Kappa- and delta-opioid receptor functional activities are increased in the caudate putamen of cannabinoid CB1 receptor knockout mice

The purpose of this study was to examine the functional interaction between endogenous opioid and cannabinoid receptor systems in the caudate putamen and nucleus accumbens. We therefore examined by autoradiography the functional activity and density of micro-, kappa- and delta-opioid receptors in both brain regions of cannabinoid CB1 receptor knockout mice. Functional activity was estimated by measuring agonist-stimulated [35S]GTPgammaS binding. Results showed that deletion of the CB1 cannabinoid receptor markedly increased kappa-opioid (50%) and delta-opioid (42%) receptor activities whereas no differences were found in micro-opioid receptor in the caudate putamen. In contrast, binding autoradiography showed a similar density of micro-, kappa- and delta-opioid receptors between mutant and wild-type mice. No differences were found in densities or activities of micro-, kappa- and delta-opioid receptors between mutant and wild-type mice in the nucleus accumbens. Taken together, our results revealed that deletion of CB1 cannabinoid receptors produced a pronounced increase in the activity of kappa- and delta-opioid receptors in the caudate putamen. This endogenous interaction between opioid and cannabinoid receptors may be relevant to further understand a variety of neuroadaptative processes involving the participation of opioid receptors, such as motor behaviour, emotional responses and drug dependence.     

Assessing level of consciousness and cognitive changes from vegetative state to full recovery

Although investigations addressing cognitive recovery from the vegetative state have been reported, to date there have been no detailed studies of these patients combining both neuropsychology and functional imaging to monitor and record the recovery of consciousness. This paper describes the recovery of a specific vegetative state (VS) case. The patient (OG) remained in the vegetative state for approximately two months, increasing her level of awareness to a minimally conscious state, where she continued for approximately 70 days. In the course of the ensuing 18 months, she was able to reach an acceptable level of cognitive functioning, with partial levels of independence. Throughout this two year period, she received continuous cognitive evaluation, for which several different tools were applied including coma and low functioning scales, full cognitive batteries, and structural and functional magnetic resonance imaging (MRI). We present here preliminary data on fMRI using a word presentation paradigm before and after recovery; we also discuss the difficulty of how to determine level of consciousness using the tools currently available, and the subsequent improvement in different cognitive domains. We confirm that accurate diagnosis and proper cognitive assessment are critical for the rehabilitation of patients with disorders of consciousness.     

The presence of glia stimulates the appearance of glycinergic synaptic transmission in spinal cord neurons

Previous studies, using electrophysiological and fluorimetric analysis with a calcium sensitive dye, have shown that 5-7 DIV developing spinal cord neurons displayed high levels of glycinergic transmission. GABAergic and AMPAergic neurotransmission contributed much less to the overall transmission. Here, we show that culturing neurons in absence of a glia cell monolayer reduced the frequency of glycinergic spontaneous IPSCs (0.1 +/- 0.01 Hz), without altering the level of overall transmission (3 +/- 1.1 Hz). The predominant transmission was mediated by GABA(A) receptors (72% of total synaptic events). In addition, combination of bicuculline and CNQX blocked synaptically mediated calcium transients recorded with fluo-3. Furthermore, application of glycine revealed the presence of extrasynaptic receptors in these neurons (25 +/- 6 pA/pF). Culturing neurons on a glial cell monolayer increased the frequency of glycinergic currents (0.4 +/- 0.02 Hz), without changing the amplitude of the current (20 +/- 4 pA). The use of a glia-conditioned media reversed the effect of growing the neurons in a glia-deprived condition. These results indicate that the establishment of glycinergic transmission is dependent on the presence of a glia derived soluble factor. However, functional GlyRs were still able to insert in the neuronal membrane in a glia-independent manner.     

Proliferation markers in the adult rodent brain: bromodeoxyuridine and proliferating cell nuclear antigen

The rostral migratory stream is one of the few regions of the adult mammalian central nervous system in which cellular migration and proliferation have been described. Most rostral migratory stream cells divide rapidly and hence different proliferation markers have been employed to identify them. Nitrogen base substitutes, such as tritiated thymidine or 5-bromo-2'-deoxyuridine (BrdU), together with endogenous molecules, such as Proliferating Cell Nuclear Antigen (PCNA), are the cell cycle markers most widely employed. Protocols for BrdU and PCNA localization are both plentiful and diverse, but to date no optimized protocol for obtaining trustworthy double staining of both markers has been described. In this work, we propose optimized protocols for achieving both single staining and the joint detection of BrdU and PCNA in the rodent brain using double-immunofluorescence procedures. The double labeling described allows the discrimination of different cell cycle stages in migratory cells from the mouse brain.     

Neuromonitoring of cochlea and auditory nerve with multiple extracted parameters during induced hypoxia and nerve manipulation

A system capable of comprehensive and detailed monitoring of the cochlea and the auditory nerve during intraoperative surgery was developed. The cochlear blood flow (CBF) and the electrocochleogram (ECochGm) were recorded at the round window (RW) niche using a specially designed otic probe. The ECochGm was further processed to obtain cochlear microphonics (CM) and compound action potentials (CAP). The amplitude and phase of the CM were used to quantify the activity of outer hair cells (OHC); CAP amplitude and latency were used to describe the auditory nerve and the synaptic activity of the inner hair cells (IHC). In addition, concurrent monitoring with a second electrophysiological channel was achieved by recording compound nerve action potential (CNAP) obtained directly from the auditory nerve. Stimulation paradigms, instrumentation and signal processing methods were developed to extract and differentiate the activity of the OHC and the IHC in response to three different frequencies. Narrow band acoustical stimuli elicited CM signals indicating mainly nonlinear operation of the mechano-electrical transduction of the OHCs. Special envelope detectors were developed and applied to the ECochGm to extract the CM fundamental component and its harmonics in real time. The system was extensively validated in experimental animal surgeries by performing nerve compressions and manipulations.     

Improved long-term allograft function in pediatric renal transplantation with mycophenolate mofetil

MMF has been shown to decrease the incidence of acute rejection in children and adults at 1 and 3 yr. Other beneficial effects of MMF have been more difficult to demonstrate. Our open-labeled study presents a 5-yr data for patients and graft survival, allograft function, and growth in MMF-treated patients. The trial included 29 patients who were treated with MMF in combination with cyclosporine and methylprednisone. Patients were compared with a preceding group of 29 patients treated with AZA instead of MMF. Patient and graft survival rate 5 yr after transplantation were 97 and 90% in the MMF group vs. 93 and 83% in the AZA group (p: NS). Acute rejection was 20.6% in the MMF group vs. 58.6% in the AZA group (p < 0.01). Chronic rejection was 10.3% in the MMF group and 25% in the AZA group (p: NS). The changes in the creatinine clearance from baseline to 5 yr (Delta) were different between groups (-6.0 +/- 5.1 mL/min/1.73 m(2) in the MMF group vs. -22.2 +/- 7.6 mL/min/1.73 m(2) in the AZA group, p < 0.05). Also, the slope of 1/Scr showed a significant lower incidence of worsening renal function after the second year of renal transplantation (p < 0.0001) in the MMF group compared with the AZA group. Delta Height SDS in prepubertal patients was 0.3 +/- 0.4 SDS in the MMF group vs. -0.8 +/- 0.2 SDS in the AZA group (p < 0.05). This study shows that long-term MMF therapy has resulted in a decrease in acute rejection and was associated with a protection against renal function deterioration. The use of MMF enables a reduction in the dose of steroids and leads to a linear growth improvement of children after renal transplantation.     

Intra-aortic filtration: capturing particulate emboli during aortic cross-clamping

Particulate emboli are an important factor in the development of complications after cardiac surgery. The use of an intra-aortic filter during aortic declamping has proved safe and effective in reducing the incidence of embolic complications. Because aortic cross-clamping is also associated with the risk of embolism, we measured the release of particulate emboli at the time of aortic cross-clamping and at declamping. We deployed intra-aortic filters (Edwards EMBOL-X Slim Protection System) at 2 different times during 15 consecutive cardiac procedures with cardiopulmonary bypass. Filter A was inserted before aortic cross-clamping and left in place for a mean time of 3 minutes, and Filter B was inserted before aortic declamping for a mean of 17 minutes. An independent institution performed histopathologic examination. All filters captured particulate emboli, independent of the patients' degree of proximal aortic atherosclerotic disease. Macroscopy showed that Filter A captured more particles (mean, 70 +/- 2.6) in 7 of 15 patients (46.7%), Filter B captured more particles (mean, 6.7 +/- 2.6) in 5 of 15 patients (33.3%), and the filters captured the same number in 3 of 15 patients (20%). Microscopy showed that both filters captured fibrous atheroma or fibrous cap in 10 of 15 patients. We observed no complications related to the use of the filters. Aortic cross-clamping is associated with a high risk of embolism. We suggest that optimal protection against embolism during cardiac surgery can be achieved by applying intra-aortic filtration not only before aortic declamping but also before aortic cross-clamping.     

Clock gene polymorphisms and narcolepsy in positive and negative HLA-DQB1*0602 patients

Narcolepsy is a chronic sleep disorder. It is linked to the HLA-DQB1*0602 allele. A recent report established a genetic linkage between narcolepsy and the chromosomal region 4p13-q21 that contains the Clock gene. We studied two SNPs in the Clock gene aiming to find any association with narcolepsy. We did not find differences in genetic frequencies in the patients group. We concluded that these two SNPs are not associated with narcolepsy.