stimulus intensity were not significantly different between BH and NBH groups (p=0,275, F(1,16)=1.276; BH: n=7 slices/ 3 mice, NBH: n=11 slices/4 mice), suggesting consistent basal synaptic strength under the conditions tested (Fig 6a2). Short-term synaptic plasticity was assessed using a standard paired-pulse stimulation protocol. Paired-pulse facilitation was generated at CA3-CA1 synapses in both tested groups. The paired-pulse ratios (PPR) were not significantly different between BH and NBH groups (p=0.8507, F(1,16)=0.036), suggesting that NBH TCP did not alter short-term plasticity (Fig 6a3). Next, we investigated the electrophysiological properties of putative Layer 5 pyramidal cells of the somatosensory cortex using whole cell patch clamp recordings (Fig 6b1). Pyramidal cells in both TCP conditions (NBH: 4 mice; BH: 3 mice) showed similar excitability profiles in response to depolarizing current steps. Both the rheobase (170 ± 25.5, n=5 in NBH vs 166.7 ± 30.73, n=6 in BH; p=0.8463) and the IO curve of the firing frequency showed no significant difference between the two groups. There was also no change in the sag potential indicative of the activation of the hyperpolarization-activated inward current (I h ) (0.2206 ± 0.03008, n=5 in NBH vs 0.2309 ± 0.03904, n=6 in BH; p=0.5368) (Fig 6b2). Furthermore, the passive membrane properties of pyramidal cells were also unaffected by TCP conditions in NBH (n=6) vs BH (n=6) groups. Membrane resting potential (-69.58 ± 2.716 vs -70.98 ± 2.875; p=0.9372), input resistance (158.7 ± 26.69 vs 153.0 ± 34.31; p=0.6991) and capacitance (205.3 ± 25.36 vs 181.2 ± 13.68; p=0.3939) showed no changes (Fig 6b3). Recordings of spontaneous postsynaptic currents (sPSCs) revealed a slightly smaller amplitude of events in the BH group (11.81 ± 0.9029, n=6 in NBH vs 9.273 ± 0.5073, n=6 in BH; p=0.0368) but the frequency remained unchanged (11.42 ± 1.337; n=6 in NBH; vs 8.46 ± 1.133; n=6 in BH; p=0.1320) (Fig b4). DISCUSSION results. A key debate in this context centers around whether intracardiac perfusion should be conducted on a beating versus non-beating heart. Our comparative analysis of xylazine/pentobarbital and xylazine/ketamine injections demonstrated that pentobarbital in our experimental study, induces a more rapid and consistent cessation of brain activity, making it a preferable agent for euthanasia. This quick onset of brain inactivity supports pentobarbital’s effectiveness in ensuring tissue quality. Importantly, regardless of the euthanasia agent used, initiating perfusion with a pump or other perfusion system remains crucial to ensuring consistent delivery of the perfusion fluid. As describe recently in a comparative study of pre- and post-mortem perfusion, the method of tissue fixation has a major impact on experimental parameters and there was not only one condition that provided the best results for all different analysis 8 . The time elapsed between death and tissue processing affects the quality of the samples and the use of a peristatic pump in all conditions (BH versus NBH) to mimic the dynamics of the heart's biological processes is essential. The preparation of living acute brain slices has long been established as a powerful model for investigating synaptic connectivity in neuronal circuits 9 . Since the 1990s, TCP has been used to reduce damage caused by decapitation and dissection, as well as to address the increased susceptibility to anoxia in older tissues. While many studies have explored protocols for slice preparation and various parameters such as physiological solutions and incubation temperature, few emphasized the importance of performing TCP on a beating heart ( 10 , 7 ). In the present study, we demonstrated that this factor does not significantly influence slice quality. By recording extracellular fPSP using MEA, we showed that the strength of synaptic transmission and short-term plasticity were not affected at CA3-CA1 hippocampal synapses by the NBH TCP. However, these results should be interpreted with caution, as other relevant pattern of synaptic activity, such as long-term plasticity, were not tested. Moreover, patch clamp recordings revealed no alteration of the passive membrane properties and excitability of Layer 5 cortical pyramidal cells. Spontaneous synaptic currents are slightly smaller in BH condition, but this could be due to the small number of cells recorded in each group and more experiments will be needed to confirm this effect. While living acute brain slices are valuable for studying synaptic connectivity, tissue preservation protocols for fixed tissues focus more on maintaining ultrastructural integrity. Previous electron microscopy studies have shown that delayed perfusion fixation causes specific alterations in postsynaptic densities and cytoplasmic structures across various neuronal cell types, although the precise timing of the delay was not specified 5 . In our experiments, we did not observe significant changes in samples from the NBH group, provided the delay did not exceed 5 minutes after the injection of euthanasia drug. This study was exclusively conducted on adult mice, which limits generalizability. Protocols and procedures may not be directly applicable to younger mice or other species, such as rats 11 . For example, perfusion rates might need adjustment for neonatal mice pups to prevent damage to their delicate tissues 12 . Future research should explore the applicability of these methods to different age groups and species to ensure optimal results and minimize adverse effects. Moreover, to enhance the removal of residual blood, we evaluated the use of heparin, which inhibits coagulation and maintains vascular patency, thereby improving perfusion efficiency. Heparin was administered either by adding it to the wash solution or through direct intracardiac injection. Although not explored in our study, intraperitoneal (IP) injection represents an alternative route of administration. Additionally, sodium nitrite (NaNO₂), a vasodilator, has been shown to significantly reduce residual blood without compromising the integrity of the blood-brain barrier 13 . Our analyzes indicate that these interventions do not affect the quality of samples for downstream applications (data not shown). However, they markedly enhance perfusion efficiency, allowing for more rapid clearance of blood and facilitating the achievement of a "white liver" state in a shorter time frame. Finally, our work received ethical approval in 2021, using xylazine and ketamine at appropriate doses for anesthetizing mice in a "beating heart" procedure. Recent advancements suggest stronger analgesia for this invasive surgery (thoracotomy and laparotomy), even if classified as non-recovery. A multimodal approach, including an opioid (e.g., fentanyl or buprenorphine), an alpha-2 agonist (e.g., xylazine or medetomidine), and possibly a local anesthetic, would likely be recommended today. Finally, from the experimenter's point of view, our study was conducted by skilled users, including a single experienced individual who performed all TCP throughout the study. Performing TCP is an invasive procedure that requires quick and precise handling, which can be emotionally challenging for the experimenter. There are few specific studies on professionals working with laboratory animals, particularly those involved in procedures such as euthanasia and TCP. However, these are essential, given that the field of laboratory animal probably poses unique challenges in terms of compassion fatigue 14 . To promote a culture of care in animal research, it’s essential to consider the wellbeing of experimenters, as there is a close link between individual performance and a supportive working environment 15 . Encouraging this type of study not only refines the procedure but also strengthens the bonds between all professionals involved in the process. By working together to refine procedures such as TCP, we increase mutual understanding and foster a collaborative environment that prioritizes both animal and human wellbeing for the best science. CONCLUSION Our findings were unexpected for researchers accustomed to the beating heart method. This study is the first to investigate the implementation of TCP procedures under both BH and NBH conditions at the onset of perfusion, for both fixed and live brain tissue, with histological, electron microscopy, and electrophysiological analyses. Our study highlights that overdose of pentobarbital with the use of peristatic pump is a better, more effective, euthanasia agent compared to xylazine/ketamine for inducing a rapid cessation of brain activity. Contrary to expectations, the tissue processing conditions including the choice of perfusion methods and fixatives did not significantly impact the histological or electrophysiological quality of the samples. These results are essential for standardizing protocols in neuroscience research, ensuring greater consistency and reliability across studies. Furthermore, we have improved our methodologies to better address animal welfare concerns in experimental procedures. Future research should focus on further optimizing these protocols and exploring the influence of other variables on tissue quality and function. MATERIALS AND METHODS Animals Manipulation of animals were carried out in accordance with the guidelines of the European Union (directive 2010/603/EU) and received approval (APAFIS n #37052) from the French Ministry for Research. Wild-type C57BL/6 mice were housed under specific-pathogen-free conditions at the PHENO-ICMice facility of the Paris Brain Institute. They were provided with food and water ad libitum and maintained on a 12-hour light/dark cycle (lights on from 8:00 AM to 8:00 PM). For each group, we used 7 to 10-week-old C57Bl6J wild type mice (half male/ female). Brain and cardiac monitoring during euthanasia: As described in previous study 16 , simultaneous EEG-ECG recordings were used to monitor time of death. Two groups of mice, reused from a previous study involving EEG electrode implantation, were studied under identical brain implantation conditions. Anesthesia and euthanasia procedures are outlined in the following section. Time of death was determined by the absence of visible EEG activity. QRS component of the ECG was recorded from mice immediately after they were positioned in decubitus following the administration of pentobarbital. Anesthesia and euthanasia For the NBH group, mice are initially sedated with a xylazine injection (Rompun,15 mg/kg, ip) followed 10 minutes after by injection of 100µL sodium pentobarbital (Euthasol®, 700 mg/kg ) to induce euthanasia. We waited until cardiac and respiratory arrest, maximum 5 minutes after pentobarbital injection, before starting TCP procedures. For the BH group, mice are subjected to deep anesthesia using a combination of ketamine and xylazine (Imalgène, 150 mg/kg, Rompun,15 mg/kg, ip). Transcardiac perfusion (TCP) After grasping the skin of the thorax with forceps, an incision was made to expose the xiphoid process, followed by lateral cuts to reveal diaphragm and liver. Incisions were then made along the diaphragm followed by two cuts on each side up of the ribcage down to the clavicles. The sternum was lifted for complete exposure of the heart and lungs. The heart was held with dissecting forceps and a 27-gauge needle was inserted into the left ventricle, angled roughly parallel to the heart's midline, advancing toward the ascending aorta. The needle was clamped in place and a small incision was made over the right atrium using fine scissors, allowing venous blood flow. The intracardiac perfusion was then initiated promptly, maintaining a constant speed of approximately 3-5 ml per minute, except for electron microscopy samples, where it was slowed down to 2ml per min. Fixative solution or aCSF was perfused for histology and in vitro electrophysiology experiments respectively (see below for details). For the histology and ME one additional group was included in which mice received heparin (Choay-Sanofi) in the saline solution at a concentration of 50 IU/ml to prepare BH and NBH+h group samples. Once the draining fluid was free of red blood cells (after 20-50 ml saline for adult mice), the liver's discoloration indicated successful perfusion, the perfusion stopped, and the brain was extracted. Histology & Immunohistochemistry For BH and NBH, mice were perfused with a fixative solution (4% paraformaldehyde powder Merck #1.04005 or liquid EMS #15714). Following extraction from the skull, brains underwent additional overnight fixation in a fresh 4% PFA/PBS solution before being embedded in paraffin. Sagittal sections (5μm thick) were then prepared using a paraffin microtome (Leica). We utilized the markers IBA1, GFAP and VGluT1 to study cell types and neuronal structures. For immunostaining, brain tissue sections were deparaffinized and subjected to antigen retrieval (in citrate buffer at 110°C in a decloaking chamber for 1min 30 for GFAP and 25min for IBA1) to reveal hidden epitopes. For observing glutamate neurons and microglia, subsequently, sections were incubated overnight at 4°C with primary antibody (Vglut1, 1/1000, Synaptic system #135304; IBA1, 1/100, Wako #01919741; GFAP, 1/500, Dako #Z0334). Following primary antibody incubation, sections were exposed to secondary biotinylated horse anti-rabbit (IBA1 and GFAP) or goat anti-guinea pig (Vglut1) for 30 minutes (Vector, dilution 1/250) and visualized using the ABC method (Vector Laboratories) with 3,3-diaminobenzidine (DAB, Sigma-Aldrich) as the chromogen. For Nissl staining, sections were dewaxed and rehydrated before being stained in a thionin solution (Sigma #T-3387). Stained sections were digitized at x20 magnification using a slide scanner (nanozoomer S60/ Hamamatsu). Two blinded experimenters evaluated BH and NBH samples using a semi-quantitative method of scoring from 0 to 3 for various criteria: general coloration, border effects, perivascular space, global vacuolization, and the presence of dark neurons. By applying these diverse criteria, we acquired a comprehensive score, reaching a maximum of 17 for the samples, and enhanced our understanding of potential variances among them. Electron microcopy For BH and NBH, mice were perfused with fixative solution (2% w/v paraformaldehyde, 2% v/v glutaraldehyde in 0.12 M phosphate buffer pH 7.4 (PB)). After one hour at 4°C brain was extracted from the skull and post-fixed in fresh fixative overnight at +4 °C. Brains were rinsed in PB and sliced in 1 mm thick coronal sections using an adult mouse brain acrylic matrix. Section of Bregma -1.58mm was selected, washed in 0.12M PB and post-fixed at room temperature (21 °C) for 1 h in 1% osmium tetroxide in PB. After rinsing with water, sections were incubated with 2% aqueous uranyl acetate overnight at +4°C. They were washed in water and dehydrated in a series of of ethanol (30%, 50%,70%, 90%, 100% (X3)) and 3 times with 10% acetone. Sections were incubated overnight in a 50:50 solution of acetone: Epon 812R (EMS, Souffelweyersheim, France). They were incubated twice with fresh Epon 812 resin for 1h at room T°, embedded in moulds. Polymerisation took place at +56°C for 48 h in a dry oven. Blocks were cut with an UC7 ultramicrotome (Leica Microsystems, Nanterre, France). Semi-thin sections (0.5–1 μm) were stained with a 1% v/v Toluidine Blue in 1% w/v Borax aqueous solution. Ultra-thin sections (60–70 nm) were cut, placed onto 200 mesh copper grids (EMS, Souffelweyersheim, France) and stained with Reynolds lead citrate for 7 min (Reynolds, 1963). Grids were examined using a Hitachi HT7700 transmission electron microscope (TEM, Milexia France) operating at 120kV. Pictures (2048x2048 pixels) were taken with an AMT41B camera. Variability among groups regarding the presence of fixation artifacts and the relative number of dark neurons. Additionally, a qualitative evaluation of the morphological changes affecting neurons, glial cells (oligodendrocyte, astrocyte and microglia), endothelial cells, pericytes notably their subcellular compartments. Slice preparation for in vitro electrophysiology TCP was performed with ice-cold solution containing (in mM): 200 sucrose, 26 NaHCO 3 , 2.5 KCl, 1.25 NaH 2 PO4, 7 MgSO 4 , 0.5 CaCl 2 , 3 pyruvic acid, 3 myo-inositol, 0.4 ascorbic acid and 16 glucose, saturated with 95% O 2 / 5% CO 2 . Brains were removed and acute coronal slices (300 μm) containing somatosensory cortex and hippocampus were cut in the same solution, using a vibratome (Leica VT1200S). Slices were then incubated in oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 2.5 KCl, 2 CaCl 2 , 1 MgSO 4 , 1.25 NaH 2 PO 4 , 26 NaHCO 3 and 16 glucose (pH 7.4), at 32°C for 30 min, and subsequently at room temperature. MEA recordings Hippocampal Slices were transferred on a polyethyleneimine coated chip (MED-R515A), continuously perfused with aCSF (34°C) and recordings started after 20 minutes recovery period. Extracellular field postsynaptic potentials (fPSPs) were recorded in CA1 stratum radiatum following stimulation of Schaffer collateral with a 64-channel multielectrode array (MEA) system (Alpha MED Scientific). Extracellular field potentials were bandpass filtered between 1Hz-10kHz and acquired at a sampling rate of 20 kHz. fPSPs were evoked by a series of stimuli of increasing intensities (10-100μA) and the related Input–output (I/O) curves of the fPSP slope were generated. The intensity of stimulation eliciting 40–50% of the maximum response was then determined from the IO curve and used to investigate short term plasticity. Paired pulse stimulation was applied at increasing inter-pulse interval (20-200ms). Paired pulse ratio (PPR) was calculated by dividing the slope of the second fPSP by the slope of the first one. Data analysis was performed using a custom R script and GraphPad Prism 9 software Patch-clamp recordings Whole cell recordings were obtained at 30°C from layer V pyramidal cells of the somatosensory cortex. Glass electrodes were filled with a solution containing (in mM): 133 K-gluconate, 11 KCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 4 Na-phosphocreatine; 290mOsm. No liquid junction potential correction was applied. Signals were acquired at a sampling rate of 20 kHz or 50Hz and filtered at 2kHz or 10Hz in voltage or current clamp, respectively (Multiclamp 700B, Molecular devices). Access resistancewas <20 MΩ. Resting membrane potential was measured 5 minutes after whole cell configuration establishment. Spontaneous postsynaptic currents (sPSCs) were recorded at -70 mV, and thus mainly excitatory AMPA-receptors mediated currents in our recording conditions. sPSCs were detected using a custom software (Wdetecta, J. R. Huguenard, Stanford University) and their mean amplitude and frequency calculated over 1-3 minutes. Voltage responses to small current steps (−30pA; 1sec; 20 times) were used to extract passive membrane properties from the averaged curve. Input resistance (R in ) was calculated at steady state, membrane time constant (t m ) was determined by fitting a single exponential function and membrane capacitance was calculated with the equation C m Voltage responses to a series of current steps (1sec; 50pA increment) were recorded at -70 mV. Evoked firing properties were determined from responses to depolarizing steps. Action potentials (AP) were detected using a threshold criterion. The rheobase is the minimum current intensity that triggers the first spike, the mean firing frequency was calculated (number of AP/s). Activation of hyperpolarization-activated cation current (I h ) was estimated from the voltage deflection (sag) in response to hyperpolarizing steps (-300/-50pA). Sag amplitudes were determined by the difference between peak and steady state potentials and normalized to the peak. The average of normalized sag potential was used as an I h estimate. Data were analyzed with pClamp10 (Molecular devices), Prism (GraphPad) software and custom-written scripts (MATLAB, MathWorks). Quantification and Statistical analysis For the statistical analysis of histology scores, the Kruskal-Wallis test was applied to compare the different groups. In vivo data : EEG data were analyzed using Neurowork or Deltamed software (Natus Medical Incorporated, Pleasanton, CA) alongside custom-written scripts in MATLAB (MathWorks). Statistical analyses were conducted using GraphPad Prism, with parameters compared between two experimental groups using Mann Whitney test. ECG data : RR intervals were analyzed using Spike2 software, and heart rate was determined by calculating the number of RR intervals in 2 seconds (Spike2 v7.06; Cambridge Electronic Design, Cambridge, UK). MEA (https://gitlab.com/icm-institute/dac/biostats/MEASpikeR): Data were analyzed using Mobius software and a custom R script. All data were analyzed using Repeated measures two-way ANOVA. All results are reported as mean + SEM with significance set at a p-value of less than 0.05 (∗). Patch Clamp : All data are presented as mean ± SEM. All statistical analyses were performed in Prism (GraphPad) and were two-tailed. Unmatched non-parametric two-way ANOVA was used for mean firing frequency (FI curve), and unpaired Mann-Whitney tests were used for other data. P values lower than 0.05 were considered statistically significant. Declarations AUTHOR CONTRIBUTION D. R., J. D., C. D., C. D., D. L., S. I., A. P., L. S., participated in experimental investigations and analyses. D. R., B.D., N.S. designed the study, D.R., C. D., C. D., D. L., A. P., B.D., N.S. wrote and revised the manuscript. All authors reviewed the manuscript before submission. ACKNOWLEDGMENTS We thank the ICM core facilities: ePHYS, PHENO-ICMice, ICM.Quant and Histomics supported by Investissements d’Avenir (ANR-10-IAIHU-06 and ANR-11-INBS-0011-NeurATRIS). We thank Francois-Xavier Lejeune (DAC, Paris Brain Institute) for the development of a data analytics tools box for MEA data analysis. We also thank Serge Marty for his review and valuable advice on the manuscript, as well as Alice Gilbert, Stéphanie Baulac and Jean-Christophe Poncer for allowing the reuse of EEG-implanted mice. References Laferriere, C. A. & Pang, D. S. 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W., Dosemeci, A. & Reese, T. S. Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. J Comp Neurol 501 , 731-740, doi:10.1002/cne.21276 (2007). Palay, S. L., McGee-Russell, S. M., Gordon, S. & Grillo, M. A. Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide. THE JOURNAL OF CELL BIOLOGY 12 , 385 - 410 (1962). Lipton, P. et al. Making the best of brain slices: comparing preparative methods. Journal of Neuroscience Methods 59 , 151 - 156 (1995). Meyer-Dilhet, G., Ellouze, S., Raineteau, O. & Courchet, J. Comparative study of pre- and post-mortem perfusion of fixative for the quality of neuronal tissue preparation. Lab Anim (NY) , doi:10.1038/s41684-025-01633-1 (2025). Yamamoto, C. & McIlwain, H. Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro. J Neurochem 12 , 1333 - 1343, doi:doi: 10.1111/j.1471-4159.1966.tb04296.x. (1966). Eguchi, K. et al. Advantages of acute brain slices prepared at physiological temperature in characterization of synaptic functions. bioRxiv doi:10.1101/845461 (2019). Zatroch, K. K., Knight, C. G., Reimer, J. N. & Pang, D. S. Refinement of intraperitoneal injection of sodium pentobarbital for euthanasia in laboratory rats (Rattus norvegicus). BMC Vet Res 13 , 60, doi:10.1186/s12917-017-0982-y (2017). Perez Arevalo, A., Lutz, A.-K., Atanasova, E. & Boeckers, T. M. Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development. PLOS ONE 17 , 1 - 8, doi: https://doi.org/10.1371/journal.pone.0275780 (2022). Noh, K., Liu, X. & Wei, C. Optimizing transcardial perfusion of small molecules and biologics for brain penetration and biodistribution studies in rodents. Biopharmaceutics & Drug Disposition 44 , 71 -83, doi:https://doi.org/10.1002/bdd.2317 (2022). Randall, M. S., Moody, C. M. & Turner, P. V. Mental Wellbeing in Laboratory Animal Professionals: A Cross-Sectional Study of compassion Fatigue, Contributing Factors, and Coping Mechanisms. Journal of the American Association for Laboratory Animal Science 60 , 54 - 63, doi:DOI: 10.30802/AALAS-JAALAS-20-000039 (2021). Ferrara, F. et al. Culture of care in animal research – Expanding the 3Rs to include people. Laboratory animals 56 , 511 - 518, doi:DOI: 10.1177/00236772221102238 (2022). Bacq, A. et al. Cardiac Investigations in Sudden Unexpected Death in DEPDC5-Related Epilepsy. ANNALS of Neurology 91 , 101 - 116, doi: 10.1002/ana.26256 (2022). Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8544231","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":576750908,"identity":"11796192-f4cd-432c-8ad1-105e20c1dd85","order_by":0,"name":"NADEGE SARRAZIN","email":"data:image/png;base64,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","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":true,"prefix":"","firstName":"NADEGE","middleName":"","lastName":"SARRAZIN","suffix":""},{"id":576750909,"identity":"4640f2fc-8645-4f63-bcbc-d93736164277","order_by":1,"name":"Delphine Roussel","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Delphine","middleName":"","lastName":"Roussel","suffix":""},{"id":576750910,"identity":"4b40e2a8-2757-447a-8200-0e5f12cec297","order_by":2,"name":"Joanna Droesbeke","email":"","orcid":"","institution":"institut Pasteur","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Droesbeke","suffix":""},{"id":576750911,"identity":"aaeaa299-1f9f-45a3-a47d-32a44c4e54c9","order_by":3,"name":"Charlotte Deleuze","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Deleuze","suffix":""},{"id":576750912,"identity":"03266189-2ae1-49fd-8459-3fa231a00405","order_by":4,"name":"Carine DALLE","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Carine","middleName":"","lastName":"DALLE","suffix":""},{"id":576750913,"identity":"3f7eca52-d8a2-47f4-a2a7-3fde9ed9bf33","order_by":5,"name":"Dominique Langui","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Dominique","middleName":"","lastName":"Langui","suffix":""},{"id":576750914,"identity":"4eb8ecb6-8ba1-497f-be5c-65ae22312cf1","order_by":6,"name":"Sonita ING","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Sonita","middleName":"","lastName":"ING","suffix":""},{"id":576750915,"identity":"29f655f7-4504-4bf6-bdc9-7063953e7349","order_by":7,"name":"Annick PRIGENT","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Annick","middleName":"","lastName":"PRIGENT","suffix":""},{"id":576750916,"identity":"a42faee6-7b9d-4e6c-9ce5-f52537902284","order_by":8,"name":"Lev STIMMER","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Lev","middleName":"","lastName":"STIMMER","suffix":""},{"id":576750917,"identity":"7fa66dd4-a8a8-468c-b2ea-743331e8af55","order_by":9,"name":"Benedicte DABOVAL","email":"","orcid":"","institution":"Paris Brain Institute","correspondingAuthor":false,"prefix":"","firstName":"Benedicte","middleName":"","lastName":"DABOVAL","suffix":""}],"badges":[],"createdAt":"2026-01-07 17:40:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8544231/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8544231/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104402045,"identity":"71ee7490-e4d2-4b45-80de-2d244c01c1bd","added_by":"auto","created_at":"2026-03-11 12:14:06","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":190670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDescription of Experimental Design and Groups. (a)\u003c/strong\u003e In the Non-Beating Heart Group (NBH), mice are sedated with xylazine (15 mg/kg) and euthanized with an overdose of pentobarbital (700 mg/kg) followed by TransCardiac Perfusion (TCP) after cardiac arrest. Brain and cardiac activity were monitored by electroencephalogram (EEG) and electrocardiogram (ECG). In the Beating Heart Group (BH), mice are anesthetized with xylazine/ketamine (15/150 mg/kg), and perfusion is performed with peristaltic pump while the heart is beating. \u003cstrong\u003e(b)\u003c/strong\u003e Fixed tissues analysis after TCP using peristaltic pump, from both groups are examined by histology and electron microscopy. \u003cstrong\u003e(c)\u003c/strong\u003eFunctional analysis by Multi-Electrode Array (MEA) and patch clamp techniques were used to record neuronal electrical activity in brain slice.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544231/v1/838d50ab6e33db511209f678.jpeg"},{"id":103957901,"identity":"6cf5a49c-8c0f-4d5e-b25c-832010e790a4","added_by":"auto","created_at":"2026-03-05 03:41:04","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":181368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectroencephalogram (EEG) and Electrocardiogram (ECG) Monitoring during euthanasia. (a)\u003c/strong\u003e Dashed lines indicate the different phases of the protocol: analgesia induced by xylazine and euthanasia induced by an overdose of pentobarbital. The color-coded fast Fourier transform (FFT) power spectrum illustrates the changes in EEG amplitude and frequency from the motor cortex (M1) in correlation with ECG recording and Heart rate (HR) changes observed during euthanasia. \u003cstrong\u003e(b)\u003c/strong\u003e Duration of EEG activity following the administration of pentobarbital (n = 19) or xylazine/ketamine (n = 5) are shown. Results are expressed as mean ± standard deviation. \u003cstrong\u003e(c)\u003c/strong\u003e Comparison of RR interval plots during euthanasia (n=3, yellow, blue and red circles) by pentobarbital overdose versus mice undergoing anesthesia (black circle). Arrows indicate time points 1, 2, 3, and 4 with corresponding ECG tracings.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544231/v1/314efd37580dd1f7fd42e582.jpeg"},{"id":103957906,"identity":"874f51cc-ec27-4b91-8d22-f31f836690ed","added_by":"auto","created_at":"2026-03-05 03:41:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":387184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistology study of fixed tissues in NBH (Non-Beating Heart) and BH (Beating Heart) groups. (a)\u003c/strong\u003e Scores used for dark neurons, perivascular space, and vacuolization range from 0 (no anomaly) to 2/3, with anomalies marked by red arrows. \u003cstrong\u003e(b)\u003c/strong\u003e Heatmaps of semi-quantitative scoring (scale: 0–15, blue to yellow) from two blinded operators showing no significant differences between NBH and BH groups, with or without heparin (+h) or powder PFA (+P). Rows correspond to brain samples, whereas columns denote experimental groups. \u003cstrong\u003e(c)\u003c/strong\u003e Immunohistochemistry showing microglia (Iba1) distribution and morphology, and astrocytes (GFAP) spatial organization with end-feet near blood vessels (black arrow) in cortex of NBH and BH groups. \u003cstrong\u003e(d)\u003c/strong\u003e Glutamatergic synapses (VGlut) localized in the cortex and cerebellum in NBH and BH groups.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544231/v1/28a492e707c7ce3dbe9ec592.jpeg"},{"id":103957905,"identity":"7b754871-63e4-45fd-bed4-d311556e14f3","added_by":"auto","created_at":"2026-03-05 03:41:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":733302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy of CA1 hippocampal region in electron\u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eic\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003emicrocopy in beating heart (BH) and non-beating heart (NBH). (a, b) \u003c/strong\u003eNeuronal profiles in the \u003cem\u003estratum pyramidal\u003c/em\u003e and \u003cstrong\u003e(c, d) \u003c/strong\u003esynapses in the\u003cem\u003estratum radiatum\u003c/em\u003e of CA1 hippocampal region showed no difference. Scale bar: 5 and 1µm respectively.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8544231/v1/99d528a0d92457c7b2918045.png"},{"id":103957904,"identity":"98c29e9d-1896-4c6d-9954-01b292a1ae3e","added_by":"auto","created_at":"2026-03-05 03:41:04","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":143659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy of Fixed Tissues in electron microcopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eTwo dark cell profiles (DC) which are in fact neurons and two profiles of normal neurons (Ne) in the CA1 region of the mouse hippocampus. Scale bar :5µm. \u003cstrong\u003e(b)\u003c/strong\u003ePartial profile of a pyramidal CA1 neuron showing subcellular compartments; Nu: nucleus; Mi: mitochondria; Go: Golgi saccules; Er: endoplasmic reticulum; Ly: lysosome. Scale bar: 1µm. \u003cstrong\u003e(c) \u003c/strong\u003eHigh magnification image showing multiple synapse profiles; sv: synaptic vesicle; psd: post-synaptic density. Scale bar: 1µm. \u003cstrong\u003e(d)\u003c/strong\u003e OL: Oligodendrocyte profile (blue contour) among numerous myelinated axons in the white matter tract between cortex and hippocampus. Scale bar: 5µm. \u003cstrong\u003e(e)\u003c/strong\u003e MI microglial cell profile (blue contour) in the neuropil. Scale bar: 5µm \u003cstrong\u003e(f)\u003c/strong\u003e VE: vessel profile with its endothelial cell EN (blue contours). Scale bar: 2µm.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544231/v1/48592257e55b647325f9bf04.jpeg"},{"id":103957902,"identity":"e7d7ffe0-944c-4de9-9444-adbe630605e6","added_by":"auto","created_at":"2026-03-05 03:41:04","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":214098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbsence of functional impact of beating (BH) and non-beating heart (NBH) TCP on neuronal activity in acute brain slices. (a) \u003c/strong\u003eBasal synaptic transmission and short-term plasticity at the CA3-CA1 synapse using MEA. (\u003cstrong\u003ea1\u003c/strong\u003e) Hippocampal brain slice positioned on the MEA chip: electrodes used for stimulation (red arrow) and for analysis (blue square). \u0026nbsp;fPSP traces obtained from stratum radiatum (SR) in response to paired pulse stimulation at 20 ms and 200ms inter-pulse interval (dash line) (\u003cstrong\u003ea2\u003c/strong\u003e) Input-output (I/O) relationship between fPSP slope and stimulus intensity. I/O curves were obtained by increasing stimulus intensities, until a maximal fPSP slope was reached, usually at ~100 μA. (\u003cstrong\u003ea3\u003c/strong\u003e) Paired-pulse ratio (PPR) curve for the slope of fPSP evoked by the 2\u003csup\u003end\u003c/sup\u003e pulse over the 1\u003csup\u003est\u003c/sup\u003e pulse, at increasing inter-stimulus intervals. \u003cstrong\u003e(b) \u003c/strong\u003eElectrophysiological properties\u003cstrong\u003e \u003c/strong\u003eof cortical pyramidal cells using patch clamp \u003cstrong\u003e(b1)\u003c/strong\u003e Acute slice with a pipette located in the layer 5 of the primary somatosensory cortex (S1). Recordings of spontaneous synaptic activity and voltage responses evoked by current steps showing I\u003csub\u003eh\u003c/sub\u003e-mediated sag and spike firing. \u003cstrong\u003e(b2) \u003c/strong\u003eAverage frequency-current (FI) curve of the mean firing frequency evoked by current steps of increasing intensity, together with the related rheobase and the I\u003csub\u003eh\u003c/sub\u003e sag box plots. Population data for passive membrane properties: resting membrane potential (RMP), input resistance (R\u003csub\u003ein\u003c/sub\u003e) and membrane capacitance (C\u003csub\u003em\u003c/sub\u003e) (\u003cstrong\u003eb3\u003c/strong\u003e) and for the mean amplitude and frequency of postsynaptic currents \u003cstrong\u003e(b4)\u003c/strong\u003e. All data are represented as mean ± SEM. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; ns, non-significant.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544231/v1/00e98514eb977c7054e3667f.jpeg"},{"id":104408226,"identity":"762e60eb-8757-4ce1-9ed9-9e4765a1e453","added_by":"auto","created_at":"2026-03-11 12:42:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2770216,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8544231/v1/d833ce73-6cf0-4c70-b71c-97613ca7dffd.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Transcardiac Perfusion in Mice: Comparing Heart-Beating and Non-Beating Conditions for Brain Histological and Electrophysiological Analyses","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn neuroscience research, TransCardiac Perfusion (TCP) is a widely used method to preserve brain tissue integrity. This technique delivers fixative or physiological solutions uniformly through the cerebral vascular system. While direct immersion treatment works for small samples, it is less reliable for larger ones due to inconsistent penetration, affecting tissue viability and morphology. Despite the risk of irreversible brain damage with delayed perfusion, many researchers argue that TCP is feasible post-mortem. Training staff in TCP presents significant challenges, requiring advanced skills and precision, while considering ethical and scientific standards under the 3Rs principles. This study, based on internal discussions among stakeholders, addresses specific questions related to TCP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePain-free methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe brain harvesting process requires careful oversight to ensure it is painless. The alkaline pH of pentobarbital can lead to peritoneal irritation and pain in mice highlighting the need for refinements \u003csup\u003e1\u003c/sup\u003e. Peritoneal local anesthesia reduces pain \u003csup\u003e2\u003c/sup\u003e, but it has limitations, offering no overall relaxation or sedation. In contrast, xylazine offers both rapid sedation and analgesic effects \u003csup\u003e3\u003c/sup\u003e. This enhancement quickly calms the animal, reducing distress for both the animal and the operator. This study aims to evaluate the physiological effects of administering xylazine prior to a pentobarbital overdose on heart and brain activity in mice, measured through Electrocardiogram (ECG) and Electroencephalogram (EEG) recordings. To ensure perfusion with a beating heart, deep anesthesia is achieved using a combination of xylazine and ketamine. This approach aims to determine the ideal timing to ensure the animal is fully dead before perfusion begins, allowing the tissue processing to be adapted to the specific requirements of the sample. It also upholds ethical and welfare standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChallenges associated with lived and fixed tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe brain sample must be carefully adapted to analysis to be performed. Tissue fixation must maintain the structure of tissues and cells as closely as possible to their natural state. Using the appropriate fixative solution is critical because it is perfused through the cerebral vasculature and diffuses into the surrounding tissue and therefore, directly affecting preservation. It is formaldehyde's ability to diffuse rapidly in tissues to preserve cellular proteins that makes it invaluable a general-purpose fixative in histology \u003csup\u003e4\u003c/sup\u003e. Fixation for ultrastructural studies is more delicate and too long perfusion time could cause structural changes in the synapses \u003csup\u003e5\u003c/sup\u003e. Fixation is achieved through a succession of treatments. First, glutaraldehyde, a fixative that reacts with proteins, preserves tissue structure. Osmium tetroxide and uranyl acetate are then applied to stabilize membrane lipids and enhance contrast, respectively \u003csup\u003e6\u003c/sup\u003e. Live tissues are used for functional studies using electrophysiology and imaging approaches. Brain slices provide important information on membrane excitability, firing profile, synaptic activity and plasticity, essential for understanding neuronal networks. Good tissue viability is crucial for successful electrophysiological recordings. However, this can be difficult to obtain, particularly in adult mice that are more susceptible to anoxia. To address this issue, TCP can be used to rapidly perfuse the brain with ice-cold artificial cerebrospinal fluid (aCSF), preserving tissue viability, integrity, and functionality, and improving the quality of electrophysiological recordings from brain slices \u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study (Fig. 1), we aimed to (1) determinate the most suitable euthanasia method and timing corresponding to various states of cardiac and brain activity until death, and (2) evaluate the effectiveness of TCP under Non-Beating Heart (NBH) and Beating Heart (BH) conditions. This evaluation was based on histological analysis of fixed tissues and the assessment of live tissue functionality using electrophysiological techniques.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eEuthanasia \u003cem\u003eby overdose of pentobarbital versus xylazine/Ketamine\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe aimed to understand the stages leading to death during the TCP-specific euthanasia protocol by performing EEG and ECG recordings, ensuring the timeline closely aligns with the animal\u0026apos;s death to minimize tissue damage. In NBH condition, mice received a xylazine administration followed by pentobarbital overdose or in BH condition a xylazine-ketamine overdose (Fig 2a). Using Fast Fourier Transform (FFT), EEG were visualized in a color-coded format, revealing a rapid onset of deep blue hues indicative of suppressed brain activity and reduced neuronal excitability. Quantitative analysis showed that in the NBH group (n= 15), the mean duration of EEG activity after injection was 133.6 \u0026plusmn; 47.49 seconds, whereas in the BH group (n= 5), it averaged 410 \u0026plusmn; 91.95 seconds (Fig 2b). This difference was statistically significant (P\u0026lt;0.0001), indicating that pentobarbital, at our concentration induces a faster and more consistent cessation of brain activity compared to xylazine/ketamine overdose. Heart rate monitoring revealed a significant decrease following xylazine administration, consistent with its expected sedative and bradycardic effects. Cardiac arrest occurred rapidly, with fibrillation persisting for extended durations. Analysis of RR intervals showed disruption within the first 100 seconds after supine positioning (Fig 2c), and residual ECG activity persisted beyond 200 seconds, reflecting isolated cardiomyocyte contractions. This loss of coordinated pump function led to ineffective circulation, evident from distinct color changes in the heart, with darker atria. These results suggest that performing post-mortem TCP within five minutes of pentobarbital administration ensures the animal is deceased, with a non-beating heart.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of Perfusion Conditions on Histological Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, we independently evaluated samples using a scoring system that assessed various criteria: overall staining, border effects, presence of dark neurons, vacuolization, and periventricular space (Fig 3a). No significant difference was observed for the NBH and NH group (P=565). Additionally, we tested the effects of heparin usage and compared samples from mice perfused with powdered PFA, no discernible structural differences were observed (Fig 3b). Secondly, we performed immunohistochemistry for Iba1, GFAP in cortex (Fig 3c), and VGluT1 in the cortex and cerebellum (Fig 3d), which showed no discernible difference between perfusion methods, confirming the consistency of our findings across different analytical techniques to ensure that the nature of perfusion had no effect. Consistent with the results obtained in histology, the samples (6NBH, 6BH) observed in electron microscopy show no difference between groups at least in the CA1 hippocampus (Fig 4). The ultrastructural integrity of the cells is maintained in both NBH and BH samples (Fig 5). Brain sections of post-mortem samples (Fig 4b, d) revealed a diversity of cell types with resolution and quality comparable to those observed in ante-mortem samples (Fig 4a, c). These included microglia, endothelial cells, oligodendrocytes, and neurons, each displaying well-defined cellular organelles such as the nucleus, mitochondria, and endoplasmic reticulum, all observed at high resolution (Fig 5a-f). We looked for synapses in the CA1 stratum radiatum region of the hippocampus because of the homogeneity of the synapses (mainly glutamatergic). The two components of synapses were observed: presynaptic boutons with numerous vesicles of homogenous size (20 to 30 nm in diameter) and post synaptic element showing a more or less thick, electron-dense zone in front of the presynaptic element (Fig 5c). Excellent visibility of the different organelles was achieved in cells: the nuclear field with it\u0026rsquo;s double layered envelope interrupted from place to place by a nuclear pore (not shown), cisternae of endoplasmic reticulum often associated with rows of ribosomes, Golgi apparatus stacks with budding vesicles, microtubules and neurofilaments (Fig 5b). There was no difference in the number of lysosomes per neuron both in NBH and BH samples. Also in both cases, the mitochondria were the same showing well-preserved cristae. With this absence of difference in number and morphology, we decided to stay at this qualitative level. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional impact of perfusion conditions on electrophysiological properties of neurons.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether NBH TCP were associated with any functional changes in neuronal activity, using electrophysiological approaches. First, basal synaptic transmission and short-term plasticity were assessed at the CA3-CA1 hippocampal synapse using Multielectrode array (MEA) technique (Fig 6a1). The input-output (I/O) relationships between evoked fPSP slope and stimulus intensity were not significantly different between BH and NBH groups (p=0,275, F(1,16)=1.276; BH: n=7 slices/ 3 mice, NBH: n=11 slices/4 mice), suggesting consistent basal synaptic strength under the conditions tested (Fig 6a2). Short-term synaptic plasticity was assessed using a standard paired-pulse stimulation protocol. Paired-pulse facilitation was generated at CA3-CA1 synapses in both tested groups. The paired-pulse ratios (PPR) were not significantly different between BH and NBH groups (p=0.8507, F(1,16)=0.036), suggesting that NBH TCP did not alter short-term plasticity (Fig 6a3).\u003c/p\u003e\n\u003cp\u003eNext, we investigated the electrophysiological properties of putative Layer 5 pyramidal cells of the somatosensory cortex using whole cell patch clamp recordings (Fig 6b1). Pyramidal cells in both TCP conditions (NBH: 4 mice; BH: 3 mice) showed similar excitability profiles in response to depolarizing current steps. Both the rheobase (170 \u0026plusmn; 25.5, n=5 in NBH \u003cem\u003evs\u003c/em\u003e 166.7 \u0026plusmn; 30.73, n=6 in BH; p=0.8463) and the IO curve of the firing frequency showed no significant difference between the two groups. There was also no change in the sag potential indicative of the activation of the hyperpolarization-activated inward current (I\u003csub\u003eh\u003c/sub\u003e) (0.2206 \u0026plusmn; 0.03008, n=5 in NBH \u003cem\u003evs\u003c/em\u003e 0.2309 \u0026plusmn; 0.03904, n=6 in BH; p=0.5368) (Fig 6b2). Furthermore, the passive membrane properties of pyramidal cells were also unaffected by TCP conditions in NBH (n=6) \u003cem\u003evs\u003c/em\u003e BH (n=6) groups. Membrane resting potential (-69.58 \u0026plusmn; 2.716 \u003cem\u003evs\u003c/em\u003e -70.98 \u0026plusmn; 2.875; p=0.9372), input resistance (158.7 \u0026plusmn; 26.69 \u003cem\u003evs\u003c/em\u003e 153.0 \u0026plusmn; 34.31; p=0.6991) and capacitance (205.3 \u0026plusmn; 25.36 \u003cem\u003evs\u003c/em\u003e 181.2 \u0026plusmn; 13.68; p=0.3939) showed no changes (Fig 6b3). Recordings of spontaneous postsynaptic currents (sPSCs) revealed a slightly smaller amplitude of events in the BH group (11.81 \u0026plusmn; 0.9029, n=6 in NBH \u003cem\u003evs\u003c/em\u003e 9.273 \u0026plusmn; 0.5073, n=6 in BH; p=0.0368) but the frequency remained unchanged (11.42 \u0026plusmn; 1.337; n=6 in NBH; \u003cem\u003evs\u003c/em\u003e 8.46 \u0026plusmn; 1.133; n=6 in BH; p=0.1320) (Fig b4).\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eresults. A key debate in this context centers around whether intracardiac perfusion should be conducted on a beating versus non-beating heart. Our comparative analysis of xylazine/pentobarbital and xylazine/ketamine injections demonstrated that pentobarbital in our experimental study, induces a more rapid and consistent cessation of brain activity, making it a preferable agent for euthanasia. This quick onset of brain inactivity supports pentobarbital’s effectiveness in ensuring tissue quality. Importantly, regardless of the euthanasia agent used, initiating perfusion with a pump or other perfusion system remains crucial to ensuring consistent delivery of the perfusion fluid. As describe recently in a comparative study of pre- and post-mortem perfusion, the method of tissue fixation has a major impact on experimental parameters and there was not only one condition that provided the best results for all different analysis \u003csup\u003e8\u003c/sup\u003e. The time elapsed between death and tissue processing affects the quality of the samples and the use of a peristatic pump in all conditions (BH versus NBH) to mimic the dynamics of the heart's biological processes is essential.\u003c/p\u003e\n\u003cp\u003eThe preparation of living acute brain slices has long been established as a powerful model for investigating synaptic connectivity in neuronal circuits \u003csup\u003e9\u003c/sup\u003e. Since the 1990s, TCP has been used to reduce damage caused by decapitation and dissection, as well as to address the increased susceptibility to anoxia in older tissues. While many studies have explored protocols for slice preparation and various parameters such as physiological solutions and incubation temperature, few emphasized the importance of performing TCP on a beating heart (\u003csup\u003e10\u003c/sup\u003e, \u003csup\u003e7\u003c/sup\u003e). In the present study, we demonstrated that this factor does not significantly influence slice quality. By recording extracellular fPSP using MEA, we showed that the strength of synaptic transmission and short-term plasticity were not affected at CA3-CA1 hippocampal synapses by the NBH TCP. However, these results should be interpreted with caution, as other relevant pattern of synaptic activity, such as long-term plasticity, were not tested. Moreover, patch clamp recordings revealed no alteration of the passive membrane properties and excitability of Layer 5 cortical pyramidal cells. Spontaneous synaptic currents are slightly smaller in BH condition, but this could be due to the small number of cells recorded in each group and more experiments will be needed to confirm this effect. While living acute brain slices are valuable for studying synaptic connectivity, tissue preservation protocols for fixed tissues focus more on maintaining ultrastructural integrity. Previous electron microscopy studies have shown that delayed perfusion fixation causes specific alterations in postsynaptic densities and cytoplasmic structures across various neuronal cell types, although the precise timing of the delay was not specified \u003csup\u003e5\u003c/sup\u003e. In our experiments, we did not observe significant changes in samples from the NBH group, provided the delay did not exceed 5 minutes after the injection of euthanasia drug.\u003c/p\u003e\n\u003cp\u003eThis study was exclusively conducted on adult mice, which limits generalizability. Protocols and procedures may not be directly applicable to younger mice or other species, such as rats\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e. For example, perfusion rates might need adjustment for neonatal mice pups to prevent damage to their delicate tissues \u003csup\u003e12\u003c/sup\u003e. Future research should explore the applicability of these methods to different age groups and species to ensure optimal results and minimize adverse effects. Moreover, to enhance the removal of residual blood, we evaluated the use of heparin, which inhibits coagulation and maintains vascular patency, thereby improving perfusion efficiency. Heparin was administered either by adding it to the wash solution or through direct intracardiac injection. Although not explored in our study, intraperitoneal (IP) injection represents an alternative route of administration. Additionally, sodium nitrite (NaNO₂), a vasodilator, has been shown to significantly reduce residual blood without compromising the integrity of the blood-brain barrier\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e. Our analyzes indicate that these interventions do not affect the quality of samples for downstream applications (data not shown). However, they markedly enhance perfusion efficiency, allowing for more rapid clearance of blood and facilitating the achievement of a \"white liver\" state in a shorter time frame. Finally, our work received ethical approval in 2021, using xylazine and ketamine at appropriate doses for anesthetizing mice in a \"beating heart\" procedure. \u0026nbsp;Recent advancements suggest stronger analgesia for this invasive surgery (thoracotomy and laparotomy), even if classified as non-recovery. A multimodal approach, including an opioid (e.g., fentanyl or buprenorphine), an alpha-2 agonist (e.g., xylazine or medetomidine), and possibly a local anesthetic, would likely be recommended today. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, from the experimenter's point of view, our study was conducted by skilled users, including a single experienced individual who performed all TCP throughout the study. Performing TCP is an invasive procedure that requires quick and precise handling, which can be emotionally challenging for the experimenter. There are few specific studies on professionals working with laboratory animals, particularly those involved in procedures such as euthanasia and TCP. However, these are essential, given that the field of laboratory animal probably poses unique challenges in terms of compassion fatigue \u003csup\u003e14\u003c/sup\u003e. To promote a culture of care in animal research, it’s essential to consider the wellbeing of experimenters, as there is a close link between individual performance and a supportive working environment \u003csup\u003e15\u003c/sup\u003e. Encouraging this type of study not only refines the procedure but also strengthens the bonds between all professionals involved in the process. By working together to refine procedures such as TCP, we increase mutual understanding and foster a collaborative environment that prioritizes both animal and human wellbeing for the best science.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur findings were unexpected for researchers accustomed to the beating heart method. This study is the first to investigate the implementation of TCP procedures under both BH and NBH conditions at the onset of perfusion, for both fixed and live brain tissue, with histological, electron microscopy, and electrophysiological analyses. Our study highlights that overdose of pentobarbital with the use of peristatic pump is a better, more effective, \u0026nbsp;euthanasia agent compared to xylazine/ketamine for inducing a rapid cessation of brain activity. Contrary to expectations, the tissue processing conditions including the choice of perfusion methods and fixatives did not significantly impact the histological or electrophysiological quality of the samples. These results are essential for standardizing protocols in neuroscience research, ensuring greater consistency and reliability across studies. Furthermore, we have improved our methodologies to better address animal welfare concerns in experimental procedures. Future research should focus on further optimizing these protocols and exploring the influence of other variables on tissue quality and function.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManipulation of animals were carried out in accordance with the guidelines of the European Union (directive 2010/603/EU) and received approval (APAFIS n #37052) from the French Ministry for Research. \u0026nbsp;Wild-type C57BL/6 mice were housed under specific-pathogen-free conditions at the PHENO-ICMice facility of the Paris Brain Institute. They were provided with food and water \u003cem\u003ead libitum\u003c/em\u003e and maintained on a 12-hour light/dark cycle (lights on from 8:00 AM to 8:00 PM). For each group, we used 7 to 10-week-old C57Bl6J wild type mice (half male/ female).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBrain and cardiac monitoring during euthanasia:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described in previous study \u003csup\u003e16\u003c/sup\u003e, simultaneous EEG-ECG recordings were used to monitor time of death. Two groups of mice, reused from a previous study involving EEG electrode implantation, were studied under identical brain implantation conditions. Anesthesia and euthanasia procedures are outlined in the following section. Time of death was determined by the absence of visible EEG activity. QRS component of the ECG was recorded from mice immediately after they were positioned in decubitus following the administration of pentobarbital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnesthesia and euthanasia\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the NBH group, mice are initially sedated with a xylazine injection (Rompun,15 mg/kg, ip) followed 10 minutes after by injection of 100µL sodium pentobarbital (Euthasol®, 700 mg/kg ) to induce euthanasia. We waited until cardiac and respiratory arrest, maximum 5 minutes after pentobarbital injection, before starting TCP procedures. For the BH group, mice are subjected to deep anesthesia using a combination of ketamine and xylazine (Imalgène, 150 mg/kg, Rompun,15 mg/kg, ip). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscardiac perfusion (TCP)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter grasping the skin of the thorax with forceps, an incision was made to expose the xiphoid process, followed by lateral cuts to reveal diaphragm and liver. Incisions were then made along the diaphragm followed by two cuts on each side up of the ribcage down to the clavicles. The sternum was lifted for complete exposure of the heart and lungs. The heart was held with dissecting forceps and a 27-gauge needle was inserted into the left ventricle, angled roughly parallel to the heart's midline, advancing toward the ascending aorta. The needle was clamped in place and a small incision was made over the right atrium using fine scissors, allowing venous blood flow. The intracardiac perfusion was then\u0026nbsp;initiated promptly, maintaining a constant speed of approximately 3-5 ml per minute, except for electron microscopy samples, where it was slowed down to 2ml per min. Fixative solution or aCSF was perfused for histology and in vitro electrophysiology experiments respectively (see below for details). For the histology and ME one additional group was included in which mice received heparin (Choay-Sanofi) in the saline solution at a concentration of 50 IU/ml to prepare BH and NBH+h group samples. Once the draining fluid was free of red blood cells (after 20-50 ml saline for adult mice), the liver's discoloration indicated successful perfusion, the perfusion stopped, and the brain was extracted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistology \u0026amp; Immunohistochemistry\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor BH and NBH, mice were perfused with a fixative solution (4% paraformaldehyde powder Merck #1.04005 or liquid EMS #15714). Following extraction from the skull, brains underwent additional overnight fixation in a fresh 4% PFA/PBS solution before being embedded in paraffin. Sagittal sections (5μm thick) were then prepared using a paraffin microtome (Leica). We utilized the markers IBA1, GFAP and VGluT1 to study cell types and neuronal structures. For immunostaining, brain tissue sections were deparaffinized and subjected to antigen retrieval (in citrate buffer at 110°C in a decloaking chamber for 1min 30 for GFAP and 25min for IBA1) to reveal hidden epitopes. For observing glutamate neurons and microglia, subsequently, sections were incubated overnight at 4°C with primary antibody (Vglut1, 1/1000, Synaptic system #135304; IBA1, 1/100, Wako #01919741; GFAP, 1/500, Dako #Z0334). Following primary antibody incubation, sections were exposed to secondary biotinylated horse anti-rabbit (IBA1 and GFAP) or goat anti-guinea pig (Vglut1) for 30 minutes (Vector, dilution 1/250) and visualized using the ABC method (Vector Laboratories) with 3,3-diaminobenzidine (DAB, Sigma-Aldrich) as the chromogen. For Nissl staining, sections were dewaxed and rehydrated before being stained in a thionin solution (Sigma #T-3387). Stained sections were digitized at x20 magnification using a slide scanner (nanozoomer S60/ Hamamatsu). Two blinded experimenters evaluated BH and NBH samples using a semi-quantitative method of scoring from 0 to 3 for various criteria: general coloration, border effects, perivascular space, global vacuolization, and the presence of dark neurons. By applying these diverse criteria, we acquired a comprehensive score, reaching a maximum of 17 for the samples, and enhanced our understanding of potential variances among them.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eElectron microcopy\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor BH and NBH, mice were perfused with fixative solution (2% w/v paraformaldehyde, 2% v/v glutaraldehyde in 0.12 M phosphate buffer pH 7.4 (PB)). After one hour at 4°C brain was extracted from the skull and post-fixed in fresh fixative overnight at +4 °C. Brains were rinsed in PB and sliced in 1 mm thick coronal sections using an adult mouse brain acrylic matrix. Section of Bregma -1.58mm was selected, washed in 0.12M PB and post-fixed at room temperature (21 °C) for 1 h in 1% osmium tetroxide in PB. After rinsing with water, sections were incubated with 2% aqueous uranyl acetate overnight at +4°C. They were washed in water and dehydrated in a series of of ethanol (30%, 50%,70%, 90%, 100% (X3)) and 3 times with 10% acetone. Sections were incubated overnight in a 50:50 solution of acetone: Epon 812R (EMS, Souffelweyersheim, France). They were incubated twice with fresh Epon 812 resin for 1h at room T°, embedded in moulds. Polymerisation took place at +56°C for 48 h in a dry oven. Blocks were cut with an UC7 ultramicrotome (Leica Microsystems, Nanterre, France). Semi-thin sections (0.5–1 μm) were stained with a 1% v/v Toluidine Blue in 1% w/v Borax aqueous solution. Ultra-thin sections (60–70 nm) were cut, placed onto 200 mesh copper grids (EMS, Souffelweyersheim, France) and stained with Reynolds lead citrate for 7 min (Reynolds, 1963). Grids were examined using a Hitachi HT7700 transmission electron microscope (TEM, Milexia France) operating at 120kV. Pictures (2048x2048 pixels) were taken with an AMT41B camera. \u0026nbsp;Variability among groups regarding the presence of fixation artifacts and the relative number of dark neurons. Additionally, a qualitative evaluation of the morphological changes affecting neurons, glial cells (oligodendrocyte, astrocyte and microglia), endothelial cells, pericytes notably their subcellular compartments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSlice preparation for in vitro electrophysiology\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTCP was performed with ice-cold solution containing (in mM): 200 sucrose, 26 NaHCO\u003csub\u003e3\u003c/sub\u003e, 2.5 KCl, 1.25 NaH\u003csub\u003e2\u003c/sub\u003ePO4, 7 MgSO\u003csub\u003e4\u003c/sub\u003e, 0.5 CaCl\u003csub\u003e2\u003c/sub\u003e, 3 pyruvic acid, 3 myo-inositol, 0.4 ascorbic acid and 16 glucose, saturated with 95% O\u003csub\u003e2\u003c/sub\u003e / 5% CO\u003csub\u003e2\u003c/sub\u003e. Brains were removed and acute coronal slices (300 μm) containing somatosensory cortex and hippocampus were cut in the same solution, using a vibratome (Leica VT1200S). Slices were then incubated in oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 2.5 KCl, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgSO\u003csub\u003e4\u003c/sub\u003e, 1.25 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 26 NaHCO\u003csub\u003e3\u003c/sub\u003e and 16 glucose (pH 7.4), at 32°C for 30 min, and subsequently at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMEA recordings\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHippocampal Slices were transferred on a polyethyleneimine coated chip (MED-R515A), continuously perfused with aCSF (34°C) and recordings started after 20 minutes recovery period. Extracellular field postsynaptic potentials (fPSPs) were recorded in CA1 stratum radiatum following stimulation of Schaffer collateral with a 64-channel multielectrode array (MEA) system (Alpha MED Scientific). Extracellular field potentials were bandpass filtered between 1Hz-10kHz and acquired at a sampling rate of 20 kHz. fPSPs were evoked by a series of stimuli of increasing intensities (10-100μA) and the related Input–output (I/O) curves of the fPSP slope were generated. The intensity of stimulation eliciting 40–50% of the maximum response was then determined from the IO curve and used to investigate short term plasticity. Paired pulse stimulation was applied at increasing inter-pulse interval (20-200ms). Paired pulse ratio (PPR) was calculated by dividing the slope of the second fPSP by the slope of the first one. Data analysis was performed using a custom R script and GraphPad Prism 9 software\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePatch-clamp recordings\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole cell recordings were obtained at 30°C from layer V pyramidal cells of the somatosensory cortex. Glass electrodes were filled with a solution containing (in mM): 133 K-gluconate, 11 KCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 4 Na-phosphocreatine; 290mOsm. No liquid junction potential correction was applied. Signals were acquired at a sampling rate of 20 kHz or 50Hz and filtered at 2kHz or 10Hz in voltage or current clamp, respectively (Multiclamp 700B, Molecular devices). Access resistancewas \u0026lt;20 MΩ. Resting membrane potential was measured 5 minutes after whole cell configuration establishment. Spontaneous postsynaptic currents (sPSCs) were recorded at -70 mV, and thus mainly excitatory AMPA-receptors mediated currents in our recording conditions. sPSCs were detected using a custom software (Wdetecta, J. R. Huguenard, Stanford University) and their mean amplitude and frequency calculated over 1-3 minutes.\u003c/p\u003e\n\u003cp\u003eVoltage responses to small current steps (−30pA; 1sec; 20 times) were used to extract passive membrane properties from the averaged curve. Input resistance (R\u003csub\u003ein\u003c/sub\u003e) was calculated at steady state, membrane time constant (t\u003csub\u003em\u003c/sub\u003e) was determined by fitting a single exponential function and membrane capacitance was calculated with the equation \u003cem\u003eC\u003csub\u003em\u003c/sub\u003e\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVoltage responses to a series of current steps (1sec; 50pA increment) were recorded at -70 mV. Evoked firing properties were determined from responses to depolarizing steps. Action potentials (AP) were detected using a threshold criterion. The rheobase is the minimum current intensity that triggers the first spike, the mean firing frequency was calculated (number of AP/s). Activation of hyperpolarization-activated cation current (I\u003csub\u003eh\u003c/sub\u003e) was estimated from the voltage deflection (sag) in response to hyperpolarizing steps (-300/-50pA). Sag amplitudes were determined by the difference between peak and steady state potentials and normalized to the peak. The average of normalized sag potential was used as an I\u003csub\u003eh\u003c/sub\u003e estimate. Data were analyzed with pClamp10 (Molecular devices), Prism (GraphPad) software and custom-written scripts (MATLAB, MathWorks).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification and Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the statistical analysis of \u003cstrong\u003ehistology\u003c/strong\u003e scores, the Kruskal-Wallis test was applied to compare the different groups. \u003cstrong\u003eIn vivo data\u003c/strong\u003e: EEG data were analyzed using Neurowork or Deltamed software (Natus Medical Incorporated, Pleasanton, CA) alongside custom-written scripts in MATLAB (MathWorks). Statistical analyses were conducted using GraphPad Prism, with parameters compared between two experimental groups using Mann Whitney test. \u003cstrong\u003eECG data\u003c/strong\u003e: RR intervals were analyzed using Spike2 software, and heart rate was determined by calculating the number of RR intervals in 2 seconds (Spike2 v7.06; Cambridge Electronic Design, Cambridge, UK). \u003cstrong\u003eMEA\u003c/strong\u003e (https://gitlab.com/icm-institute/dac/biostats/MEASpikeR):\u0026nbsp;Data were analyzed using Mobius software and a custom R script.\u0026nbsp;All data were analyzed using Repeated measures two-way ANOVA. All results are reported as mean + SEM with significance set at a p-value of less than 0.05 (∗).\u0026nbsp;\u003cstrong\u003ePatch Clamp\u003c/strong\u003e: All data are presented as mean ± SEM. All statistical analyses were performed in Prism (GraphPad) and were two-tailed. Unmatched non-parametric two-way ANOVA was used for mean firing frequency (FI curve), and unpaired Mann-Whitney tests were used for other data. \u003cem\u003eP\u003c/em\u003e values lower than 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAUTHOR CONTRIBUTION\u003c/h2\u003e\n\u003cp\u003eD. R., J. D., C. D., C. D., D. L., S. I., A. P., L. S., participated in experimental investigations and analyses. D. R., B.D., N.S. designed the study, D.R., C. D., C. D., D. L., A. P., B.D., N.S. wrote and revised the manuscript. \u0026nbsp;All authors reviewed the manuscript before submission.\u003c/p\u003e\n\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e\n\u003cp\u003eWe thank the ICM core facilities: ePHYS, PHENO-ICMice, ICM.Quant and Histomics supported by \u003cem\u003eInvestissements d’Avenir\u003c/em\u003e (ANR-10-IAIHU-06 and ANR-11-INBS-0011-NeurATRIS). We thank Francois-Xavier Lejeune (DAC, Paris Brain Institute) for the development of a data analytics tools box for MEA data analysis. We also thank Serge Marty for his review and valuable advice on the manuscript, as well as Alice Gilbert, Stéphanie Baulac and Jean-Christophe Poncer for allowing the reuse of EEG-implanted mice.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLaferriere, C. A. \u0026amp; Pang, D. S. Review of Intraperitoneal Injection of Sodium Pentobarbital as a Method of euthanasia in Laboratory Rodents \u003cem\u003eJournal of the American Association for Laboratory Animal Science\u003c/em\u003e\u003cstrong\u003e59\u003c/strong\u003e, 254 - 263, doi: 10.30802/AALAS-JAALAS-19-000081 (2020).\u003c/li\u003e\n\u003cli\u003eKhoo, S. Y.-S., Lay, B. P. P., Joya, J. \u0026amp; McNally, G. P. Local anaesthetic refinement of pentobarbital euthanasia reduces abdominal writhing without affecting immunohistochemical endpoints in rats. \u003cem\u003eLaboratory Animals\u003c/em\u003e\u003cstrong\u003e52\u003c/strong\u003e, 152\u0026ndash;162 (2018).\u003c/li\u003e\n\u003cli\u003eKitano, T., Kobayashi, T., Yamaguchi, S. \u0026amp; Otsuguro, K.-I. The \u0026alpha;2A-adrenoceptor subtype plays a key role in the analgesic and sedative effects of xylazine. \u003cem\u003eJournal of Veterinary Pharmacology and Therapeutics\u003c/em\u003e\u003cstrong\u003e42\u003c/strong\u003e, 243 - 247, doi:https://doi.org/10.1111/jvp.12724 (2018).\u003c/li\u003e\n\u003cli\u003eHowat, W. J. \u0026amp; Wilson, B. A. Tissue fixation and the effect of molecular fixatives on downstream staining procedures. \u003cem\u003eMethods\u003c/em\u003e\u003cstrong\u003e70\u003c/strong\u003e, 12 - 19, doi:http://dx.doi.org/10.1016/j.ymeth.2014.01.022 (2014).\u003c/li\u003e\n\u003cli\u003eTao-Cheng, J. H., Gallant, P. E., Brightman, M. W., Dosemeci, A. \u0026amp; Reese, T. S. Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. \u003cem\u003eJ Comp Neurol\u003c/em\u003e\u003cstrong\u003e501\u003c/strong\u003e, 731-740, doi:10.1002/cne.21276 (2007).\u003c/li\u003e\n\u003cli\u003ePalay, S. L., McGee-Russell, S. M., Gordon, S. \u0026amp; Grillo, M. A. Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide. \u003cem\u003eTHE JOURNAL OF CELL BIOLOGY\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 385 - 410 (1962).\u003c/li\u003e\n\u003cli\u003eLipton, P.\u003cem\u003e et al.\u003c/em\u003e Making the best of brain slices: comparing preparative methods. \u003cem\u003eJournal of Neuroscience Methods\u003c/em\u003e\u003cstrong\u003e59\u003c/strong\u003e, 151 - 156 (1995).\u003c/li\u003e\n\u003cli\u003eMeyer-Dilhet, G., Ellouze, S., Raineteau, O. \u0026amp; Courchet, J. Comparative study of pre- and post-mortem perfusion of fixative for the quality of neuronal tissue preparation. \u003cem\u003eLab Anim (NY)\u003c/em\u003e, doi:10.1038/s41684-025-01633-1 (2025).\u003c/li\u003e\n\u003cli\u003eYamamoto, C. \u0026amp; McIlwain, H. Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro. \u003cem\u003eJ Neurochem\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 1333 - 1343, doi:doi: 10.1111/j.1471-4159.1966.tb04296.x. (1966).\u003c/li\u003e\n\u003cli\u003e Eguchi, K.\u003cem\u003e et al.\u003c/em\u003e Advantages of acute brain slices prepared at physiological temperature in characterization of synaptic functions. \u003cem\u003ebioRxiv \u003c/em\u003edoi:10.1101/845461 (2019).\u003c/li\u003e\n\u003cli\u003e Zatroch, K. K., Knight, C. G., Reimer, J. N. \u0026amp; Pang, D. S. 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Mental Wellbeing in Laboratory Animal Professionals: A Cross-Sectional Study of compassion Fatigue, Contributing Factors, and Coping Mechanisms. \u003cem\u003eJournal of the American Association for Laboratory Animal Science\u003c/em\u003e\u003cstrong\u003e60\u003c/strong\u003e, 54 - 63, doi:DOI: 10.30802/AALAS-JAALAS-20-000039 (2021).\u003c/li\u003e\n\u003cli\u003e Ferrara, F.\u003cem\u003e et al.\u003c/em\u003e Culture of care in animal research \u0026ndash; Expanding the 3Rs to include people. \u003cem\u003eLaboratory animals\u003c/em\u003e\u003cstrong\u003e56\u003c/strong\u003e, 511 - 518, doi:DOI: 10.1177/00236772221102238 (2022).\u003c/li\u003e\n\u003cli\u003e Bacq, A.\u003cem\u003e et al.\u003c/em\u003e Cardiac Investigations in Sudden Unexpected Death in DEPDC5-Related Epilepsy. \u003cem\u003eANNALS of Neurology\u003c/em\u003e\u003cstrong\u003e91\u003c/strong\u003e, 101 - 116, doi: 10.1002/ana.26256 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"transcardiac perfusion, electrophysiology, histology, animal welfare, ethics, euthanasia methods, mice, 3R, wellbeing","lastPublishedDoi":"10.21203/rs.3.rs-8544231/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8544231/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn vivo and ex vivo animal model experiments are essential for understanding the mechanisms underlying neurological diseases. These investigations bridge the gap between organism behavior and cellular pathways, providing a holistic view of brain function. A key factor is the quality of tissue, which depends on the efficacy of the perfusion process. Rapid and uniform delivery of fixative or physiological solutions is crucial to preserve tissue integrity, yet the effectiveness of perfusion under non-beating heart conditions remains debated.\u003c/p\u003e\n\u003cp\u003eOur study aimed to optimize perfusion conditions for quality brain tissue samples, complying with ethical standards. We tested both beating heart conditions with xylazine/ketamine and non-beating heart conditions with xylazine/pentobarbital, with perfusion following cardiorespiratory arrest, occurring within 5 minutes in our conditions. Electroencephalography and electrocardiography measurements following pentobarbital injection demonstrated a rapid cessation of brain activity coinciding with the onset of irregular, non-sinusoidal cardiac rhythms. Death was confirmed at the time of thoracotomy, performed after cardiorespiratory arrest, which consistently occurred within a 5 minutes window post-injection. Semi-quantitative histology and neurotransmitter immunohistochemistry showed no significant differences between the 2 conditions. Electron microscopy confirmed good tissue quality in both, with similar results in functional studies using electrophysiological approaches.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study demonstrates that post-mortem transcardiac perfusion in mice, performed under conditions where perfusion is carried out very quickly, reliably yields high-quality brain samples for histology, cytology, and electrophysiology. Our findings help address controversies regarding perfusion efficacy and highlight the need to reconsider euthanasia practices to ensure sample quality while minimizing impact on animals and researchers.\u003c/p\u003e","manuscriptTitle":"Transcardiac Perfusion in Mice: Comparing Heart-Beating and Non-Beating Conditions for Brain Histological and Electrophysiological Analyses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 03:40:55","doi":"10.21203/rs.3.rs-8544231/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"lab-animal","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"laban","sideBox":"Learn more about [Lab Animal](http://www.nature.com/laban/)","snPcode":"","submissionUrl":"","title":"Lab Animal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b8529e50-dad7-4745-a834-43331cff4a71","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61360229,"name":"Biological sciences/Biological techniques/Experimental organisms/Model vertebrates/Mouse"},{"id":61360230,"name":"Biological sciences/Biological techniques/Biological models/Neurological models"}],"tags":[],"updatedAt":"2026-03-05T03:40:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 03:40:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8544231","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8544231","identity":"rs-8544231","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}