BackgroundNeuropeptide Y (NPY) Y2 receptor (Y2) antagonist BIIE0246 can both inhibit and facilitate nociception. The authors hypothesized that Y2 function depends on inflammation or nerve injury status. MethodsThe authors implemented a battery of behavioral tests in mice of both sexes that received (1) no injury; (2) an incision model of postoperative pain; (3) a spared nerve injury model of neuropathic pain; and (4) a latent sensitization model of chronic postsurgical pain. In addition to Y2 gene expression assays, spinal Y2 G-protein coupling was studied with guanosine-5 '-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding assays. ResultsThe authors report that intrathecal BIIE0246 increased mechanical and cold hypersensitivity, produced behavioral signs of spontaneous nociception and itch, and produced conditioned place aversion and preference in normal, uninjured mice. BIIE0246 did not change heat hypersensitivity or motor coordination. Conditional (sensory neuron-specific) Y2 deletion prevented BIIE0246-induced mechanical and cold hypersensitivity, nocifensive behaviors, and aversion. Both conditional deletion and pharmacologic blockade of Y2 reduced mechanical and thermal hypersensitivity after incision or nerve injury. SNI did not change the sensitivity of Y2 G-protein coupling with the Y2 agonist peptide YY (3-36) (PYY3-36), but increased the population of Y2 that effectively coupled G-proteins. Intrathecal PYY3-36 failed to reduce spared nerve injury- or incision-induced hypersensitivity in C57BL/6N mice. Incision did not change Npy2r gene expression in dorsal root ganglion. ConclusionsThe authors conclude that Y2 at central terminals of primary afferent neurons provides tonic inhibition of mechanical and cold nociception and itch. This switches to the promotion of mechanical and thermal hyperalgesia in models of acute and chronic postsurgical and neuropathic pain, perhaps due to an increase in the population of Y2 that effectively couples to G-proteins. These results support the development of Y2 antagonists for the treatment of chronic postsurgical and neuropathic pain.

Ketamine is a potent sedative and dissociative anesthetic agent that has been used clinically for over 50 years since it was first developed in the 1960 s as an alternative to phencyclidine (PCP). When compared to PCP, ketamine exhibited a much lower incidence of severe side effects, including hallucinations, leading to its increased popularity in clinical practice. Ketamine was initially used as an anesthetic agent, especially in emergency medicine and in surgical procedures where rapid induction and recovery was necessary. However, over the last few decades, ketamine was found to have additional clinically useful properties making it effective in the treatment of a variety of other conditions. Presently, ketamine has a wide range of clinical uses beyond anesthesia including management of acute and chronic pain, as well as treatment of psychiatric disorders such as major depression. In addition to various clinical uses, ketamine is also recognized as a common drug of abuse sought for its hallucinogenic and sedative effects. This review focuses on exploring the different clinical and non-clinical uses of ketamine and its overall impact on patient care.

Alcohol consumption is believed to affect Alzheimer’s disease (AD) risk, but the contributing mechanisms are not well understood. A potential mediator of the proposed alcohol-AD connection is autophagy, a degradation pathway that maintains organelle and protein homeostasis. Autophagy is regulated through the activity of Transcription factor EB (TFEB), which promotes lysosome and autophagy-related gene expression. The purpose of this study is to explore whether chronic alcohol consumption worsens the age-related decline in TFEB-mediated lysosomal biogenesis in the brain and exacerbates cognitive decline associated with aging. To explore the effect of alcohol on brain TFEB and autophagy, we exposed young (3-month-old) and aged (23-month-old) mice to two alcohol-feeding paradigms and assessed biochemical, transcriptome, histology, and behavioral endpoints. In young mice, alcohol decreased hippocampal nuclear TFEB staining but increased SQSTM1/p62, LC3-II, ubiquitinated proteins, and phosphorylated Tau. Hippocampal TFEB activity was lower in aged mice than it was in young mice, and Gao-binge alcohol feeding did not worsen the age-related reduction in TFEB activity. Morris Water and Barnes Maze spatial memory tasks were used to characterize the effects of aging and chronic alcohol exposure (mice fed alcohol for 4 weeks). The aged mice showed worse spatial memory acquisition in both tests. Alcohol feeding slightly impaired spatial memory in the young mice, but had little effect or even slightly improved spatial memory acquisition in the aged mice. In conclusion, aging produces greater reductions in brain autophagy flux and impairment of spatial memory than alcohol consumption.

Clinical reports indicate a bidirectional relationship between mental illness and chronic systemic diseases. However, brain mechanisms linking chronic stress and development of mood disorders to accompanying peripheral organ dysfunction are still not well characterized in animal models. In the current study, we investigated whether activation of hippocampal mitogen-activated protein kinase phosphatase-1 (MKP-1), a key factor in depression pathophysiology, also acts as a mediator of systemic effects of stress. First, we demonstrated that treatment with the glucocorticoid receptor (GR) agonist dexamethasone or acute restraint stress (ARS) significantly increased Mkp-1 mRNA levels within the rat hippocampus. Conversely, administration of the GR antagonist mifepristone 30 min before ARS produced a partial blockade of Mkp-1 upregulation, suggesting that stress activates MKP-1, at least in part, through upstream GR signaling. Chronic corticosterone (CORT) administration evoked comparable increases in hippocampal MKP-1 protein levels and produced a robust increase in behavioral emotionality. In addition to behavioral deficits, chronic CORT treatment also produced systemic pathophysiological effects. Elevated levels of renal inflammation protein markers (NGAL and IL18) were observed suggesting tissue damage and early kidney impairment. In a rescue experiment, the effects of CORT on development of depressive-like behaviors and increased NGAL and IL18 protein levels in the kidney were blocked by CRISPRmediated knockdown of hippocampal Mkp-1 prior to CORT exposure. In sum, these findings further demonstrate that MKP-1 is necessary for development of enhanced behavioral emotionality, while also suggesting a role in stress mechanisms linking brain dysfunction and systemic illness such as kidney disease.

Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the processing of pain sensation at CNS sites, the anti-inflammatory properties of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. Detailed in this unit are methods to elicit and measure carrageenan- and complete Freund's adjuvant (CFA)-induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections into either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) or by the trigeminal ganglion neurons (vibrissal pad).

The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this article has been extensively used to identify potential anti-inflammatory drugs. © 2021 Wiley Periodicals LLC. Basic Protocol: Air pouch model in the rat Alternate Protocol: Air pouch model in the mouse.

The techniques and protocols to modify the mouse genome described in this volume allow researchers to produce genetic models of a remarkable number and breadth of human disease. The generation of gene-modified mice offers profoundly powerful approaches for bringing known or purported human gene disruptions into mouse models, but the degree to which the resultant mutant mouse recapitulates the complex physiological and behavioral features of the human disease state is a key variable in the ultimate usefulness of the mouse model organism. Accordingly, the behavioral characterization of mice with novel targeted gene mutations is an important initial step in determining the potential impact of a novel mouse model. This chapter addresses strategies useful in the initial observations of the animal that assist in directing the choice of secondary tests to assess more detailed aspects of potentially disrupted behaviors that may be relevant to the disease being modeled. An initial standardized, comprehensive screen that assesses general health, reflexes, and sensorimotor functions is the first step in characterizing behavioral phenotype, and results often suggest areas where more complex complementary behavioral assays may reveal more detailed disruption of normal behavior. This sequential, standardized approach reduces variability between subjects; this chapter also addresses approaches to reducing experimental artifacts due to handling, test order, testing facility environment, and other sources. This brief overview of behavioral phenotyping approaches is intended to provide practical information to streamline initial characterization of new mouse models and maximize the usefulness of efforts to use these models to study human health and disease.

Acute pain is a prevalent and debilitating complication following burn injury and has the potential in certain individuals to become chronic pain. There is a paucity of preclinical and clinical research that has addressed this large clinical problem. Our recent work has been focused on developing a burn injury model that will allow for mechanistic investigations for acute and chronic burn injury pain. Our group has previously reported that mice given a high fat/rich carbohydrate diet (54% kcal from fat, 25% kcal from carbohydrate (CHO))develop mechanical allodynia. The present work sought to examine whether this high fat, high CHO diet may affect pain behaviors related to acute burn injury and whether it would lead to prolonged pain behaviors consistent with chronic burn pain. Mice were given either a normal chow or a high fat/high CHO diet and then either were given burn or sham injury on the right hind paw. Pain behaviors were observed over a 28 day period after the burn injury and included mechanical and thermal assessments. Results indicate that both normal chow and high fat diet burn injured mice exhibited significantly reduced mechanical pain thresholds for two weeks after burn injury. After 14 days, both burn injury groups displayed increased mechanical pain thresholds that by 28 days, were surprisingly higher than baseline. Overall, the high fat diet burn group was not found to display greater levels of allodynia compared to the normal chow burn group and there was no evidence that the current model or the high fat diet led to pain behaviors consistent with chronic pain. Preliminary investigations into proportions of mast cell degranulation show that burn injury led to a higher percentage of degranulated mast cells compared to sham injury and that dietary influence may also have a small impact.

Cytosolic NADH­cytochrome­b5­oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues. We have previously reported that global ablation of NCB5OR in mice results in early-onset lean diabetes with decreased serum leptin levels and increased metabolic and feeding activities. The conditional deletion of NCB5OR in the mouse cerebellum and midbrain (conditional knock out, CKO mice) results in local iron dyshomeostasis and altered locomotor activity. It has been established that lesion to or removal of the cerebellum leads to changes in nutrient organization, visceral response, feeding behavior, and body weight. This study assessed whether loss of NCB5OR in the cerebellum and midbrain altered feeding or metabolic activity and had an effect on serum T3, cortisol, prolactin, and leptin levels. Metabolic cage data revealed that 16 week old male CKO mice had elevated respiratory quotients and decreased respiratory water expulsion, decreased voluntary exercise, and altered feeding and drinking behavior compared to wild-type littermate controls. Most notably, male CKO mice displayed higher consumption of food during refeeding after a 48­h fast. Echo MRI revealed normal body composition but decreased total water content and hydration ratios in CKO mice. Increased serum osmolality measurements confirmed the dehydration status of male CKO mice. Serum leptin levels were significantly elevated in male CKO mice while prolactin, T3, and cortisol levels remain unchanged relative to wild-type controls, consistent with elevated transcript levels for leptin receptors (short form) in the male CKO mouse cerebellum. Taken together, these findings suggest altered feeding response post starvation as a result of NCB5OR deficiency in the cerebellum.

Behavioral phenotyping is a crucial step in validating animal models of human disease. Most traditional behavioral analyses rely on investigator observation of animal subjects, which can be confounded by inter-observer variability, scoring consistency, and the ability to observe extremely rapid, small, or repetitive movements. Force-Plate Actimeter (FPA)-based assessments can quantify locomotor activity and detailed motor activity with an incredibly rich data stream that can reveal details of movement unobservable by the naked eye. This report describes four specific examples of FPA analysis of behavior that have been useful in specific rat or mouse models of human neurological disease, which show how FPA analysis can be used to capture and quantify specific features of the complex behavioral phenotypes of these animal models. The first example quantifies nociceptive behavior of the rat following injection of formalin into the footpad as a common model of persistent inflammatory pain. The second uses actimetry to quantify intense, rapid circling behaviors in a transgenic mouse that overexpresses human laminin α5, a basement membrane protein. The third example assesses place preference behaviors in a rat model of migraine headache modeling phonophobia and photophobia. In the fourth example, FPA analysis revealed a unique movement signature emerged with age in a digenic mutant mouse model of Tourette Syndrome. Taken together, these approaches demonstrate the power and usefulness of the FPA in the examination and quantification of minute details of motor behaviors, greatly expanding the scope and detail of behavioral phenotyping of preclinical models of human disease.

Acute and chronic pain is a prevalent and debilitating complication following burn injury. However, burn injury pain remains largely an untreatable condition and current strategies only address ongoing pain control. Little research has addressed this large clinical problem, with only a few studies using an animal model to explore molecular and cellular mechanisms underlying this form of acute and chronic pain. This paucity of information has hampered clinicians in developing effective treatment strategies for patients that suffer burn-related pain. Currently, there are a number of proposed mechanisms that lead to acute and chronic pain in burn injuries. Alterations in inflammation or neurotrophic factor expression are thought to modulate both acute and chronic pain. Our studies have shown that exercise intervention can modify each of these factors and exercise can correct alterations in pro-nociceptive peripheral nervous system adaptations. Together, our multidisciplinary team has developed a murine model of burn injury that leads to robust pain behaviors and pathological changes consistent with acute and chronic burn injury neuropathic pain. Furthermore, our preliminary studies show that mice with burn-injured hind paws voluntarily exercise on running wheels. Future studies will identify mechanisms of how exercise can modulate these acute and chronic neuropathic pain associated with burn injury. Importantly, our approaches are readily translatable to human studies and may be applicable to other forms of chronic pain. These studies will also aid future experiments to investigate predictive features regarding diet, lifestyle, and activity that will be important in treating chronic pain associated with burn injury.

Diet, activity, and inflammation are increasingly recognized as important modulators of nervous system disease, including pain. Sex differences have also been well established in the development of chronic pain. Experimental studies of exercise consistently reveal a positive benefit of exercise on pain. Here, we have assessed a genetic rat model that has selected for traits that promote high exercise capacity or low exercise capacity. Low capacity runners (LCR) have diverged from high capacity runners (HCR) based on rats' inability to exercise, leading to many differences in their metabolic status. This study focused on female rats in order to understand differences related to metabolic status and peripheral nerve function in females. Our analysis of a number of parameters of peripheral nerve function relevant to pain and neuropathy have revealed select differences between LCR and HCR female rats. LCR rats have reduced mechanical sensitivity, higher intraepidermal nerve fiber density and TrkA-positive epidermal axons, increased numbers of Langerhans and mast cells in cutaneous tissues, and a higher fat content despite similar overall body weights between LCR and HCR rats. Other aspects, including sensory and motor nerve conduction velocity, thermal sensitivity, and mRNA expression of selected genes relevant to peripheral sensation were not different. Overall, these results suggest that the differences in metabolic state and inflammation can lead to important changes in sensitivity and peripheral tissues. These baseline differences of the LCR rat that could lead to poor responses to tissue damage and painful stimuli with alterations in diet or disease state. The LCR and HCR rats should prove useful in future studies to assess how the interaction of metabolic status and inflammation impacts pain.

INTRODUCTION:Diet and activity are recognized as modulators of nervous system disease, including pain. Studies of exercise consistently reveal a benefit on pain. This study focused on female rats to understand differences related to metabolic status and peripheral nerve function in females.METHODS:Here, we investigated parameters of peripheral nerve function relevant to pain in rats selectively bred for high (high-capacity runners; HCR) or low endurance exercise capacity (low-capacity runners; LCR) resulting in divergent intrinsic aerobic capacities and susceptibility for metabolic conditions.RESULTS:LCR female rats have reduced mechanical sensitivity, higher intraepidermal nerve fiber density and TrkA-positive epidermal axons, increased numbers of Langerhans and mast cells in cutaneous tissues, and a higher fat content despite similar overall body weights compared to female HCR rats. Sensory and motor nerve conduction velocities, thermal sensitivity, and mRNA expression of selected genes relevant to peripheral sensation were not different.CONCLUSIONS:These results suggest that aerobic capacity and metabolic status influence sensory sensitivity and aspects of inflammation in peripheral tissues that could lead to poor responses to tissue damage and painful stimuli. The LCR and HCR rats should prove useful as models to assess how the metabolic status impacts pain.