Effect of chronic intermittent hypoxia on ocular and intraoral mechanical allodynia mediated via the calcitonin gene-related peptide in a rat.

STUDY OBJECTIVES: Obstructive sleep apnea, a significant hypoxic condition, may exacerbate several orofacial pain conditions. The study aims to define the involvement of calcitonin gene-related peptide (CGRP) in peripheral and central sensitization and in evoking orofacial mechanical allodynia under chronic intermittent hypoxia (CIH). METHODS: Male rats were exposed to CIH. Orofacial mechanical allodynia was assessed using the eyeblink test and the two-bottle preference drinking test. The CGRP-immunoreactive neurons in the trigeminal ganglion (TG), CGRP-positive primary afferents projecting to laminae I-II of the trigeminal spinal subnucleus caudalis (Vc), and neural responses in the second-order neurons of the Vc were determined by immunohistochemistry. CGRP receptor antagonist was administrated in the TG. RESULTS: CIH-induced ocular and intraoral mechanical allodynia. CGRP-immunoreactive neurons and activated satellite glial cells (SGCs) were significantly increased in the TG and the number of cFos-immunoreactive cells in laminae I-II of the Vc were significantly higher in CIH rats compared to normoxic rats. Local administration of the CGRP receptor antagonist in the TG of CIH rats attenuated orofacial mechanical allodynia; the number of CGRP-immunoreactive neurons and activated SGCs in the TG, and the density of CGRP-positive primary afferent terminals and the number of cFos-immunoreactive cells in laminae I-II of the Vc were significantly lower compared to vehicle-administrated CIH rats. CONCLUSIONS: An increase in CGRP in the TG induced by CIH, as well as orofacial mechanical allodynia and central sensitization of second-order neurons in the Vc, supported the notion that CGRP plays a critical role in CIH-induced orofacial mechanical allodynia.

Long-lasting adverse effects of short-term stress during the suckling-mastication transition period on masticatory function and intraoral sensation in rats.

Early-life stress affects brain development, eventually resulting in adverse behavioral and physical health consequences in adulthood. The present study assessed the hypothesis that short-term early-life stress during infancy before weaning, a period for the maturation of mastication and sleep, poses long-lasting adverse effects on masticatory function and intraoral sensations later in life.Rat pups were exposed to either maternal separation (MS) or intermittent hypoxia (IH-Infancy) for 6 h/day in the light/sleep phase from postnatal day (P)17 to P20 to generate "neglect" and "pediatric obstructive sleep apnea" models, respectively. The remaining rats were exposed to IH during P45-P48 (IH-Adult). Masticatory ability was evaluated based on the rats' ability to chew pellets and bite pasta throughout the growth period (P21-P70). Intraoral chemical and mechanical sensitivities were assessed using two-bottle preference drinking tests, and hind paw pain thresholds were measured in adulthood (after P60).No differences were found in body weight, grip force, and hind paw sensitivity in MS, IH-Infancy, and IH-Adult rats compared with naïve rats. Masticatory ability was lower in MS and IH-Infancy rats from P28 to P70 than in naïve rats. MS and IH-Infancy rats exhibited intraoral hypersensitivity to capsaicin and mechanical stimulations in adulthood. The IH-Adult rats did not display inferior masticatory ability or intraoral hypersensitivity.In conclusion, short-term early-life stress during the suckling-mastication transition period potentially causes a persistent decrease in masticatory ability and intraoral hypersensitivity in adulthood. The period is a "critical window" for the maturation of oral motor and sensory functions.

STRA8-RB interaction is required for timely entry of meiosis in mouse female germ cells.

Meiosis is differently regulated in males and females. In females, germ cells initiate meiosis within a limited time period in the fetal ovary and undergo a prolonged meiotic arrest until puberty. However, how meiosis initiation is coordinated with the cell cycle to coincide with S phase remains elusive. Here, we demonstrate that STRA8 binds to RB via the LXCXE motif. Mutation of the RB-binding site of STRA8 in female mice delays meiotic entry, which consequently delays progression of meiotic prophase and leads to precocious depletion of the oocyte pool. Single-cell RNA-sequencing analysis reveals that the STRA8-RB interaction is required for S phase entry and meiotic gene activation, ensuring precise timing of meiosis initiation in oocytes. Strikingly, the results suggest STRA8 could sequester RB from E2F during pre-meiotic G1/S transition. This study highlights the gene regulatory mechanisms underlying the female-specific mode of meiotic initiation in mice.

The role of Zfp352 in the regulation of transient expression of 2-cell specific genes in mouse embryonic stem cells.

Mouse ES cell populations contain a minor sub-population that expresses genes specifically expressed in 2-cell stage embryos. This sub-population consists of 2-cell-gene labeled cells (2CLCs) generated by the transient activation of the 2-cell specific genes initiated by the master regulator, Dux. However, the mechanism regulating the transient expression remains largely unclear. Here we reported a novel function of Zfp352, one of the 2-cell specific genes, in regulating the 2CLC sub-population. Zfp352 encodes zinc-finger transcription factor belonging to the Klf family. Dux transiently activates Zfp352 after the activation of Zscan4c in a subset of the 2CLC subpopulation. Interestingly, in the reporter assay, the transcriptional activation of Zscan4c by Dux is strongly repressed by the co-expression of Zfp352. However, the knockout of Zfp352 resulted in the repression of a subset of the 2-cell-specific genes. These data suggest the dual roles of Zfp352 in regulating the transient activation of the 2-cell-specific genes.

Cerebellar Nuclei Receiving Orofacial Proprioceptive Signals through the Mossy Fiber Pathway from the Supratrigeminal Nucleus in Rats.

Proprioception from muscle spindles is necessary for motor function executed by the cerebellum. In particular, cerebellar nuclear neurons that receive proprioceptive signals and send projections to the lower brainstem or spinal cord play key roles in motor control. However, little is known about which cerebellar nuclear regions receive orofacial proprioception. Here, we investigated projections to the cerebellar nuclei from the supratrigeminal nucleus (Su5), which conveys the orofacial proprioception arising from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 resulted in a large number of labeled axon terminals bilaterally in the dorsolateral hump (IntDL) of the cerebellar interposed nucleus (Int) and the dorsolateral protuberance (MedDL) of the cerebellar medial nucleus. In addition, a moderate number of axon terminals were ipsilaterally labeled in the vestibular group Y nucleus (group Y). We electrophysiologically detected JCMS proprioceptive signals in the IntDL and MedDL. Retrograde tracing analysis confirmed bilateral projections from the Su5 to the IntDL and MedDL. Furthermore, anterograde tracer injections into the external cuneate nucleus (ECu), which receives other proprioceptive input from forelimb/neck muscles, resulted in only a limited number of ipsilaterally labeled terminals, mainly in the dorsomedial crest of the Int and the group Y. Taken together, the Su5 and ECu axons almost separately terminated in the cerebellar nuclei (except for partial overlap in the group Y). These data suggest that orofacial proprioception is differently processed in the cerebellar circuits in comparison to other body-part proprioception, thus contributing to the executive function of orofacial motor control.

Initial loading of dexmedetomidine and continuous propofol sedation for prevention of delayed recovery: A randomized controlled trial.

BACKGROUND: Sedation with continuous dexmedetomidine and bolus midazolam administration provides a lower incidence of unacceptable patient movement during procedures but requires a longer recovery time. The authors aimed to compare recovery time and unacceptable patient movement during sedation with initial loading of dexmedetomidine followed by continuous propofol infusion with those during sedation with continuous dexmedetomidine and bolus midazolam administration. METHODS: In this prospective randomized controlled trial, 54 patients undergoing dental surgery and requiring intravenous sedation were assigned to either the dexmedetomidine and propofol group (n = 27, dexmedetomidine administered at 6 µg/kg/h for 5 minutes, followed by continuous propofol infusion using a target-controlled infusion) or the dexmedetomidine and midazolam group (n = 27, dexmedetomidine administered at 0.2-0.7 µg/kg/h continuously after the same initial loading dose with bolus midazolam). A bispectral index of 70 through 80 was maintained during the procedure. Patient movement that interfered with the procedure and time from the end of sedation to achieving a negative Romberg sign were assessed. RESULTS: Times from the end of sedation to achieving a negative Romberg sign in the dexmedetomidine and propofol group (median, 14 minutes [interquartile range, 12-15 minutes]) were significantly shorter (P < .001) than in the dexmedetomidine and midazolam group (median, 22 minutes [interquartile range, 17.5-30.5 minutes]). The incidence of unacceptable patient movement was comparable between groups (n = 3 in the dexmedetomidine and propofol group, n = 4 in the dexmedetomidine and midazolam group; P = .999). CONCLUSIONS: Sedation with a single loading dose of dexmedetomidine followed by continuous propofol infusion can prevent delayed recovery without increasing unacceptable patient movement. PRACTICAL IMPLICATIONS: The combination of dexmedetomidine and propofol may provide high-quality sedation for ambulatory dental practice. This clinical trial was registered in the University Hospital Medical Information Network Clinical Trials Registry. The registration number is UMIN000039668.

Antagonist of transient receptor potential melastatin 2 suppresses mechanical hypersensitivity and activation of microglia induced by infraorbital nerve ligation in male rats.

Activation of microglia is known to be involved in neuropathic pain. However, the pathway that regulates the microglial activation is not completely understood. Transient receptor potential (TRP) melastatin 2 (TRPM2), which is part of the TRP superfamily, is reportedly expressed on microglia and is suggested to be involved in neuropathic pain. To explore the effect of a TRPM2 antagonist on orofacial neuropathic pain and the relationship between TRPM2 and the activation of microglia, experiments were conducted using male rats that underwent infraorbital nerve (ION) ligation as orofacial neuropathic pain models. TRPM2 expression was detected on microglia in the trigeminal spinal subnucleus caudalis (Vc). The immunoreactivity of TRPM2 in the Vc increased after ION ligation. Mechanical threshold for head-withdrawal response was measured using von Frey filament, and it decreased after ION ligation. When the TRPM2 antagonist was administered to the ION-ligated rats, the low mechanical threshold for head-withdrawal response increased, and the number of phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive cells in the Vc decreased. The number of CD68-immunoreactive cells in the Vc also decreased after the administration of the TRPM2 antagonist in the ION-ligated rats. These findings suggest that TRPM2 antagonist administration suppresses hypersensitivity to mechanical stimulation induced by ION ligation and microglial activation, and TRPM2 is also involved in microglial activation in orofacial neuropathic pain.

Longitudinal electromyographic analysis of jaw-closing muscle activities during ingestive behaviors from pre-weaning to juvenile periods in rats.

This longitudinal study investigated developmental changes in jaw-closing muscle activities during ingestive behaviors in rats. On postnatal day (P) 10, electromyography (EMG) electrodes were inserted into the masseter and temporalis muscles of rat pups. EMG activities were recorded for the following ingestive behaviors between P14 and P49: for suckling, including nipple attachment and rhythmic sucking on P14 and for pasta biting, pellet chewing, and milk licking between P21 and P49. Burst rhythms and muscle coordination (i.e., the correlation and time lag) between masseter and temporalis activities were assessed for each behavior. The burst rhythms of nipple attachment and rhythmic sucking on P14 were significantly slower than those of pasta biting, pellet chewing, and milk licking on P21. Muscle coordination differed between suckling on P14 and mastication and licking on P21. Between P21 and P49, increases were observed in burst rhythms for pasta biting and pellet chewing. The rate of increases in burst rhythms was higher for pasta biting than for pellet chewing. Muscle coordination between the two muscle activities for pasta biting did not significantly change between P21 and P49, whereas that for pellet chewing significantly changed between P21 and P24 and stabilized after P24. Burst rhythms for milk licking did not significantly change over time, while muscle coordination between the two muscle activities changed from agonist to antagonist muscle-like activity on approximately P35. The present results demonstrate that distinct patterns of rhythmic jaw-closing muscle activities emerge before weaning, they continue to change over time, and they exhibit unique developmental dynamics for each behavior after weaning.

Effect of midazolam in autism spectrum disorder: A retrospective observational analysis.

BACKGROUND: While midazolam is commonly used as premedication for uncooperative patients, its effects are difficult to predict in patients with autism spectrum disorder for whom abnormalities in gamma-aminobutyric acid have been reported. This study aimed to investigate the influence of autism spectrum disorder on the effect of midazolam when used as premedication. METHODS: This retrospective observational study was performed between April 2017 and August 2018. Before inducing general anesthesia with sevoflurane for dental treatment, 390 uncooperative patients received premedication with midazolam. Ordinal logistic regression analysis was performed with the Observer's Assessment of Alertness/Sedation score 30 min after premedication as the objective variable. Age, sex, American Society of Anesthesiologists physical status class, premedication route, dose per body weight, presence of specific disorders (autism spectrum disorder, intellectual disability, epilepsy, cerebral palsy, and other psychiatric disorders), and regular benzodiazepine or non-benzodiazepine psychotropic administration were included as explanatory variables. Kendall's rank correlation coefficient was used to assess the correlation between the Observer's Assessment of Alertness/Sedation score and cooperation level (1, obvious negative response; 2, negative response; 3, positive reaction; 4, obvious positive reaction) during admission and inhalation induction. All data were extracted from anesthesia and medical records. RESULTS: Age (odds ratio 1.437 [95% confidence interval (CI) 1.213-1.708], P < .001), autism spectrum disorder (1.318 [1.079-1.612], P = .007), benzodiazepine medication (0.574 [0.396-0.827], P = .002), and intramuscular route (1.478 [1.137-1.924], P = .004) were significantly associated with the Observer's Assessment of Alertness/Sedation score, while the score was negatively associated with cooperation levels during admission (τ = -0.714, P < .001) and inhalation induction (τ = -0.606, P < .001). CONCLUSIONS: Patients with autism spectrum disorder may be susceptible to premedication with midazolam; however, regular benzodiazepine administration may reduce the effect.

Evaluation of the effects of manual acupuncture and electroacupuncture at LI4 and LI11 on perception thresholds: a prospective crossover trial.

OBJECTIVE: The objective of the study was to investigate and compare the effects of manual acupuncture (MA) and electroacupuncture (EA) on current perception thresholds (CPTs) using quantitative methods. METHODS: Twenty-nine healthy volunteers participated in this prospective crossover trial, in which three acupuncture methods were compared: control, MA, and EA. Acupuncture needles were inserted to a depth of 15 mm at LI4 and LI11 on the left side and retained for 30 min with or without electrical stimulation at a frequency of 2 Hz (EA and MA, respectively). The needles were removed and participants rested for 30 min. CPT in the left mental region was measured at 2000, 250, and 5 Hz, corresponding to the activation of Aß, Aδ, and C-fibers, respectively, at four time points: baseline, T0; 15 min after needle application, T1; immediately after needle removal, T2; and 30 min after needle removal, T3. In the control session, only a sensory test was performed (without acupuncture). RESULTS: Significant effects of time course on CPT were observed (p < 0.001). CPT values increased significantly at T1, T2, and T3, compared with those at T0, at all stimulation frequencies during MA and EA. Changes in CPT values with EA were not significantly greater than those with MA. CONCLUSIONS: Both MA and EA increased the sensory thresholds of Aß, Aδ, and C-fibers in the mental region for ⩾30 min after needle removal. Additional of electrical stimulation may not confer additional benefits over needling alone. TRIAL REGISTRATION NUMBER: UMIN000017983 (University Hospital Medical Information Network Clinical Trials Registry).

Ensemble of old and new techniques escorts ESCs to bona fide embryo-like structures.

Three recent studies from the Zernicka-Goetz and Hanna groups (Amadei et al., 2022; Tarazi et al., 2022; Lau et al., 2022) report the development of mouse embryo-like structures from stem cells. Using a combination of stem cell-derived sources, they show that mouse embryo-like structures develop key features mimicking post-gastrulation embryos.

A Case of Wide QRS Tachycardia After the Local Administration of Epinephrine to Reduce Bleeding During General Anesthesia.

We report a case of wide QRS tachycardia or ventricular tachycardia with a pulse after the administration of epinephrine under general anesthesia. After induction and achieving a sufficiently deep plane of general anesthesia, gauze soaked in a 1:100,000 epinephrine solution was applied to the patient's nasal mucosa and 1% lidocaine with 1:100,000 epinephrine was administered via intraoral infiltration. Several minutes after the start of surgery, the patient's blood pressure and heart rate suddenly increased and a wide QRS tachycardia was observed on the electrocardiogram, which then reverted to a normal sinus rhythm. According to the past reports, similar arrhythmias have occurred after administration of epinephrine in the head and neck. These findings suggest that anesthesia providers must be aware of the risks associated with epinephrine and local anesthetic use, particularly in the head and neck region.

Stepwise pluripotency transitions in mouse stem cells.

Pluripotent cells in mouse embryos, which first emerge in the inner cell mass of the blastocyst, undergo gradual transition marked by changes in gene expression, developmental potential, polarity, and morphology as they develop from the pre-implantation until post-implantation gastrula stage. Recent studies of cultured mouse pluripotent stem cells (PSCs) have clarified the presence of intermediate pluripotent stages between the naïve pluripotent state represented by embryonic stem cells (ESCs-equivalent to the pre-implantation epiblast) and the primed pluripotent state represented by epiblast stem cells (EpiSCs-equivalent to the late post-implantation gastrula epiblast). In this review, we discuss these recent findings in light of our knowledge on peri-implantation mouse development and consider the implications of these new PSCs to understand their temporal sequence and the feasibility of using them as model system for pluripotency.

The polyol pathway is an evolutionarily conserved system for sensing glucose uptake.

Cells must adjust the expression levels of metabolic enzymes in response to fluctuating nutrient supply. For glucose, such metabolic remodeling is highly dependent on a master transcription factor ChREBP/MondoA. However, it remains elusive how glucose fluctuations are sensed by ChREBP/MondoA despite the stability of major glycolytic pathways. Here, we show that in both flies and mice, ChREBP/MondoA activation in response to glucose ingestion involves an evolutionarily conserved glucose-metabolizing pathway: the polyol pathway. The polyol pathway converts glucose to fructose via sorbitol. It has been believed that this pathway is almost silent, and its activation in hyperglycemic conditions has deleterious effects on human health. We show that the polyol pathway regulates the glucose-responsive nuclear translocation of Mondo, a Drosophila homologue of ChREBP/MondoA, which directs gene expression for organismal growth and metabolism. Likewise, inhibition of the polyol pathway in mice impairs ChREBP's nuclear localization and reduces glucose tolerance. We propose that the polyol pathway is an evolutionarily conserved sensing system for glucose uptake that allows metabolic remodeling.

Cerebroventricular administration of anti-calcitonin gene-related peptide antibody reduces the increase of dopamine D2 receptor observed in the trigeminal spinal subnucleus caudalis following infraorbital nerve ligation.

A dopamine D2 receptor (D2R) agonist and an anti-calcitonin gene-related peptide (CGRP) antibody were separately reported to reduce neuropathic pain. To further attenuate neuropathic pain, co-administration of a D2R agonist and an anti-CGRP antibody was performed in a rat with the infraorbital nerve (ION) ligation. However, this co-administration showed no further attenuation of mechanical hypersensitivity compared to the administration of anti-CGRP antibody alone. Our results also revealed that D2R immunoreactivity in the trigeminal spinal subnucleus caudalis (Vc) increased following the nerve ligation and decreased following administration of an anti-CGRP antibody. The ratio of immunoreactive neurons of phosphorylated cyclic adenosine monophosphate-response-element-binding protein in the Vc also increased following nerve ligation and decreased with the anti-CGRP antibody. Our results suggest that a decrease in D2R immunoreactivity reduces the effect of a D2R agonist, and transcription of D2R is activated following the ION ligation and suppressed by treatment with an anti-CGRP antibody.

Enhanced Ocular Surface and Intraoral Nociception via a Transient Receptor Potential Vanilloid 1 Mechanism in a Rat Model of Obstructive Sleep Apnea.

Obstructive sleep apnea (OSA), characterized by low arterial oxygen saturation during sleep, is associated with an increased risk of orofacial pain. In this study, we simulated chronic intermittent hypoxia (CIH) during the sleep/rest phase (light phase) to determine the role of transient receptor potential vanilloid 1 (TRPV1) in mediating enhanced orofacial nocifensive behavior and trigeminal spinal subnucleus caudalis (Vc) neuronal responses to capsaicin (a TRPV1 agonist) stimulation in a rat model of OSA. Rats were subjected to CIH (nadir O2, 5%) during the light phase for 8 or 16 consecutive days. CIH yielded enhanced behavioral responses to capsaicin after application to the ocular surface and intraoral mucosa, which was reversed under normoxic conditions. The percentage of TRPV1-immunoreactive trigeminal ganglion neurons was greater in CIH rats than in normoxic rats and recovered under normoxic conditions after CIH. The ratio of large-sized TRPV1-immunoreactive trigeminal ganglion neurons increased in CIH rats. The density of TRPV1 positive primary afferent terminals in the superficial laminae of Vc was higher in CIH rats. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive cells intermingled with the central terminal of TRPV1-positive afferents in the Vc. The number of pERK-immunoreactive cells following low-dose capsaicin (0.33 µM) application to the tongue was significantly greater in the middle portion of the Vc of CIH rats than of normoxic rats and recovered under normoxic conditions after CIH. These data suggest that CIH during the sleep (light) phase is sufficient to transiently enhance pain on the ocular surface and intraoral mucosa via TRPV1-dependent mechanisms.

Isoflurane induces Art2-Rsp5-dependent endocytosis of Bap2 in yeast.

Although general anesthesia is indispensable during modern surgical procedures, the mechanism by which inhalation anesthetics act on the synaptic membrane at the molecular and cellular level is largely unknown. In this study, we used yeast cells to examine the effect of isoflurane, an inhalation anesthetic, on membrane proteins. Bap2, an amino acid transporter localized on the plasma membrane, was endocytosed when yeast cells were treated with isoflurane. Depletion of RSP5, an E3 ligase, prevented this endocytosis and Bap2 was ubiquitinated in response to isoflurane, indicating an ubiquitin-dependent process. Screening all the Rsp5 binding adaptors showed that Art2 plays a central role in this process. These results suggest that isoflurane affects Bap2 via an Art2-Rsp5-dependent ubiquitination system.

Anti-calcitonin gene-related peptide antibody attenuates orofacial mechanical and heat hypersensitivities induced by infraorbital nerve injury.

Currently, limited information regarding the role of calcitonin gene-related peptide (CGRP) in neuropathic pain is available. Intracerebroventricular administrations of an anti-CGRP antibody were performed in rats with infraorbital nerve ligation. Anti-CGRP antibody administration attenuated mechanical and heat hypersensitivities induced by nerve ligation and decreased the phosphorylated extracellular signal-regulated kinase expression levels in the trigeminal spinal subnucleus caudalis (Vc) following mechanical or heat stimulation. An increased CGRP immunoreactivity in the Vc appeared after nerve ligation. A decreased CGRP immunoreactivity resulted from anti-CGRP antibody administration. Our findings suggest that anti-CGRP antibody administration attenuates the symptoms of trigeminal neuropathic pain by acting on CGRP in the Vc.

Meiosis-specific ZFP541 repressor complex promotes developmental progression of meiotic prophase towards completion during mouse spermatogenesis.

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.

Generation of Mitochondrial Toxin Rodent Models of Parkinson's Disease Using 6-OHDA , MPTP , and Rotenone.

Several animal models are employed to discover novel treatments for the symptoms of Parkinson's disease (PD). PD models can be divided into two models: neurotoxin models and genetic models. Among neurotoxins to produce PD models, 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone, which inhibit the mitochondrial complex I, are widely used. Animal models of PD using these neurotoxins are also known as mitochondrial toxin models. Here this chapter describes the preparation of these mitochondrial toxin models.