Maria Albani

Reorganization of motor units in reinnervated muscles of the rat

Changes in motor unit organisation following nerve injury in adult and neonatal rats were compared. Motor units were studied in extensor digitorum longus muscles reinnervated after nerve injury in either neonatal or adult rats. The force developed by individual motor units was measured by stimulating ventral root filaments. After nerve section in adult rats the distribution of motor unit force was restored to normal but this did not occur following nerve crush in neonatal animals. Thus following nerve injury during the neonatal period the muscles were not only permanently weaker, but the distribution of motor unit sizes was also abnormal. Muscle fibres belonging to a single motor unit were identified histologically by the glycogen depletion method, and their fibre type and cross-sectional area measured. Although all the fibres of the same unit became histochemically homogeneous, they showed greater variation in size than normal units, suggesting that factors other than the influence of the axons control the size of muscle fibres.

Alcohol Affects the Brain's Resting-State Network in Social Drinkers

Acute alcohol intake is known to enhance inhibition through facilitation of GABAA receptors, which are present in 40% of the synapses all over the brain. Evidence suggests that enhanced GABAergic transmission leads to increased large-scale brain connectivity. Our hypothesis is that acute alcohol intake would increase the functional connectivity of the human brain resting-state network (RSN). To test our hypothesis, electroencephalographic (EEG) measurements were recorded from healthy social drinkers at rest, during eyes-open and eyes-closed sessions, after administering to them an alcoholic beverage or placebo respectively. Salivary alcohol and cortisol served to measure the inebriation and stress levels. By calculating Magnitude Square Coherence (MSC) on standardized Low Resolution Electromagnetic Tomography (sLORETA) solutions, we formed cortical networks over several frequency bands, which were then analyzed in the context of functional connectivity and graph theory. MSC was increased (p<0.05, corrected with False Discovery Rate, FDR corrected) in alpha, beta (eyes-open) and theta bands (eyes-closed) following acute alcohol intake. Graph parameters were accordingly altered in these bands quantifying the effect of alcohol on the structure of brain networks; global efficiency and density were higher and path length was lower during alcohol (vs. placebo, p<0.05). Salivary alcohol concentration was positively correlated with the density of the network in beta band. The degree of specific nodes was elevated following alcohol (vs. placebo). Our findings support the hypothesis that short-term inebriation considerably increases large-scale connectivity in the RSN. The increased baseline functional connectivity can -at least partially- be attributed to the alcohol-induced disruption of the delicate balance between inhibitory and excitatory neurotransmission in favor of inhibitory influences. Thus, it is suggested that short-term inebriation is associated, as expected, to increased GABA transmission and functional connectivity, while long-term alcohol consumption may be linked to exactly the opposite effect.

How does the metric choice affect brain functional connectivity networks

Abstract Brain functional connectivity has gained increasing interest over the last few years. The application of Graph Theory on functional connectivity networks (FCNs) has shed light into different topics related to physiology as well as pathology. To this end, different connectivity metrics may be used; however, some concerns are often raised related with inconsistency of results and their associated neurophysiological interpretations depending on the metric used. This paper examines how the use of different connectivity metrics affects the small-world-ness of the FCNs and eventually the neuroscientific evidences and their interpretation; to achieve this, electroencephalography (EEG) data recorded from healthy subjects during an emotional paradigm are utilized. Participants passively viewed emotional stimuli from the international affective picture system (IAPS), categorized in four groups ranging in pleasure (valence) and arousal. Four different pair-wise metrics were used to estimate the connectivity between each pair of EEG channels: the magnitude square coherence (MSC), cross-correlation (CCOR), normalized mutual information (NMI) and normalized joint entropy (NJE). The small-world-ness is found to be varying among the connectivity metrics, while it was also affected by the choice of the threshold level. The use of different connectivity metrics affected the significance of the neurophysiological results. However, the results from different metrics were to the same direction: pleasant images exhibited shorter characteristic path length than unpleasant ones, while high arousing images were related to lower local efficiency as compared to the low arousing ones. Our findings suggest that the choice of different metrics modulates the small-world-ness of the FCNs as well as the neurophysiological results and should be taken into account when studying brain functional connectivity using graph theory.

Effect of Acute Limb Ischaemia on Neuromuscular Function in Rats

OBJECTIVE To locate the exact site of the primary lesion in the neuromuscular system in acutely ischaemic extremities. DESIGN Experimental study. SETTING University hospital, Greece. ANIMALS 22 adult rats. INTERVENTIONS Isometric tensions of extensor digitorum longus muscles were recorded before ischaemia and every 5 minutes after the arterial occlusions by indirect stimulation. When no contractile activity was elicited, the muscle was stimulated directly and recordings made every 5 minutes. The sciatic nerve function was checked by recordings of nerve conduction velocity. Specimens from the muscles were examined under electron microscopy. MAIN OUTCOME MEASURES Muscle contractile properties, conduction velocity, and electron microscopic appearance. RESULTS After a period of about 50 minutes neuromuscular function under indirect stimulation in the ischaemic limbs was lost, whilst under direct stimulation the extensor digitorum longus muscles and the sciatic nerves still functioned. Electron microscopic study showed distinct alterations at the neuromuscular junctions. CONCLUSIONS The response of the neuromuscular system to acute ischaemia indicated that the neuromuscular junction is probably the site most susceptible to acute ischaemia.

Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases

Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N2/CO2 (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

Morphological, histochemical, and interstitial pressure changes in the tibialis anterior muscle before and after aortofemoral bypass in patients with peripheral arterial occlusive disease

BackgroundMorphological and electrophysiological studies of ischemic muscles in peripheral arterial disease disclosed evidence of denervation and fibre atrophy. The purpose of the present study is to describe morphological changes in ischemic muscles before and after reperfusion surgery in patients with peripheral occlusive arterial disease, and to provide an insight into the effect of reperfusion on the histochemistry of the reperfused muscle.MethodsMuscle biopsies were obtained from the tibialis anterior of 9 patients with chronic peripheral arterial occlusive disease of the lower extremities, before and after aortofemoral bypass, in order to evaluate the extent and type of muscle fibre changes during ischemia and after revascularization. Fibre type content and muscle fibre areas were quantified using standard histological and histochemical methods and morphometric analysis. Each patient underwent concentric needle electromyography, nerve conduction velocity studies, and interstitial pressure measurements.ResultsPreoperatively all patients showed muscle fibre atrophy of both types, type II fibre area being more affected. The mean fibre cross sectional area of type I was 3,745 μm2 and of type II 4,654 μm2 . Fibre-type grouping, great variation in fibre size and angular fibres were indicative of chronic dennervation-reinnervation, in the absence of any clinical evidence of a neuropathic process. Seven days after the reperfusion the areas of both fibre types were even more reduced, being 3,086 μm2 for type I and 4,009 μm2 for type II, the proportion of type I fibres, and the interstitial pressure of tibialis anterior were increased.ConclusionsThe findings suggest that chronic ischemia of the leg muscles causes compensatory histochemical changes in muscle fibres resulting from muscle hypoxia, and chronic dennervation-reinnervation changes, resulting possibly from ischemic neuropathy. Reperfusion seems to bring the oxidative capacity of the previously ischemic muscle closer to normal.

Magnesium administration provokes motor unit survival, after sciatic nerve injury in neonatal rats

BackgroundWe examined the time course of the functional alterations in two types of muscles following sciatic nerve crush in neonatal rats and the neuroprotective effect of Mg2+.MethodsThe nerve crush was performed on the 2nd postnatal day. MgSO4*7H2O was administered daily for two weeks. Animals were examined for the contractile properties and for the number of motor units of extensor digitorum longus and soleus muscles at three postnatal stages and adulthood. Four experimental groups were included in this study: i) controls, ii) axotomized rats, iii) magnesium treated controls and iv) axotomized and Mg2+-treated rats.ResultsAxotomy resulted in 20% MU survival in EDL and 50% in soleus. In contrast, magnesium treatment resulted in a significant motor unit survival (40% survival in EDL and 80% in soleus). The neuroprotective effects of Mg2+ were evident immediately after the Mg2+-treatment. Immature EDL and soleus muscles were slow and fatigueable. Soleus gradually became fatigue resistant, whereas, after axotomy, soleus remained fatigueable up to adulthood. EDL gradually became fastcontracting. Tetanic contraction in axotomized EDL was just 3,3% of the control side, compared to 15,2% in Mg2+-treated adult rats. The same parameter for axotomized soleus was 12% compared to 97% in Mg2+-treated adult rats.ConclusionsThese results demonstrate that motoneuron death occurs mostly within two weeks of axotomy. Magnesium administration rescues motoneurons and increases the number of motor units surviving into adulthood. Fast and slow muscles respond differently to axotomy and to subsequent Mg2+ treatment in vivo.

Oxygen–Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen–glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.

Low dose administration of glutamate triggers a non-apoptotic, autophagic response in PC12 cells

Background/Aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

Histochemical and ultrastructural characteristics of leg muscle fibres in patients with repairative abdominal aortic aneurysm (AAA)

Tibialis anterior (ta) muscle biopsies before and after elective abdominal aortic aneurysm (AAA) repair operation were obtained, in order to observe possible changes after the aortic declamping reperfusion. Open muscle biopsies were taken from each of eight patients (60–75 years old) which were processed for enzyme histochemistry, and for transmission electron microscopy (EM). Morphometric analysis was applied to estimate the number and the area of muscle fibres of each fibre type. Rectus abdominis muscle biopsies were served as controls. Before the operation the predominant elements found were the presence of atrophic muscle fibres, fibre size diversity, localised cellular reactions, increased extent of connective tissue, disappearance, in many cases, of the mosaic pattern, predominance of type I and oxidative fibres, and existence of fibres with core‐like structures in the sarcoplasm. Type I fibres consisted of 66.95 ± 9% of all muscle fibres, the mean cross sectional area of which was 3,372.8 ± 1,016 μm2 and of type II fibres was 3,786.5 ± 6,046 μm2. After the aortic clamping was performed mitochondrial swelling was found, as well as disorganisation of sarcomeres. After declamping of the aorta, there were also severe edema, local fibre necrosis, and adhesion of leucocytes, whereas muscle fibre areas became 3,935.18 μ 531 μm2 for type I and 5,804 ± 1,075 μm2 for type II. The short ischemic period during aortic clamping and the subsequent reperfusion resulted mainly in ultrastructural changes. Anat Rec 260:1–15, 2000.