A biological tube technique for the repair of peripheral nerve defects using 'stuffed nerves'.

BACKGROUND: Presently described is research examining the "stuffed nerve" technique to repair peripheral nerve defects. METHODS: Twenty-one male Wistar Albino rats were divided into 3 groups of 7, and standard 10-mm defects were created in the sciatic nerve of all subjects. Rats were treated with autogenous nerve graft (Group 1), hollow vein graft (Group 2), or vein graft stuffed with shredded nerves (Group 3). After 12 weeks, electrophysiological and histomorphological analyses were performed to evaluate axonal regeneration. RESULTS: Rat groups were compared in terms of latency period and peak-to-peak potential. Latency period was significantly shorter and peak-to-peak potential was significantly greater in Group 1 than in Group 2. However, latency period and peak-to-peak potential did not differ significantly between Groups 1 and 3 or between Groups 2 and 3. To evaluate axonal regeneration, number of axons, axon diameter and myelin sheath thickness was compared between groups. Results indicated that axonal regeneration was similar in Groups 1 and 3, and was better than results seen in Group 2. CONCLUSION: The stuffed nerve technique is an alternative to autogenous nerve grafting and produces similar electrophysiological and histomorphological properties.

Anti-inflammatory and Anti-apoptotic Effect of Valproic Acid and Doxycycline Independent from MMP Inhibition in Early Radiation Damage.

BACKGROUND: Matrix metalloproteinase (MMP) inhibitors decrease inflammation in normal tissues and suppress cancer progress in normal tissues. Valproic acid (VA) and doxycycline (DX) are MMP inhibitors that have radio-protective effects. Their ability to inhibit MMPs in irradiated tissue is unknown and the role of MMPs in radio-protective effects has not been tested to date. AIMS: The purpose of this study was to examine whether administration of VA and DX to rats before irradiation affects tissue inflammation and apoptosis in the early phase of radiation, and whether the effect of these drugs is mediated by MMP inhibition. STUDY DESIGN: Animal experimentation. METHODS: Twenty-six Wistar rats were randomized into four groups: control (CTRL), radiation (RT), VA plus radiation (VA+RT), and DX plus radiation (DX+RT). Three study groups were exposed to a single dose of abdominal 10 Gy gamma radiation; the CTRL group received no radiation. Single doses of VA 300 mg/kg and DX 100 mg/kg were administered to each rat before radiation and all rats were sacrificed 8 hours after irradiation, at which point small intestine tissue samples were taken for analyses. Levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and matrix metal-loproteinases (MMP-2 and MMP 9) were measured by ELISA, MMP activities were measured by gelatin and casein zymography and apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: VA decreased the levels of TNF-α and IL-1ß proteins insignificantly and decreased apoptosis significantly in the irradiated tissue, but did not inhibit MMPs. In contrast, VA protected the basal MMP activities, which decreased in response to irradiation. No effect of DX was observed on the levels of inflammatory cytokines or activities of MMPs in the early phases of radiation apoptosis. CONCLUSION: Our findings indicated that VA protects against inflammation and apoptosis, and DX exhibits anti-apoptotic effects in early radiation and these effects are independent from MMP inhibition.

DAMP signaling is a key pathway inducing immune modulation after brain injury.

Acute brain lesions induce profound alterations of the peripheral immune response comprising the opposing phenomena of early immune activation and subsequent immunosuppression. The mechanisms underlying this brain-immune signaling are largely unknown. We used animal models for experimental brain ischemia as a paradigm of acute brain lesions and additionally investigated a large cohort of stroke patients. We analyzed release of HMGB1 isoforms by mass spectrometry and investigated its inflammatory potency and signaling pathways by immunological in vivo and in vitro techniques. Features of the complex behavioral sickness behavior syndrome were characterized by homecage behavior analysis. HMGB1 downstream signaling, particularly with RAGE, was studied in various transgenic animal models and by pharmacological blockade. Our results indicate that the cytokine-inducing, fully reduced isoform of HMGB1 was released from the ischemic brain in the hyperacute phase of stroke in mice and patients. Cytokines secreted in the periphery in response to brain injury induced sickness behavior, which could be abrogated by inhibition of the HMGB1-RAGE pathway or direct cytokine neutralization. Subsequently, HMGB1-release induced bone marrow egress and splenic proliferation of bone marrow-derived suppressor cells, inhibiting the adaptive immune responses in vivo and vitro. Furthermore, HMGB1-RAGE signaling resulted in functional exhaustion of mature monocytes and lymphopenia, the hallmarks of immune suppression after extensive ischemia. This study introduces the HMGB1-RAGE-mediated pathway as a key mechanism explaining the complex postischemic brain-immune interactions.

Double-blind, placebo-controlled first in human study to investigate an oral vaccine aimed to elicit an immune reaction against the VEGF-Receptor 2 in patients with stage IV and locally advanced pancreatic cancer.

BACKGROUND: The investigational oral DNA vaccine VXM01 targets the vascular endothelial growth factor receptor 2 (VEGFR-2) and uses Salmonella typhi Ty21a as a vector. The immune reaction elicited by VXM01 is expected to disrupt the tumor neovasculature and, consequently, inhibit tumor growth. VXM01 potentially combines the advantages of anti-angiogenic therapy and active immunotherapy. METHODS/DESIGN: This phase I trial examines the safety, tolerability, and immunological and clinical responses to VXM01. The randomized, placebo-controlled, double blind dose-escalation study includes up to 45 patients with locally advanced and stage IV pancreatic cancer. The patients will receive four doses of VXM01 or placebo in addition to gemcitabine as standard of care. Doses from 106 cfu up to 1010 cfu of VXM01 will be evaluated in the study. An independent data safety monitoring board (DSMB) will be involved in the dose-escalation decisions. In addition to safety as primary endpoint, the VXM01-specific immune reaction, as well as clinical response parameters will be evaluated. DISCUSSION: The results of this study shall provide the first data regarding the safety and immunogenicity of the oral anti-VEGFR-2 vaccine VXM01 in cancer patients. They will also define the recommended dose for phase II and provide the basis for further clinical evaluation, which may also include additional cancer indications. TRIAL REGISTRATION: EudraCT No.: 2011-000222-29, NCT01486329, ISRCTN68809279.

Protective effects of methylxanthines on hypoxia-induced apoptotic neurodegeneration and long-term cognitive functions in the developing rat brain.

Aminophylline is widely used in the management of premature apnea. The methylxanthines aminophylline, theophylline and caffeine are nonspecific inhibitors of adenosine receptors. There are no proven effects of methylxanthines on acute brain injury and long-term cognitive functions. This study is aimed at investigating the effects of methylxanthines on brain injury and cognitive functions. Newborn rats were allocated to form four groups, which contained at least 21 pups: two groups were exposed to room air and two groups were exposed to intermittent hypoxia. Intraperitoneal aminophylline was administered to treatment groups during postnatal day 1 through postnatal day 7. All rats were sacrificed on postnatal day 8 via intraperitoneal pentobarbital and the effects of the administered drug on brain injury and adenosine receptor expression were determined. Cognitive functions of rats were evaluated via water maze test. Histopathological evaluation demonstrated that aminophylline significantly diminished the number of 'apoptotic cells' in the hippocampal CA1, CA2, CA3 and gyrus dentatus regions in the brain. Aminophylline treatment immediately after hypoxic insult significantly improved long-term neurobehavioral achievements. In conclusion, aminophylline administration immediately after neonatal hypoxic insult provides benefit over a prolonged period in the developing rat brain.

Does antioxidant supplementation alter the effects of acute exercise on erythrocyte aggregation, deformability and endothelium adhesion in untrained rats?

This study aimed to determine whether high-dose antioxidant supplementation had an impact on the acute exercise effects related to erythrocyte membrane mechanics. Experimental animals (n=32) were divided into four groups as control, exercised, supplemented, and supplemented + exercise. Four-week antioxidant supplementation (vitamin C, vitamin E, and zinc) was applied to experimental animals. Following acute exercise on a motor-driven rodent treadmill, erythrocyte aggregation and deformability, erythrocyte adhesion to endothelial cells, superoxide dismutase (SOD), and glutathione peroxidase activities of the erythrocytes were analyzed. In both supplemented and non-supplemented exercised groups, there was a significant decrease in SOD activities and erythrocyte aggregation, and an increase in adhesion to endothelial cell although there was no change on erythrocyte deformability. There were no differences in the responses to the exercise of supplemented and nonsupplemented rats. The data suggested that high-dose antioxidant supplementation did not alter the effects of acute exercise on erythrocyte membrane mechanics.

The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system.

The physiological effects of pineal melatonin are primarily mediated by melatonin receptors located in the brain and periphery. Even though there are a number of studies demonstrating the regulatory role of melatonin in the development of dopaminergic behaviors, such as psychostimulant-induced diurnal locomotor sensitization or drug seeking, little is known about the contribution of melatonin receptors (i.e., MT1) to this role. Therefore, as a first step in understanding the functional role of melatonin receptors in dopaminergic behaviors, we focused on determining the expression pattern of MT1 receptors in the dopaminergic system of the human and rodent brain. Regional (e.g., nucleus accumbens shell) and cellular (e.g., tyrosine hydroxylase immunopositive cells) expression of MT1 mRNA was characterized by applying the immuno-laser capture microdissection (immuno-LCM) technique coupled with nested RT-PCR. Moreover, employing quantitative Western immunoblotting and RT-PCR, we found that the mouse MT1 receptor expression presents diurnal variations (i.e., low mRNA and high protein levels at night, ZT21). The dopaminergic system-based presence of MT1 receptor proteins was not limited to rodents; we found these receptors in postmortem human brain as well. Further research is needed to understand the regional/cellular functional role of melatonin receptors in the regulation of dopaminergic behaviors, using models such as melatonin receptor knockout mice.

Diurnal rhythms in quinpirole-induced locomotor behaviors and striatal D2/D3 receptor levels in mice.

Dopaminergic drugs, including the D2/D3 agonist quinpirole, produce lasting changes in the brain that lead to altered behavioral responses. The action of these drugs is dosing time-dependent; in fruit flies, behavioral response to quinpirole shows a marked circadian variability. Here we demonstrate diurnal rhythm-dependent variations both in quinpirole-induced locomotor behaviors and in striatal D2 and D3 protein levels in mice. We found opposing diurnal rhythms in striatal D2 and D3 protein levels, resulting in a high D2/D3 ratio during the day and a low D2/D3 ratio at night. Protracted quinpirole treatment differentially altered striatal D2/D3 rhythms depending on the time of injection (i.e., day or night). When quinpirole-induced locomotor activity was analyzed for 90 min, we found hypomotility after the first day or nighttime drug injection. By the seventh injection, daytime quinpirole treatment produced clear hyperactivity while nighttime quinpirole treatment continued to induce a significant initial hypoactivity followed by a hyperactivity period. Our data indicate that quinpirole-induced long-term alterations in the brain include dosing time-dependent changes in dopamine receptor rhythms. Therefore, we propose that diurnal mechanisms, which participate in drug-induced long-term changes in the dopamine system, are important for the development of dopaminergic behaviors.

Diurnal rhythms in cocaine sensitization and in Period1 levels are common across rodent species.

Circadian and seasonal rhythms in psychostimulant-induced behaviors have been reported in different species including humans. Using inbred mice, we recently reported that both cocaine sensitization and striatal "clock" gene Period1 (PER1 for protein) levels demonstrate a diurnal pattern that is maintained by the rhythm of pineal products N-acetylserotonin (NAS) and melatonin. It is well known that genetic background differences in inbred mice affect their behavioral traits. Therefore, to test whether our initial observations were limited to these mouse strains or whether these traits are common across rodent species we have tested additional strains of mice (CBA/J and AKR/J) and rats (Sprague-Dawley). We found that regardless of the species/strains, subjects with regular NAS and melatonin rhythms present diurnal cocaine sensitization and striatal PER1 rhythm. Since there is a growing interest in clock gene-mediated circadian mechanisms, these results may be important in designing experiments (e.g., time of day and subject strain) to study the role of these genes in psychiatric disorders such as addiction and depression.

Involvement of the pineal gland in diurnal cocaine reward in mice.

Contribution of circadian mechanisms to the psychostimulant-induced behaviors has been suggested. The pineal gland is important component of circadian mechanisms. Using pinealectomized mice and sham-operated controls, we tested the contribution of pineal gland to the rewarding effects of cocaine in conditioned place preference test. Experiments were performed both during the day and at night. Controls with intact pineal glands demonstrated significant decrease in cocaine-induced conditioned place preference at night compared to daytime, whereas pinealectomized mice did not show any diurnal differences. Circadian mechanisms regulated by the pineal gland thus appear critically involved in cocaine-induced reward.

The pineal gland and anxiogenic-like action of fluoxetine in mice.

Fluoxetine produces initial paradoxical anxiogenic effect in some patients. In an elevated plus-maze (EPM), fluoxetine triggers an anxiogenic-like effect in rodents. Behavioral responses to psychoactive drugs can be modified by the pineal gland. We assessed the actions of fluoxetine in the EPM in melatonin-proficient C3H mice, melatonin-deficient C57BL6 mice, and in sham-operated and pinealectomized mice. Mice were assayed 30 min after the first injection and on day 14. Protracted fluoxetine treatment reduced the time on the anxiogenic open arms and increased the entries into the safe closed arms in sham-operated C3H mice. Fluoxetine was ineffective in pinealectomized C3H or C57BL6 mice. It is possible that the pineal system contributes to the previously observed anxiogenic action of fluoxetine in humans.

Both aging and chronic fluoxetine increase S100B content in the mouse hippocampus.

S100B is a cytokine with neurotrophic and neurite-extending activity that has been implicated in the mechanism of action of anti-depressants and in the pathobiology of aging associated disorders such as Alzheimer's disease. The antidepressant fluoxetine increases hippocampal S100B content in young adult rats. In humans, brain levels of S100B mRNA and protein increase with advancing age. We assayed hippocampal S100B protein content in young (2 month) and old (24 month) mice, and in old mice treated for 2 weeks with fluoxetine. Using quantitative Western immunoblotting and an immunoassay kit we found higher S100B content in the hippocampus of old mice. Fluoxetine treatment of old mice further increased hippocampal S100B, suggesting that aging does not interfere with fluoxetine's action on hippocampal S100B.

Erythropoietin exerts neuroprotective effect in neonatal rat model of hypoxic-ischemic brain injury.

Hypoxic-ischemic encephalopathy seen in survivors of perinatal asphyxia is a frequently encountered and a major clinical problem for which there is currently no effective treatment. Hematopoietic neuroprotective agents, such as erythropoietin (EPO) may rescue neurons from cell death in this setting. EPO is a cytokine hormone that has neuroprotective effect in vitro and in vivo. In this study, we evaluated the effect of posthypoxic EPO administration in an animal model of neonatal hypoxic-ischemic injury. Our results show that a single intracerebroventricular injection of EPO immediately after hypoxic-ischemic insult in neonatal rat model of hypoxic-ischemia reduced the extent of hypoxic-ischemic brain damage. The mean infarct volume assessed 7 days after hypoxia was significantly smaller in EPO-treated group than in the control group. These findings suggest that EPO may provide benefit after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.

The pineal gland is critical for circadian Period1 expression in the striatum and for circadian cocaine sensitization in mice.

Sensitization to psychostimulants can be influenced by circadian rhythms. The pineal gland, the main source of circadian melatonin synthesis, may influence behavioral sensitization to cocaine; mice with normal melatonin rhythms do not get sensitized at night. Clock genes such as Period1 (Per1) show rhythmic region- and strain-dependent expression in the mouse brain, and mice mutant for the Per1 gene lack cocaine sensitization. Here, for the first time we show circadian changes of PER1 protein levels in the mouse striatum, a brain region crucial for the development of locomotor sensitization to cocaine. In male C3H/HeJ mice, we found peak striatal PER1 protein levels during the day; this was preceded by a Per1 mRNA peak 16 h earlier. Pinealectomized mice did not show this circadian pattern. We analyzed circadian cocaine sensitization at times when striatal PER1 protein levels in control mice (naive and sham-pinealectomized) were high and low, respectively. Only mice with circadian changes in striatal Per1 expression showed the night-time absence of cocaine sensitization, whereas pinealectomized mice were without circadian changes in striatal Per1 and were sensitized to cocaine regardless of diurnal rhythm. Our results indicate that both the striatal circadian Per1 expression and diurnal locomotor cocaine sensitization are strongly influenced by pineal products. Since we found evidence for the expression of melatonin receptor mRNA in the striatum, we suggest that further studies on pineal-driven mechanisms will help us better understand the mechanisms of drug abuse and identify novel targets for the prevention and/or treatment of addictions.

Neuroprotective effect of erythropoietin on hypoxic-ischemic brain injury in neonatal rats.

Erythropoietin (Epo) prevents ischemia and hypoxia-induced neuronal death in vitro. Recent studies have shown that this cytokine also has in vivo neuroprotective effects in cerebral and spinal ischemia in adult rodents. In this study, we aimed to investigate the effect of systemically administered recombinant human Epo on infarct volume and apoptotic neuronal death in a newborn rat hypoxic-ischemic brain injury model. Our results showed that a single dose of intraperitoneal Epo treatment (1,000 U/kg) significantly decreased the mean infarct volume as compared to the control group. In contrast to the Epo-treated group, histopathological examination by positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of the affected brain in control animals revealed widespread neuronal injury associated with numerous apoptotic cells. Morphometric analysis to determine the extent of damage quantitatively ascertained that the mean infarct volume was significantly lower in the Epo-treated group (p < 0.003). These results suggest the beneficial neuroprotective effect of Epo in this model of neonatal hypoxic-ischemic brain injury. To our knowledge, this is the first study that demonstrates a protective effect of Epo against hypoxia-ischemia in the developing brain.

Erythropoietin restores glutathione peroxidase activity in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced neurotoxicity in C57BL mice and stimulates murine astroglial glutathione peroxidase production in vitro.

Recently, we have reported that erythropoietin (Epo) provides neuroprotection in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-induced neurotoxicity in vivo. In the present study, we investigated the effects of single Epo administration on brain antioxidant enzyme (superoxide dismutase (SOD) and glutathion peroxidase (GSHPx)) activities in this model in C57BL/6 mice. We found that MPTP treatment decreased GSHPx activity in both substantia nigra and striatum, and Epo restores nigral GSHPx activity decreased by MPTP. SOD enzyme activity was not significantly changed by MPTP and Epo treatment. Further, Epo stimulated astroglial GSHPx production in neonatal murine astroglial cell culture suggesting that the possible cell source for the stimulation of GSHPx activity by Epo in the MPTP-induced neurotoxicity model are astroglia. In conclusion, modulation of the astroglial antioxidant defense system might be one of the mechanisms by which Epo exerts a beneficial effect in MPTP-induced Parkinsonism.

Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury.

The cytokine erythropoietin (EPO) possesses potent neuroprotective activity against a variety of potential brain injuries, including transient ischemia and reperfusion. It is currently unknown whether EPO will also ameliorate spinal cord injury. Immunocytochemistry performed using human spinal cord sections showed abundant EPO receptor immunoreactivity of capillaries, especially in white matter, and motor neurons within the ventral horn. We used a transient global spinal ischemia model in rabbits to test whether exogenous EPO can cross the blood-spinal cord barrier and protect these motor neurons. Spinal cord ischemia was produced in rabbits by occlusion of the abdominal aorta for 20 min, followed by saline or recombinant human (rHu)-EPO (350, 800, or 1,000 units/kg of body weight) administered intravenously immediately after the onset of reperfusion. The functional neurological status of animals was better for rHu-EPO-treated animals 1 h after recovery from anesthesia, and improved dramatically over the next 48 h. In contrast, saline-treated animals exhibited a poorer neurological score at 1 h and did not significantly improve. Histopathological examination of the affected spinal cord revealed widespread motor neuron injury associated with positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling in control but not in rHu-EPO-treated animals. These observations suggest both an acute as well as a delayed beneficial action of rHu-EPO in ischemic spinal cord injury. Because rHu-EPO is currently used widely with an excellent safety profile, clinical trials evaluating its potential to prevent motor neuron apoptosis and the neurological deficits that occur as a consequence of ischemic injury are warranted.