Saku Ruohonen

Contralateral non-operated nerve to transected rat sciatic nerve shows increased expression of IL-1β, TGF-β1, TNF-α, and IL-10

Abstract Recent reports indicate that after a peripheral nerve injury, the uninjured contralateral nerve is also affected. Because cytokines play an important role in the peripheral nerve injury, we studied the expression of five different mRNAs (interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1) and interleukin-4 (IL-4)) in the contralateral, non-operated, left sciatic nerve when the right rat sciatic nerve was transected. This study extended up to 42 days after the transection. No IL-4 expression was noted. During the first 3 days, high expression of the other studied cytokines was noted in the endoneurium. At day 7, the expression diminished to the control levels. After this, a cyclic expression pattern appeared, which was most pronounced in the endoneurium at 35 days. We also show that the expression pattern in the endoneurium is different from that in the surrounding epi- and perineurium. Also, our present study shows clearly that contralateral nerves are poor controls after injury.

Temporal profiles of age-dependent changes in cytokine mRNA expression and glial cell activation after status epilepticus in postnatal rat hippocampus

BackgroundStatus epilepticus (SE) is proposed to lead to an age-dependent acute activation of a repertoire of inflammatory processes, which may contribute to neuronal damage in the hippocampus. The extent and temporal profiles of activation of these processes are well known in the adult brain, but less so in the developing brain. We have now further elucidated to what extent inflammation is activated by SE by investigating the acute expression of several cytokines and subacute glial reactivity in the postnatal rat hippocampus.MethodsSE was induced by an intraperitoneal (i.p.) injection of kainic acid (KA) in 9- and 21-day-old (P9 and P21) rats. The mRNA expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9 (MMP-9), glial-derived neurotrophic factor (GDNF), interferon gamma (IFN-γ), and transforming growth factor-beta 1 (TGF-β1) were measured from 4 h up to 3 days after KA injection with real-time quantitative PCR (qPCR). IL-1β protein expression was studied with ELISA, GFAP expression with western blotting, and microglial and astrocyte morphology with immunohistochemistry 3 days after SE.ResultsSE increased mRNA expression of IL-1β, TNF-α and IL-10 mRNA in hippocampus of both P9 and P21 rats, their induction being more rapid and pronounced in P21 than in P9 rats. MMP-9 expression was augmented similarly in both age groups and GDNF expression augmented only in P21 rats, whereas neither IFN-γ nor TGF-β1 expression was induced in either age group. Microglia and astrocytes exhibited activated morphology in the hippocampus of P21 rats, but not in P9 rats 3 d after SE. Microglial activation was most pronounced in the CA1 region and also detected in the basomedial amygdala.ConclusionOur results suggest that SE provokes an age-specific cytokine expression in the acute phase, and age-specific glial cell activation in the subacute phase as verified now in the postnatal rat hippocampus. In the juvenile hippocampus, transient increases in cytokine mRNA expression after SE, in contrast to prolonged glial reactivity and region-specific microglial activity after SE, suggest that the inflammatory response is changed from a fulminant and general initial phase to a more moderate and specific subacute response.

Cytokine responses during chronic denervation

BackgroundThe aim of the present study was to examine inflammatory responses during Wallerian degeneration in rat peripheral nerve when the regrowth of axons was prevented by suturing.MethodsTransected rat sciatic nerve was sutured and ligated to prevent reinnervation. The samples were collected from the left sciatic nerve distally and proximally from the point of transection. The endoneurium was separated from the surrounding epi- and perineurium to examine the expression of cytokines in both of these compartments. Macrophage invasion into endoneurium was investigated and Schwann cell proliferation was followed as well as the expression of cytokines IL-1β, IL-10, IFN-γ and TNF-α mRNA. The samples were collected from 1 day up to 5 weeks after the primary operation.ResultsAt days 1 to 3 after injury in the epi-/perineurium of the proximal and distal stump, a marked expression of the pro-inflammatory cytokines TNF-α and IL-1β and of the anti-inflammatory cytokine IL-10 was observed. Concurrently, numerous macrophages started to gather into the epineurium of both proximal and distal stumps. At day 7 the number of macrophages decreased in the perineurium and increased markedly in the endoneurium of both stumps. At this time point marked expression of TNF-α and IFN-γ mRNA was observed in the endo- and epi-/perineurium of the proximal stump. At day 14 a marked increase in the expression of IL-1β could be noted in the proximal stump epi-/perineurium and in the distal stump endoneurium. At that time point many macrophages were observed in the longitudinally sectioned epineurium of the proximal 2 area as well as in the cross-section slides from the distal stump. At day 35 TNF-α, IL-1β and IL-10 mRNA appeared abundantly in the proximal epi-/perineurium together with macrophages.ConclusionThe present studies show that even during chronic denervation there is a cyclic expression pattern for the studied cytokines. Contrary to the previous findings on reinnervating nerves the studied cytokines show increased expression up to 35 days. The high expressions of pro-inflammatory and anti-inflammatory cytokines in the proximal epi-/perineurial area at day 35 may be involved in the formation of fibrosis due to irreversible nerve injury and thus may have relevance to the formation of traumatic neuroma.

The effects of equal caloric high fat and western diet on metabolic syndrome, oxidative stress and vascular endothelial function in mice

Nutrition contributes to increased adiposity, but it remains to be determined whether high fat rather than Western diet exacerbates the development of obesity and other characteristics of metabolic syndrome and vascular function.

α-Melanocyte-stimulating hormone regulates vascular NO availability and protects against endothelial dysfunction

AIMS α-Melanocyte-stimulating hormone (α-MSH), derived from the precursor molecule pro-opiomelanocortin, exerts potent anti-inflammatory actions in the vasculature, but its role in circulatory regulation remains unclear. Therefore, we sought to investigate whether α-MSH could regulate the local control of blood vessel tone. METHODS AND RESULTS Using in vivo and ex vivo methods to assess vascular reactivity, we found that α-MSH improved endothelium-dependent vasodilatation in the mouse aorta and coronary circulation without directly contracting or relaxing blood vessels. α-MSH promoted vasodilatation by enhancing endothelial nitric oxide (NO) formation and by improving sensitivity to endothelium-independent blood vessel relaxation. Using cultured human endothelial cells to elucidate the involved molecular mechanisms, we show that α-MSH increased the expression and phosphorylation of endothelial NO synthase in these cells. The observed effects were regulated by melanocortin 1 (MC1) receptors expressed in the endothelium. In keeping with the vascular protective role of α-MSH, in vivo treatment with stable analogues of α-MSH ameliorated endothelial dysfunction associated with aging and diet-induced obesity in mice. CONCLUSION The present study identifies α-MSH and endothelial MC1 receptors as a new signalling pathway contributing to the regulation of NO availability and vascular function. These findings suggest applicability of α-MSH analogues for therapeutic use in pathological conditions that are characterized by vascular dysfunction.

Neuropeptide Y in the noradrenergic neurones induces obesity and inhibits sympathetic tone in mice

Neuropeptide Y (NPY) co‐localized with noradrenaline in central and sympathetic nervous systems seems to play a role in the control of energy metabolism. In this study, the aim was to elucidate the effects and pathophysiological mechanisms of increased NPY in catecholaminergic neurones on accumulation of body adiposity.

Dietary sodium modulates the interaction between efferent and afferent renal nerve activity by altering activation of α2-adrenoceptors on renal sensory nerves

Activation of efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which then reflexively decreases ERSNA via activation of the renorenal reflexes to maintain low ERSNA. The ERSNA-ARNA interaction is mediated by norepinephrine (NE) that increases and decreases ARNA by activation of renal α(1)-and α(2)-adrenoceptors (AR), respectively. The ERSNA-induced increases in ARNA are suppressed during a low-sodium (2,470 ± 770% s) and enhanced during a high-sodium diet (5,670 ± 1,260% s). We examined the role of α(2)-AR in modulating the responsiveness of renal sensory nerves during low- and high-sodium diets. Immunohistochemical analysis suggested the presence of α(2A)-AR and α(2C)-AR subtypes on renal sensory nerves. During the low-sodium diet, renal pelvic administration of the α(2)-AR antagonist rauwolscine or the AT1 receptor antagonist losartan alone failed to alter the ARNA responses to reflex increases in ERSNA. Likewise, renal pelvic release of substance P produced by 250 pM NE (from 8.0 ± 1.3 to 8.5 ± 1.6 pg/min) was not affected by rauwolscine or losartan alone. However, rauwolscine+losartan enhanced the ARNA responses to reflex increases in ERSNA (4,680 ± 1,240%·s), and renal pelvic release of substance P by 250 pM NE, from 8.3 ± 0.6 to 14.2 ± 0.8 pg/min. During a high-sodium diet, rauwolscine had no effect on the ARNA response to reflex increases in ERSNA or renal pelvic release of substance P produced by NE. Losartan was not examined because of low endogenous ANG II levels in renal pelvic tissue during a high-sodium diet. Increased activation of α(2)-AR contributes to the reduced interaction between ERSNA and ARNA during low-sodium intake, whereas no/minimal activation of α(2)-AR contributes to the enhanced ERSNA-ARNA interaction under conditions of high sodium intake.

Sympathetic nervous system-targeted neuropeptide Y overexpression in mice enhances neointimal formation in response to vascular injury

Sympathetic neurotransmitter neuropeptide Y (NPY) is associated with vascular remodelling, neointimal hyperplasia and atherosclerosis in experimental animal models and clinical studies. In order to study the role of sympathetic nerve-produced NPY in vascular diseases, transgenic mouse model overexpressing NPY in central and peripheral noradrenergic neurons under the dopamine-beta-hydroxylase (DBH) promoter was recently created (OE-NPY(DBH) mouse). This study aimed to examine the effect of NPY overexpression on arterial neointimal hyperplasia in an experimental model of vascular injury. Transgenic OE-NPY(DBH) mice and wildtype control mice of two different inbred strains (C57BL/6 and FVB/n) underwent a femoral artery surgery with a transluminar injury by a 0.38-mm guide wire insertion. Arteries were harvested 4 weeks from the surgery, and they were stained for basic morphology. Both strains of OE-NPY(DBH) mice, as compared with wildtype control mice, showed on average 50% greater formation of the neointima (P<0.01) and an increase in the medial area (P=0.05). The results suggest that moderately increased neuronal NPY causes the arteries to be more susceptible to femoral artery thickening after endothelial injury. The OE-NPY(DBH) mouse provides a novel tool to explore the role of NPY in the development of vascular disease related to metabolic disorders.

Subtle increases in heart size persist into adulthood in growth restricted babies: the Cardiovascular Risk in Young Finns Study

Background and objectives Impaired fetal growth is associated with increased cardiovascular morbidity and mortality in adulthood. We sought to determine whether adults born with intrauterine growth restriction have primary maladaptive changes in cardiac structure. Methods Study participants were adults (34–49 years) who attended the 31-year follow-up of the Cardiovascular Risk in Young Finns Study (longitudinal cohort). Transthoracic echocardiograms and demographic and cardiovascular risk surveys were completed for 157 adults born small for gestational age (SGA, birth weight <10th population centile) and 627 born average for gestational age (average for gestational age (AGA), birth weight 50th–90th population centile). Results Those born growth restricted had subtly enlarged hearts with indexed left ventricular (LV) end-systolic and end-diastolic diameters slightly greater in the SGA individuals than the AGA group (LVESD 18.7 mm/m2 SGA vs 18.1 mm/m2 AGA, p<0.01; LVEDD 27.5 mm/m2 SGA vs 26.6 mm/m2 AGA, p<0.01); LV base-to-apex length (47.4 mm/m2 SGA vs 46.0 mm/m2 AGA, p<0.01); LV basal diameter (26.4 mm/m2 SGA vs 25.7 mm/m2 AGA, p<0.01); and right ventricular base-to-apex length (40.1 mm/m2 SGA vs 39.2 mm/m2 AGA, p=0.02). LV stroke volume was greater in those born AGA (74.5 mL SGA vs 78.8 mL AGA, p<0.01), with no significant difference in cardiac output (5 L/min SGA vs 5.2 L/min AGA, p=0.06), heart rate, diastolic indices or sphericity index. Conclusions Adults born SGA have some statistically significant but subtle changes in cardiac structure and function, which are less marked than have been described in childhood, and are unlikely to play a pathogenic role in their elevated cardiovascular risk.

Lentivirus-mediated α-melanocyte-stimulating hormone overexpression in the hypothalamus decreases diet induced obesity in mice.

Melanocyte stimulating hormone (MSH) derived from the pro‐hormone pro‐opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long‐term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long‐term anti‐obesity effects of hypothalamic α‐MSH overexpression in mice. An α‐MSH lentivirus (LVi‐α‐MSH‐EGFP) vector carrying the N‘‐terminal part of POMC and the α‐MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi‐α‐MSH‐EGFP or control LVi‐EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high‐fat diet. The effects of 6‐week‐treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14‐day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi‐α‐MSH‐EGFP treatment is dependent on decreased food intake. The 6‐week LVi‐α‐MSH‐EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi‐EGFP treated mice. Food intake was decreased during the first week in the LVi‐α‐MSH‐EGFP treated mice but subsequently increased to the level of LVi‐EGFP treated mice. The LVi‐EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi‐α‐MSH‐EGFP treated mice. We demonstrate that gene transfer of α‐MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α‐MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site‐specific effects of POMC peptides.