Patrick Burns

Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation

Objective: Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood. Methods: Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation. Results: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia. Discussion: We identified a unique HMOX1down and FBPup phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

Evaluation of oscillometric and Doppler ultrasonic devices for blood pressure measurements in anesthetized and conscious dogs.

Two non-invasive blood pressure (NIBP) devices (oscillometry and Doppler) were compared to invasive blood pressure using a Bland-Altman analysis, in anesthetized and conscious dogs. When considering the systolic arterial pressure only during general anesthesia, both NIBP devices slightly underestimated the systolic arterial blood pressure however the precision and the limits of agreement for the Doppler were of a greater magnitude. This indicates a worse clinical performance by the Doppler. The performance of both NIBP devices deteriorated as measured in conscious animals. In general, for the oscillometric device, determination of invasive diastolic and mean arterial pressures was better than the invasive systolic arterial pressure. Overall, the oscillometric device satisfied more of the criteria set by the American College of Veterinary Internal Medicine consensus statement. Based upon these results, the oscillometric device is more reliable than the Doppler in the determination of blood pressure in healthy medium to large breed dogs.

Intraperitoneal bilirubin administration decreases infarct area in a rat coronary ischemia/reperfusion model

Bilirubin was previously considered a toxin byproduct of heme catabolism. However, a mounting body of evidence suggests that at physiological doses, bilirubin is a powerful antioxidant and anti-atherosclerotic agent. Recent clinical studies have shown that human beings with genetically-induced hyperbilirubinemia (Gilbert Syndrome) are protected against coronary heart disease. The purpose of this study was to investigate whether administration of exogenous bilirubin to normal rats would convey similar protective effects in an experimental model of coronary ischemia. We hypothesized that intraperitoneal bilirubin administration 1 h before injury would decrease infarct area and preserve left ventricular (LV) systolic function when compared to non-treated rats. Coronary ischemia was induced by temporary (30 min) ligation of the left anterior descending coronary artery in control or bilirubin treated rats, followed by a 1-h period of reperfusion. LV function was estimated by measurements of fractional shortening (FS) and fractional area shortening using echocardiography. LV function decreased in both experimental groups after ischemia and reperfusion, although in bilirubin-treated rats FS was less depressed during the period of ischemia (18.8 vs. 25.8%, p = 0.034). Infarct size was significantly reduced in the bilirubin treated group compared to the non-treated group (13.34 vs. 25.5%, p = 0.0067). Based on the results of this study, bilirubin supplementation appears to provide significant decrease in infarct size although protective effects on LV function were noted only during the period of ischemia. This result also suggests that lipid soluble antioxidant bilirubin prevents the oxidation of cardiolipin and decreases the infarct size in the heart during ischemia.

Does heart rate variability reflect the systemic inflammatory response in a fetal sheep model of lipopolysaccharide-induced sepsis?

Fetal inflammatory response occurs during chorioamnionitis, a frequent and often subclinical inflammation associated with increased risk for brain injury and life-lasting neurologic deficits. No means of early detection exist. We hypothesized that systemic fetal inflammation without septic shock will be reflected in alterations of fetal heart rate (FHR) variability (fHRV) distinguishing baseline versus inflammatory response states. In chronically instrumented near-term fetal sheep (n = 24), we induced an inflammatory response with lipopolysaccharide (LPS) injected intravenously (n = 14). Ten additional fetuses served as controls. We measured fetal plasma inflammatory cytokine IL-6 at baseline, 1, 3, 6, 24 and 48 h. 44 fHRV measures were determined continuously every 5 min using continuous individualized multi-organ variability analysis (CIMVA). CIMVA creates an fHRV measures matrix across five signal-analytical domains, thus describing complementary properties of fHRV. Using principal component analysis (PCA), a widely used technique for dimensionality reduction, we derived and quantitatively compared the CIMVA fHRV PCA signatures of inflammatory response in LPS and control groups. In the LPS group, IL-6 peaked at 3 h. In parallel, PCA-derived fHRV composite measures revealed a significant difference between LPS and control group at different time points. For the LPS group, a sharp increase compared to baseline levels was observed between 3 h and 6 h, and then abating to baseline levels, thus tracking closely the IL-6 inflammatory profile. This pattern was not observed in the control group. We also show that a preselection of fHRV measures prior to the PCA can potentially increase the difference between LPS and control groups, as early as 1 h post LPS injection. We propose a fHRV composite measure that correlates well with levels of inflammation and tracks well its temporal profile. Our results highlight the potential role of HRV to study and monitor the inflammatory response non-invasively over time.

Can Monitoring Fetal Intestinal Inflammation Using Heart Rate Variability Analysis Signal Incipient Necrotizing Enterocolitis of the Neonate

Objective: Necrotizing enterocolitis of the neonate is an acute inflammatory intestinal disease that can cause necrosis and sepsis. Chorioamnionitis is a risk factor of necrotizing enterocolitis. The gut represents the biggest vagus-innervated organ. Vagal activity can be measured via fetal heart rate variability. We hypothesized that fetal heart rate variability can detect fetuses with incipient gut inflammation. Design: Prospective animal study. Setting: University research laboratory. Subjects: Chronically instrumented near-term fetal sheep (n = 21). Measurements and Main Results: Animals were surgically instrumented with vascular catheters and electrocardiogram to allow manipulation and recording from nonanesthetized animals. In 14 fetal sheep, inflammation was induced with lipopolysaccharide (IV) to mimic chorioamnionitis. Fetal arterial blood samples were drawn at selected time points over 54 hours post lipopolysaccharide for blood gas and cytokines (interleukin-6 and tumor necrosis factor-&agr; enzymelinked immunosorbent assay). Fetal heart rateV was quantified throughout the experiment. The time-matched fetal heart rate variability measures were correlated to the levels of interleukin-6 and tumor necrosis factor-&agr;. Upon necropsy, ionized calcium binding adaptor molecule 1+ (Iba1+), CD11c+ (M1), CD206+ (M2 macrophages), and occludin (leakiness marker) immunofluorescence in the terminal ileum was quantified along with regional Iba1+ signal in the brain (microglia). Interleukin-6 peaked at 3 hours post lipopolysaccharide accompanied by mild cardiovascular signs of sepsis. At 54 hours, we identified an increase in Iba1+ and, specifically, M1 macrophages in the ileum accompanied by increased leakiness, with no change in Iba1 signal in the brain. Preceding this change on tissue level, at 24 hours, a subset of nine fetal heart rate variability measures correlated exclusively to the Iba+ markers of ileal, but not brain, inflammation. An additional fetal heart rate variability measure, mean of the differences of R-R intervals, correlated uniquely to M1 ileum macrophages increasing due to lipopolysaccharide. Conclusions: We identified a unique subset of fetal heart rate variability measures reflecting 1.5 days ahead of time the levels of macrophage activation and increased leakiness in terminal ileum. We propose that such subset of fetal heart rate variability measures reflects brain-gut communication via the vagus nerve. Detecting such noninvasively obtainable organ-specific fetal heart rate variability signature of inflammation would alarm neonatologists about neonates at risk of developing necrotizing enterocolitis and sepsis. Clinical validation studies are required.

Physiological, pharmacokinetic and liver metabolism comparisons between 3-, 6-, 12- and 18-month-old male Sprague Dawley rats under ketamine-xylazine anesthesia

The main objective of this study was to compare the physiological changes (withdrawal and corneal reflexes, respiratory and cardiac frequency, blood oxygen saturation, and rectal temperature) following intraperitoneal administration of ketamine (80 mg/kg) and xylazine (10 mg/kg) to 3-, 6-, 12- and 18-month-old male Sprague Dawley rats (n=6/age group). Plasma pharmacokinetics, liver metabolism, and blood biochemistry were examined for a limited number of animals to better explain anesthetic drug effects. Selected organs were collected for histopathology. The results for the withdrawal and corneal reflexes suggest a shorter duration and decreased depth of anesthesia with aging. Significant cardiac and respiratory depression, as well as decreased blood oxygen saturation, occurred in all age groups however, cardiac frequency was the most affected parameter with aging, since the 6-, 12-, and 18-month-old animals did not recuperate to normal values during recovery from anesthesia. Pharmacokinetic parameters (T1/2 and AUC) increased and drug clearance decreased with aging, which strongly suggests that drug exposure is associated with the physiological results. The findings for liver S9 fractions of 18-month-old rats compared with the other age groups suggest that following a normal ketamine anesthetic dose (80 mg/kg), drug metabolism is impaired, leading to a significant increase of drug exposure. In conclusion, age and related factors have a substantial effect on ketamine and xylazine availability, which is reflected by significant changes in pharmacokinetics and liver metabolism of these drugs, and this translates into shorter and less effective anesthesia with increasing age.

Clinical effects of a constant rate infusion of remifentanil, alone or in combination with ketamine, in cats anesthetized with isoflurane

OBJECTIVE To evaluate the effects of a constant rate infusion of remifentanil, alone or in combination with ketamine, in healthy cats anesthetized with isoflurane. DESIGN Randomized, controlled, clinical trial. ANIMALS 23 cats undergoing elective ovariohysterectomy. PROCEDURES Cats were premedicated with acepromazine and morphine; anesthesia was induced with propofol and maintained with isoflurane. Cats were given constant rate infusions of remifentanil (20 μg/kg/h [9 μg/lb/h], IV; n = 8), remifentanil and ketamine (0.5 mg/kg [0.23 mg/lb], then 1.8 mg/kg/h [0.82 mg/lb/h], IV; 7), or crystalloid fluids (8). The anesthesiologist was blinded to treatment group, end-tidal isoflurane concentration, and vaporizer setting. Heart rate, systolic arterial blood pressure, respiratory rate, end-tidal partial pressure of CO2, temperature, and end-tidal isoflurane concentration were monitored; recovery scores were assigned. RESULTS There were no significant differences among treatment groups with respect to age, body weight, surgery time, anesthesia time, time to extubation, recovery score, or cardiorespiratory variables. End-tidal isoflurane concentration was significantly reduced in cats given remifentanil and ketamine (mean ± SD, 0.63 ± 0.4%), compared with concentration in cats given crystalloid fluids (1.22 ± 0.5%) but not compared with concentration in cats given remifentanil alone (1.03 ± 0.4%). Compared with cats given crystalloid fluids, mean isoflurane requirement was reduced by 48.3% in cats given remifentanil-ketamine and 15.6% in cats given remifentanil alone. CONCLUSIONS AND CLINICAL RELEVANCE At the dosages administered, a constant rate infusion of remifentanil-ketamine resulted in a significant decrease in the isoflurane requirement in healthy cats undergoing ovariohysterectomy. However, significant differences in cardiovascular variables were not observed among treatment groups.

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

Ex Vivo Biomechanical Comparison of 4 Suture Materials for Laparoscopic Bladder Closure in the Horse.

OBJECTIVE To compare a knotless, barbed suture to standard suture using laparoscopic suturing methods in an ex vivo model of equine bladder repair. STUDY DESIGN Cadaveric study. SAMPLE POPULATION Equine cadaver bladders (n=42). METHODS A 5-cm incision was created and repaired in a laparoscopic training box with 4 different suture materials. Groups 1 and 2 used 2-0 poliglecaprone and 2-0 glycomer knotless, barbed suture, respectively, placed using laparoscopic instruments. Groups 3 and 4 used 0 and 2-0 polyglyconate knotless, barbed suture, respectively, placed using an automated laparoscopic suturing device. All groups used a double-layer inverting pattern. Time for suture placement was recorded. Bladders were inflated with water and bursting strength pressures recorded, including a control group of intact bladders. Statistical analysis using a linear model and taking into account the unequal variances was followed by a post-hoc Tukeys test. Significance was set at P<.05. RESULTS Bursting strength did not vary significantly between treatment groups, but was significantly decreased compared to the control group (P<.001). Time to place the sutures with the 2 automated suture device groups (groups 3 and 4) was significantly faster than those in which the suture was placed using laparoscopic needle holders and forceps (groups 1 and 2; P=.001). CONCLUSION Knotless, barbed suture may be a viable alternative to standard suture material for laparoscopic closure of the urinary bladder in horses. Further cyclic and in vivo testing should be performed before use in clinical cases.

Anesthetic and pathological changes following high doses of ketamine and xylazine in Sprague Dawley rats.

The main objective of this study was to compare the effects of ketamine and xylazine in aging rats when coadministered intraperitoneally at high anesthetic doses. Three groups (n=6 rats/group) consisting of rats at 3, 6 and 12 months of age were used. During anesthesia, animals were monitored for heart rate, respiratory frequency, blood oxygen saturation, and rectal temperature. The corneal and paw withdrawal reflex were also examined during anesthesia. During anesthesia, withdrawal and corneal reflexes were absent for progressively longer durations with increasing age. Significant decreases in cardiac and respiratory frequency and, blood oxygen saturation occurred for the 6- and 12-month-old animals. Respiratory frequency and blood oxygen saturation returned to normal at the end of the anesthesia; however, the significant decrease in cardiac frequency persisted in the 6- and 12-month-old animals. Rectal temperature was decreased significantly only in the 3-month-old animals. Pulmonary edema and effusion occurred in 50% of the 12-month-old animals. In conclusion, if ketamine-xylazine are used for anesthesia, the doses should be optimized for the age of the subjects prior to initiation of the research project.