An ethogram analysis of cutaneous thermal pain sensitivity and oxycodone reward-related behaviors in rats.

Inter-relationships between pain sensitivity, drug reward, and drug misuse are of considerable interest given that many analgesics exhibit misuse potential. Here we studied rats as they underwent a series of pain- and reward-related tests: cutaneous thermal reflex pain, induction and extinction of conditioned place preference to oxycodone (0.56 mg/kg), and finally the impact of neuropathic pain on reflex pain and reinstatement of conditioned place preference. Oxycodone induced a significant conditioned place preference that extinguished throughout repeated testing. Correlations identified of particular interest included an association between reflex pain and oxycodone-induced behavioral sensitization, and between rates of behavioral sensitization and extinction of conditioned place preference. Multidimensional scaling analysis followed by k-clustering identified three clusters: (1) reflex pain, rate of behavioral sensitization and rate of extinction of conditioned place preference (2) basal locomotion, locomotor habituation, acute oxycodone-stimulated locomotion and rate of change in reflex pain during repeated testing, and (3) magnitude of conditioned place preference. Nerve constriction injury markedly enhanced reflex pain but did not reinstate conditioned place preference. These results suggest that high rates of behavioral sensitization predicts faster rates of extinction of oxycodone seeking/reward, and suggest that cutaneous thermal reflex pain may be predictive of both.

An ethogram analysis of cutaneous thermal pain sensitivity and oxycodone reward-related behaviors in rats.

Inter-relationships between pain sensitivity, drug reward, and drug misuse are of considerable interest given that many analgesics exhibit misuse potential. Here we studied rats as they underwent a series of pain- and reward-related tests: cutaneous thermal reflex pain, induction and extinction of conditioned place preference to oxycodone (0.56 mg/kg), and finally the impact of neuropathic pain on reflex pain and reinstatement of conditioned place preference. Oxycodone induced a significant conditioned place preference that was extinguished throughout repeated testing. Correlations identified of particular interest included an association between reflex pain and oxycodone-induced behavioral sensitization, and between rates of behavioral sensitization and extinction of conditioned place preference. Multidimensional scaling analysis followed by k-clustering identified three clusters: (1) reflex pain and the rate of change in reflex pain response throughout repeated testing, (2) basal locomotion, locomotor habituation, and acute oxycodone-stimulated locomotion, and (3) behavioral sensitization, strength of conditioned place preference, and rate of extinction. Nerve constriction injury markedly enhanced reflex pain but did not reinstate conditioned place preference. These results support the notion that behavioral sensitization relates to the acquisition and extinction of oxycodone seeking/reward, but suggest that generally cutaneous thermal reflex pain poorly predicts oxycodone reward-related behaviors except for behavioral sensitization.

Assessment of Nerve Injury-induced Mechanical Hypersensitivity in Rats using an Orofacial Operant Pain Assay.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube. In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures. Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

Central Administration of Hydrogen Sulfide Donor NaHS Reduces Iba1-Positive Cells in the PVN and Attenuates Rodent Angiotensin II Hypertension.

Hydrogen sulfide (H2S) is a gaseous signaling molecule with neuromodulatory, anti-inflammatory, and anti-hypertensive effects. Here, we investigate whether chronic intracerebroventricular (ICV) infusion of sodium hydrosulfide (NaHS), an H2S donor, can alleviate angiotensin II (Ang II)-induced hypertension (HTN), improve autonomic function, and impact microglia in the paraventricular nucleus (PVN) of the hypothalamus, a brain region associated with autonomic control of blood pressure (BP) and neuroinflammation in HTN. Chronic delivery of Ang II (200 ng/kg/min, subcutaneous) for 4 weeks produced a typical increase in BP and sympathetic drive and elevated the number of ionized calcium binding adaptor molecule 1-positive (Iba1+) cells in the PVN of male, Sprague-Dawley rats. ICV co-infusion of NaHS (at 30 and/or 60 nmol/h) significantly attenuated these effects of Ang II. Ang II also increased the abundance of cecal Deltaproteobacteria and Desulfovibrionales, among others, which was prevented by ICV NaHS co-infusion at 30 and 60 nmol/h. We observed no differences in circulating H2S between the groups. Our results suggest that central H2S may alleviate rodent HTN independently from circulating H2S via effects on autonomic nervous system and PVN microglia.

Comparison of Isoflurane, Ketamine-Dexmedetomidine, and Ketamine-Xylazine for General Anesthesia during Oral Procedures in Rice Rats (Oryzomys palustris).

Rice rats (Oryzomys palustris) are an unconventional laboratory species that has been used to study photoperiodicity, periodontitis, and osteonecrosis of the jaw. Interventional procedures that require anesthesia, including oral procedures, are sometimes necessary in preclinical settings. The use of anesthetics including isoflurane and ketamine combined with α2-adrenoreceptor agonists, such as dexmedetomidine and xylazine, is well-established for laboratory rodents. However, their effects have been studied only modestly in rice rats. The aims of this study were to 1) determine the safety and consistency of 3 common anesthetic modalities in rice rats; 2) compare the physiologic and clinical responses to these anesthetics, and 3) verify the effectiveness of the most successful modality by testing it during an oral procedure (tooth extraction). Isoflurane, intraperitoneal ketamine-dexmedetomidine, and intraperitoneal ketamine-xylazine were evaluated by using a crossover design, in which each rat received all of the anesthetics. Compared with ketamine-dexmedetomidine and ketamine-xylazine, isoflurane inhalation through a nose cone produced more rapid induction, entry to a surgical plane of anesthesia, and initial recovery. In addition, isoflurane produced optimal anesthesia throughout the procedure for most rats. Unlike ketamine-dexmedetomidine and ketamine-xylazine, isoflurane did not alter rectal temperature, SpO2, or respiratory rate during the surgical tolerance period, whereas ketamine-dexmedetomidine and ketamine-xylazine decreased rectal temperature during the last stage of anesthesia and induced cardiorespiratory depression. Furthermore, 2 rats experienced negative outcomes warranting euthanasia: one after receiving ketamine-dexmedetomidine, and the other after ketamine-xylazine anesthesia. In conclusion, isoflurane was the most reliable and effective anesthetic in rice rats and maintained a surgical depth of anesthesia for as long as 30 min, thus supporting successful tooth extractions.

Increased Abundance of Lactobacillales in the Colon of Beta-Adrenergic Receptor Knock Out Mouse Is Associated With Increased Gut Bacterial Production of Short Chain Fatty Acids and Reduced IL17 Expression in Circulating CD4+ Immune Cells.

Emerging evidence suggests an associative link between gut dysbiosis, the autonomic nervous system (ANS) and the immune system in pathophysiology of neurogenic hypertension (HTN). However, the close interplay between these three systems presents us with difficulties in deciphering the cause-effect relationship in disease. The present study utilized beta 1 and 2 adrenergic receptor knock out (AdrB1tm1BkkAdrB2tm1Bkk/J KO) mice to isolate the effects of reduced overall sympathetic drive on gut microbiota and systemic immune system. We observed the following: (i) Diminished beta adrenergic signaling mainly reflects in shifts in the Firmicutes phyla, with a significant increase in abundance of largely beneficial Bacilli Lactobacillales in the KO mice; (ii) This was associated with increased colonic production of beneficial short chain fatty acids (SCFAs) butyrate, acetate and propionate, confirming functional microbiota shifts in the KO mice; (iii) Dampened systemic immune responses in the KO mice reflected in reduction on circulating CD4+.IL17+ T cells and increase in young neutrophils, both previously associated with shifts in the gut microbiota. Taken together, these observations demonstrate that reduced expression of beta adrenergic receptors may lead to beneficial shifts in the gut microbiota and dampened systemic immune responses. Considering the role of both in hypertension, this suggests that dietary intervention may be a viable option for manipulation of blood pressure via correcting gut dysbiosis.

Butyrate regulates inflammatory cytokine expression without affecting oxidative respiration in primary astrocytes from spontaneously hypertensive rats.

Neurons and glia exhibit metabolic imbalances in hypertensive animal models, and loss of metabolic homeostasis can lead to neuroinflammation and oxidative stress. The objective of this study was to determine the effects of the microbial metabolite butyrate on mitochondrial bioenergetics and inflammatory markers in mixed brainstem and hypothalamic primary cultures of astrocytes between normotensive (Sprague-Dawley, S-D) and spontaneously hypertensive (SHR) rats. Bioenergetics of mitochondria in astrocytes from normotensive S-D rats were modified with butyrate, but this was not the case in astrocytes derived from SHR, suggesting aberrant mitochondrial function. Transcripts related to oxidative stress, butyrate transporters, butyrate metabolism, and neuroinflammation were quantified in astrocyte cultures treated with butyrate at 0, 200, 600, and 1000 µmol/L. Butyrate decreased catalase and monocarboxylate transporter 1 mRNA in astrocytes of S-D rats but not in the SHR. Moreover, while butyrate did not directly regulate the expression of 3-hydroxybutyrate dehydrogenase 1 and 2 in astrocytes of either strain, the expression levels for these transcripts in untreated cultures were lower in the SHR compared to S-D. We observed higher levels of specific inflammatory cytokines in astrocytes of SHR, and treatment with butyrate decreased expression of Ccl2 and Tlr4 in SHR astrocytes only. Conversely, butyrate treatment increased expression of tumor necrosis factor in astrocytes from SHR but not from the S-D rats. This study improves our understanding of the role of microbial metabolites in regulating astrocyte function, and provides support that butyrate differentially regulates both the bioenergetics and transcripts related to neuroinflammation in astrocytes from SHR versus S-D rats.

A Single Angiotensin II Hypertensive Stimulus Is Associated with Prolonged Neuronal and Immune System Activation in Wistar-Kyoto Rats.

Activation of autonomic neural pathways by chronic hypertensive stimuli plays a significant role in pathogenesis of hypertension. Here, we proposed that even a single acute hypertensive stimulus will activate neural and immune pathways that may be important in initiation of memory imprinting seen in chronic hypertension. We investigated the effects of acute angiotensin II (Ang II) administration on blood pressure, neural activation in cardioregulatory brain regions, and central and systemic immune responses, at 1 and 24 h post-injection. Administration of a single bolus intra-peritoneal (I.P.) injection of Ang II (36 µg/kg) resulted in a transient increase in the mean arterial pressure (MAP) (by 22 ± 4 mmHg vs saline), which returned to baseline within 1 h. However, in contrast to MAP, neuronal activity, as measured by manganese-enhanced magnetic resonance (MEMRI), remained elevated in several cardioregulatory brain regions over 24 h. The increase was predominant in autonomic regions, such as the subfornical organ (SFO; ~20%), paraventricular nucleus of the hypothalamus (PVN; ~20%) and rostral ventrolateral medulla (RVLM; ~900%), among others. Similarly, systemic and central immune responses, as evidenced by circulating levels of CD4+/IL17+ T cells, and increased IL17 levels and activation of microglia in the PVN, respectively, remained elevated at 24 h following Ang II challenge. Elevated Fos expression in the PVN was also present at 24 h (by 73 ± 11%) following Ang II compared to control saline injections, confirming persistent activation of PVN. Thus, even a single Ang II hypertensive stimulus will initiate changes in neuronal and immune cells that play a role in the developing hypertensive phenotype.