Neuroprotective Effects of Ozone Therapy After Sciatic Nerve Cut Injury.

A number of antioxidants have been used to treat peripheral nerve injury. However, there are few definitive experimental studies of ozone therapy for peripheral nerve cut injury. We aimed to examine the effects of mild level ozone therapy on sciatic nerve regeneration. One hundred adult male Wistar albino rats were randomly divided into four groups: group 1 (n=20) no cut injury or therapy; group 2 (n=20) sham; group 3 (n=30) nerve cut injury, no therapy; group 4 (n=30) nerve cut injury and ozone therapy. Sciatic functional index (SFI) and withdrawal reflex (WDR) were measured for all groups before nerve cut, at postoperative day 1, and at weeks 2, 4, 6 and 8. More myelinated (M) nerve fibers were observed after nerve cut injury in the ozone-therapy group. Significant differences were seen in plasma SOD (superoxide dismutase), CAT (catalase) and GPx (glutathione peroxidase) activities (p<0.05), and significant functional improvement was observed at postoperative weeks 2 and 4 (p<0.05) after ozone treatment. This is the first study conducted for the purpose of examining the effects of ozone therapy on sciatic nerve cut injury.

The inflammatory response to sciatic nerve injury in a familial amyloidotic polyneuropathy mouse model.

Inflammation is a hallmark of several neurodegenerative disorders including familial amyloidotic polyneuropathy (FAP). FAP is associated with extracellular deposition of mutant transthyretin (TTR), leading to degeneration of cells and tissues, particularly in the peripheral nervous system (PNS). With this work, our goal was to characterize the expression/deposition of TTR and the associated inflammatory immune response, induced by nerve injury, in WT mice and in a mouse model carrying the most common TTR mutation in FAP (V30M). Our results indicate that upon nerve injury TTR is significantly produced by Schwann cells and is dynamically regulated over time in V30M mice, accompanying a peak of inflammation. Strikingly, V30M TTR deposition in nerve tissue occurred, suggesting that inflammation contributes to TTR polymerization. In response to nerve injury, V30M mice display a downregulated innate immune response when compared to WT mice. More specifically, we saw decreased expression of cytokines and chemokines important for the recruitment of immune cells like macrophages and neutrophils, known to be important for the tissue regenerative process which was found impaired in V30M mice. In conclusion, with this work we were able to characterize the biology of TTR both in WT and V30M animals, upon nerve injury, and found that V30M TTR impairs the inflammatory response necessary for nerve regeneration. Taken together, our findings suggest that inflammation is an important target to be considered in therapeutic strategies for FAP.

Multimodal assessment of nervous and immune system responses following sciatic nerve injury.

Subsequent to peripheral nerve compression and irritation, pathophysiological processes take place within nervous and immune systems. Here, we utilized a multimodal approach to comprehend peripheral and central soft tissue changes as well as alterations occurring in systemic analytes following unilateral chronic constriction injury (CCI) of the sciatic nerve in rodents. Using magnetic resonance imaging and [18F]-2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography, we demonstrated robust structural abnormalities and enhanced FDG uptake within the injured nerve and surrounding muscle, respectively. To assess whether central morphological changes were induced by nerve injury, diffusion tenor imaging was performed. A decrease in fractional anisotropy in primary motor cortex contralateral to the injury site was observed. Evaluation of a panel of circulating cytokines, chemokines, and growth factors showed decreased levels of interleukin-1ß and Fractalkine in CCI animals. Area under the receiver operating curve (ROC) calculations of analyte levels, imaging, and behavioral end points ranged from 0.786 to 1, where behavioral and peripheral imaging end points (eg, FDG uptake in muscle) were observed to have the highest discriminatory capabilities (maximum area under ROC = 1) between nerve injury and sham conditions. Lastly, performance of correlation analysis involving all analyte, behavioral, and imaging data provided an understanding of the overall association amongst these end points, and importantly, a distinction in correlation patterns was observed between CCI and sham conditions. These findings demonstrate the multidimensional pathophysiology of sciatic nerve injury and how a combined analyte, behavioral, and imaging assessment can be implemented to probe this complexity.

Dynamic changes of ICAM-1 expression in peripheral nervous system following sciatic nerve injury.

The injuries of the peripheral nerve are the results of trauma, disease, and surgical procedures that require section of peripheral nerves to gain access to the surgical site. Unlike elements of the central nervous system, peripheral nerves can regenerate when damaged. Recent studies provide evidence that intercellular adhesion molecule-1 (ICAM-1) is involved not only in inflammation, but also in cell recruitment during Wallerian degeneration after peripheral nerve injury. In our studies, two different injury models (crush and transect) for peripheral nerve regeneration were used. We investigated the dynamic changes of ICAM-1 expression in peripheral nervous system, including sciatic nerves and the dorsal root ganglia (DRG) following sciatic nerve injury. Double labeling demonstrated the presence of ICAM-1 within both Schwann cells and nerve blood vein endothelia cells in sciatic nerve after injury. In ipsilateral DRG, a few ICAM-1 signals were found in satellite cells, and ICAM-1 staining was mainly distributed in neuron. The results implied that ICAM-1 might be correlated with the function of Schwann cells about myelinogenesis and nerve regeneration.

Methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle and promotes nerve regeneration in a rat sciatic nerve injury model.

Methylcobalamin is a vitamin B12 analog and is necessary for the maintenance of the nervous system. Although some previous studies have referred to the effects of methylcobalamin on neurons, the precise mechanism of this effect remains obscure. Here we show that methylcobalamin at concentrations above 100 nM promotes neurite outgrowth and neuronal survival and that these effects are mediated by the methylation cycle, a metabolic pathway involving methylation reactions. We also demonstrate that methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle. In a rat sciatic nerve injury model, continuous administration of high doses of methylcobalamin improves nerve regeneration and functional recovery. Therefore, methylcobalamin may provide the basis for better treatments of nervous disorders through effective systemic or local delivery of high doses of methylcobalamin to target organs.

Dietary supplement with fermented soybeans, natto, improved the neurobehavioral deficits after sciatic nerve injury in rats.

Clearance of fibrin and associated inflammatory cytokines by tissue-type plasminogen activator (t-PA) is related to improved regeneration in neurological disorder. The biological activity of fermented soybean (natto) is very similar to that of t-PA. We investigated the effect of the dietary supplement of natto on peripheral nerve regeneration. The peripheral nerve injury was produced by crushing the left sciatic nerve with a vessel clamp in Sprague-Dawley rats. The injured animals were fed orally either with saline or natto (16 mg/day) for seven consecutive days after injury. Increased functional outcome such as sciatic nerve functional index, angle of ankle, compound muscle action potential and conduction latency were observed in natto-treated group. Histological examination demonstrated that natto treatment improved injury-induced vacuole formation, S-100 and vessel immunoreactivities and axon loss. Oral intake of natto prolonged prothrombin time and reduced fibrinogen but did not change activated partial thromboplastin time and bleeding time. Furthermore, natto decreased injury-induced fibrin deposition, indicating a tolerant fibrinolytic activity. The treatment of natto significantly improved injury-induced disruption of blood-nerve barrier and loss of matrix component such as laminin and fibronectin. Sciatic nerve crush injury induced elevation of tumor necrosis factor alpha (TNF-alpha) production and caused apoptosis. The increased production of TNF-alpha and apoptosis were attenuated by natto treatment. These findings indicate that oral intake of natto has the potential to augment regeneration in peripheral nerve injury, possibly mediated by the clearance of fibrin and decreased production of TNF-alpha.

Changes in cardiovascular parameters and plasma norepinephrine level in rats after chronic constriction injury on the sciatic nerve.

To evaluate whether neuropathic pain affects autonomic nervous activities, we investigated daily change in cardiovascular parameters and plasma norepinephrine (NE) in free-moving rats after chronic constriction injury (CCI) on the sciatic nerve. Arterial blood pressure (BP), heart rate (HR), and the power spectrum of pulse interval variability were analyzed. Daily change in motor activity and nociceptive behavior was also measured from some CCI rats. In others, NE from daily blood samples was quantified and spontaneous pain was evaluated by daily monitoring of foot guarding behavior. We identified three stages in the daily change of cardiovascular parameters and plasma NE level over 3 weeks following CCI. The first stage (up to 3 days after the surgery) was characterized by increased MAP and HR, especially in the daytime, even though plasma NE was unchanged and motor activity decreased. The second stage (mid first to mid second postoperative weeks) was characterized by increased daytime MAP and HR, and the animals developed punctate hyperalgesia in the affected hindpaw. An NE surge that may have been related to spontaneous pain was present 3-5 days after CCI. The third stage, which appeared after the second postoperative week, was characterized by normalized MAP and decreased HR, and increased high-frequency (0.8-3.0Hz) power in pulse interval variability, which is an index of cardiac parasympathetic tone. These results demonstrated that cardiovascular function was kept high through sympathetic and non-sympathetic activity for 2 weeks after CCI, followed by a predominance of parasympathetic tone.

Antiphospholipid antibodies and ischemic neuropathy following cardiac surgery.

Sciatic nerve palsy is an uncommon complication of cardiac surgery and is thought to be induced by a combination of reduced femoral artery blood flow, small vessel vascular disease or prolonged hypoxia. We here describe a new case which is the first described with transient elevation of antiphospholipid antibodies. Although transient elevation of lupus coagulation inhibitor is known to occur frequently in patients treated in an intensive care unit, there are very few data about the possible role of antiphospholipid antibodies in the generation of ischemic neuropathies. We can not prove that the ischemic neuropathy in our case has been favored by the presence of lupus coagulation inhibitor and antiphospholipid antibodies as the occurrence of the symptoms seemed to precede the transient elevation of lupus coagulation inhibitor. This case suggests that antiphospholipid antibodies and lupus coagulation inhibitor should be included in the work up of patients who present nerve damage after cardiac surgery but further studies are needed to ascertain this association.

Inhibitory effect of anandamide on resiniferatoxin-induced sensory neuropeptide release in vivo and neuropathic hyperalgesia in the rat.

Anandamide (AEA) is an endogenous cannabinoid ligand acting predominantly on the cannabinoid 1 (CB(1)) receptor, but it is also an agonist on the capsaicin VR(1)/TRPV(1) receptor. In the present study we examined the effects of AEA and the naturally occurring cannabinoid 2 (CB(2)) receptor agonist palmitylethanolamide (PEA) on basal and resiniferatoxin (RTX)-induced release of calcitonin gene-related peptide (CGRP) and somatostatin in vivo. Since these sensory neuropeptides play important role in the development of neuropathic hyperalgesia, the effect of AEA and PEA was also examined on mechanonociceptive threshold changes after partial ligation of the sciatic nerve. Neither AEA nor PEA affected basal plasma peptide concentrations, but both of them inhibited RTX-induced release. The inhibitory effect of AEA was prevented by the CB(1) receptor antagonist SR141716A. AEA abolished and PEA significantly decreased neuropathic mechanical hyperalgesia 7 days after unilateral sciatic nerve ligation, which was antagonized by SR141716A and the CB(2) receptor antagonist SR144528, respectively. Both SR141716A and SR144528 increased hyperalgesia, indicating that endogenous cannabinoids acting on CB(1) and peripheral CB(2)-like receptors play substantial role in neuropathic conditions to diminish hyperalgesia. AEA and PEA exert inhibitory effect on mechanonociceptive hyperalgesia and sensory neuropeptide release in vivo suggesting their potential therapeutical use to treat chronic neuropathic pain.

A chronic-constriction injury of the sciatic nerve reduces bilaterally the responsiveness to formalin in rats: a behavioral and hormonal evaluation.

UNLABELLED: Application of four loose ligatures to the sciatic nerve of a rat (chronic constriction injury [CCI]) induces clear hypersensitivity to non-noxious stimulation and chemical irritants. However, in this study, an injection of formalin in the hind paw of a rat with CCI-induced mononeuropathy resulted in an ipsilateral decreased flinching and licking or biting behavior in both phases of the formalin testing. The effect was independent of the formalin concentration used. This altered behavior was accompanied with smaller plasma levels of adrenocorticotrope hormone and corticosterone compared with sham and non-operated animals. Formalin injection in the contralateral nonligated hind paw of CCI rats also reduced the licking or biting behavior as compared with sham-operated and non-operated control animals only in the second phase of the formalin test. Thus, CCI reduces the pain reactivity and hypothalamic-pituitary-adrenal-axis activation to ipsilateral and contralateral formalin injection. Further research should investigate whether the decreased pain reactivity by CCI is situated at the peripheral, spinal, or supraspinal level or is result of changes in the stress reactivity and coping strategies. IMPLICATIONS: We evaluated the changes in the behavioral reactions and the hormonal effects of a noxious chemical stimulus, i.e., formalin injection in animals with previously induced chronic constriction injury to the sciatic nerve. The effect in animals injected at the ipsilateral and contralateral site, sham-operated and controls, were compared.

Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model.

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.