Ketogenic diet mitigates opioid-induced hyperalgesia by restoring short-chain fatty acids-producing bacteria in the gut.

ABSTRACT: Opioids are commonly prescribed to patients with chronic pain. Chronic opioid usage comes with a slew of serious side effects, including opioid-induced hyperalgesia (OIH). The patients with long-term opioid treatment experience paradoxical increases in nociceptive hypersensitivity, namely, OIH. Currently, treatment options for OIH are extremely lacking. In this study, we show that the ketogenic diet recovers the abnormal pain behavior caused by chronic morphine treatment in male mice, and we further show that the therapeutic effect of the ketogenic diet is mediated through gut microbiome. Our 16S rRNA sequencing demonstrates that chronic morphine treatment causes changes in mouse gut microbiota, specifically a decrease in short-chain fatty acids-producing bacteria, and the sequencing data also show that the ketogenic diet rescues those bacteria in the mouse gut. More importantly, we show that supplementation with short-chain fatty acids (butyrate, propionate, and acetate) can delay the onset of OIH, indicating that short-chain fatty acids play a direct role in the development of OIH. Our findings suggest that gut microbiome could be targeted to treat OIH, and the ketogenic diet can be used as a complementary approach for pain relief in patients with chronic opioid treatment. We only used male mice in this study, and thus, our findings cannot be generalized to both sexes.

Gut-Brain Crosstalk and the Central Mechanisms of Orofacial Pain.

Accumulated evidence has demonstrated that the gut microbiome can contribute to pain modulation through the microbiome-gut-brain axis. Various relevant microbiome metabolites in the gut are involved in the regulation of pain signaling in the central nervous system. In this review, we summarize recent advances in gut-brain interactions by which the microbiome metabolites modulate pain, with a focus on orofacial pain, and we further discuss the role of gut-brain crosstalk in the central mechanisms of orofacial pain whereby the gut microbiome modulates orofacial pain via the vagus nerve-mediated direct pathway and the gut metabolites/molecules-mediated indirect pathway. The direct and indirect pathways both contribute to the central regulation of orofacial pain through different brain structures (such as the nucleus tractus solitarius and the parabrachial nucleus) and signaling transmission across the blood-brain barrier, respectively. Understanding the gut microbiome-regulated pain mechanisms in the brain could help us to develop non-opioid novel therapies for orofacial pain.

A female-specific role for trigeminal dynorphin in orofacial pain comorbidity.

ABSTRACT: Migraine is commonly reported in patients with temporomandibular disorders (TMDs), but little is known about the mechanisms underlying the comorbid condition. Here, we prepared a mouse model to investigate this comorbidity, in which masseter muscle tendon ligation (MMTL) was performed to induce a myogenic TMD, and the pre-existing TMD enabled a subthreshold dose of nitroglycerin (NTG) to produce migraine-like pain in mice. RNA sequencing followed by real-time quantitative polymerase chain reaction confirmation showed that MMTL plus NTG treatment increased prodynorphin ( Pdyn ) mRNA expression in the spinal trigeminal nucleus caudalis (Sp5C) of female mice but not in male mice. Chemogenetic inhibition of Pdyn -expressing neurons or microinjection of antidynorphin antiserum in the Sp5C alleviated MMTL-induced masseter hypersensitivity and diminished the MMTL-enabled migraine-like pain in female mice but not in male mice. Moreover, chemogenetic activation of Pdyn -expressing neurons or microinjection of dynorphin A (1-17) peptide in the Sp5C enabled a subthreshold dose of NTG to induce migraine-like pain in female mice but not in male mice. Taken together, our results suggest that trigeminal dynorphin has a female-specific role in the modulation of comorbid TMDs and migraine.

Neuronal Histone Methyltransferase EZH2 Regulates Neuronal Morphogenesis, Synaptic Plasticity, and Cognitive Behavior in Mice.

The histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)-mediated trimethylation of histone H3 lysine 27 (H3K27me3) regulates neural stem cell proliferation and fate specificity through silencing different gene sets in the central nervous system. Here, we explored the function of EZH2 in early post-mitotic neurons by generating a neuron-specific Ezh2 conditional knockout mouse line. The results showed that a lack of neuronal EZH2 led to delayed neuronal migration, more complex dendritic arborization, and increased dendritic spine density. Transcriptome analysis revealed that neuronal EZH2-regulated genes are related to neuronal morphogenesis. In particular, the gene encoding p21-activated kinase 3 (Pak3) was identified as a target gene suppressed by EZH2 and H3K27me3, and expression of the dominant negative Pak3 reversed Ezh2 knockout-induced higher dendritic spine density. Finally, the lack of neuronal EZH2 resulted in impaired memory behaviors in adult mice. Our results demonstrated that neuronal EZH2 acts to control multiple steps of neuronal morphogenesis during development, and has long-lasting effects on cognitive function in adult mice.

Assessing Orofacial Pain Behaviors in Animal Models: A Review.

Orofacial pain refers to pain occurring in the head and face, which is highly prevalent and represents a challenge to clinicians, but its underlying mechanisms are not fully understood, and more studies using animal models are urgently needed. Currently, there are different assessment methods for analyzing orofacial pain behaviors in animal models. In order to minimize the number of animals used and maximize animal welfare, selecting appropriate assessment methods can avoid repeated testing and improve the reliability and accuracy of research data. Here, we summarize different methods for assessing spontaneous pain, evoked pain, and relevant accompanying dysfunction, and discuss their advantages and disadvantages. While the behaviors of orofacial pain in rodents are not exactly equivalent to the symptoms displayed in patients with orofacial pain, animal models and pain behavioral assessments have advanced our understanding of the pathogenesis of such pain.

Gut microbiota and migraine.

Migraine is a leading cause of disability among the adult population and is a significant burden on the economies of the world. Studies into the underlying causes of migraine have spanned centuries but its underlying mechanisms are still not fully understood. In recent years, accumulating evidence implicates that microbiota-mediated gut-brain crosstalk may contribute to the pathogenesis of migraine. This review provides a brief account of the history of migraine theories and summarizes the recent studies showing how gut microbiota is involved in the pathophysiology of migraine. Future research perspectives for better understanding the role of the gut microbiota in migraine are also discussed.

A Multidisciplinary Approach to Simultaneously Monitoring Real-Time Neuronal Activity and Pain Behaviors During Optogenetic Stimulation of Brain Neurons in Freely Moving Mice.

BACKGROUND: Highlighted by the current opioid epidemic, identifying novel therapies to treat chronic trigeminal neuropathic pain is a critical need. To develop these treatments, it is necessary to have viable targets in the brain to act on. Historically, neural tracing studies have been extremely useful in determining connections between brain areas but do not provide information about the functionality of these connections. Combining optogenetics and behavioral observation allows researchers to determine whether a particular brain area is involved in the regulation of such behavior. The addition of multi-channel electrophysiological recording provides information on real-time neuronal activity in the specific neuronal pathway. METHODS: Male C57/BL/6J mice (8-week-old) underwent either chronic constriction injury of infraorbital nerve (CCI-ION) or a sham surgery and were injected with either channelrhodopsin (ChR2) or a control virus in the hypothalamic A11 nucleus. Two weeks after CCI-ION, they were tested in real-time place preference (RTPP), while neuronal activity in the spinal trigeminal nucleus caudalis (Sp5C) was recorded. RESULTS: Optogenetic excitation of the A11 neurons results in more time spent in the stimulation chamber during RTPP testing. Additionally, stimulation of the A11 results in a greater number of neuronal activity increase in the Sp5C in animals with the injection of AAV carrying ChR2 compared to animals injected with a control virus or that underwent a sham surgery. CONCLUSION: In vivo multi-channel electrophysiological recording, optogenetic stimulation, and behavioral observation can be combined in a mouse model of chronic trigeminal neuropathic pain to validate brain areas involved in the modulation of such pain.

Optogenetic Activation of Dopamine Receptor D1 and D2 Neurons in Anterior Cingulate Cortex Differentially Modulates Trigeminal Neuropathic Pain.

Anterior cingulate cortex (ACC) is a critical brain center for chronic pain processing. Dopamine signaling in the brain has been demonstrated to contribute to descending pain modulation. However, the role of ACC dopamine receptors in chronic neuropathic pain remains unclear. In this study, we investigated the effect of optogenetic activation of ACC dopamine receptors D1- and D2-expressing neurons on trigeminal neuropathic pain. Chronic constriction injury of infraorbital nerve (CCI-ION) was carried out to induce trigeminal neuropathic pain in mice. We conducted optogenetic stimulation to specifically activate D1- and D2-expressing neurons in the ACC. Western blotting and immunofluorescence staining were used to examine ACC D1 and D2 expression and localization. The von Frey and real-time place preference tests were performed to measure evoked mechanical pain and nonreflexive emotional pain behaviors, respectively. We observed that dopamine receptors D1 and D2 in the ACC are primarily expressed in excitatory neurons and that the D2 receptor is differentially regulated in the early and late phases of trigeminal neuropathic pain. Optogenetic activation of D1-expressing neurons in the ACC markedly exacerbates CCI-ION-induced trigeminal neuropathic pain in both early and late phases, but optogenetic activation of D2-expressing neurons in the ACC robustly ameliorates such pain in its late phase. Our results suggest that dopamine receptors D1 and D2 in the ACC play different roles in the modulation of trigeminal neuropathic pain.

Resveratrol alleviates temporomandibular joint inflammatory pain by recovering disturbed gut microbiota.

Patients with temporomandibular disorders (TMDs) often experience persistent facial pain. However, the treatment of TMD pain is still inadequate. In recent years, the disturbance of gut microbiota has been shown to play an important role in the pathogenesis of different neurological diseases including chronic pain. In the present study, we investigated the involvement of gut microbiota in the development of temporomandibular joint (TMJ) inflammation. Intra-temporomandibular joint injection of complete Freund's adjuvant (CFA) was employed to induce TMJ inflammation. Resveratrol (RSV), a natural bioactive compound with anti-inflammatory property, was used to treat the CFA-induced TMJ inflammation. We observed that CFA injection not only induces persistent joint pain, but also causes the reduction of short-chain fatty acids (SCFAs, including acetic acid, propionic acid and butyric acid) in the gut as well as decreases relevant gut bacteria Bacteroidetes and Lachnospiraceae. Interestingly, systemic administration of RSV (i.p.) dose-dependently inhibits CFA-induced TMJ inflammation, reverses CFA-caused reduction of SCFAs and these gut bacteria. Moreover, CFA injection causes blood-brain barrier (BBB) leakage, activates microglia and enhances tumor necrosis factor alpha (TNFα) release in the spinal trigeminal nucleus caudalis (Sp5C). The RSV treatment restores the BBB integrity, inhibits microglial activation and decreases the release of TNFα in the Sp5C. Furthermore, fecal microbiota transplantation with feces from RSV-treated mice significantly diminishes the CFA-induced TMJ inflammation. Taken together, our results suggest that gut microbiome perturbation is critical for the development of TMJ inflammation and that recovering gut microbiome to normal levels could be a new therapeutic approach for treating such pain.

Biosimilars and implications for pharmacy practice: Ready or not, here they come

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Dopamine receptor D2, but not D1, mediates descending dopaminergic pathway-produced analgesic effect in a trigeminal neuropathic pain mouse model.

Neuropathic pain represents a challenge to clinicians because it is resistant to commonly prescribed analgesics due to its largely unknown mechanisms. Here, we investigated a descending dopaminergic pathway-mediated modulation of trigeminal neuropathic pain. We performed chronic constriction injury of the infraorbital nerve from the maxillary branch of trigeminal nerve to induce trigeminal neuropathic pain in mice. Our retrograde tracing showed that the descending dopaminergic projection from hypothalamic A11 nucleus to spinal trigeminal nucleus caudalis is bilateral. Optogenetic/chemogenetic manipulation of dopamine receptors D1 and D2 in the spinal trigeminal nucleus caudalis produced opposite effects on the nerve injury-induced trigeminal neuropathic pain. Specific excitation of dopaminergic neurons in the A11 nucleus attenuated the trigeminal neuropathic pain through the activation of D2 receptors in the spinal trigeminal nucleus caudalis. Conversely, specific ablation of the A11 dopaminergic neurons exacerbated such pain. Our results suggest that the descending A11-spinal trigeminal nucleus caudalis dopaminergic projection is critical for the modulation of trigeminal neuropathic pain and could be manipulated to treat such pain.

Cognitive impairments induced by necrotizing enterocolitis can be prevented by inhibiting microglial activation in mouse brain.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease of the premature infant. One of the most important long-term complications observed in children who survive NEC early in life is the development of profound neurological impairments. However, the pathways leading to NEC-associated neurological impairments remain unknown, thus limiting the development of prevention strategies. We have recently shown that NEC development is dependent on the expression of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium, whose activation by bacteria in the newborn gut leads to mucosal inflammation. Here, we hypothesized that damage-induced production of TLR4 endogenous ligands in the intestine might lead to activation of microglial cells in the brain and promote cognitive impairments. We identified a gut-brain signaling axis in an NEC mouse model in which activation of intestinal TLR4 signaling led to release of high-mobility group box 1 in the intestine that, in turn, promoted microglial activation in the brain and neurological dysfunction. We further demonstrated that an orally administered dendrimer-based nanotherapeutic approach to targeting activated microglia could prevent NEC-associated neurological dysfunction in neonatal mice. These findings shed light on the molecular pathways leading to the development of NEC-associated brain injury, provide a rationale for early removal of diseased intestine in NEC, and indicate the potential of targeted therapies that protect the developing brain in the treatment of NEC in early childhood.

Ventricular Volume Dynamics During the Development of Adult Chronic Communicating Hydrocephalus in a Rodent Model.

INTRODUCTION: The pathophysiology of normal-pressure hydrocephalus and the correlation with its symptomatology is not well understood. OBJECTIVE: To monitor and evaluate the enlargement patterns of the ventricular system for each ventricle and its correlation with the presenting symptoms. METHODS: Bilateral kaolin injection into the subarachnoid space overlying the cranial convexities was done in 18 adult rats. Magnetic resonance imaging was performed on an 11.7-T scanner 15, 60, 90, and 120 days after injection. Volumes of the ventricular system were measured for each ventricle and correlated with biweekly behavioral findings. RESULTS: There was a progressive increase in the ventricular volume for the lateral ventricles since day 15 in the kaolin-injected animals. There was a nonsignificant trend in volume growth for the third ventricle, but its enlargement was synchronous with the lateral ventricles. No significant change for the fourth ventricle. No symptoms were detected in the first 60 days. Association was found between the ventricular volume and locomotor changes. In addition, the odds of locomotor symptoms increased by 3% for every additional cubic millimeter of volume in the left (P < 0.001) and right (P = 0.023) ventricles, and for the total magnetic resonance imaging volume by 1% (P = 0.013). CONCLUSIONS: Expansion of the lateral ventricles maintained similar proportions over time, accompanied by a synchronous third ventricular expansion with less proportion and a nonsignificant fourth enlargement. Lateral ventricles enlarged most in those animals that were to develop late locomotor deterioration. Further research using this animal model combined with different radiologic imaging techniques, such as diffusion tensor imaging and perfusion studies, is recommended.

Improved rate of substrate oxidation catalyzed by genetically-engineered myoglobin.

This study showcases the potential of unnatural amino acids to enable non-natural functions when incorporated in the protein scaffold of heme metalloproteins. For this purpose, a genetically-engineered myoglobin (Mb) mutant was created by incorporating redox-active 3-amino-l-tyrosine (NH2Tyr) into its active site, replacing the distal histidine (H64) with NH2Tyr. In peroxide-shunt assays, this variant exhibits an increased rate of turnover for thioanisole and benzaldehyde oxidation as compared to the wild-type (WT) Mb. Indeed, in the presence of excess hydrogen peroxide (H2O2), a 9-fold and 81-fold increase in activity was observed over multiple turnovers for thioanisole sulfoxidation and benzoic acid formation, respectively. The increased oxidation activity in the H64NH2Tyr Mb mutant underlined the role of NH2Tyr in the distal active-site scaffold in peroxide activation. Kinetic, electrochemical, and EPR spectroscopic experiments were performed. On the basis of these studies, it is argued that the single NH2Tyr residue within the Mb variant simultaneously serves the role of the conserved His/Arg-pair within the distal pocket of horseradish peroxidase.

Thorase variants are associated with defects in glutamatergic neurotransmission that can be rescued by Perampanel.

The AAA+ adenosine triphosphatase (ATPase) Thorase plays a critical role in controlling synaptic plasticity by regulating the expression of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Bidirectional sequencing of exons of ATAD1, the gene encoding Thorase, in a cohort of patients with schizophrenia and healthy controls revealed rare Thorase variants. These variants caused defects in glutamatergic signaling by impairing AMPAR internalization and recycling in mouse primary cortical neurons. This contributed to increased surface expression of the AMPAR subunit GluA2 and enhanced synaptic transmission. Heterozygous Thorase-deficient mice engineered to express these Thorase variants showed altered synaptic transmission and several behavioral deficits compared to heterozygous Thorase-deficient mice expressing wild-type Thorase. These behavioral impairments were rescued by the competitive AMPAR antagonist Perampanel, a U.S. Food and Drug Administration-approved drug. These findings suggest that Perampanel may be useful for treating disorders involving compromised AMPAR-mediated glutamatergic neurotransmission.

DISC1 in Astrocytes Influences Adult Neurogenesis and Hippocampus-Dependent Behaviors in Mice.

The functional role of genetic variants in glia in the pathogenesis of psychiatric disorders remains poorly studied. Disrupted-In-Schizophrenia 1 (DISC1), a genetic risk factor implicated in major mental disorders, has been implicated in regulation of astrocyte functions. As both astrocytes and DISC1 influence adult neurogenesis in the dentate gyrus (DG) of the hippocampus, we hypothesized that selective expression of dominant-negative C-terminus-truncated human DISC1 (mutant DISC1) in astrocytes would affect adult hippocampal neurogenesis and hippocampus-dependent behaviors. A series of behavioral tests were performed in mice with or without expression of mutant DISC1 in astrocytes during late postnatal development. In conjunction with behavioral tests, we evaluated adult neurogenesis, including neural progenitor proliferation and dendrite development of newborn neurons in the DG. The ameliorative effects of D-serine on mutant DISC1-associated behaviors and abnormal adult neurogenesis were also examined. Expression of mutant DISC1 in astrocytes decreased neural progenitor proliferation and dendrite growth of newborn neurons, and produced elevated anxiety, attenuated social behaviors, and impaired hippocampus-dependent learning and memory. Chronic treatment with D-serine ameliorated the behavioral alterations and rescued abnormal adult neurogenesis in mutant DISC1 mice. Our findings suggest that psychiatric genetic risk factors expressed in astrocytes could affect adult hippocampal neurogenesis and contribute to aspects of psychiatric disease through abnormal production of D-serine.

A Novel Experimental Animal Model of Adult Chronic Hydrocephalus.

BACKGROUND: The pathogenesis of adult chronic hydrocephalus is not fully understood, and the temporal relationship between development of the radiological changes and neurological deterioration is unknown. OBJECTIVE: To clarify the progression of radiological-histological changes and subsequent clinical manifestations of adult chronic hydrocephalus. METHODS: Kaolin was injected bilaterally into the subarachnoid space overlying the cranial convexities in 20 adult rats. Magnetic resonance imaging (MRI) was obtained by using an 11.7 T scanner at 14, 60, 90, and 120 days after kaolin injection. Locomotor, gait, and cognitive evaluations were performed independently. Kaolin distribution and the associated inflammatory and fibrotic responses were histologically analyzed. RESULTS: Evans index of ventriculomegaly showed significant progressive growth in ventricular size over all time points examined. The greatest enlargement occurred within the first 2 months. Evans index also correlated with the extent of kaolin distribution by MRI and by pathological examination at all time points. First gait changes occurred at 69 days, anxiety at 80, cognitive impairment at 81, and locomotor difficulties after 120 days. Only locomotor deterioration was associated with Evans index or the radiological evaluation of kaolin extension. Inflammatory/fibrotic response was histologically confirmed over the cranial convexities in all rats, and its extension was associated with ventricular size and with the rate of ventricular enlargement. CONCLUSION: Kaolin injected into the subarachnoid space over the cerebral hemispheres of adult rats produces an inflammatory/fibrotic response leading in a slow-onset communicating hydrocephalus that is initially asymptomatic. Increased ventricular size eventually leads to gait, memory, and locomotor impairment closely resembling the course of human adult chronic hydrocephalus. ABBREVIATION: NPH, normal pressure hydrocephalus.

Anti-NMDA receptor autoantibodies and associated neurobehavioral pathology in mice are dependent on age of first exposure to Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is a pathogen implicated in psychiatric disorders. As elevated antibodies to T. gondii are also present in non-symptomatic individuals, we hypothesized that the age during first exposure to the pathogen may affect symptom manifestation. We tested this hypothesis by evaluating neurobehavioral abnormalities and the immune response in mice following adolescent or adult T. gondii infection. METHODS: Mice were infected with T. gondii at postnatal day 33 (adolescent/juvenile) or 61 (adult). At 8weeks post-infection (wpi), pre-pulse inhibition of the acoustic startle (PPI) in mice administered MK-801 (0.1 and 0.3mg/kg) and amphetamine (5 and 10mg/kg) was assessed. Peripheral (anti-T. gondii, C1q-associated IgG and anti-GLUN2 antibodies) and central (C1q and Iba1) markers of the immune response were also evaluated. In addition, regional brain expression of N-methyl-d-aspartate receptor (NMDAR) subunits (GLUN1 and GLUN2A), glutamatergic (vGLUT1, PSD95) and GABAergic (GAD67) markers, and monoamines (DA, NE, 5-HT) and their metabolites were measured. RESULTS: Juvenile and adult infected mice exhibited opposite effects of MK-801 on PPI, with decreased PPI in juveniles and increased PPI in adults. There was a significantly greater elevation of GLUN2 autoantibodies in juvenile-compared to adult-infected mice. In addition, age-dependent differences were found in regional expression of NMDAR subunits and markers of glutamatergic, GABAergic, and monoaminergic systems. Activated microglia and C1q elevations were found in both juvenile- and adult-T. gondii infected mice. CONCLUSIONS: Our study demonstrates that the age at first exposure to T. gondii is an important factor in shaping distinct behavioral and neurobiological abnormalities. Elevation in GLUN2 autoantibodies or complement protein C1q may be a potential underlying mechanism. A better understanding of these age-related differences may lead to more efficient treatments of behavioral disorders associated with T. gondii infection.

Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin.

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT: We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function.