High-fat diet, microbiome-gut-brain axis signaling, and anxiety-like behavior in male rats.

Obesity, associated with the intake of a high-fat diet (HFD), and anxiety are common among those living in modern urban societies. Recent studies suggest a role of microbiome-gut-brain axis signaling, including a role for brain serotonergic systems in the relationship between HFD and anxiety. Evidence suggests the gut microbiome and the serotonergic brain system together may play an important role in this response. Here we conducted a nine-week HFD protocol in male rats, followed by an analysis of the gut microbiome diversity and community composition, brainstem serotonergic gene expression (tph2, htr1a, and slc6a4), and anxiety-related defensive behavioral responses. We show that HFD intake decreased alpha diversity and altered the community composition of the gut microbiome in association with obesity, increased brainstem tph2, htr1a and slc6a4 mRNA expression, including in the caudal part of the dorsomedial dorsal raphe nucleus (cDRD), a subregion previously associated with stress- and anxiety-related behavioral responses, and, finally, increased anxiety-related defensive behavioral responses. The HFD increased the Firmicutes/Bacteroidetes ratio relative to control diet, as well as higher relative abundances of Blautia, and decreases in Prevotella. We found that tph2, htr1a and slc6a4 mRNA expression were increased in subregions of the dorsal raphe nucleus in the HFD, relative to control diet. Specific bacterial taxa were associated with increased serotonergic gene expression in the cDRD. Thus, we propose that HFD-induced obesity is associated with altered microbiome-gut-serotonergic brain axis signaling, leading to increased anxiety-related defensive behavioral responses in rats.

Image-Free Robotic-Assisted Total Knee Arthroplasty Results in Quicker Recovery but Equivalent One-Year Outcomes Compared to Conventional Total Knee Arthroplasty.

BACKGROUND: Few studies have addressed whether robotic-assisted total knee arthroplasty (RA-TKA) significantly impacts functional outcomes. This study was conducted to determine whether image-free RA-TKA improves function compared to conventional total knee arthroplasty (C-TKA), performed without the utilization of robotics or navigation, using the Minimal Clinically Important Difference (MCID) and Patient Acceptable Symptom State (PASS) as measures of meaningful clinical improvement. METHODS: A multicenter propensity score-matched retrospective study was conducted of RA-TKA using an image-free robotic system and C-TKA cases at an average follow-up of 14 months (range, 12 months to 20 months). Consecutive patients who underwent primary unilateral TKA and had a preoperative and postoperative Knee Injury and Osteoarthritis Outcome Score-Joint Replacement (KOOS-JR) were included. The primary outcomes were the MCID and PASS for KOOS-JR. 254 RA-TKA and 762 C-TKA patients were included, with no significant differences in sex, age, body mass index, or comorbidities. RESULTS: Preoperative KOOS-JR scores were similar in the RA-TKA and C-TKA cohorts. Significantly greater improvement in KOOS-JR scores were achieved at 4 to 6 weeks postoperatively with RA-TKA compared to C-TKA. While the mean 1-year postoperative KOOS-JR was significantly higher in the RA-TKA cohort, no significant differences were found in the Delta KOOS-JR scores between the cohorts, when comparing preoperative and 1-year postoperative. No significant differences existed in the rates of MCID or PASS being achieved. CONCLUSION: Image-free RA-TKA reduces pain and improves early functional recovery compared to C-TKA at 4 to 6 weeks, but functional outcomes at 1 year are equivalent based on the MCID and PASS for KOOS-JR.

A Comparative Study of Thumb Metacarpal Morphology and Shape Categorization.

PURPOSE: To describe the shape of a biplanar thumb metacarpal (MC) head and identify how it differs morphologically from previously described flat and round MC heads. METHODS: Lateral radiographs of the thumb were collected retrospectively from our patient database. Patients were included in the study if they had an appropriate lateral radiograph, met the age criteria (range; 18-75 years), and did not have severe metacarpophalangeal (MCP) joint arthritis. Metacarpal heads were categorized as flat or round by dividing the distance measured from the volar to the dorsal edge of the articular surface by the radius of curvature of the articular surface. A ratio of 1.7 or greater indicated a round MC head, whereas a ratio of less than 1.7 indicated a flat MC head. RESULTS: Among the 210 study participants, 110 were female and the average age was 47.3 years. During the measurement and classification process, it was determined by a board-certified hand surgeon that a subset of MCs did not meet the criteria for being categorized as either flat or round because of the inability to appropriately measure the radius of curvature of the MCP joint. Of the participants 113, 79, and 18, were classified as having either round, flat, or biplanar MC head shapes, respectively. CONCLUSIONS: We have identified a third, biplanar MC head shape. The biplanar head shape is more triangular and has two distinct planes on the articular surface that converge into an apex. CLINICAL RELEVANCE: The shape of the MC head has been shown to influence the range of motion of the MCP joint which may have an influence on the types of injuries that occur at the MCP joint. Further studies are required to understand how shape classification of MC heads may be useful and relevant to range of motion and risk of injury.

Ultrasound-guided bilateral erector spinae plane nerve blocks: a novel application for the management of acute postoperative pain in awake spine surgery. Illustrative case.

BACKGROUND: Postoperative pain management is a limiting factor for early ambulation and discharge following spine fusion surgery. Awake spinal surgery, when combined with minimally invasive transforaminal lumbar interbody fusion, is associated with enhanced recovery in well-selected patients. Some neurosurgeons have recently aimed to further improve outcomes by utilizing erector spinae plane block catheters, allowing for a continuous infusion of local anesthetic to improve the management of acute postoperative pain following minimally invasive transforaminal lumbar interbody fusion. OBSERVATIONS: A patient who underwent a minimally invasive transforaminal lumbar interbody fusion with perioperatively placed erector spinae plane catheters at the T12 level ambulated 30 minutes after surgery and was discharged the same day (length of stay, 4.6 hours). The total amount of narcotics administered during the hospital stay was 127.5 morphine milligram equivalents. LESSONS: The placement of bilateral erector spine plane nerve block catheters at the T12 level with an ambulatory infusion pump may help to improve acute postoperative pain management for patients undergoing lumbar spinal fusion.

Effects of paternal high-fat diet and maternal rearing environment on the gut microbiota and behavior.

Exposing a male rat to an obesogenic high-fat diet (HFD) influences attractiveness to potential female mates, the subsequent interaction of female mates with infant offspring, and the development of stress-related behavioral and neural responses in offspring. To examine the stomach and fecal microbiome's potential roles, fecal samples from 44 offspring and stomach samples from offspring and their fathers were collected and bacterial community composition was studied by 16 small subunit ribosomal RNA (16S rRNA) gene sequencing. Paternal diet (control, high-fat), maternal housing conditions (standard or semi-naturalistic housing), and maternal care (quality of nursing and other maternal behaviors) affected the within-subjects alpha-diversity of the offspring stomach and fecal microbiomes. We provide evidence from beta-diversity analyses that paternal diet and maternal behavior induced community-wide shifts to the adult offspring gut microbiome. Additionally, we show that paternal HFD significantly altered the adult offspring Firmicutes to Bacteroidetes ratio, an indicator of obesogenic potential in the gut microbiome. Additional machine-learning analyses indicated that microbial species driving these differences converged on Bifidobacterium pseudolongum. These results suggest that differences in early-life care induced by paternal diet and maternal care significantly influence the microbiota composition of offspring through the microbiota-gut-brain axis, having implications for adult stress reactivity.

Characterization of gut microbiome and metabolome in Helicobacter pylori patients in an underprivileged community in the United States.

BACKGROUND: Helicobacter pylori (H. pylori), a bacterium that infects approximately half of the world's population, is associated with various gastrointestinal diseases, including peptic ulcers, non-ulcer dyspepsia, gastric adenocarcinoma, and gastric lymphoma. As the burden of antibiotic resistance increases, the need for new adjunct therapies designed to facilitate H. pylori eradication and reduce negative distal outcomes associated with infection has become more pressing. Characterization of the interactions between H. pylori, the fecal microbiome, and fecal fatty acid metabolism, as well as the mechanisms underlying these interactions, may offer new therapeutic approaches. AIM: To characterize the gut microbiome and metabolome in H. pylori patients in a socioeconomically challenged and underprivileged inner-city community. METHODS: Stool samples from 19 H. pylori patients and 16 control subjects were analyzed. 16S rRNA gene sequencing was performed on normalized pooled amplicons using the Illumina MiSeq System using a MiSeq reagent kit v2. Alpha and beta diversity analyses were performed in QIIME 2. Non-targeted fatty acid analysis of the samples was carried out using gas chromatography-mass spectrometry, which measures the total content of 30 fatty acids in stool after conversion into their corresponding fatty acid methyl esters. Multi-dimensional scaling (MDS) was performed on Bray-Curtis distance matrices created from both the metabolomics and microbiome datasets and a Procrustes test was performed on the metabolomics and microbiome MDS coordinates. RESULTS: Fecal microbiome analysis showed that alpha diversity was lowest in H. pylori patients over 40 years of age compared to control subjects of similar age group. Beta diversity analysis of the samples revealed significant differences in microbial community structure between H. pylori patients and control subjects across all ages. Thirty-eight and six taxa had lower and higher relative abundance in H. pylori patients, respectively. Taxa that were enriched in H. pylori patients included Atopobium, Gemellaceae, Micrococcaceae, Gemellales and Rothia (R. mucilaginosa). Notably, relative abundance of the phylum Verrucomicrobia was decreased in H. pylori patients compared to control subjects. Procrustes analysis showed a significant relationship between the microbiome and metabolome datasets. Stool samples from H. pylori patients showed increases in several fatty acids including the polyunsaturated fatty acids (PUFAs) 22:4n6, 22:5n3, 20:3n6 and 22:2n6, while decreases were noted in other fatty acids including the PUFA 18:3n6. The pattern of changes in fatty acid concentration correlated to the Bacteroidetes:Firmicutes ratio determined by 16S rRNA gene analysis. CONCLUSION: This exploratory study demonstrates H. pylori-associated changes to the fecal microbiome and fecal fatty acid metabolism. Such changes may have implications for improving eradication rates and minimizing associated negative distal outcomes.

Evaluation of the gut microbiome in association with biological signatures of inflammation in murine polytrauma and shock.

Severe injuries are frequently accompanied by hemorrhagic shock and harbor an increased risk for complications. Local or systemic inflammation after trauma/hemorrhage may lead to a leaky intestinal epithelial barrier and subsequent translocation of gut microbiota, potentially worsening outcomes. To evaluate the extent with which trauma affects the gut microbiota composition, we performed a post hoc analysis of a murine model of polytrauma and hemorrhage. Four hours after injury, organs and plasma samples were collected, and the diversity and composition of the cecal microbiome were evaluated using 16S rRNA gene sequencing. Although cecal microbial alpha diversity and microbial community composition were not found to be different between experimental groups, norepinephrine support in shock animals resulted in increased alpha diversity, as indicated by higher numbers of distinct microbial features. We observed that the concentrations of proinflammatory mediators in plasma and intestinal tissue were associated with measures of microbial alpha and beta diversity and the presence of specific microbial drivers of inflammation, suggesting that the composition of the gut microbiome at the time of trauma, or shortly after trauma exposure, may play an important role in determining physiological outcomes. In conclusion, we found associations between measures of gut microbial alpha and beta diversity and the severity of systemic and local gut inflammation. Furthermore, our data suggest that four hours following injury is too early for development of global changes in the alpha diversity or community composition of the intestinal microbiome. Future investigations with increased temporal-spatial resolution are needed in order to fully elucidate the effects of trauma and shock on the gut microbiome, biological signatures of inflammation, and proximal and distal outcomes.

Effects of immunization with heat-killed Mycobacterium vaccae on autism spectrum disorder-like behavior and epileptogenesis in a rat model of comorbid autism and epilepsy.

Autism spectrum disorders (ASDs) and epilepsy are often comorbid. The basis for this co-occurrence remains unknown; however, inflammatory stressors during development are a shared risk factor. To explore this association, we tested the effect of repeated immunizations using a heat-killed preparation of the stress-protective immunoregulatory microbe Mycobacterium vaccae NCTC 11,659 (M. vaccae) on the behavioral and epileptogenic consequences of the combined stress-terbutaline (ST) rat model of ASD-like behavior/epilepsy. Repeated immunization of the dam with M. vaccae during pregnancy, followed by immunization of the pups after terbutaline injections, prevented the expression of ASD-like behavior but did not appear to protect against, and may have even enhanced, the spontaneous epileptogenic effects of ST. Maternal M. vaccae injections transferred an anti-inflammatory immunophenotype to offspring, and repeated injections across development prevented ST-induced increases in microglial density at early developmental time points in a region-specific manner. Despite epidemiological comorbidity between ASD/epileptic conditions and shared environmental risk factors, our results suggest that the expression of ASD-like behaviors, but perhaps not epileptogenesis, is sensitive to early anti-inflammatory intervention. These data provide support for the exploration of immunoregulatory strategies to prevent the negative neurodevelopmental behavioral effects of stressors during early critical periods.

Association of the Salivary Microbiome With Animal Contact During Early Life and Stress-Induced Immune Activation in Healthy Participants.

The prevalence of stress-associated somatic and psychiatric disorders is increased in environments offering a narrow relative to a wide range of microbial exposure. Moreover, different animal and human studies suggest that an overreactive immune system not only accompanies stress-associated disorders, but might even be causally involved in their pathogenesis. In support of this hypothesis, we recently showed that urban upbringing in the absence of daily contact with pets, compared to rural upbringing in the presence of daily contact with farm animals, is associated with a more pronounced immune activation following acute psychosocial stressor exposure induced by the Trier Social Stress Test (TSST). Here we employed 16S rRNA gene sequencing to test whether this difference in TSST-induced immune activation between urban upbringing in the absence of daily contact with pets (n = 20) compared with rural upbringing in the presence of daily contact with farm animals (n = 20) is associated with differences in the composition of the salivary microbiome. Although we did not detect any differences in alpha or beta diversity measures of the salivary microbiome between the two experimental groups, statistical analysis revealed that the salivary microbial beta diversity was significantly higher in participants with absolutely no animal contact (n = 5, urban participants) until the age of 15 compared to all other participants (n = 35) reporting either daily contact with farm animals (n = 20, rural participants) or occasional pet contact (n = 15, urban participants). Interestingly, when comparing these urban participants with absolutely no pet contact to the remaining urban participants with occasional pet contact, the former also displayed a significantly higher immune, but not hypothalamic-pituitary-adrenal (HPA) axis or sympathetic nervous system (SNS) activation, following TSST exposure. In summary, we conclude that only urban upbringing with absolutely no animal contact had long-lasting effects on the composition of the salivary microbiome and potentiates the negative consequences of urban upbringing on stress-induced immune activation.

Effects of Immunization With the Soil-Derived Bacterium Mycobacterium vaccae on Stress Coping Behaviors and Cognitive Performance in a "Two Hit" Stressor Model.

Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a "two hit" stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day -28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days -21, -14, -7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0-56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days -1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.

Interactions between whole-body heating and citalopram on body temperature, antidepressant-like behaviour, and neurochemistry in adolescent male rats.

Evidence suggests that affective disorders are associated with altered thermoregulation, and it has been hypothesized that therapeutic strategies targeting body-to-brain thermosensory systems may be effective for treating depression. Consistent with this hypothesis, a recent randomized, double blind, placebo-controlled clinical trial has suggested that infrared whole-body hyperthermia has therapeutic potential for the treatment of depression. Preclinical models may help uncover the mechanism(s) underlying the antidepressant-like effects of whole-body heating. We have previously shown that exposure to whole-body heating potentiates antidepressant-like behavioural responses following administration of a behaviourally subthreshold dose of the selective serotonin reuptake inhibitor citalopram, but the neurochemical and behavioural interactions between whole body heating and behaviourally effective doses of citalopram are not known. In these experiments, we examined the effects of whole-body heating, either with or without treatment of a suprathreshold dose of citalopram (20 mg/kg, s.c.), on body temperature, antidepressant-like behavioural responses in the forced swim test, and tissue concentrations of serotonin and its metabolite, 5-hydoxyindoleacetic acid (5-HIAA), in the prefrontal cortex of adolescent male Wistar rats. Although whole-body heating did not potentiate the behavioural effects of suprathreshold citalopram, citalopram was observed to increase body temperature and potentiate the effects of whole-body heating on body temperature. Whole-body heating, by itself, decreased serotonin concentrations in the infralimbic cortex to a level similar to that observed following treatment with citalopram, suggesting that these treatments have convergent effects on a mesolimbocortical system innervating the medial prefrontal cortex, an effect that was correlated with effects of treatment on body temperature.

Military-Related Exposures, Social Determinants of Health, and Dysbiosis: The United States-Veteran Microbiome Project (US-VMP).

Significant effort has been put forth to increase understanding regarding the role of the human microbiome in health- and disease-related processes. In turn, the United States (US) Veteran Microbiome Project (US-VMP) was conceptualized as a means by which to serially collect microbiome and health-related data from those seeking care within the Veterans Health Administration (VHA). In this manuscript, exposures related to military experiences, as well as conditions and health-related factors among patients seen in VHA clinical settings are discussed in relation to common psychological and physical outcomes. Upon enrollment in the study, Veterans complete psychometrically sound (i.e., reliable and valid) measures regarding their past and current medical history. Participants also provide skin, oral, and gut microbiome samples, and permission to track their health status via the VHA electronic medical record. To date, data collection efforts have been cross-diagnostic. Within this manuscript, we describe current data collection practices and procedures, as well as highlight demographic, military, and psychiatric characteristics of the first 188 Veterans enrolled in the study. Based on these findings, we assert that this cohort is unique as compared to those enrolled in recent large-scale studies of the microbiome. To increase understanding regarding disease and health among diverse cohorts, efforts such as the US-VMP are vital. Ongoing barriers and facilitators to data collection are discussed, as well as future research directions, with an emphasis on the importance of shifting current thinking regarding the microbiome from a focus on normalcy and dysbiosis to health promotion and disease prevention.

Mycobacterium vaccae immunization protects aged rats from surgery-elicited neuroinflammation and cognitive dysfunction.

Aging is a major risk factor for developing postoperative cognitive dysfunction. Neuroinflammatory processes, which can play a causal role in the etiology of postoperative cognitive dysfunction, are potentiated or primed as a function of aging. Here we explored whether exposure to a microorganism with immunoregulatory and anti-inflammatory properties, Mycobacterium vaccae NCTC 11659 (M. vaccae), could ameliorate age-associated neuroinflammatory priming. Aged (24 months) and adult (3 months) male F344XBN rats were immunized with heat-killed M. vaccae (3 injections, once per week) before undergoing a laparotomy or anesthesia control procedure. Aged, but not young rats, showed postoperative learning/memory deficits in a fear-conditioning paradigm. Importantly, M. vaccae immunization protected aged rats from these surgery-induced cognitive impairments. M. vaccae immunization also shifted the aged proinflammatory hippocampal microenvironment toward an anti-inflammatory phenotype. Furthermore, M. vaccae immunization reduced age-related hyperinflammatory responses in isolated hippocampal microglia. Overall, our novel data suggest that M. vaccae can induce an anti-inflammatory milieu in the aged brain and thus mitigate the neuroinflammatory and cognitive impairments induced by surgery.

Two models of inescapable stress increase tph2 mRNA expression in the anxiety-related dorsomedial part of the dorsal raphe nucleus.

Expression of TPH2, the rate-limiting enzyme for brain serotonin synthesis, is elevated in the dorsal raphe nucleus (DR) of depressed suicide victims. One hypothesis is that this increase in TPH2 expression is stress-induced. Here, we used an established animal model to address whether exposure to an acute stressor, inescapable tail shock (IS), increases tph2 mRNA and Tph2 protein expression, and if IS sensitizes the DR to a subsequent, heterotypic stressor. In Experiment 1, we measured tph2 mRNA expression 4 h after IS or home cage (HC) control conditions in male rats, using in situ hybridization histochemistry. In Experiment 2, we measured Tph2 protein expression 12 h or 24 h after IS using western blot. In Experiment 3, we measured tph2 mRNA expression following IS on Day 1, and cold swim stress (10 min, 15 °C) on Day 2. Inescapable tail shock was sufficient to increase tph2 mRNA expression 4 h and 28 h later, selectively in the dorsomedial DR (caudal aspect of the dorsal DR, cDRD; an area just rostral to the caudal DR, DRC) and increased Tph2 protein expression in the DRD (rostral and caudal aspects of the dorsal DR combined) 24 h later. Cold swim increased tph2 mRNA expression in the dorsomedial DR (cDRD) 4 h later. These effects were associated with increased immobility during cold swim, elevated plasma corticosterone, and a proinflammatory plasma cytokine milieu (increased interleukin (IL)-6, decreased IL-10). Our data demonstrate that two models of inescapable stress, IS and cold swim, increase tph2 mRNA expression selectively in the anxiety-related dorsomedial DR (cDRD).

Acute Administration of the Nonpathogenic, Saprophytic Bacterium, Mycobacterium vaccae, Induces Activation of Serotonergic Neurons in the Dorsal Raphe Nucleus and Antidepressant-Like Behavior in Association with Mild Hypothermia.

Peripheral immune activation can have profound physiologic and behavioral effects. One mechanism through which immune activation may affect physiology and behavior is through actions on brainstem neuromodulatory systems, such as serotonergic systems. To test this hypothesis, in Experiment 1, adult male BALB/c mice were implanted with telemetric recording devices and then immunized with Mycobacterium vaccae NCTC 11659 (0.1 mg, s.c.; Days - 28, - 14; N = 36). On Day 1, mice received an acute challenge with M. vaccae (0.1 mg, s.c.) or borate-buffered saline vehicle. Core body temperature and locomotor activity recordings were conducted during a 36 h period beginning 24 h prior to challenge; 12 h following acute challenge, mice were either tested in a 6-min forced swim test, or served as home cage controls (n = 9 per group). In Experiment 2, the protocol was repeated, but with the aim of assessing c-Fos expression in brainstem serotonergic neurons, assessed 90 min following exposure to forced swim (N = 32; n = 8 per group). In Experiment 1, acute M. vaccae challenge in M. vaccae-immunized mice, relative to vehicle-challenged controls, decreased locomotor activity and core body temperature measured 3 h following challenge, as measured by continuous telemetric recordings, and decreased immobility in the forced swim test measured 12 h following challenge. In Experiment 2, acute M. vaccae challenge in M. vaccae-immunized mice decreased home cage locomotion, in alignment with findings in Experiment 1, as measured by video-based behavioral analysis, and, among mice exposed to the forced swim test, increased c-Fos expression in subsets of serotonergic neurons within the dorsal raphe nucleus (DR) measured 13.5 h following challenge. Together, these data are consistent with the hypothesis that acute peripheral immune activation with a heat-killed preparation of M. vaccae transiently induces mild hypothermia in association with suppression of locomotor activity, activates subsets of serotonergic neurons in the DR, and induces antidepressant-like behavioral responses.