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Equipment required for intracerebral injections in mice. (A) Surgical tools. 1, 1 mL syringe for anesthesia; 2 and 3, fine forceps; 4, surgical scissors; 5, surgical hook; 6, needle holder with surgical braided absorbable suture; 7, black leader for human spinal anesthesia; 8, 10 μL calibrated micropipette. (B) 10 μL injection syringes with 1, flexible or 2, rigid 33-gauge needle. (C) Surgical area. 1 and 2, Stereotaxic apparatus with stereotaxic arm; 3, Stereotaxic injection system; 4, High-speed dental drill with small drill-bits. (D) Temperature-controlled heating cage.
Source publication
Intracerebral injections of tracers or viral constructs in rodents are now commonly used in the neurosciences and must be executed perfectly. The purpose of this article is to update existing protocols for intracerebral injections in adult and neonatal mice. Our procedure for stereotaxic injections in adult mice allows the investigator to improve t...
Contexts in source publication
Context 1
... (Fig. 1) - Stereotaxic apparatus (e.g., Stoelting , Digital Lab Standard for mouse, or David Kopf Instruments for mouse or rat) - Stereotaxic injection system (Harvard Apparatus , Pump 11 Elite) - Dissecting microscope (Leica S6E) - Temperature-controlled heating cage (VetTech Solutions HE011), - Surgical tools: surgical scissors, fine forceps, surgical hook or 26-gauge needle, black leader for human spinal anesthesia (Braun, Spinocan 4509900), needle holder - Surgical braided absorbable suture (Vicryl 3-0, Ethicon®) - High-speed dental drill with small drill-bits (Foredom 38,000 rpm) - 10 μL injection syringe (Hamilton® 1700) with flexible (silica) 33-gauge needle (Phymep ), or rigid 33-gauge needle (Hamilton® 7762-03) - 10 μL calibrated micropipettes (Eppendorf® Research Plus) - Container of crushed ice to preserve injection fluid - Cotton swab - Laboratory film (Parafilm®) - Precision wipes (Kimtech Science®) - 10 mL-syringes, - Isoflurane vaporizer (optional) - Electric shaver ...
Context 2
... new-born mice (postnatal days 0-1 (P0-P1); use of live mice must conform to institutional regulations), -Crushed ice -Sterile saline (NaCl 0.9%) -Distilled water -Injection fluid: virus, enzyme, tracer... -Iodine-based wash (Betadine®; optional) -Mineral oil (optional) EQUIPMENT (Fig. 1) -Stereotaxic apparatus (Stoelting , Digital Lab Standard) -Stereotaxic injection system (Harvard Apparatus , Pump 11 Elite) -Dissecting microscope (Leica S6E) -Temperature-controlled heating cage (VetTech Solutions HE011) -Surgical tools: 30-gauge needle -10 μL injection syringe (Hamilton® 1700) with rigid 33-gauge needle (Hamilton® 7762-03) -10 μL calibrated micropipettes (Eppendorf® Research Plus) -Polystyrene foam stage -Container of crushed ice to preserve the injection fluid and induce anesthesia -Petri dish -Cotton swab -Laboratory film (Parafilm®) -Precision wipes (Kimtech Science®) -Aluminum foil -Syringes Weigh the animal and calculate the appropriate dose for anesthesia. We use a mixture of ketamine and xylazine given intraperitoneally. Doses are summarized in Table 1. To inject the ketamine-xylazine mixture, restrain the animal with one hand, abdomen up. Inject the mixture intraperitoneally through a 21-gauge needle inserted into the lower left or right abdominal quadrant (Fig. 2A). Immediately place the animal in the heating cage ...
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Background:
Botulinum toxin A (BoNT-A) is an effective treatment for cervical dystonia. Nevertheless, up to 30% to 40% patients discontinue treatment, often because of poor response. The British Neurotoxin Network (BNN) recently published guidelines on the management of poor response to BoNT-A in cervical dystonia, but adherence to these guideline...
Citations
... A newborn mouse model was used for the neurovirulence assessment. One-day-old suckling C57BL/6 mice (n = 4-6) were inoculated with 10 4 and 10 5 tissue culture infective doses (TCID 50 ) of the F10 and F30 viruses by intracerebral (i.c) injection in a 10 µL volume using a 31-gauge needle as previously described [19]. The inoculation site was located in the right parietal area of the skull approximately 1 mm to the right of the midline. ...
Mumps virus (MuV) causes an acute contagious human disease characterized by swelling of the parotid glands. Despite the near elimination of mumps in many countries, the disease has recurred, even in vaccinated populations, especially adolescents. Immunization effectivity of the genotype A vaccine strain Jeryl Lynn (JL) is declining as genotype A is no longer predominant; therefore, a new vaccine strain and booster vaccine are required. We generated a cell culture-adapted MuV genotype F called F30 and evaluated its immunogenicity and cross-protective activity against diverse genotypes. F30 genome nucleotide sequence determination revealed changes in the NP, L, SH, and HN genes, leading to five amino acid changes compared to a minimally passaged stock (F10). F30 showed delayed growth, smaller plaque size in Vero cells, and lower neurotoxicity in neonatal mice than F10. Furthermore, F30 was immunogenic to other genotypes, including the JL vaccine strain, with higher efficacy than that of JL for homologous and heterologous immunization. Further, F30 exhibited cross-protective immunity against MuV genotypes F and G in Ifnar−/− mice after a third immunization with F30 following two doses of JL. Our data suggest that the live-attenuated virus F30 could be an effective booster vaccine to control breakthrough infections and mumps epidemics.
... In addition, we modified the radiotherapy protocol, previously described by Bolcaen et al., to enhance the clinical relevance of the model and to suppress the progression of extracranial and extra-axial tumor growth [32]. To this end, all steps in the intracerebral inoculation procedure should be executed with the highest precision, given that each step has the potential to influence the accuracy and reproducibility of the rodent model, subsequently compromising the integrity of scientific outcomes [23,30,33]. The utilization of a stereotactic frame and microinfusion pump is of major significance for obtaining accurate and consistent tumors, a technique elucidated as early as 1980 by Kobayashi et al. [23]. ...
Glioblastoma (GB) is the most common and malignant primary brain tumor in adults with a median survival of 12–15 months. The F98 Fischer rat model is one of the most frequently used animal models for GB studies. However, suboptimal inoculation leads to extra-axial and extracranial tumor formations, affecting its translational value. We aim to improve the F98 rat model by incorporating MRI-guided (hypo)fractionated radiotherapy (3 x 9 Gy) and concomitant temozolomide chemotherapy, mimicking the current standard of care. To minimize undesired tumor growth, we reduced the number of inoculated cells (starting from 20 000 to 500 F98 cells), slowed the withdrawal of the syringe post-inoculation, and irradiated the inoculation track separately. Our results reveal that reducing the number of F98 GB cells correlates with a diminished risk of extra-axial and extracranial tumor growth. However, this introduces higher variability in days until GB confirmation and uniformity in GB growth. To strike a balance, the model inoculated with 5000 F98 cells displayed the best results and was chosen as the most favorable. In conclusion, our improved model offers enhanced translational potential, paving the way for more accurate and reliable assessments of novel adjuvant therapeutic approaches for GB.
... Viral injections were performed unilaterally (Mathon et al., 2015 ;Richevaux et al., 2019 ) at the coordinates −0.82 AP, 0.75 ML and −3.2 mm DV for the ADN, and at −2.1 to −2.15 AP, 0.65 ML and −0.65 mm DV for the RSC. Volumes of 200 to 250 nl were injected with a 10 µL Hamilton syringe equipped with 33ga needle over a time of 10 min. ...
Head-direction (HD) signals function as the brain’s internal compass. They are organized as an attractor, and anchor to the environment via visual landmarks. Here we examine how thalamic HD signals and visual landmark information from the retrosplenial cortex combine in the presubiculum. We find that monosynaptic excitatory connections from anterior thalamic nucleus and from retrosplenial cortex converge on single layer 3 pyramidal neurons in the dorsal portion of mouse presubiculum. Independent dual wavelength photostimulation of these inputs in slices leads to action potential generation preferentially for near-coincident inputs, indicating that layer 3 neurons can transmit a visually matched HD signal to medial entorhinal cortex. Layer 4 neurons, which innervate the lateral mammillary nucleus, form a second step in the association of HD and landmark signals. They receive little direct input from thalamic and retrosplenial axons. We show that layer 4 cells are excited di-synaptically, transforming regular spiking activity into bursts of action potentials, and that their firing is enhanced by cholinergic agonists. Thus, a coherent sense of orientation involves projection specific translaminar processing in the presubiculum, where neuromodulation facilitates landmark updating of HD signals in the lateral mammillary nucleus.
... The injection speed must also be slow so that the liquid coming out of the syringe can diffuse into the tissue without displacing it and damaging it. Thus, the maximum injection rate should not exceed 250 nl/min [18][19][20]. Since the microsyringe's needle will move through the brain tissue [21], its characteristics are also important, so that minimum damage is caused to the nervous tissue. ...
Stereotaxic surgery is a less invasive form of surgery that uses a three-dimensional coordinate system to place instruments at a specific location in the brain. Through this type of surgery, one can place needles among other tools within the structures of the brain. Therefore, injections can be given in order to deliver substances that cannot cross the blood-brain barrier. The two most important parameters of the microinjection to control are volume and speed. The volume should not be so large that it displaces the brain tissue and tears it. The injection speed must also be slow so that the liquid that comes out of the syringe can diffuse into the tissue without displacing it and damaging it. Thus, the objectives of the present work are: 1) To develop not a 3D printed prototype but an end-user device. 2) The device must be for animal research only. 3) It must have the same precision in volume and speed as commercial devices. 4) It must be adjustable for microsyringes from 0.5 µl to 1 ml. 5) It must be possible to place it directly on the stereotaxic surgery apparatus and to use it separately. 6) The price must be substantially lower than that of the commercial devices.
... In rodent models, this is commonly achieved via precise microinjection of chemicals, implantation of cannulas, electrodes and optic fibers, or delivery of viral vectors into the brain using stereotaxic surgery [1,2]. For this purpose, animals are secured into a stereotaxic frame while anesthetics are delivered via intraperitoneal injection or inhalation through a mouthpiece [3]. ...
... 1. Connect the 12 V power supply to the barrel jack (Fig. 5a, b). 2. Power up using the ON/OFF switch (Fig. 5b). 3. The display will show three parameters: the temperature from the external thermocouple (T1), the temperature from the bed (T2) and the desired temperature of the bed (DT). ...
Studying the development of neural circuits in rodent models requires surgical access to the neonatal brain. Since commercially available stereotaxic and anesthetic equipment is designed for use in adults, reliable targeting of brain structures in such young animals can be challenging. Hypothermic cooling (cryoanesthesia) has been used as a preferred anesthesia approach in neonates. This commonly involves submerging neonates in ice, an approach that is poorly controllable. We have developed an affordable, simple to construct device - CryoPup - that allows for fast and robust cryoanesthesia of rodent pups. CryoPup consists of a microcontroller controlling a Peltier element and a heat exchanger. It is capable of both cooling and heating, thereby also functioning as a heating pad during recovery. Importantly, it has been designed for size compatibility with common stereotaxic frames. We validate CryoPup in neonatal mice, demonstrating that it allows for rapid, reliable and safe cryoanesthesia and subsequent recovery. This open-source device will facilitate future studies into the development of neural circuits in the postnatal brain.
... Fluorescent beads for retrograde tracing were injected (300-500 nl) into the PrS at the coordinates: -4.06 antero-posterior (AP), 2.00 medio-lateral (ML) and -2.15 mm dorso-ventral (DV) and into the LMN (-2.8 AP, 0.75 ML, -5.35 DV) with respect to the bregma. Viral injections were performed unilaterally (Mathon et al., 2015;Richevaux et al., 2019) at the coordinates -0.82 AP, 0.75 ML and -3.2 mm DV for the ADN, and at -2.1 to -2.15 AP, 0.65 ML and -0.65 mm DV for the RSC. Volumes of 200 to 250 nl were injected with a 10 µL Hamilton syringe equipped with 33ga needle over a time of 10 min. ...
Head-direction (HD) signals function as the brain’s internal compass. They are organized as an attractor, and anchor to the environment via visual landmarks. Here we test the hypothesis that HD and landmark signals are associated in the dorsal presubiculum. We find that monosynaptic excitatory connections from anterior thalamic nucleus and from retrosplenial cortex converge on single layer 3 presubicular neurons. Independent dual wavelength photostimulation of these inputs in slices leads to action potential generation preferentially for near-coincident inputs, sending an integrated HD signal to medial entorhinal cortex. Layer 4 neurons, which innervate the lateral mammillary nucleus, form a second step in the association of HD and landmark signals. They are not directly contacted by thalamic and retrosplenial axons. Instead, layer 4 neurons receive a di-synaptic excitation from layer 3, providing a distinct landmark updating of HD signals for transmission to the lateral mammillary nucleus. Thus, a coherent sense of orientation involves projection specific translaminar processing in the presubiculum.
Visual abstract
... Mice were anesthetized with 2%−4% isoflurane under analgesia (0.1 mg/kg Buprecare for 48 h) and placed in a stereotaxic frame. A solution of 50 nL of kainate (20 nM in phosphate-buffered saline [PBS]) or PBS for control mice was injected (10 nL/min) into the right dorsal hippocampus (anterior−posterior, −1.8 mm; mediolateral, −1.8 mm; dorsoventral, −1.8 mm, from bregma) according to the previously described methodology [22]. Six weeks after the intrahippocampal injection, mice were sonicated. ...
Objective:
It is unknown whether ultrasound-induced blood-brain barrier (BBB) disruption can promote epileptogenesis and how BBB integrity changes over time after sonication.
Methods:
To gain more insight into the safety profile of ultrasound (US)-induced BBB opening, we determined BBB permeability as well as histological modifications in C57BL/6 adult control mice and in the kainate (KA) model for mesial temporal lobe epilepsy in mice after sonication with low-intensity pulsed ultrasound (LIPU). Microglial and astroglial changes in ipsilateral hippocampus were examined at different time points following BBB disruption by respectively analyzing Iba1 and glial fibrillary acidic protein immunoreactivity. Using intracerebral EEG recordings, we further studied the possible electrophysiological repercussions of a repeated disrupted BBB for seizure generation in nine non-epileptic mice.
Results:
LIPU-induced BBB opening led to transient albumin extravasation and reversible mild astrogliosis, but not to microglial activation in the hippocampus of non-epileptic mice. In KA mice, the transient albumin extravasation into the hippocampus mediated by LIPU-induced BBB opening did not aggravate inflammatory processes and histologic changes that characterize the hippocampal sclerosis. Three LIPU-induced BBB opening did not induce epileptogenicity in non-epileptic mice implanted with depth EEG electrodes.
Conclusion:
Our experiments in mice provide persuasive evidence of the safety of LIPU-induced BBB opening as a therapeutic modality for neurological diseases.
... Such a heating is, indeed, insufficient for effective hyperthermia and suggests that the NFs within the OMNF are not efficient enough in mouse brain models, where effective thermal treatments require local temperature increments above 7°C, and injection volumes below 5 µL. [42][43][44][45] The low magnetic heating released by the studied OMNF in tissue phantoms, compared with those obtained previously by the dispersions in Eppendorf tubes (i.e. ΔT ≈ 15°C, Fig. S3A †), could stem from different reasons including NF agglomeration/immobilization (which can also occur in biological matrices), viscosity changes and the reduced injectable volumes. ...
... 54,56,57 (iii) Due to the reduced volume of the mouse brain, the maximum recommended injection volume is restricted to about 5 μL. [42][43][44][45] This is the reason why 4 μL was chosen at the upper limit in terms of OMNF injected volume. This is much smaller than the volume of the ferrofluid typically injected in other works dealing with the mouse prostate or human brain tumors (typically ≥100 μL at similar or higher concentrations). ...
Optomagnetic nanofluids (OMNFs) are colloidal dispersions of nanoparticles (NPs) with combined magnetic and optical properties. They are especially appealing in biomedicine since they can be used as minimally invasive platforms for controlled hyperthermia treatment of otherwise difficultly accessible tumors such as intracranial ones. On the one hand, magnetic NPs act as heating mediators when subjected to alternating magnetic fields or light irradiation. On the other hand, suitably tailored luminescent NPs can provide a precise and remote thermal readout in real time. The combination of heating and thermometric properties allows, in principle, to precisely monitor the increase in the temperature of brain tumors up to the therapeutic level, without causing undesired collateral damage. In this work we demonstrate that this view is an oversimplification since it ignores the presence of relevant interactions between magnetic (γ-Fe2O3 nanoflowers) and luminescent nanoparticles (Ag2S NPs) that result in a detrimental alteration of their physicochemical properties. The magnitude of such interactions depends on the interparticle distance and on the surface properties of nanoparticles. Experiments performed in mouse brains (phantoms and ex vivo) revealed that OMNFs cannot induce relevant heating under alternating magnetic fields and fail to provide reliable temperature reading. In contrast, we demonstrate that the use of luminescent nanofluids (containing only Ag2S NPs acting as both photothermal agents and nanothermometers) stands out as a better alternative for thermally monitored hyperthermia treatment of brain tumors in small animal models.
... Afin d'obtenir des modèles animaux réussis, l'injection intracérébrale de virus, de cellules tumorales ou de molécules doit être parfaitement exécutée. Au fur et à mesure des connaissances et des avancées technologiques, les protocoles chirurgicaux existants sont mis à jour pour permettre aux chercheurs d'améliorer l'efficacité et la sécurité de l'injection intracérébrale par une aiguille [Mathon et al. (2015)]. ...
L’IRM et la SRM, deux techniques issues de la RMN, sont de plus en plus utilisées pour étudier lefonctionnement et l’anatomie du cerveau et pour diagnostiquer et évaluer un grand nombre de pathologiescérébrales. Toutefois, la faible sensibilité inhérente de la RMN fait encore obstacle à l’analyse précisede régions cérébrales de très faible volume. Cette thèse a eu pour but d’explorer une solution pouraméliorer cette dernière et qui a consisté à miniaturiser et à implanter l’antenne de détection RF auplus près de la région d’intérêt. Les performances électromagnétiques, notamment caractérisées par unimportant facteur de gain en sensibilité comparé à l’utilisation d’antennes conventionnelles, et le caractèremini-invasif des micro-antennes RMN implantables développées ici ont permis de mettre en avant, avecune bonne reproductibilité et fiabilité, les différences métaboliques et morphologiques entre un tissucérébral sain et un tissu cérébral tumoral sur des échantillons biologiques de très faible volume (del’ordre du μL) chez des rats sains et chez des rats porteurs d’un gliome C6. D’autres applications ont puêtre, par la suite, envisagées comme l’association d’une micro-antenne RMN et d’un système d’injectiond’un composé d’intérêt permettant de suivre, en temps réel, l’effet de ce dernier sur le métabolisme del’échantillon biologique implanté. Par l’implantation de ce dispositif chez des rats porteurs d’un gliomeC6, l’efficacité thérapeutique de deux molécules antitumorales a pu être évaluée, en temps réel, parl’évolution de biomarqueurs spectroscopiques caractéristiques d’une réponse thérapeutique observablessur les spectres RMN 1H acquis avec la micro-antenne RMN. Ces diverses applications ont ainsi démontrétout le potentiel de ces micro-antennes RMN implantables dans l’analyse d’échantillons de faible volume.Mots-clés : Micro-antenne RMN, SRM et IRM in vivo, métabolisme tumoral, efficacité thérapeutique
... PGC-1α-specific shRNA were CTGCGAACATATTTGAGAATTCAAGAGATTCT-CAAATATGTTCGCAGTTTTTT. We made a tailored stage for stereotactic injection according to the literature (Mathon et al., 2015). After anesthetized by a freeze, PND3 mice were placed into the tailored stage on a stereotactic frame (KOPF, KD Scientific) for injection. ...
Background
Developing brain is highly plastic and can be easily affected. Growing pediatric usage of anesthetics during painless procedures has raised concerns about the effect of low-dose anesthetics on neurodevelopment. It is urgent to ascertain the neuronal effect of low-dose Propofol, a widely used anesthetic in pediatrics, on developing brains.
Methods
The behavioral tests after neonatal exposure to low-dose/high-dose Propofol in mice were conducted to clarify the cognitive effect. The nascent cells undergoing proliferation and differentiation stage in the hippocampus and cultured neural stem cells (NSCs) were further identified. In addition, single-nuclei RNA sequencing (snRNA-seq), NSCs bulk RNA-seq, and metabolism trials were performed for pathway investigation. Furthermore, small interfering RNA and stereotactic adenovirus injection were, respectively, used in NSCs and hippocampal to confirm the underlying mechanism.
Results
Behavioral tests in mice showed enhanced spatial cognitive ability after being exposed to low-dose Propofol. Activated neurogenesis was observed both in hippocampal and cultured NSCs. Moreover, transcriptome analysis of snRNA-seq, bulk RNA-seq, and metabolism trials revealed a significantly enhanced oxidative phosphorylation (OXPHOS) level in NSCs. Furthermore, PGC-1α, a master regulator in mitochondria metabolism, was found upregulated after Propofol exposure both in vivo and in vitro . Importantly, downregulation of PGC-1α remarkably prevented the effects of low-dose Propofol in activating OXPHOS and neurogenesis.
Conclusions
Taken together, this study demonstrates a novel alteration of mitochondrial function in hippocampal neurogenesis after low-dose Propofol exposure, suggesting the safety, even potentially beneficial effect, of low-dose Propofol in pediatric use.
