Involvement of kinin B1 receptor and oxidative stress in sensory abnormalities and arterial hypertension in an experimental rat model of insulin resistance

Diabetes Mellitus leads to pain neuropathy and cardiovascular complications which remain resistant to current therapies involving the control of glycaemia. This study aims at defining the contribution of kinin B(1) receptor (B(1)R) and the oxidative stress on sensory abnormalities and arterial hypertension in a rat model of insulin resistance. Rats were fed with 10% d-glucose for a chronic period of 12-14 weeks and the impact of a diet supplemented with alpha-lipoic acid, a potent antioxidant, was determined on tactile and cold allodynia, arterial hypertension and the expression of kinin B(1)R (real-time PCR and autoradiography) in several tissues. Acute effects of brain penetrant (LF22-0542) and peripherally acting (R-715) B(1)R antagonists were also assessed. Glucose-fed rats exhibited tactile and cold allodynia along with increases in systolic blood pressure between 4 and 12 weeks; these alterations were alleviated by alpha-lipoic acid. The latter regimen also decreased significantly increased plasma levels of insulin and glucose and insulin resistance (HOMA index) at 14 weeks. B(1)R mRNA was virtually absent in liver, aorta, lung, kidney and spinal cord isolated from control rats, yet B(1)R mRNA was markedly increased in all tissues in glucose-fed rats. Up-regulated B(1)R mRNA and B(1)R binding sites (spinal cord) were significantly reduced by alpha-lipoic acid in glucose-fed rats. LF22-0542 reduced tactile and cold allodynia (3h) and reversed arterial hypertension (3-48h) in glucose-fed rats. R-715 abolished tactile and cold allodynia but had not effect on blood pressure. Data suggest that the oxidative stress contributes to the induction and up-regulation of B(1)R in the model of insulin resistance induced by glucose feeding. The over expressed B(1)R contributes centrally to arterial hypertension and in the periphery to sensory abnormalities.     

Endogenous Activation of adenosine A1 receptors promotes post-ischemic electrocortical burst suppression

Following transient global cerebral ischemia (GCI), spontaneous electrocortical activity resumes from the isoelectric line through a sequence of “bursts” of activity alternating with periods of electrical “suppression,” commonly referred to as the post-ischemic burst suppression (BS) pattern. Several lines of evidence suggest that BS reflects an impairment of neocortical connectivity. Here we tested in vivo whether synaptic depression by adenosine A₁ receptor (A₁R) activation contributes to BS patterns following GCI. Male Wistar rats were subjected to 1, 5 or 10 min of GCI using a “four-vessel occlusion” model under chloral hydrate anesthesia. Quantification of BS recovery was carried out using BS ratio. During GCI full electrocortical suppression was attained (BS ratio reached 100%). During the following reperfusion the BS ratio returned to 0. The time course of the decay was exponential after 1 and 5-min GCI and bi-exponential after 10-min GCI. The BS recovery was progressively delayed with the duration of ischemia. Administration of the A₁R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 1.25 mg/kg i.p.) accelerated the post-ischemic BS recovery for all GCI durations. Following the 10-min GCI the effect of DPCPX was only apparent on the initial fast decay of the BS ratio. These data suggest that endogenous adenosine release promotes BS patterns during reperfusion following transient cerebral ischemia. Furthermore, the endogenous A₁R activation may be the primary underlying cause of post-ischemic BS patterns following brief ischemic episodes. It is likely that synaptic depression by post-ischemic A₁R activation functionally disrupts the connectivity within the cortical networks to an extent that promotes BS patterns.     

Intratumoural level of SDF-1 correlates with survival in head and neck squamous cell carcinoma

The SDF-1/CXCR4 pathway has been suggested to play a role in the metastatic dissemination of various tumours. We assessed the prognostic impact of SDF-1 and CXCR4 expression in head and neck squamous cell carcinoma (HNSCC). Seventy-one HNSCC samples collected at the time of initial diagnosis were retrospectively analysed. SDF-1 and CXCR4 expression levels were measured using real-time RT-PCR and correlated to survival. After a median follow-up of 45 months, 25 patients (35%) died of cancer (group D), and 46 patients (65%) were alive or dead without evidence of HSNCC evolution (group A). The median level of CXCR4 expression was 0.33 and 0.29 in groups A and D, respectively (P=0.93), showing no correlation with recurrence or survival. By contrast, the median level of SDF-1 expression was significantly different in the A and D groups (2.41 vs 1.16, respectively, P=0.018). Using the median level as a cut-off, patients with low SDF-1 had poorer metastasis-free (P=0.026), disease-free (P=0.006) and overall specific survival rates (P=0.002). The prognostic value of SDF-1 was confirmed by a multivariate analysis. In this series of 71 HNSCC patients, the SDF-1 expression level correlated significantly with metastatic evolution and overall survival.     

Multiple sclerosis patients show sexual dimorphism in cytokine responses to myelin antigens

Multiple sclerosis affects more women than men. The reasons for this are unknown. Previously, we have shown significant differences in women versus men in inflammatory cytokine responses to the major protein component of myelin, proteolipid protein (PLP), which is thought to be a target in MS patients. Here, using the ELISPOT assay, we examined sex differences in single-cell secretion of Th1 and Th2 cytokines from freshly isolated PBMC between relapsing remitting (RR) MS patients and healthy individuals. Cells were stimulated with MS-associated antigens including proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and non-disease related antigens. Our data show a sex bias in the cytokine responses to multiple MS-relevant myelin antigens: Women with MS show IFNgamma-skewed responses and men with MS show IL-5-skewed responses. These data extend our previous findings [Pelfrey, C.M., Cotleur, A.C., Lee, J.C., Rudick, R.A. 2002. Sex differences in cytokine responses to myelin peptides in multiple sclerosis. J. Neuroimmunol. 130, 211-223.]: (1) by demonstrating gender skewing in cytokine responses to an expanded myelin antigen repertoire, which includes MBP, MOG and PLP; (2) by showing TNFalpha and IL-10 do not display comparable gender skewing compared to IFNgamma and IL5; (3) by defining the patient population as early, untreated RRMS patients to avoid confounding factors, such as different disease stages/disability and immunomodulatory therapy; and (4) by showing HLA type does not appear to underlie the gender differences. These findings may explain increased susceptibility to MS in women and could contribute to the differences in disease severity between men and women.     

Acute energy restriction triggers Wallerian degeneration in mouse

Acute exposure of peripheral axons to the free radical Nitric Oxide (NO) may trigger conduction block and, if prolonged, Wallerian degeneration. It was hypothesized that this neurotoxic effect of NO may be due primarily to energy restriction by inhibition of mitochondrial respiration. We compared the neurotoxic effect of NO with the effect of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) on electrically active axons of mouse sciatic nerve. The right tibial nerve was stimulated at the ankle. Muscle responses were recorded from plantar muscles and ascending nerve action potentials were recorded form the exposed sciatic nerve by means of a hook electrode. The sciatic nerve was focally immersed over a length of 1 cm in either phosphate buffered saline (PBS), a solution of ∼ 4 μM NO obtained from 10 mM of the NO-donor DETA NONOate, or a solution of up to 1 mM DNP. Following 3 hours of 200 Hz stimulation, the nerves were washed in PBS for 1 hour, the surgical wounds were closed and the mice were left to recover. Following repetitive stimulation in PBS, the nerve responses recovered within 1 hour and the muscle responses within 1 day. The effects of focal acute exposure to NO or DNP were similar: (i) a transient conduction failure that rapidly normalized within one hour of washout and (ii) subsequent Wallerian degeneration of some axons confirmed at morphological studies. Taken together, these data support the hypothesis that neurotoxicity may be caused by energy restriction. Since the pharmacologic effect of NO and DNP was only transient, our data suggest that even a brief period of focal energy restriction can trigger Wallerian degeneration.     

In vivo transfer of interleukin-1 receptor antagonist gene in osteoarthritic rabbit knee joints: prevention of osteoarthritis progression

The goal of this study was to determine the efficacy of local IL-1Ra gene therapy by intra-articular plasmid injections on structural changes in the meniscectomy rabbit model of osteoarthritis. A partial meniscectomy of the right knee was performed on the rabbits through a medial parapatellar incision. The rabbits were then divided into four experimental groups. Group 1 received no treatment. Group 2 received three consecutive intra-articular injections at 24-hour intervals of 0.9% saline containing a lipid, gammaAP-DLRIE/DOPE, and a DNA plasmid, VR1012. Group 3 received three consecutive injections of saline containing 1000 microg of canine IL-1Ra plasmid and lipid. The injections were given starting 4 weeks post-surgery. Rabbits from Group 1 were killed 4 weeks post-surgery, and all other rabbits 8 weeks post-surgery. The severity of macroscopic and microscopic changes on cartilage on the medial and femoral condyles and tibial plateaus and synovium were graded separately. Specimens were also processed for immunohistochemical staining using a rabbit polyclonal antibody against canine IL-1Ra. The level of canine IL-1Ra in synovial fluid was determined using enzyme-linked immunosorbent assay. The presence of the DNA plasmid in the synovium was tested by polymerase chain reaction. A significant reduction in the width of osteophytes and size of macroscopic lesions (P < 0.04) was observed, and was dependent on the amount of IL-1Ra plasmid injected. A significant reduction was also noted in the severity of histologic cartilage lesions (P < 0.01) in the group that received the highest dosage (1000 microg) of IL-1Ra plasmid. IL-1Ra was detected in synovial fluid by enzyme-linked immunosorbent assay and by immunohistochemical staining in the synovium and cartilage of rabbits that received injections containing the IL-1Ra plasmid. Polymerase chain reaction analysis of synovial DNA revealed the presence of the cloned cDNA dog IL-1Ra up to 4 weeks after the first intra-articular injection. This study demonstrates that direct in vivo transfer of the IL-1Ra gene into osteoarthritis knee cells using intra-articular injections of a plasmid vector and lipids can significantly reduce the progression of experimental osteoarthritis. This avenue may therefore represent a promising future treatment for osteoarthritis.     

Mechanisms of hyperpolarization in regenerated mature motor axons in cat

We found persistent abnormalities in the recovery of membrane excitability in long-term regenerated motor nerve fibres in the cat as indicated in the companion paper. These abnormalities could partly be explained by membrane hyperpolarization. To further investigate this possibility, we compared the changes in excitability in control nerves and long-term regenerated cat nerves (3–5 years after tibial nerve crush) during manoeuvres known to alter axonal membrane Na⁺–K⁺ pump function: polarization, cooling to 20°C, reperfusion after 10 min ischaemia, and up to 60 s of repetitive stimulation at 200 Hz. The abnormalities in excitability of regenerated nerves were reduced by depolarization and cooling and increased by hyperpolarization and during postischaemia. Moreover, the time course of recovery of excitability from repetitive stimulation and ischaemia was prolonged in regenerated nerves. Our data are consistent with an increased demand for electrogenic Na⁺–K⁺ pumping in regenerated nerves leading to membrane hyperpolarization. Such persistent hyperpolarization may influence the ability of the axon to compensate for changes in membrane potential following normal repetitive activity.     

Exact one-sided confidence limits for the difference between two correlated proportions

We construct exact and optimal one-sided upper and lower confidence bounds for the difference between two probabilities based on matched binary pairs using well-established optimality theory of Buehler. Starting with five different approximate lower and upper limits, we adjust them to have coverage probability exactly equal to the desired nominal level and then compare the resulting exact limits by their mean size. Exact limits based on the signed root likelihood ratio statistic are preferred and recommended for practical use.