Search

Background Phlebotomy-induced anemia (PIA) is universal and variable in degree among preterm infants and may contribute to neurodevelopmental risk. In mice, PIA causes brain tissue hypoxia, iron deficiency, and long-term sex-dependent neurobehavioral abnormalities. The neuroregulatory molecular pathways disrupted by PIA underlying these effects are unknown. Methods Male and female pups were phlebotomized daily from postnatal day (P)3-P14 via facial venipuncture to target hematocrits of 25% (moderate, mPIA) and 18% (severe, sPIA). P14 hippocampal RNA from non-bled control and PIA mice was sequenced by next-generation sequencing to identify differentially expressed genes (DEGs) that were analyzed using Ingenuity Pathway Analysis. Results mPIA females showed the least DEGs (0.5% of >22,000 genes) whereas sPIA females had the most (8.6%), indicating a dose-dependent effect. mPIA and sPIA males showed similar changes in gene expression (5.3% and 4.7%, respectively), indicating a threshold effect at mPIA. The pattern of altered genes induced by PIA indicates sex-specific and anemia-dose-dependent effects with increased pro-inflammation in females and decreased neurodevelopment in males. Conclusion These gene-expression changes may underlie the reduced recognition memory function in male and abnormal social-cognitive behavior in female adult mice following neonatal PIA. These results parallel clinical studies demonstrating sex-specific behavioral outcomes as a function of neonatal anemia. Impact Phlebotomy-induced anemia (PIA) in neonatal mice results in an altered hippocampal transcriptome and the severity of changes are dependent upon degree of anemia and sex of neonatal mice. The reported findings provide context to the sex-specific outcomes that have been reported in transfusion threshold clinical trials of preterm infants and therefore may inform treatment strategies that may be based on sex. These data advance the field by showing that consequences of PIA may be based in sex-specific transcriptomic alterations. Such changes may also result from other causes of neonatal anemia that also affect term infants.

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task state. This protocol describes a preclinical survival method for obtaining rs-fMRI data. Combining low dose isoflurane with continuous infusion of the α(2) adrenergic receptor agonist dexmedetomidine provides a robust option for stable, high-quality data acquisition while preserving brain network function. Furthermore, this procedure allows for spontaneous breathing and near-normal physiology in the rat. Additional imaging sequences can be combined with resting-state acquisition creating experimental protocols with anesthetic stability of up to 5 hours using this method. This protocol describes the setup of equipment, monitoring of rat physiology during four distinct phases of anesthesia, acquisition of resting-state scans, quality assessment of data, recovery of the animal, and a brief discussion of post-processing data analysis. This protocol can be used across a wide variety of preclinical rodent models to help reveal the resulting brain network changes that occur at rest.

BackgroundPhlebotomy-induced anemia (PIA) is common in premature infants and affects neurodevelopment. PIA alters hippocampal metabolism in neonatal mice through tissue hypoxia and iron deficiency. The mammalian target of rapamycin (mTOR) pathway senses the status of critical metabolites (e.g., oxygen, iron), thereby regulating hippocampal growth and function. We determined the effect of PIA and recombinant human erythropoietin (rHuEpo) treatment on mTOR signaling and expression of genes related to mTOR pathway functions.MethodsMice receiving an iron-supplemented diet were phlebotomized from postnatal day (P)3 to a target hematocrit of25% by P7. Half were maintained at25% until P14; half received rHuEpo from P7 to increase the hematocrit to 25-28%. Hippocampal phosphorylated to total protein ratios of four key mTOR pathway proteins were measured by western blotting at P14 and compared with non-phlebotomized, non-anemic control mice. mRNA levels of genes regulated by mTOR were measured by quantitative PCR.ResultsPIA suppressed phosphorylation of all mTOR proteins. rHuEpo restored AMP-activated protein kinase (AMPK) and AKT status, and partially rescued the mTOR output protein S6K. PIA and rHuEpo treatment also altered the expression of genes regulated by S6K.ConclusionPIA compromises and rHuEpo treatment partially rescues a pathway regulating neuronal DNA transcription, protein translation, and structural complexity.

Phlebotomy-induced anemia (PIA) is common in preterm infants. The hippocampus undergoes rapid differentiation during late fetal/early neonatal life and relies on adequate oxygen and iron to support oxidative metabolism necessary for development. Anemia shortchanges these two critical substrates, potentially altering hippocampal development and function. PIA (hematocrit

Pharmacological magnetic resonance imaging (phMRI) is one method by which a drug's pharmacodynamic effects in the brain can be assessed. Although phMRI has been frequently used in preclinical and clinical settings, the extent to which a phMRI signature for a compound translates between rodents and humans has not been systematically examined. In the current investigation, we aimed to build on recent clinical work in which the functional response to 0.1 and 0.2 mg/70 kg i.v. buprenorphine (partial µ-opioid receptor agonist) was measured in healthy humans. Here, we measured the phMRI response to 0.04 and 0.1 mg/kg i.v. buprenorphine in conscious, naive rats to establish the parallelism of the phMRI signature of buprenorphine across species. PhMRI of 0.04 and 0.1 mg/kg i.v. buprenorphine yielded dose-dependent activation in a brain network composed of the somatosensory cortex, cingulate, insula, striatum, thalamus, periaqueductal gray, and cerebellum. Similar dose-dependent phMRI activation was observed in the human phMRI studies. These observations indicate an overall preservation of pharmacodynamic responses to buprenorphine between conscious, naive rodents and healthy human subjects, particularly in brain regions implicated in pain and analgesia. This investigation further demonstrates the usefulness of phMRI as a translational tool in neuroscience research that can provide mechanistic insight and guide dose selection in drug development.

The behavioral response to pain is driven by sensory and affective components, each of which is mediated by the CNS. Subjective pain ratings are used as readouts when appraising potential analgesics; however, pain ratings alone cannot enable a characterization of CNS pain circuitry during pain processing or how this circuitry is modulated pharmacologically. Having a more objective readout of potential analgesic effects may allow improved understanding and detection of pharmacological efficacy for pain. The pharmacological/functional magnetic resonance imaging (phMRI/fMRI) methodology can be used to objectively evaluate drug action on the CNS. In this context, we aimed to evaluate two drugs that had been developed as analgesics: one that is efficacious for pain (buprenorphine (BUP)) and one that failed as an analgesic in clinical trials aprepitant (APREP). Using phMRI, we observed that activation induced solely by BUP was present in regions with μ-opioid receptors, whereas APREP-induced activation was seen in regions expressing NK(1) receptors. However, significant pharmacological modulation of functional connectivity in pain-processing pathways was only observed following BUP administration. By implementing an evoked pain fMRI paradigm, these drugs could also be differentiated by comparing the respective fMRI signals in CNS circuits mediating sensory and affective components of pain. We report a correlation of functional connectivity and evoked pain fMRI measures with pain ratings as well as peak drug concentration. This investigation demonstrates how CNS-acting drugs can be compared, and how the phMRI/fMRI methodology may be used with conventional measures to better evaluate candidate analgesics in small subject cohorts.

A dramatic increase in the use and dependence of prescription opioids has occurred within the last 10 years. The consequences of long-term prescription opioid use and dependence on the brain are largely unknown, and any speculation is inferred from heroin and methadone studies. Thus, no data have directly demonstrated the effects of prescription opioid use on brain structure and function in humans. To pursue this issue, we used structural magnetic resonance imaging, diffusion tensor imaging and resting-state functional magnetic resonance imaging in a highly enriched group of prescription opioid-dependent patients [(n = 10); from a larger study on prescription opioid dependent patients (n = 133)] and matched healthy individuals (n = 10) to characterize possible brain alterations that may be caused by long-term prescription opioid use. Criteria for patient selection included: (i) no dependence on alcohol or other drugs; (ii) no comorbid psychiatric or neurological disease; and (iii) no medical conditions, including pain. In comparison to control subjects, individuals with opioid dependence displayed bilateral volumetric loss in the amygdala. Prescription opioid-dependent subjects had significantly decreased anisotropy in axonal pathways specific to the amygdala (i.e. stria terminalis, ventral amygdalofugal pathway and uncinate fasciculus) as well as the internal and external capsules. In the patient group, significant decreases in functional connectivity were observed for seed regions that included the anterior insula, nucleus accumbens and amygdala subdivisions. Correlation analyses revealed that longer duration of prescription opioid exposure was associated with greater changes in functional connectivity. Finally, changes in amygdala functional connectivity were observed to have a significant dependence on amygdala volume and white matter anisotropy of efferent and afferent pathways of the amygdala. These findings suggest that prescription opioid dependence is associated with structural and functional changes in brain regions implicated in the regulation of affect and impulse control, as well as in reward and motivational functions. These results may have important clinical implications for uncovering the effects of long-term prescription opioid use on brain structure and function.

Using functional magnetic resonance imaging (fMRI) methods, we evaluated the effects of lamotrigine vs placebo in a double-blind 1:1 randomized trial. Six patients with neuropathic pain were recruited for the study. All subjects had baseline pain >4/10 on a visual analog scale (VAS) and allodynia to brush as inclusion criteria for the study. Patients underwent two fMRI sessions, with half of the subjects receiving placebo first and half receiving drug first (based on the blinding protocol). Lamotrigine decreased their average pain intensity level from 5.6 to 3.5 on a VAS. All subjects had brush, cold, and heat applied to the affected and mirror-unaffected sides of their face. The results show: 1) in a small cohort, lamotrigine had a significant effect on heat VAS but not on the other stimuli; and 2) contrast analysis of fMRI results for heat stimuli applied to the affected face for lamotrigine vs placebo produced an overall decrease in blood oxygen dependent level signal, suggesting a potential inhibitory effect of the drug on predominantly cortical regions (frontal, parietal, and temporal).

Complex regional pain syndrome (CRPS) in paediatric patients is clinically distinct from the adult condition in which there is often complete resolution of its signs and symptoms within several months to a few years. The ability to compare the symptomatic and asymptomatic condition in the same individuals makes this population interesting for the investigation of mechanisms underlying pain and other symptoms of CRPS. We used fMRI to evaluate CNS activation in paediatric patients (9-18 years) with CRPS affecting the lower extremity. Each patient underwent two scanning sessions: once during an active period of pain (CRPS(+)), and once after symptomatic recovery (CRPS(-)). In each session, mechanical (brush) and thermal (cold) stimuli were applied to the affected region of the involved limb and the corresponding mirror region of the unaffected limb. Two fundamental fMRI analyses were performed: (i) within-group analysis for CRPS(+) state and CRPS(-) state for brush and cold for the affected and unaffected limbs in each case; (ii) between-group (contrast) analysis for activations in affected and unaffected limbs in CRPS or post-CRPS states. We found: (i) in the CRPS(+) state, stimuli that evoked mechanical or cold allodynia produced patterns of CNS activation similar to those reported in adult CRPS; (ii) in the CRPS(+) state, stimuli that evoked mechanical or cold allodynia produced significant decreases in BOLD signal, suggesting pain-induced activation of endogenous pain modulatory systems; (iii) cold- or brush-induced activations in regions such as the basal ganglia and parietal lobe may explain some CNS-related symptoms in CRPS, including movement disorders and hemineglect/inattention; (iv) in the CRPS(-) state, significant activation differences persisted despite nearly complete elimination of evoked pain; (v) although non-noxious stimuli to the unaffected limb were perceived as equivalent in CRPS(+) and CRPS(-) states, the same stimulus produced different patterns of activation in the two states, suggesting that the 'CRPS brain' responds differently to normal stimuli applied to unaffected regions. Our results suggest significant changes in CNS circuitry in patients with CRPS.

Background. Previous reports have indicated that ketamine anesthesia may produce significant improvement if not complete recovery of patients with complex regional pain syndrome (CRPS). Aims. Here we report on a patient who had CRPS affecting mainly the right side of her body who underwent functional magnetic resonance imaging (fMRI) scans prior to and in the months following apparent successful treatment with anesthetic doses of ketamine. Materials and Methods. The patient underwent two imaging sessions: one during her pain state (CRPS+) and 1 month after her ketamine treatment in her pain-free state (CRPS-). Both spontaneous and evoked (brush, cold, and heat) pain scores decreased from 7–9/10 on a visual analog scale prior to the treatment to 0–1 immediately following and for months after the treatment. For each imaging session, the identical mechanical (brush) and thermal (cold and heat) stimuli were applied to the same location (the skin of the dorsum of the right hand). Results. Comparison of CRPS+ vs CRPS- for the three stimuli showed significant changes throughout the cerebral cortex (frontal, parietal, temporal, cingulate, and hippocampus), in subcortical regions such as caudate nucleus, and in the cerebellum. In addition, resting state network analysis showed a reversal of brain network state, and the recovered state paralleled specific default networks in healthy volunteers. Discussion. The observed changes in brain response to evoked stimuli provide a readout for the subjective response. Conclusion. Future studies of brain function in these patients may provide novel insight into brain plasticity in response to this treatment for chronic pain.

Nalbuphine, an agonist-antagonist kappa-opioid, produces brief analgesia followed by enhanced pain/hyperalgesia in male postsurgical patients. However, it produces profound analgesia without pain enhancement when co-administration with low dose naloxone. To examine the effect of nalbuphine or nalbuphine plus naloxone on activity in brain regions that may explain these differences, we employed pharmacological magnetic resonance imaging (phMRI) in a double blind cross-over study with 13 healthy male volunteers. In separate imaging sessions subjects were administered nalbuphine (5 mg/70 kg) preceded by either saline (Sal-Nalb) or naloxone 0.4 mg (Nalox-Nalb). Blood oxygen level-dependent (BOLD) activation maps followed by contrast and connectivity analyses revealed marked differences. Sal-Nalb produced significantly increased activity in 60 brain regions and decreased activity in 9; in contrast, Nalox-Nalb activated only 14 regions and deactivated only 3. Nalbuphine, like morphine in a previous study, attenuated activity in the inferior orbital cortex, and, like noxious stimulation, increased activity in temporal cortex, insula, pulvinar, caudate, and pons. Co-administration/pretreatment of naloxone selectively blocked activity in pulvinar, pons and posterior insula. Nalbuphine induced functional connectivity between caudate and regions in the frontal, occipital, temporal, insular, middle cingulate cortices, and putamen; naloxone co-admistration reduced all connectivity to non-significant levels, and, like phMRI measures of morphine, increased activation in other areas (e.g., putamen). Naloxone pretreatment to nalbuphine produced changes in brain activity possess characteristics of both analgesia and algesia; naloxone selectively blocks activity in areas associated with algesia. Given these findings, we suggest that nalbuphine interacts with a pain salience system, which can modulate perceived pain intensity.

Buprenorphine (BUP) is a partial agonist at μ-, δ- and ORL1 (opioid receptor-like)/nociceptin receptors and antagonist at the κ-opioid receptor site. BUP is known to have both analgesic as well as antihyperalgesic effects via its central activity, and is used in the treatment of moderate to severe chronic pain conditions. Recently, it was shown that intravenous (IV) administration of 0.2mg/70 kg BUP modulates the blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) response to acute noxious stimuli in healthy human subjects. The present study extends these observations by investigating the effects of BUP dose and route of administration on central nervous system (CNS) pain circuitry. Specifically, the modulation of evoked pain BOLD responses and resting state functional connectivity was measured following IV (0.1 and 0.2mg/70 kg) and sublingual (SL) (2mg) BUP administration in healthy human subjects. While 0.1mg/70 kg IV BUP is sub-analgesic, both 0.2mg/70 kg IV BUP and 2.0mg SL BUP are analgesic doses of the drug. Evoked BOLD responses were clearly modulated in a dose-dependent manner. The analgesic doses of BUP by both routes of administration yielded a potentiation in limbic/mesolimbic circuitry and attenuation in sensorimotor/sensory-discriminative circuitry. In addition, robust decreases in functional connectivity between the putamen and the sensorimotor/sensory-discriminative structures were observed at the two analgesic doses subsequent to measuring the maximum plasma BUP concentrations (C(max)). The decreases in functional connectivity within the sensorimotor/sensory-discriminative circuitry were also observed to be dose-dependent in the IV administration cohorts. These reproducible and consistent functional CNS measures at clinically effective doses of BUP demonstrate the potential of evoked pain fMRI and resting-state functional connectivity as objective tools that can inform the process of dose selection. Such methods may be useful during early clinical phase evaluation of potential analgesics in drug development.