Gabriele Campana

CNF1 increases brain energy level, counteracts neuroinflammatory markers and rescues cognitive deficits in a murine model of Alzheimer's disease.

Overexpression of pro-inflammatory cytokines and cellular energy failure are associated with neuroinflammatory disorders, such as Alzheimers disease. Transgenic mice homozygous for human ApoE4 gene, a well known AD and atherosclerosis animal model, show decreased levels of ATP, increased inflammatory cytokines level and accumulation of beta amyloid in the brain. All these findings are considered responsible for triggering cognitive decline. We have demonstrated that a single administration of the bacterial E. coli protein toxin CNF1 to aged apoE4 mice, beside inducing a strong amelioration of both spatial and emotional memory deficits, favored the cell energy restore through an increment of ATP content. This was accompanied by a modulation of cerebral Rho and Rac1 activity. Furthermore, CNF1 decreased the levels of beta amyloid accumulation and interleukin-1β expression in the hippocampus. Altogether, these data suggest that the pharmacological modulation of Rho GTPases by CNF1 can improve memory performances in an animal model of Alzheimers disease via a control of neuroinflammation and a rescue of systemic energy homeostasis.

Traumatic subclavian arterial rupture: a case report and review of literature

Subclavian artery injuries represent an uncommon complication of blunt chest trauma, this structure being protected by subclavius muscle, the clavicle, the first rib, and the deep cervical fascia as well as the costo-coracoid ligament, a clavi-coraco-axillary fascia portion. Subclavian artery injury appears early after trauma, and arterial rupture may cause life-treatening haemorrages, pseudo-aneurysm formation and compression of brachial plexus. These clinical eveniences must be carefully worked out by accurate physical examination of the upper limb: skin color, temperature, sensation as well as radial pulse and hand motility represent the key points of physical examination in this setting. The presence of large hematomas and pulsatile palpable mass in supraclavicular region should raise the suspicion of serious vascular injury. Since the first reports of endovascular treatment for traumatic vascular injuries in the 90’s, an increasing number of vascular lesions have been treated this way. We report a case of traumatic subclavian arterial rupture after blunt chest trauma due to a 4 meters fall, treated by endovascular stent grafting, providing a complete review of the past twenty years’ literature.

Post-natal stress-induced endocrine and metabolic alterations in mice at adulthood involve different pro-opiomelanocortin-derived peptides

In previous investigations we added a physical stress (mild pain) to the classical post-natal psychological stress in male mice, and we found that this combination produced a series of dysmetabolic signs very similar to mild human type-2 diabetes. Here, for the first time we demonstrate that within this diabetes model at least two groups of signs depend on the unbalance of two different endogenous systems. Newborn male mice were daily exposed to stressful procedures for 21 days (brief mother separation plus sham injection). Other groups underwent the same procedure, and also received naloxone (Na) to block μ-δ endogenous receptors, or a phosphorothioate antisense oligonucleotide (AS) directed against pro-opiomelanocortin (POMC)-mRNA [to block adrenocorticotropin (ACTH)- and POMC-derived opioid peptides]. Adult mice which received only post-natal stress increased body weight (+7.5%), abdominal overweight (+74%), fasting glycemia (+43%), plasma corticosterone (+110%), plasma (+169%) and pituitary (+153%) ACTH levels. Conversely, hypothalamic ACTH and corticotropin-releasing hormone (CRH) were reduced (-70% and -75%, respectively). Neonatal AS administration reverted all parameters to control values. Neonatal naloxone had little or no influence on glucose, corticosterone, ACTH, CRH levels, whereas it prevented body overweight and abdominal overweight. We conclude that, within this type-2 diabetes model in male mice at least two endocrino-neurohumoral systems are damaged, one concerning the opioid system, and the other concerning HPA hormones. The use of the two drugs was of primary importance to demonstrate this statement, and to demonstrate that these two groups of signs could be defined as separate entities following our complex post-natal stress model.

Long-term intrathecal morphine and bupivacaine upregulate MOR gene expression in lymphocytes

BACKGROUNDnSeveral studies have shown that opioids may suppress the immune system either by binding to mu-opioid receptors (MOR) expressed in immune cells or by activating these receptors within the central nervous system.nnnOBJECTIVEnTo assess the level of lymphocyte expression of MOR mRNA in patients with chronic non-cancer pain, who were treated with intrathecal morphine or with morphine plus bupivacaine via an intrathecal drug delivery system, and to investigate whether intrathecal morphine and the associated local anesthetic administration influences lymphocyte subpopulations.nnnMETHODSnIn total, 29 people [10 controls (age range 59-85 years) and 19 patients (age range 47-89 years) with various chronic non-malignant pain conditions] were enrolled in the study. MOR mRNA levels were evaluated in peripheral lymphocytes, and lymphocyte subsets were determined by direct immunofluorescence using flow cytometry.nnnRESULTSnAfter 12 months of treatment with intrathecal morphine (1.5-4 mg/day), there was an increase in MOR mRNA levels in lymphocytes of 65% compared with controls and 47% with pretreatment values. Even higher levels (increase of 142% compared with controls and 135% with pretreatment values) were observed in the patients treated with morphine plus bupivacaine (0.2-0.4 mg/day). Elevation of MOR mRNA levels was confirmed in patients after 24 months of treatment. At this time point, the percentage of natural killer cells was significantly decreased.nnnCONCLUSIONnThis preliminary study suggests that opioids must be used with care in patients who are already immunosuppressed by disease or by other, concurrently administered drugs.

Enhanced brain performance in mice following postnatal stress

The double postnatal stress model (brief maternal separation plus sham injection daily applied from birth to weaning) induces metabolic alterations similar to type 2 diabetes in young-adult male mice. We verify whether 1) the stress also induces brain metabolic-functional alterations connected to diabetes and 2) different alterations are modulated selectively by two stress-damaged endogenous systems (opioid- and/or ACTH-corticosteroid-linked). Here, diabetes-like metabolic plus neurophysiologic-neurometabolic parameters are studied in adult mice following postnatal stress and drug treatment. Surprisingly, together with classic diabetes-like alterations, the stress model induces in young-adult mice significantly enhanced brain neurometabolic-neurophysiologic performances, consisting of decreased latency to flash-visual evoked potentials (- ~8%); increased level (+ ~40%) and reduced latency (- ~30%) of NAD(P)H autofluorescence postsynaptic signals following electric stimuli; enhanced passive avoidance learning (+ ~135% latency); and enhanced brain-derived neurotrophic factor level (+ ~70%). Postnatal treatment with the opioid receptor antagonist naloxone prevents some alterations, moreover the treatment with antisense (AS; AS vs proopiomelanocortin mRNA) draws all parameters to control levels, thus showing that some alterations are bound to endogenous opioid-system hyper-functioning, while others depend on ACTH-corticosterone system hyper-functioning. Our stress model induces diabetes-like metabolic alterations coupled to enhanced brain neurometabolic-neurophysiologic performances. Taken all together, these findings are compatible with an enduring acute-stress reaction, which puts mice in favorable survival situations vs controls. However, prolonged hormonal-metabolic imbalances are expected to also produce diabetes-like complications at later ages in stressed mice.

Evidence that AMP-activated protein kinase can negatively modulate Ornithine decarboxylase activity in cardiac myoblasts

The responses of AMP-activated protein kinase (AMPK) and Ornithine decarboxylase (ODC) to isoproterenol have been examined in H9c2 cardiomyoblasts, AMPK represents the link between cell growth and energy availability whereas ODC, the key enzyme in polyamine biosynthesis, is essential for all growth processes and it is thought to have a role in the development of cardiac hypertrophy. Isoproterenol rapidly induced ODC activity in H9c2 cardiomyoblasts by promoting the synthesis of the enzyme protein and this effect was counteracted by inhibitors of the PI3K/Akt pathway. The increase in enzyme activity became significant between 15 and 30min after the treatment. At the same time, isoproterenol stimulated the phosphorylation of AMPKα catalytic subunits (Thr172), that was associated to an increase in acetyl coenzyme A carboxylase (Ser72) phosphorylation. Downregulation of both α1 and α2 isoforms of the AMPK catalytic subunit by siRNA to knockdown AMPK enzymatic activity, led to superinduction of ODC in isoproterenol-treated cardiomyoblasts. Downregulation of AMPKα increased ODC activity even in cells treated with other adrenergic agonists and in control cells. Analogue results were obtained in SH-SY5Y neuroblastoma cells transfected with a shRNA construct against AMPKα. In conclusion, isoproterenol quickly activates in H9c2 cardiomyoblasts two events that seem to contrast one another. The first one, an increase in ODC activity, is linked to cell growth, whereas the second, AMPK activation, is a homeostatic mechanism that negatively modulates the first. The modulation of ODC activity by AMPK represents a mechanism that may contribute to control cell growth processes.

Antisense versus proopiomelanocortin mRNA reduces vascular risk in a murine model of type-2 diabetes following stress exposure in early post-natal life

Mechanisms of vascular complications in type-2 diabetes patients and animal models are matter of debate. We previously demonstrated that a double-stress model applied to male mice during nursing period produces enduring hyperfunction of endogenous opioid and adrenocorticotropin (ACTH)-corticosteroid systems, accompanied by type-2 diabetes-like alterations in adult animals. Administration of the opioid receptor antagonist naloxone, or of an antisense oligodeoxynucleotide versus proopiomelanocortin mRNA, capable to block the pro-opiomelanocortin-derived peptides β-endorphin and ACTH, selectively prevent these alterations. Here, we investigated alterations produced by our stress model on aorta endothelium-dependent relaxation and contractile responses. Mice, stressed during nursing period, showed in the adulthood hormonal and metabolic type-2 diabetes-like alterations, including hyperglycemia, increased body weight and increased plasma ACTH and corticosterone levels. Ex vivo isolated aorta rings, gathered from stressed mice, were less sensitive to noradrenaline-induced contractions versus controls. This effect was blocked by nitric-oxide synthase-inhibitor l-N(G)-nitroarginine added to bath organ solution. Aorta rings relaxation caused by acetylcholine was enhanced in stressed mice versus controls, but following treatment with the nitric-oxide donor sodium nitroprusside, concentration-relaxation curves in aorta from stressed groups were similar to controls. Therefore, vascular response alterations to physiologic-pharmacologic stimuli were apparently due to nitric-oxide hyperfunction-dependent mechanisms. Aorta functional alterations, and plasma stress hormones enhancement, were prevented in mice stressed and treated with antisense oligodeoxinucleotide, addressed to reduce ACTH- and corticosteroid-mediated hyperfunction. This study demonstrates the key role of ACTH-corticosteroid axis hyperfunction for the triggering of vascular conditions in male adult rodents following postnatal stress in a type-2 diabetes model.

CNF1 Enhances Brain Energy Content and Counteracts Spontaneous Epileptiform Phenomena in Aged DBA/2J Mice.

Epilepsy, one of the most common conditions affecting the brain, is characterized by neuroplasticity and brain cell energy defects. In this work, we demonstrate the ability of the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) to counteract epileptiform phenomena in inbred DBA/2J mice, an animal model displaying genetic background with an high susceptibility to induced- and spontaneous seizures. Via modulation of the Rho GTPases, CNF1 regulates actin dynamics with a consequent increase in spine density and length in pyramidal neurons of rat visual cortex, and influences the mitochondrial homeostasis with remarkable changes in the mitochondrial network architecture. In addition, CNF1 improves cognitive performances and increases ATP brain content in mouse models of Rett syndrome and Alzheimers disease. The results herein reported show that a single dose of CNF1 induces a remarkable amelioration of the seizure phenotype, with a significant augmentation in neuroplasticity markers and in cortex mitochondrial ATP content. This latter effect is accompanied by a decrease in the expression of mitochondrial fission proteins, suggesting a role of mitochondrial dynamics in the CNF1-induced beneficial effects on this epileptiform phenotype. Our results strongly support the crucial role of brain energy homeostasis in the pathogenesis of certain neurological diseases, and suggest that CNF1 could represent a putative new therapeutic tool for epilepsy.

Impact of admission to hospice on pain intensity and type of pain therapies administered

PurposeThe primary aim of this study was to evaluate pain intensity changes in patients admitted to a hospice. The secondary objective was to evaluate whether these changes in pain were accompanied by modifications in therapies and drugs used to treat pain.Patients and methodsThis retrospective study included 96 patients admitted to a hospice for a minimum of 7xa0days who received pain therapy. An 11-point (0–10) numerical rating scale (NRS) was used to assess pain on a daily basis. A repeated measures analysis of variance was performed to evaluate pain intensity changes over time.ResultsMeanu2009±u2009SD pain NRS values of the entire group were 2.58u2009±u20092.61 on day 1 and 1.40u2009±u20091.72 on day 7 (Pu2009=u20090.002). Restricting the analysis to patients with moderate to severe pain at the time of hospice admission, results were even more significant. In fact, meanu2009±u2009SD pain NRS was 5.51u2009±u20091.24 for patients with pain ≥4 at admission and 1.76u2009±u20091.91 for the same patients after 7xa0days (Pu2009<u20090.001). A significant increase in the number of patients receiving morphine was observed from day 1 to day 7 (24 to 41, respectively, Pu2009=u20090.001) and in those receiving drugs via parenteral routes (subcutaneous or intravenous) from 10 to 27 (Pu2009=u20090.002)ConclusionsAdmission to a hospice and the hospice environment led to a significant reduction in reported pain intensity for the patients included in this study, mainly those with moderate to severe pain at the time of admission. This decrease in pain was accompanied by a significant increase in the use of morphine, especially via parenteral routes, but not by a higher mean equivalent daily dose of oral morphine per patient.

Mechanical Nociception Measurement in Mice and Rats with Automated Von Frey Equipment

Von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used Von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.