Neurons have been long regarded as the basic functional cells of the brain, whereas astrocytes and microglia have been regarded only as elements of support. However, proper intercommunication among neurons–astrocytes–microglia is of fundamental importance for the functional organization of the brain. Perturbation in the regulation of brain energy metabolism not only in neurons but also in astrocytes and microglia may be one of the pathophysiological mechanisms of neurodegeneration, especially in hypoxia/ischemia. Glial activation has long been considered detrimental for survival of neurons, but recently it appears that glial responses to an insult are not equal but vary in different brain areas. In this review, we first take into consideration the modifications of the vascular unit of the glymphatic system and glial metabolism in hypoxic conditions. Using the method of triple-labeling fluorescent immunohistochemistry coupled with confocal microscopy (TIC), we recently studied the interplay among neurons, astrocytes, and microglia in chronic brain hypoperfusion. We evaluated the quantitative and morpho-functional alterations of the neuron–astrocyte–microglia triads comparing the hippocampal CA1 area, more vulnerable to ischemia, to the CA3 area, less vulnerable. In these contiguous and interconnected areas, in the same experimental hypoxic conditions, astrocytes and microglia show differential, finely regulated, region-specific reactivities. In both areas, astrocytes and microglia form triad clusters with apoptotic, degenerating neurons. In the neuron–astrocyte–microglia triads, the cell body of a damaged neuron is infiltrated and bisected by branches of astrocyte that create a microscar around it while a microglial cell phagocytoses the damaged neuron. These coordinated actions are consistent with the scavenging and protective activities of microglia. In hypoxia, the neuron–astrocyte–microglia triads are more numerous in CA3 than in CA1, further indicating their protective effects. These data, taken from contiguous and interconnected hippocampal areas, demonstrate that glial response to the same hypoxic insult is not equal but varies significantly. Understanding the differences of glial reactivity is of great interest to explain the differential susceptibility of hippocampal areas to hypoxia/ischemia. Further studies may evidence the differential reactivity of glia in different brain areas, explaining the higher or lower sensitivity of these areas to different insults and whether glia may represent a target for future therapeutic interventions.

For over a century, neurons have been considered the basic functional units of the brain while glia only elements of support. Activation of glia has been long regarded detrimental for survival of neurons but more it appears that this is not the case in all circumstances. In this review, we report and discuss the recent literature on the alterations of astrocytes and microglia during inflammaging, the low-grade, slow, chronic inflammatory response that characterizes normal brain aging, and in acute inflammation. Becoming reactive, astrocytes and microglia undergo transcriptional, functional, and morphological changes that transform them into cells with different properties and functions, such as A1 and A2 astrocytes, and M1 and M2 microglia. This classification of microglia and astrocytes in two different, all-or-none states seems too simplistic, and does not correspond to the diverse variety of phenotypes so far found in the brain. Different interactions occur among the many cell populations of the central nervous system in health and disease conditions. Such interactions give rise to networks of morphological and functional reciprocal reliance and dependency. Alterations affecting one cell population reverberate to the others, favoring or dysregulating their activities. In the last part of this review, we present the modifications of the interplay between neurons and glia in rat models of brain aging and acute inflammation, focusing on the differences between CA1 and CA3 areas of the hippocampus, one of the brain regions most susceptible to different insults. With triple labeling fluorescent immunohistochemistry and confocal microscopy (TIC), it is possible to evaluate and compare quantitatively the morphological and functional alterations of the components of the neuron-astrocyte-microglia triad. In the contiguous and interconnected regions of rat hippocampus, CA1 and CA3 Stratum Radiatum, astrocytes and microglia show a different, finely regulated, and region-specific reactivity, demonstrating that glia responses vary in a significant manner from area to area. It will be of great interest to verify whether these differential reactivities of glia explain the diverse vulnerability of the hippocampal areas to aging or to different damaging insults, and particularly the higher sensitivity of CA1 pyramidal neurons to inflammatory stimuli.

Increasing incidence of skin cancer combined with a shortage of dermatopathologists has increased the workload of pathology departments worldwide. In addition, the high intraobserver and interobserver variability in the assessment of melanocytic skin lesions can result in underestimated or overestimated diagnosis of melanoma. Thus, the development of new techniques for skin tumor diagnosis is essential to assist pathologists to standardize diagnoses and plan accurate patient treatment. Here, we describe the development of an artificial intelligence (AI) system that recognizes cutaneous melanoma from histopathological digitalized slides with clinically acceptable accuracy. Whole-slide digital images from 100 formalin-fixed paraffin-embedded primary cutaneous melanoma were used to train a convolutional neural network (CNN) based on a pretrained Inception-ResNet-v2 to accurately and automatically differentiate tumoral areas from healthy tissue. The CNN was trained by using 60 digital slides in which regions of interest (ROIs) of tumoral and healthy tissue were extracted by experienced dermatopathologists, while the other 40 slides were used as test datasets. A total of 1377 patches of healthy tissue and 2141 patches of melanoma were assessed in the training/validation set, while 791 patches of healthy tissue and 1122 patches of pathological tissue were evaluated in the test dataset. Considering the classification by expert dermatopathologists as reference, the trained deep net showed high accuracy (96.5%), sensitivity (95.7%), specificity (97.7%), F₁ score (96.5%), and a Cohen’s kappa of 0.929. Our data show that a deep learning system can be trained to recognize melanoma samples, achieving accuracies comparable to experienced dermatopathologists. Such an approach can offer a valuable aid in improving diagnostic efficiency when expert consultation is not available, as well as reducing interobserver variability. Further studies in larger data sets are necessary to verify whether the deep learning algorithm allows subclassification of different melanoma subtypes.

We investigated the different patterns of neurodegeneration and glia activation in CA1 and CA3 hippocampal areas of TgCRND8 mice. The main feature of this transgenic model is the rapid development of the amyloid pathology, which starts already at 3 months of age. We performed immunohistochemical analyses to compare the different sensibility of the two hippocampal regions to neurodegeneration. We performed qualitative and quantitative evaluations by fluorescence immunohistochemistry with double or triple staining, followed by confocal microscopy and digital image analysis in stratum pyramidale (SP) and stratum radiatum (SR) of CA1 and CA3, separately. We evaluated time-dependent Aβ plaques deposition, expression of inflammatory markers, as well as quantitative and morphological alterations of neurons and glia in transgenic mice at 3 (Tg 3M) and 6 (Tg 6M) months of age, compared to WT mice. In CA1 SR of Tg 6M mice, we found significantly more Medium and Large plaques than in CA3. The pattern of neurodegeneration and astrocytes activation was different in the two areas, indicating higher sensitivity of CA1. In the CA1 SP of Tg 6M mice, we found signs of reactive astrogliosis, such as increase of astrocytes density in SP, increase of GFAP expression in SR, and elongation of astrocytes branches. We found also common patterns of glia activation and neurodegenerative processes in CA1 and CA3 of Tg 6M mice: significant increase of total and reactive microglia density in SP and SR, increased expression of TNFα, of iNOS, and IL1β in astrocytes and increased density of neurons–astrocytes–microglia triads. In CA1 SP, we found decrease of volume and number of pyramidal neurons, paralleled by increase of apoptosis, and, consequently, shrinkage of CA1 SP. These data demonstrate that in TgCRND8 mice, the responses of neurons and glia to neurodegenerative patterns induced by Aβ plaques deposition is not uniform in the two hippocampal areas, and in CA1 pyramidal neurons, the higher sensitivity may be related to the different plaque distribution in this area. All these modifications may be at the basis of memory loss, the peculiar symptom of AD, which was demonstrated in this transgenic mouse model of Aβ deposition, even at early stages.

Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A₁, A_2A, A_2B, and A₃. Although adenosine exerts clear neuroprotective effects through A₁ receptors during ischemia, the use of selective A₁ receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A_2B receptors in cerebral ischemia. This study explored the role of adenosine A_2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A_2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A_2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A_2Breceptor antagonism significantly prevented astrocyte modifications. Both A_2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A_2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.