Several reports have demonstrated that attenuation of microglial activation by minocycline, an antimicrobial drug with anti-inflammatory properties, delays disease progression in a mouse model of ...ALS. However, the negative results obtained in recent clinical trials raised some questions regarding the role of inflammatory response and glial cells as a therapeutic target in ALS. To investigate this controversy we took advantage of a mouse model for live imaging of neuroinflammatory responses in ALS (GFAP-luc/ SOD1
G93A reporter mouse) and analyzed in real time the effects of minocycline treatment initiated at different stages of the disease. To our surprise, unlike neuroprotection that is conferred when minocycline is administered pre-symptomatically, treatment with minocycline initiated after the disease onset significantly altered glial responses and exaggerated neuroinflammation. Further analysis revealed that the late minocycline treatment was associated with significant induction of the end-stage GFAP-biophotonic signals, expression levels of connexin 43, a major protein of astrocytic gap junction and markers of microglial activation, such as Iba1 and CD68. The results of our study suggest that when administered at later stages of disease, once microglial cells are chronically reactive, minocycline may not have anti-inflammatory properties, and contrary to expectations, may alter astrocyte reactivity and increase microgliosis. Finally, our results further suggest the existence of close interactions/communication between activated microglia and astrocytes in late stage ALS.
► Previous reports showed that minocycline is neuroprotective in a mouse model of ALS. ► Recent clinical trials raised some questions regarding the efficacy of minocycline. ► Using live imaging of neuroinflammation we analyzed in real time the effects of minocycline. ► Treatment with minocycline initiated after disease onset exaggerated neuroinflammation.
Background:
Disinhibition of neurons in the superficial spinal dorsal horn, via microglia – neuron signaling leading to disruption of chloride homeostasis, is a potential cellular substrate for ...neuropathic pain. But, a central unresolved question is whether this disinhibition can transform the activity and responses of spinal nociceptive output neurons to account for the symptoms of neuropathic pain.
Results:
Here we show that peripheral nerve injury, local spinal administration of ATP-stimulated microglia or pharmacological disruption of chloride transport change the phenotype of spinal lamina I output neurons, causing them to 1) increase the gain of nociceptive responsiveness, 2) relay innocuous mechanical input and 3) generate spontaneous bursts of activity. The changes in the electrophysiological phenotype of lamina I neurons may account for three principal components of neuropathic pain: hyperalgesia, mechanical allodynia and spontaneous pain, respectively.
Conclusion:
The transformation of discharge activity and sensory specificity provides an aberrant signal in a primarily nociceptive ascending pathway that may serve as a basis for the symptoms of neuropathic pain.
Modern laboratory techniques allow studying NMDA receptors (NMDAR) either anatomically with specific antibodies coupled to sophisticated confocal microscopy, or physiologically by live imaging or ...electrophysiological techniques. However, NMDARs are not fixed in time and space and changes in their composition and/or distribution on the post-synaptic membrane may significantly impact the synaptic strength and overall function. The computational modeling approach therefore constitutes a complementary tool for investigating the properties of biological systems based on the knowledge provided by the lab experiments.Here, we describe a general computational method aiming at developing kinetic Markov-chain based models of NMDARs subtypes capable of reproducing various experimental results. These models are then used to make predictions on additional (non-obvious) properties and on their role in synaptic function under various physiological and pharmacological conditions. For the purpose of this book chapter, we will focus on the method used to develop a NMDAR model that includes pharmacological site of action of different compounds. Notably, this elementary model can subsequently be included in a neuron model (not described in detail here) to explore the impact of their differential distribution on synaptic functions.
continuous positive airway pressure (CPAP) and high-flow nasal oxygen (HFNO) provide enhanced oxygen delivery and respiratory support for patients with severe COVID-19. CPAP and HFNO are currently ...designated as aerosol-generating procedures despite limited high-quality experimental data. We aimed to characterise aerosol emission from HFNO and CPAP and compare with breathing, speaking and coughing.
Healthy volunteers were recruited to breathe, speak and cough in ultra-clean, laminar flow theatres followed by using CPAP and HFNO. Aerosol emission was measured using two discrete methodologies, simultaneously. Hospitalised patients with COVID-19 had cough recorded using the same methodology on the infectious diseases ward.
In healthy volunteers (n=25 subjects; 531 measures), CPAP (with exhalation port filter) produced less aerosol than breathing, speaking and coughing (even with large >50 L/min face mask leaks). Coughing was associated with the highest aerosol emissions of any recorded activity. HFNO was associated with aerosol emission, however, this was from the machine. Generated particles were small (<1 µm), passing from the machine through the patient and to the detector without coalescence with respiratory aerosol, thereby unlikely to carry viral particles. More aerosol was generated in cough from patients with COVID-19 (n=8) than volunteers.
In healthy volunteers, standard non-humidified CPAP is associated with less aerosol emission than breathing, speaking or coughing. Aerosol emission from the respiratory tract does not appear to be increased by HFNO. Although direct comparisons are complex, cough appears to be the main aerosol-generating risk out of all measured activities.
In the lithiumâpilocarpine model (Li-pilocarpine) of temporal lobe epilepsy, GABA A receptor-mediated inhibitory postsynaptic currents (GABA A IPSCs) were recorded in dentate gyrus granule cells ...(GCs) from adult rat hippocampal slices. The properties of GABA A IPSCs were compared before and after superfusion of modulators in control conditions (Li-saline rats) and in Li-pilocarpine
rats 24â48 h and 3â5 months (epileptic rats) after status epilepticus ( SE ). The mean peak amplitude of GABA A IPSCs increased by about 40% over Li-saline values in GCs 24â48 h after SE and remained higher in epileptic rats. In Li-pilocarpine rats, studied at 24â48 h after SE , diazepam (1 μ m ) lost 65% of its effectiveness at increasing the half-decay time ( T 50% ) of GABA A miniature IPSCs (mIPSCs). Diazepam had no effects on mIPSC T 50% in epileptic rats. The benzodiazepine ligand flumazenil (1 μ m ), acting as an antagonist in Li-saline rats, exhibited a potent inverse agonistic effect on GABA A mIPSCs of GCs from Li-pilocarpine rats 24â48 h and 3â5 months after SE. The neurosteroid allopregnanolone (100 n m ), which considerably prolonged GABA A mIPSCs in Li-saline rats, totally lost its effect in rats studied 24â48 h after SE. However, this decrease in effectiveness
was transient and was totally restored in epileptic rats. In addition to the up-regulation in the number of receptors at individual
GC synapses, we propose that these âepilepticâ GABA A receptors possess benzodiazepine binding sites with altered allosteric properties. The failure of benzodiazepine and neurosteroid
to potentiate inhibition early after SE may be a critical factor in the development of epileptogenesis and occurrence of seizures.
To determine if oesophago-gastro-duodenoscopy (OGD) generates increased levels of aerosol in conscious patients and identify the source events.
A prospective, environmental aerosol monitoring study, ...undertaken in an ultraclean environment, on patients undergoing OGD. Sampling was performed 20 cm away from the patient's mouth using an optical particle sizer. Aerosol levels during OGD were compared with tidal breathing and voluntary coughs within subject.
Patients undergoing bariatric surgical assessment were recruited (mean body mass index 44 and mean age 40 years, n=15). A low background particle concentration in theatres (3 L
) enabled detection of aerosol generation by tidal breathing (mean particle concentration 118 L
). Aerosol recording during OGD showed an average particle number concentration of 595 L
with a wide range (3-4320 L
). Bioaerosol-generating events, namely, coughing or burping, were common. Coughing was evoked in 60% of the endoscopies, with a greater peak concentration and a greater total number of sampled particles than the patient's reference voluntary coughs (11 710 vs 2320 L
and 780 vs 191 particles, n=9 and p=0.008). Endoscopies with coughs generated a higher level of aerosol than tidal breathing, whereas those without coughs were not different to the background. Burps also generated increased aerosol concentration, similar to those recorded during voluntary coughs. The insertion and removal of the endoscope were not aerosol generating unless a cough was triggered.
Coughing evoked during OGD is the main source of the increased aerosol levels, and therefore, OGD should be regarded as a procedure with high risk of producing respiratory aerosols. OGD should be conducted with airborne personal protective equipment and appropriate precautions in those patients who are at risk of having COVID-19 or other respiratory pathogens.
In the lithium-pilocarpine model (Li-pilocarpine) of temporal lobe epilepsy, GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A) IPSCs) were recorded in dentate gyrus granule cells ...(GCs) from adult rat hippocampal slices. The properties of GABA(A) IPSCs were compared before and after superfusion of modulators in control conditions (Li-saline rats) and in Li-pilocarpine rats 24-48 h and 3-5 months (epileptic rats) after status epilepticus (SE). The mean peak amplitude of GABA(A) IPSCs increased by about 40% over Li-saline values in GCs 24-48 h after SE and remained higher in epileptic rats. In Li-pilocarpine rats, studied at 24-48 h after SE, diazepam (1 microm) lost 65% of its effectiveness at increasing the half-decay time (T(50%)) of GABA(A) miniature IPSCs (mIPSCs). Diazepam had no effects on mIPSC T(50%) in epileptic rats. The benzodiazepine ligand flumazenil (1 microm), acting as an antagonist in Li-saline rats, exhibited a potent inverse agonistic effect on GABA(A) mIPSCs of GCs from Li-pilocarpine rats 24-48 h and 3-5 months after SE. The neurosteroid allopregnanolone (100 nm), which considerably prolonged GABA(A) mIPSCs in Li-saline rats, totally lost its effect in rats studied 24-48 h after SE. However, this decrease in effectiveness was transient and was totally restored in epileptic rats. In addition to the up-regulation in the number of receptors at individual GC synapses, we propose that these 'epileptic' GABA(A) receptors possess benzodiazepine binding sites with altered allosteric properties. The failure of benzodiazepine and neurosteroid to potentiate inhibition early after SE may be a critical factor in the development of epileptogenesis and occurrence of seizures.
The spinal dorsal horn is the first level of the CNS in which nociceptive input from sensory afferents is integrated and transmitted. Although inhibitory control in this region has a crucial impact ...on pain transmission, the respective contribution of GABA and glycine to this inhibition remains elusive. We have previously documented co-release of GABA and glycine at the same inhibitory synapse in spinal laminas I-II of adult rats older than postnatal day 30 (P30). However, despite this co-release, individual miniature inhibitory postsynaptic currents (mIPSCs) were mediated by either glycine receptors (GlyR) or GABA(A) receptors (GABA(A)R), yet never by the two together. In contrast, recent studies of ventral horn immature inhibitory synapses (</=P21) reported individual mIPSCs that were mediated by both GABA(A)Rs and GlyRs. This raises the question of whether mixed mIPSCs are present in immature lamina I-II neurons yet are lost through a maturation-dependent synaptic specialization. To test this, we recorded mIPSCs using patch-clamp techniques in lamina I-II neurons in spinal slices taken at different stages of development. We found that, in neurons younger than P23, both GlyR-only and GABA(A)R-only mIPSCs could be recorded, in addition to mixed GABA(A)R-GlyR mIPSCs. With maturation however, both lamina I-II neurons gradually discontinued exhibiting mixed mIPSCs, although with differing patterns of specialization. Yet, at all developmental stages, benzodiazepine administration could unmask mixed mIPSCs. Together, these findings indicate that, although GABA and glycine are continually co-released throughout development, junctional codetection ceases by adulthood. This indicates an age-dependent postsynaptic tuning of inhibitory synapses that occurs in a region-specific manner.
Pulmonary function tests are fundamental to the diagnosis and monitoring of respiratory diseases. There is uncertainty around whether potentially infectious aerosols are produced during testing and ...there are limited data on mitigation strategies to reduce risk to staff. Healthy volunteers and patients with lung disease underwent standardised spirometry, peak flow and FE
assessments. Aerosol number concentration was sampled using an aerodynamic particle sizer and an optical particle sizer. Measured aerosol concentrations were compared with breathing, speaking and voluntary coughing. Mitigation strategies included a standard viral filter and a full-face mask normally used for exercise testing (to mitigate induced coughing). 147 measures were collected from 33 healthy volunteers and 10 patients with lung disease. The aerosol number concentration was highest in coughs (1.45-1.61 particles/cm
), followed by unfiltered peak flow (0.37-0.76 particles/cm
). Addition of a viral filter to peak flow reduced aerosol emission by a factor of 10 without affecting the results. On average, coughs produced 22 times more aerosols than standard spirometry (with filter) in patients and 56 times more aerosols in healthy volunteers. FE
measurement produced negligible aerosols. Cardiopulmonary exercise test (CPET) masks reduced aerosol emission when breathing, speaking and coughing significantly. Lung function testing produces less aerosols than voluntary coughing. CPET masks may be used to reduce aerosol emission from induced coughing. Standard viral filters are sufficiently effective to allow guidelines to remove lung function testing from the list of aerosol-generating procedures.
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