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Chronic macrophage activation was implicated as one of the main culprits for chronical, low-grade inflammation which significantly contributes to development of age-related diseases. Microglia as the brain macrophages have been recently implicated as key players in neuroinflammation and neurodegeneration in the aged brain. Microglial cell functions are indispensable in early development, however, activation or senescence of microglia in aging cells may be detrimental. Depletion of microglia using genetical or pharmacological approaches leads to opposite results regarding effects on brain cognition. In this study we pharmacologically depleted microglia using orally delivered low and high doses of the CSF1R inhibitor PLX5622 and assessed the expression levels of known inflammation markers (TNF-α, IL1-β, IL-6, IL-10), glia markers (Iba-1 and Gfap) and specific senescence marker p16 Ink4a in the aged murine brain. Our results indicate that treatment with low and high doses of PLX5622 leads to a dose-dependent depletion of microglial cells with similar levels in young and aged mice. We also show that treatment with low and high PLX5622 differentially affected cytokine levels in young and old brains. By using low doses we could achieve reduction in inflammation circumventing the astrocyte activation. Removal of microglia cells led to decreased expression of the senescence marker p16 Ink4a in the aged brain, indicating a relevant contribution of these cells to the expression of this marker and their senescent status in the healthy aging brain. Our results indicate that increased and detrimental brain inflammation in aged murine brain can be impaired by selectively reducing the microglial cell population., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aberrations in intracellular calcium (Ca 2+ ) have been well established within amyotrophic lateral sclerosis (ALS), a severe motor neuron disease. Intracellular Ca 2+ concentration is controlled in part through the endoplasmic reticulum (ER) mitochondria Ca 2+ cycle (ERMCC). The ER supplies Ca 2+ to the mitochondria at close contacts between the two organelles, i.e. the mitochondria-associated ER membranes (MAMs). The Sigma 1 receptor (Sig1R) is enriched at MAMs, where it acts as an inter-organelle signaling modulator. However, its impact on intracellular Ca 2+ at the cellular level remains to be thoroughly investigated. Here, we used cultured embryonic mice spinal neurons to investigate the influence of Sig1R activation on intracellular Ca 2+ homeostasis in the presence of G93A hSOD1 (G93A), an established ALS-causing mutation. Sig1R expression was increased in G93A motor neurons relative to non-transgenic (nontg) controls. Furthermore, we demonstrated significantly reduced bradykinin-sensitive intracellular Ca 2+ stores in G93A spinal neurons, which were normalized by the Sig1R agonist SA4503. Moreover, SA4503 accelerated cytosolic Ca 2+ clearance following a) AMPAR activation by kainate and b) IP 3 R-mediated ER Ca 2+ release following bradykinin stimulation in both genotypes. PRE-084 (another Sig1R agonist) did not exert any significant effects on cytosolic Ca 2+ . Both Sig1R expression and functionality were altered by the G93A mutation, indicating the centrality of Sig1R in ALS pathology. Here, we showed that intracellular Ca 2+ shuttling can be manipulated by Sig1R activation, thus demonstrating the value of using the pharmacological manipulation of Sig1R to understand Ca 2+ homeostasis., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

The capability of the adult brain to generate new hippocampal neurons after brain insults like stroke is decreasing during the aging process. Recent evidence further indicates that the proliferative properties of the precursor cells change in the aged brain. We therefore analyzed the early proliferative response of distinct precursor cell populations in the subgranular zone of the dentate gyrus in 3 and 16 months old transgenic nestin-green-fluorescent protein mice 4 days after ischemic cortical infarcts. A detailed immunocytochemical analysis of proliferating precursors revealed a significant infarct-induced activation of the earliest radial glia-like precursor cells (type 1 cells) and the more differentiated precursor cell subtypes (type 2b cells) in young mice. In contrast the proliferation of early neuronal precursor cells (type 2a cells) was stimulated in the aged brain. Additional long-term experiments further demonstrated that this differential proliferative response of distinct precursor cells is associated with an enhanced number of newborn neurons in the young DG after stroke whereas this increase in neurogenesis was absent in the aged brain. However, our study demonstrates that even precursor cells in the aged hippocampus possess the ability to respond to remote cortical infarcts., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Although stroke can affect cerebral structure and function, the brain has a potential for plasticity thanks to which some degree of function can be restored. The pathways of such recovery are of great interest, since the dynamics of rewiring of the injured brain may become the basis for designing appropriate strategies of rehabilitation. We investigated the spontaneous plasticity of cortical somatosensory representations following a focal unilateral stroke in the barrel cortex of rats. Ischemic lesions were produced with the photothrombotic technique in the cortical representation of vibrissae. Functional activation of the brain in response to the stimulation of vibrissae with destroyed cortical representation was monitored through the 2 months post-stroke survival period with [(14)C] 2-deoxyglucose (2DG) autoradiographic brain mapping (1, 7, 28, 56 days after the stroke). 2DG uptake was measured on autoradiograms of tangential sections in several regions of somatosensory cortex and in motor, auditory and prefrontal cortex. Behavioral deficit was assessed by the gap-crossing test 3, 28, 56 days after the stroke. Changes in the activation pattern of the intact hemisphere and non-vibrissal somatosensory representations of the lesioned hemisphere evolved during the observation period. Full recovery of the behavioral function was reached 2 months after the stroke and at the same time, new foci of activation were observed in the lesioned hemisphere. At that time, hyperactivation of the somatosensory areas in the intact hemisphere subsided. The new activation foci located in representations of anterior vibrissae, front paw and hind paw were specific for the vibrissae stimulation and were most probably a new functional representation of the vibrissae. We demonstrated spatial and temporal remodeling of the brain induced by cortical stroke, leading to vicariation of function.

Rehabilitation therapy is known to drive motor improvement in stroke patients. However, the interplay of functional recovery and compensation in postischemic motor behavior is poorly understood. This study focused on the time course of functional recovery versus motor compensation in skilled forelimb movements after cerebral ischemia in rats. Young adult male rats underwent a focal cerebral ischemia by unilateral photothrombotic lesion of the motor cortex related to the preferred forelimb. In a first set of experiments animals were exposed to small cortical lesions comprising the forelimb motor cortex (n=8) or to larger lesions additionally extending into the hind limb motor area (n=8). In a second set of experiments animals with large lesion were either housed in standard (n=10) or enriched environment (n=14). Skilled reaching was assessed for 25 to 28 days postischemia. This task allows the distinction between recovery and compensation by parallel quantitative (reaching success) and qualitative (movement pattern) analysis. The results reveal that lesion size determines the initial magnitude of motor deficits, but not the degree of chronic impairments in movement pattern in all experimental groups. Compensatory movements represent the major mechanism of functional improvement and were accompanied by a partial functional restitution. Enriched environment facilitates effective compensation in skilled reaching, while it does not promote restitution of function. In particular, rotating movements of the forelimb during reaching were permanently impaired and required functional compensation through intensified use of the upper body. We conclude an activity dependent postischemic restoration of movement success. Enriched environment provides benefit by increased motor activity mainly due to compensation. Furthermore, these findings emphasize the power of comprehensive movement analysis to gain insight into recovery processes after stroke.

Following focal ischemic injury, several mechanisms lead to secondary expansion of the affected area and therefore increase the initial damage. We thoroughly investigated the expression of astrocytic connexin 43 (Cx43) after photothrombosis in rat brain. The temporal profile of Cx43 mRNA as well as protein expression was studied in remote, structurally uninjured cortical and hippocampal areas. The hippocampal formation revealed an increased number of Cx43 mRNA positive astrocytes and an up-regulated protein expression exclusively in the ipsilateral stratum oriens. We assume a participation of this region in glia scar formation. While Cx43 mRNA positive cells were transiently increased, immunoreactivity was reduced in the somatosensory cortex of injured hemispheres. The observed decrease of Cx43 protein in the post-ischemic cerebral cortex implies an impairment of gap junctional intercellular communication which might be detrimental to the brain.

Before exocytotic release of the inhibitory neurotransmitter GABA, this amino acid has to be stored in synaptic vesicles. Accumulation of GABA in vesicles is achieved by a specific membrane-integrated transporter termed vesicular GABA transporter. This vesicular protein is mainly located at presynaptic terminals of GABAergic interneurons. In the present study we investigated the effects of focal ischemia on the expression of the vesicular GABA transporter. Vesicular GABA transporter mRNA and protein expression was examined after photothrombosis in different cortical and hippocampal brain regions of Wistar rats. In situ hybridization and quantitative real-time RT-PCR were performed to analyze vesicular GABA transporter mRNA. Both vesicular GABA transporter mRNA-stained perikarya and mRNA expression levels remained unaffected. Vesicular GABA transporter protein-containing synaptic terminals and somata were visualized by immunohistochemistry. The pattern of vesicular GABA transporter immunoreactivity as well as the protein expression level revealed by semiquantitative image analysis and by Western blot remained stable after stroke. The steady expression of vesicular GABA transporter mRNA and protein after photothrombosis indicates that the exocytotic release mechanism of GABA is not affected by ischemia.

During sensorimotor recovery following stroke ipsi- and contralesional alterations in brain function have been characterized in patients as well as animal models of focal ischemia, but the contribution of these bilateral processes to the functional improvement is only poorly understood. Here we examined the role of the homotopic contralateral cortex for sensorimotor recovery after focal ischemic infarcts at different time periods after the insult. One group of animals received a unilateral single photothrombotic infarct in the forelimb sensorimotor cortex, while four additional groups received a second lesion in the contralateral homotopic cortex either immediately or 2 days, 7 days, or 10 days after the first infarct. The time course of functional recovery of the impaired forelimbs was assessed using different sensorimotor scores: forelimb-activity during exploratory behavior and frequency of forelimb-sliding in the glass cylinder as well as forelimb misplacement during grid walking. Focal infarcts in the forelimb sensorimotor cortex area significantly impaired the function of the contralateral forelimb in these different behavioral tests. The subsequent damage of the contralateral homotopic forelimb sensorimotor cortex only affected the forelimb opposite to the new lesion but did not reinstate the original deficit. The time course of sensorimotor recovery after bilateral sequential cortical infarcts did not significantly differ from animals with unilateral single lesions. These data indicate that following small ischemic cortical infarcts in the forelimb sensorimotor cortex the contralateral cortex homotopic to the lesion plays only a minor role for functional recovery.

Stimulation of cell proliferation and neurogenesis in the adult dentate gyrus has been observed after focal and global brain ischemia but only little is known about the underlying mechanisms. We here analyzed neurogenesis in the dentate gyrus after small cortical infarcts leaving the hippocampal formation and subcortical regions intact. Using the photothrombosis model in adult rats, focal ischemic infarcts were induced in different cortical areas (sensorimotor forelimb and hindlimb cortex) and proliferating cells were labeled at days 3-14 after infarct induction with bromodeoxyuridine. At 2, 4, and 10 weeks after ischemia, immunocytochemistry was performed with immature neuronal (doublecortin), mature neuronal (neuronal nuclei antigen) and glial (calcium-binding protein beta S100beta) markers. When compared with sham-operated controls, animals with infarcts in the forelimb as well as hindlimb cortex revealed an increase in survival of newborn progenitor cells at four and 10 weeks after the insult with predominance at the ipsilateral side. Triple immunofluorescence and confocal laser scanning microscopy revealed an increase in neurogenesis in all groups that was more pronounced 10 weeks after the infarct. Application of the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 during lesion induction significantly enhanced neurogenesis in the dentate gyrus. An even stronger increase in newborn neurons was observed after anti-inflammatory treatment with indomethacine during the first 16 days of the experiment. The present study demonstrates that small cortical infarcts leaving subcortical structures intact increase neurogenesis in the dentate gyrus and that these processes can be stimulated by N-methyl-D-aspartate receptor blockade and anti-inflammatory treatment.

Focal cerebral lesions in rat brain induced by photothrombosis lead to an impaired inhibitory neurotransmission. A reduced gamma-aminobutyric acid (GABA)-mediated inhibition has been revealed by electrophysiological recordings associated with a diminished immunostaining of GABA handling proteins. Changes were found in ipsi- as well as in contralateral brain areas. Inhibition is mediated by interneurons using GABA as neurotransmitter. These cells use GAD (glutamate decarboxylase) to synthesize GABA. To analyze the vulnerability of GABAergic neurons in rats with a lesioned hindlimb area, cells expressing GAD65/67 mRNA were labeled using in situ hybridization. Positive somata were counted 7 and 30 days after focal ischemia in different cortical (hindlimb cortex, frontal cortex, primary and secondary somatosensory cortex) and hippocampal subsectors (pyramidal cell layer, stratum oriens and stratum radiatum/lacunosum-moleculare). The lesioned hemispheres were compared with the intact brain sides and with control brains. GABAergic interneurons survived the injury for up to 30 days in all investigated brain regions. Therefore it is unlikely that a loss of GABAergic neurons contributes to the reduced inhibition.

Electrophysiological studies in humans and animal models have revealed an intrinsic epileptogenicity of cortical dysplasias which are a frequent cause of drug-resistant epilepsy. An imbalance of inhibition and excitation has been causative related. Receptor-binding studies in rodents demonstrated reduced binding to GABA and increased binding to glutamate receptors within cortical dysplasias and increments of AMPA- and kainate-receptor binding in its surround. Immunohistochemically a differential downregulation of GABA(A) receptor subunits could be demonstrated in widespread areas within and around dysplasias. As receptor binding critically depends on receptor subunit composition the observed changes in binding properties might be related to this. Here, we immunohistochemically analyzed the regional expression of four NMDA receptor subunits and two major AMPA- and kainate-receptor complexes in adult rats after neonatal freeze lesions. These lesions are characterized by a three- to four-layered cortex and a microsulcus which mimic human polymicrogyria. Using antibodies against NR1, NR2A, NR2B, NR2D, GluR2,3, and GluR5,6,7 receptor subunits we demonstrated a pronounced disturbance of cortical immunostaining pattern in the cortical malformation. These changes reflected the structural disorganization of the microgyrus with some distortion of the apical dendrites of paramicrogyral pyramidal cells, a decrease and disorganization of cells at the bottom of the microsulcus, and an inflection of apical dendrites toward the microsulcus. The neuronal staining pattern of large pyramidal cells in the neighborhood of the dysplasia did not differ for any subunit investigated. No remote or widespread changes of glutamate-receptor subunit distribution could be detected. The lack of gross and/or widespread alterations of glutamate-receptor subunit distribution in the surround of focal cortical dysplasia suggests the presence of other or additional mechanisms underlying the increased excitatory neurotransmitter binding and excitability in cortical malformations.

Experimental cortical photothrombosis leads to pronounced alterations in the binding density of [3H]muscimol and [3H]baclofen to GABA(A) and GABA(B) receptors, both in the lesioned and the structurally intact cortex. The binding density of [3H]muscimol to GABA(A) receptors was markedly increased in the "core" of the lesion during the first week, reaching a maximum on the third day post-lesion. Simultaneously, it dropped in the exofocal primary somatosensory cortex. Reductions in the binding density of [3H]muscimol were also found in remote cortical areas of the contralateral hemisphere and lasted for several weeks. In contrast to the down-regulation of apparent binding density of [3H]muscimol, a long-lasting up-regulation of that of [3H]baclofen to GABA(B) receptors was measured in the exofocal primary somatosensory cortex and in remote cortical areas of both hemispheres. The greatest increase in the binding density of [3H]baclofen was seen on the seventh day in the surroundings of the lesion. Our findings indicate that widespread alterations in the concentrations of GABA(A) and GABA(B) receptors are induced in remote cortical areas by a focal ischaemic lesion. Since GABA(A) receptor affinity is regulated by nitric oxide, we suggest that the observed down-regulation of GABA(A) receptors may be correlated with a lesion-induced increase in nitric oxide, whereas the up-regulation of GABAB receptors might be caused by other mechanisms, e.g., compensatory processes. In the centre of the lesion, however, a GABA(A) receptor-mediated mechanism, which limits the spread of lesion-induced hyperexcitability, is thought to be involved.

This study examined microglial responses after photochemically induced focal ischemia of the rat cortex. Microglial activation exceeded by far the area of the ischemic lesion. Based on morphological criteria and expression of immunomolecules three distinct patterns could be distinguished. (1) In the infarct core and the border zone microglia transformed into phagocytes and removed debris with the aid of hematogeneous macrophages. Exclusively in this area a subpopulation of CD8+ microglia/mnacrophages was present. (2) In secondarily degenerating fibre tracts and nuclei with retrograde neuronal loss, microglia were activated with a delay of days and showed increased expression of complement receptor 3, major histocompatibility complex class II and CD4 molecules, but only low phagocytic activity. (3) In remote ipsilateral cortex devoid of neuronal damage, microglia transiently responded by increased complement receptor 3, but not by major histocompatibility complex class II and CD4 expression. Furthermore, the total number of microglia had increased. This remote response could partly be blocked by dizocilpine maleate, a non-competitive N-methyl-D-aspartate receptor antagonist, implicating a functional role of spreading depression. Taken together, our findings point to a tight and differential regulation of microglial responses in the infarct core, degenerating fibre tracts and remote brain regions without neuronal loss.

Deficits of GABAergic transmission have been reported to occur in tissue surrounding ischemic cortical lesions between a few days and several weeks after the insult. In the present experiments, we used immunohistochemistry with antibodies against parvalbumin and two major subunits of the GABA(A) receptor (alpha1, alpha2) to characterize the events that underlie these changes at different levels of circuit organization. Neocortical infarcts (2 mm diameter) consistently affecting medial parts of the primary somatosensory cortex were induced photochemically in adult male Wistar rats; animals were allowed to recover for one week before perfusion-fixation. When compared to controls the pattern of immunoreactivity had changed for the al subunit of the GABA(A) receptor seven days after the insult. Ipsilateral to the ischemic lesions, we found a decrease in staining intensity reaching up to 4 mm laterally, resulting in a partial or complete absence of the normal laminar staining pattern. No consistent changes were observed for the alpha2 subunit. Parvalbumin staining revealed pathological alterations in a rim of tissue surrounding the infarct, measuring up to 1 mm from the border of the infarcts. Parvalbumin-positive interneurons in this region showed signs of degeneration; both a reduction of the number of dendrites and, to a lesser extent and only immediately adjacent to the ischemic lesions, a reduction of the number of parvalbumin-positive neurons was readily apparent. The results provide evidence for both a differential regulation of two GABA(A) receptor subunits and degenerative changes of parvalbumin-containing interneurons ipsilateral to cortical infarcts. The relevance of these findings for mechanisms underlying long-term recovery, transient functional deficits and postinfarct seizures warrants further investigation.

In order to study structural alterations which occur after a defined unilateral cortical infarct, the hindlimb region of the rat cortex was photochemically lesioned. The infarcts caused edema restricted to the perilesional cortex which affected allocortical and isocortical areas differently. Postlesional changes in cytoskeletal marker proteins such as microtubule-associated protein 2, non-phosphorylated (SMI32) and phosphorylated (SMI35, SMI31 and 200,000 mol. wt) neurofilaments and 146,000 mol. wt glycoprotein Py as well as changes in proteoglycans visualized with Wisteria floribunda lectin binding (WFA) were studied at various time points and related to glial scar formation. The results obtained by the combination of these markers revealed six distinct regions in which transient, epitope-specific changes occurred: the core, demarcation zone, rim, perilesional cortex, ipsilateral thalamus and contralateral homotopic cortical area. Within the core immunoreactivity for microtubule-associated protein 2 and SMI32 decreased and the cellular components showed structural disintegration 4 h post lesion, but partial recovery of somatodendritic staining was seen after 24 h. Microtubule-associated protein 2 and SMI32 persisted up to days 7 and 5 respectively in the core, whereas the number of glial fibrillary acidic protein- and WFA-positive cells decreased between days 7 and 14. The demarcation zone showed a dramatic loss of immunoreactivity for all epitopes 4 h post lesion which was not followed by a phase of recovery. In the inner region of the demarcation zone there was an invasion and accumulation of non-neuronal WFA-positive cells which formed a tight capsule around the core. Neuronal immunoreactivities for microtubule-associated protein 2, SMI31 and Py as well as astrocytic glial fibrillary acidic protein increased strongly within an approximately 0.4-1.0 mm-wide rim region directly bordering the demarcation zone. Py immunoreactivity increased significantly in the perilesional cortex, whereas glial fibrillary acidic protein-positive astrocytes became transiently more numerous in the entire lesioned hemisphere including strongly enhanced immunoreactivity in the thalamus by days 5-7 post lesion. Glial fibrillary acidic protein immunoreactivity increased in the corpus callosum and the homotopic cortical area of the unlesioned hemisphere by days 5-7. In this homotopic area additional changes in SMI31 immunoreactivity occurred. Our results showed that a cortical infarct is not only a locally restricted lesion, but leads to a variety of cytoskeletal and other structural changes in widely-distributed functionally-related areas of the brain.

Since ischemic insults lead to a deregulation of nitric oxide production which contributes to delayed neuronal death, we investigated changes in the distribution and amount of nitric oxide synthases I and II and in the appearance of nitrotyrosine caused by small, well-defined photothrombic lesions (2 mm in diameter) in the somatosensory cortex of rats. Four hours after lesioning, cell loss was evident in the core of the lesion and no nitric oxide synthase was present within this area, indicating that neurons expressing nitric oxide synthase I were lost or that nitric oxide synthase I was degraded. No increase in the number of neurons expressing nitric oxide synthase I was visible in the area surrounding the lesion, nor in other parts of the brain. One day after lesioning, NADPH-diaphorase- and nitric oxide synthase II-positive leucocytes had invaded the perilesional cortex and were accumulated in injured blood vessels. By two to three days post-lesion, layer V and VI pyramidal neurons, microglia, astrocytes and invading leucocytes had become strongly immunoreactive for nitric oxide synthase II within a perilesional rim. The number of cells expressing nitric oxide synthase I remained stable. Nitric oxide synthase II immunoreactivity and related NADPH-diaphorase had decreased by seven days post-lesion in most animals. However, the number of activated microglia or macrophages and astrocytes, as revealed by other markers, remained elevated. In addition, nitrotyrosine immunoreactivity was evident in the blood vessels close to the lesion, as well as in the ipsilateral hippocampus and thalamus. These findings indicate that no perilesional changes in the number of neurons expressing nitric oxide synthase I occur, but that a transient increase in nitric oxide synthase II does take place in the aftermath of small cortical lesions. The results suggest that increased nitric oxide production is limited to certain post-lesional intervals in this experimental model. It is also obvious that the vast majority of nitric oxide synthase-positive cells are nitric oxide synthase II-containing astrocytes three days after lesioning, suggesting that astrocyte-derived nitric oxide plays a significant role in delayed neuronal death. Such a condition points to an important aspect of post-lesional astrocytosis.