Sarcina sp. as a presumptive cause of fatal acute gastric dilation and gastric emphysema in rhesus macaques.

A 4-y-old female and 3-y-old male rhesus macaque (Macaca mulatta), both housed in the same facility, died unexpectedly within 2 wk. Postmortem examination revealed severe gastric dilation in both macaques and gastric emphysema in the female macaque. Histologically, bacteria consistent with Sarcina sp. were present in both macaques within the lungs and lumen of the trachea, esophagus, and gastrointestinal (GI) tract without associated inflammation. Additionally, in the female macaque, the bacteria were found in the gastric mucosa and associated with emphysematous spaces in the gastric wall without associated inflammation. PCR and Sanger sequencing of amplicons were subsequently performed on GI contents and non-alimentary tissues from the 2 affected monkeys and on comparative samples from unaffected rhesus monkeys in the same facility and an adjacent primate facility. The cases were compared using the 2-tailed Fisher exact test (p-value at 95% confidence). PCR identified Sarcina in GI contents of both affected and unaffected monkeys (p = 0.6084) and in non-alimentary tissues of affected monkeys only (p = 0.0083). These results suggest that the presence of Sarcina sp. in non-alimentary tissues is associated with gastric distension, gas accumulation, and unexpected death in nonhuman primates.

Phase relationship between micro-electrocorticography and cortical neurons.

OBJECTIVE: Electrocorticography (ECoG) is commonly used to map epileptic foci and to implement brain-computer interfaces. Understanding the spatiotemporal correspondence between potentials recorded from the brain's surface and the firing patterns of neurons within the cortex would inform the interpretation of ECoG signals and the design of (microfabricated) micro-ECoG electrode arrays. Based on the theory that synaptic potentials generated by neurons firing in synchrony superimpose to generate local field potentials (LFPs), we hypothesized that neurons in the cortex would fire at preferential phases of the micro-ECoG signal in a spatially dependent way. APPROACH: We custom fabricated micro-ECoG electrode arrays with a small opening for silicon arrays (NeuroNexus) to be inserted into the cortex. MAIN RESULTS: We found that the spectral coherence between micro-ECoG signals and intracortical LFPs decreased with distance and frequency, but the coherence with spiking units did not simply decrease over distance, likely due to the structure of the cortex. The majority of sorted units spiked during a preferred phase (usually downward) and frequency (usually below 20 Hz) of the micro-ECoG signal. Their preferred frequency decreased with administration of dexmeditomidine, a sedative commonly used for cortical mapping in patients with epilepsy prior to surgical resection. Dexmedetomidine concomitantly shifted the micro-ECoG spectral density towards lower frequencies. Therefore, the phase relationship between micro-ECoG signals and cortical spiking depends on the state of the brain, and spectrum shifts towards lower frequencies in the electrocorticography signal are a signature of increased spike-phase coupling. However, spike-phase coupling is not a static property since visual stimuli were found to modulate the magnitude of phase coupling at gamma frequency ranges (30-80 Hz), providing empirical evidence that neurons transiently phase-lock. SIGNIFICANCE: The phase relationship between intracortical spikes and micro-ECoG signals depends on brain state, site separation, cortical structure, and external stimuli.

Behavioral Characteristics of Adult Zebrafish (Danio rerio) after MS222 Anesthesia for Fin Excision.

The health of laboratory animals is an ethical responsibility of researchers and a critical determinant of experimental outcome. Therefore, all husbandry procedures should be evaluated for their effects on mortality, behavior, and physiology to maximize animal welfare and minimize experimental variability. For adult zebrafish, the excision of a small portion of the caudal fin (that is, 'fin clipping') under MS222 anesthesia is a common procedure to obtain tissue for genotyping. The potential effect of this procedure on behavioral and physiologic assays of feeding, anxiety, and stress has not previously been assessed. Here, we evaluated feeding behavior, anxiety-associated behaviors, and physiologic indicators of stress at multiple time points within 24 h after performing a standard fin-clip procedure under MS222 anesthesia. Within 1 h of the procedure, fin-clipped fish showed a mild increase in anxiety and exhibited reduced feeding; however, these effects were short-lived, and the fish exhibited baseline levels of anxiety and feeding by 6 and 24 h after fin clipping. Together with the zebrafish's ability to regenerate fin tissue and the low mortality associated with fin clipping, our data support the continued practice of this technique under MS222 anesthesia as a routine husbandry procedure that is unlikely to alter experimental outcomes related to feeding, anxiety, or stress.

A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model of Mycobacterium smegmatis Infection.

Doxycycline (doxy) is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow low-frequency dosing and extended efficacy compared with standard (STD) formulations. We developed a novel sulfuric acid-loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least 2 weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ml doxycycline loaded for 24 hours achieved 97.77% capture in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 43.87% in sphingomyelin (sphing). Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using high-performance liquid chromatography. Liver and injection site skin samples were collected at euthanasia (4 weeks postinjection). Minimal histologic tissue reactions occurred after injection of STD (nonliposomal), DPPC, or sphing-doxy. DPPC-doxy had slightly faster in vitro leakage than sphing liposomes, although both were detectable at 264 hours. The mean residence time for DPPC was the highest (111.78 hours), followed by sphing (56.00 hours) and STD (6.86 hours). DPPC and sphing-doxy were detectable at 0.2 µg/ml in serum at 336 hours postadministration. LE-doxy was not toxic to J774A.1 cells in vitro and produced inhibition of viable Mycobacterium smegmatis at 24 and 48 hours. LE-doxy will require further testing in in vivo infection models.

Patterned optogenetic modulation of neurovascular and metabolic signals.

The hemodynamic and metabolic response of the cortex depends spatially and temporally on the activity of multiple cell types. Optogenetics enables specific cell types to be modulated with high temporal precision and is therefore an emerging method for studying neurovascular and neurometabolic coupling. Going beyond temporal investigations, we developed a microprojection system to apply spatial photostimulus patterns in vivo. We monitored vascular and metabolic fluorescence signals after photostimulation in Thy1-channelrhodopsin-2 mice. Cerebral arteries increased in diameter rapidly after photostimulation, while nearby veins showed a slower smaller response. The amplitude of the arterial response was depended on the area of cortex stimulated. The fluorescence signal emitted at 450/100 nm and excited with ultraviolet is indicative of reduced nicotinamide adenine dinucleotide, an endogenous fluorescent enzyme involved in glycolysis and the citric acid cycle. This fluorescence signal decreased quickly and transiently after optogenetic stimulation, suggesting that glucose metabolism is tightly locked to optogenetic stimulation. To verify optogenetic stimulation of the cortex, we used a transparent substrate microelectrode array to map cortical potentials resulting from optogenetic stimulation. Spatial optogenetic stimulation is a new tool for studying neurovascular and neurometabolic coupling.

Optogenetic micro-electrocorticography for modulating and localizing cerebral cortex activity.

OBJECTIVE: Spatial localization of neural activity from within the brain with electrocorticography (ECoG) and electroencephalography remains a challenge in clinical and research settings, and while microfabricated ECoG (micro-ECoG) array technology continues to improve, complementary methods to simultaneously modulate cortical activity while recording are needed. APPROACH: We developed a neural interface utilizing optogenetics, cranial windowing, and micro-ECoG arrays fabricated on a transparent polymer. This approach enabled us to directly modulate neural activity at known locations around micro-ECoG arrays in mice expressing Channelrhodopsin-2. We applied photostimuli varying in time, space and frequency to the cortical surface, and we targeted multiple depths within the cortex using an optical fiber while recording micro-ECoG signals. MAIN RESULTS: Negative potentials of up to 1.5 mV were evoked by photostimuli applied to the entire cortical window, while focally applied photostimuli evoked spatially localized micro-ECoG potentials. Two simultaneously applied focal stimuli could be separated, depending on the distance between them. Photostimuli applied within the cortex with an optical fiber evoked more complex micro-ECoG potentials with multiple positive and negative peaks whose relative amplitudes depended on the depth of the fiber. SIGNIFICANCE: Optogenetic ECoG has potential applications in the study of epilepsy, cortical dynamics, and neuroprostheses.

Pharmacokinetics of ammonium sulfate gradient loaded liposome-encapsulated oxymorphone and hydromorphone in healthy dogs.

OBJECTIVE: To evaluate the pharmacokinetics, in dogs, of liposome-encapsulated oxymorphone and hydromorphone made by the ammonium sulfate gradient loading technique (ASG). ANIMALS: Four healthy purpose-bred Beagles aged 9.5 ± 3.2 months and weighing 13.4 ± 2.3 kg. STUDY DESIGN: Randomized cross-over design. METHODS: Each dog was given either 4.0 mg kg(-1) of ASG-oxymorphone or 8.0 mg kg(-1) of ASG-hydromorphone SC on separate occasions with a 3-month washout period. Blood was collected at baseline and at serial time points up to 1032 hours (43 days) after injection for determination of serum opioid concentrations. Serum opioid concentrations were measured with HPLC-MS and pharmacokinetic parameters were calculated using commercial software and non-compartmental methods. RESULTS: Serum concentrations of oxymorphone remained above the limit of quantification for 21 days, while those for hydromorphone remained above the limit of quantification for 29 days. Cmax for ASG-oxymorphone was 7.5 ng mL(-1) ; Cmax for ASG-hydromorphone was 5.7 ng mL(-1) . CONCLUSIONS AND CLINICAL RELEVANCE: Oxymorphone and hydromorphone, when encapsulated into liposomes using the ammonium sulfate gradient loading technique, result in measureable serum concentrations for between 3 to 4 weeks. This formulation may have promise in the convenient use of opioids for clinical treatment of chronically painful conditions in dogs.

Pharmacokinetics of long-acting nalbuphine decanoate after intramuscular administration to Hispaniolan Amazon parrots (Amazona ventralis).

OBJECTIVE: To evaluate the pharmacokinetics of nalbuphine decanoate after IM administration to Hispaniolan Amazon parrots (Amazona ventralis). ANIMALS: 9 healthy adult Hispaniolan Amazon parrots of unknown sex. PROCEDURES: Nalbuphine decanoate (37.5 mg/kg) was administered IM to all birds. Plasma samples were obtained from blood collected before (time 0) and 0.25, 1, 2, 3, 6, 12, 24, 48, and 96 hours after drug administration. Plasma samples were used for measurement of nalbuphine concentrations via liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were estimated with computer software. RESULTS: Plasma concentrations of nalbuphine increased rapidly after IM administration, with a mean concentration of 46.1 ng/mL at 0.25 hours after administration. Plasma concentrations of nalbuphine remained > 20 ng/mL for at least 24 hours in all birds. The maximum plasma concentration was 109.4 ng/mL at 2.15 hours. The mean terminal half-life was 20.4 hours. CONCLUSIONS AND CLINICAL RELEVANCE: In Hispaniolan Amazon parrots, plasma concentrations of nalbuphine were prolonged after IM administration of nalbuphine decanoate, compared with previously reported results after administration of nalbuphine hydrochloride. Plasma concentrations that could be associated with antinociception were maintained for 24 hours after IM administration of 37.5 mg of nalbuphine decanoate/kg. Safety and analgesic efficacy of nalbuphine treatments in this species require further investigation to determine the potential for clinical use in pain management in psittacine species.

Antinociceptive effects of long-acting nalbuphine decanoate after intramuscular administration to Hispaniolan Amazon parrots (Amazona ventralis).

OBJECTIVE: To evaluate the thermal antinociceptive effects and duration of action of nalbuphine decanoate after IM administration to Hispaniolan Amazon parrots (Amazona ventralis). ANIMALS: 10 healthy adult Hispaniolan Amazon parrots of unknown sex. PROCEDURES: Nalbuphine decanoate (33.7 mg/kg) or saline (0.9% NaCl) solution was administered IM in a randomized complete crossover experimental design (periods 1 and 2). Foot withdrawal threshold to a noxious thermal stimulus was used to evaluate responses. Baseline thermal withdrawal threshold was recorded 1 hour before drug or saline solution administration, and thermal foot withdrawal threshold measurements were repeated 1, 2, 3, 6, 12, 24, 48, and 72 hours after drug administration. RESULTS: Nalbuphine decanoate administered IM at a dose of 33.7 mg/kg significantly increased thermal foot withdrawal threshold, compared with results after administration of saline solution during period 2, and also caused a significant change in withdrawal threshold for up to 12 hours, compared with baseline values. CONCLUSIONS AND CLINICAL RELEVANCE: Nalbuphine decanoate increased the foot withdrawal threshold to a noxious thermal stimulus in Hispaniolan Amazon parrots for up to 12 hours and provided a longer duration of action than has been reported for other nalbuphine formulations. Further studies with other types of nociceptive stimulation, dosages, and dosing intervals as well as clinical trials are needed to fully evaluate the analgesic effects of nalbuphine decanoate in psittacine birds.

Flexible thin film electrode arrays for minimally-invasive neurological monitoring.

We present approaches for using thin film polymeric electrode arrays for use in applications of minimally invasive neurological monitoring. The flexibility and unique surface properties of the thin-film polyimide substrate in combination with a compact device platform make them amenable to a variety of surgical implantation procedures. Using a rapid-prototyping and fabrication technique, arrays of various geometries can be fabricated within a week. In this paper we test two different approaches for deploying electrode arrays through small cranial openings.

Analgesic effects of carprofen and liposome-encapsulated butorphanol tartrate in Hispaniolan parrots (Amazona ventralis) with experimentally induced arthritis.

OBJECTIVE: To evaluate the microcrystalline sodium urate (MSU) method for inducing arthritis in parrots and to compare the analgesic efficacy of long-acting liposome-encapsulated butorphanol (LEBT), carprofen, or a combination of both. ANIMALS: 20 Hispaniolan parrots. PROCEDURES: MSU was injected into a tibiotarsal-tarsometatarsal (intertarsal) joint to induce arthritis (time 0). Four treatments were compared (LEBT [15 mg/kg, SC] administered once at time 0; injections of carprofen [3 mg/kg, IM, q 12 h] starting at time 0; administration of LEBT plus carprofen; and a control treatment of saline [0.9% NaCl] solution). Weight load testing and behavioral scoring were conducted at 0, 2, 6, 26, and 30 hours. RESULTS: Injection of MSU into the intertarsal joint induced arthritis, which resolved within 30 hours. Treatment with LEBT or LEBT plus carprofen resulted in significantly greater weight-bearing load on the limb with induced arthritis, compared with the control treatment. Treatment with carprofen alone caused a slight but nonsignificant improvement in weight-bearing load on the arthritic limb, compared with the control treatment. Behaviors associated with motor activity and weight bearing differed between the control and analgesic treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Butorphanol was an effective treatment for pain associated with arthritis, but carprofen administered every 12 hours was insufficient. Injection of MSU to induce arthritis in a single joint was a good method for evaluating tonic pain in parrots, and measurement of the weight-bearing load was accurate for assessment of arthritic pain; however, behavioral changes associated with pain were subtle.

Evaluation of liposome-encapsulated butorphanol tartrate for alleviation of experimentally induced arthritic pain in green-cheeked conures (Pyrrhura molinae).

OBJECTIVE: To evaluate injection of microcrystalline sodium urate (MSU) for inducing articular pain in green-cheeked conures (Pyrrhura molinae) and the analgesic efficacy of liposome-encapsulated butorphanol tartrate (LEBT) by use of weight load data, behavioral scores, and fecal corticosterone concentration. ANIMALS: 8 conures. PROCEDURES: In a crossover study, conures were randomly assigned to receive LEBT (15 mg/kg) or liposomal vehicle subsequent to experimental induction of arthritis or sham injection. The MSU was injected into 1 tibiotarsal-tarsometatarsal (intertarsal) joint to induce arthritis (time 0). weight-bearing load and behavioral scores were determined at 0, 2, 6, 26, and 30 hours. RESULTS: MSU injection into 1 intertarsal joint caused a temporary decrease in weight bearing on the affected limb. Treatment of arthritic conures with LEBT resulted in significantly more weight bearing on the arthritic limb than treatment with vehicle. Administration of vehicle to arthritic conures caused a decrease in activity and feeding behaviors during the induction phase of arthritis, but as the arthritis resolved, there was a significant increase in voluntary activity at 30 hours and feeding behaviors at 26 and 30 hours, compared with results for LEBT treatment of arthritic birds. Treatment with LEBT or vehicle in conures without arthritis resulted in similar measurements for weight bearing and voluntary and motivated behaviors. CONCLUSIONS AND CLINICAL RELEVANCE: Experimental induction of arthritis in conures was a good method for evaluating tonic pain. Weight-bearing load was the most sensitive measure of pain associated with induced arthritis. Pain associated with MSU-induced arthritis was alleviated by administration of LEBT.

Pharmacokinetics of hydromorphone hydrochloride in healthy dogs.

OBJECTIVE: To assess the pharmacokinetics of hydromorphone administered intravenously (IV) or subcutaneously (SC) to dogs. STUDY DESIGN: Randomized experimental trial. ANIMALS: Seven healthy male neutered Beagles aged 12.13 +/- 1.2 months and weighing 11.72 +/- 1.10 kg. METHODS: The study was a randomized Latin square block design. Dogs were randomly assigned to receive hydromorphone hydrochloride 0.1 mg kg(-1) or 0.5 mg kg(-1) IV (n = 4 dogs) or 0.1 mg kg(-1) (n = 6) or 0.5 mg kg(-1) (n = 5) SC on separate occasions with a minimum 14-day washout between experiments. Blood was sampled via a vascular access port at serial intervals after drug administration. Serum was analyzed by mass spectrometry. Pharmacokinetic parameters were determined with computer software. RESULTS: Serum concentrations of hydromorphone decreased quickly after both routes of administration of either dose. The serum half-life, clearance, and volume of distribution after IV hydromorphone at 0.1 mg kg(-1) were 0.57 hours (geometric mean), 106.28 mL minute(-1) kg(-1), and 5.35 L kg(-1), and at 0.5 mg kg(-1) were 1.00 hour, 60.30 mL minute(-1) kg(-1), and 5.23 L kg(-1), respectively. The serum half-life after SC hydromorphone at 0.1 mg kg(-1) and 0.5 mg kg(-1) was 0.66 hours and 1.11 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Hydromorphone has a short half-life, suggesting that frequent dosing intervals are needed. Based on pharmacokinetic parameters calculated in this study, 0.1 mg kg(-1) IV or SC q 2 hours or a constant rate infusion of hydromorphone at 0.03 mg kg(-1) hour(-1) are suggested for future studies to assess the analgesic effect of hydromorphone.

A single dose of liposome-encapsulated hydromorphone provides extended analgesia in a rat model of neuropathic pain.

Opioids have been shown to relieve thermal hyperalgesia associated with neuropathic pain. We used a novel technique to produce liposome-encapsulated hydromorphone (LEH), which we then tested in a chronic constriction injury (CCI) thermal hyperalgesia model of neuropathic pain. Rats were divided into sham-operated and CCI groups. Treatments consisted of LEH or standard hydromorphone, administered at surgery or 3 d after surgery, when thermal hyperalgesia had developed in the CCI rats. We measured thermal withdrawal latencies on days 0, 3, and 5. CCI rats given liposome-encapsulated vehicle or standard hydromorphone at surgery developed full thermal hyperalgesia. CCI rats given LEH at surgery exhibited no significant change compared with baseline values in thermal withdrawal latency, indicating that this preparation prevented hyperalgesia after a single injection. CCI rats given LEH on day 3 (that is, after they had developed hyperalgesia) showed reversal of hyperalgesia that persisted to day 5, whereas CCI rats given standard hydromorphone on day 3 showed only brief (approximately 90 min) reversal of hyperalgesia. Preemptive injection of LEH prevented hyperalgesia in this model for as long as 5 d. In addition, hyperalgesia was alleviated for at least 2 d after injection of a single dose of LEH. These results suggest that liposome-encapsulation of hydromorphone offers a convenient and effective means to provide relief from neuropathic pain in this rodent model.