Longitudinal in vivo imaging of perineuronal nets.

Significance: Perineuronal nets (PNNs) are extracellular matrix structures implicated in learning, memory, information processing, synaptic plasticity, and neuroprotection. However, our understanding of mechanisms governing the evidently important contribution of PNNs to central nervous system function is lacking. A primary cause for this gap of knowledge is the absence of direct experimental tools to study their role in vivo. Aim: We introduce a robust approach for quantitative longitudinal imaging of PNNs in brains of awake mice at subcellular resolution. Approach: We label PNNs in vivo with commercially available compounds and monitor their dynamics with two-photon imaging. Results: Using our approach, we show that it is possible to longitudinally follow the same PNNs in vivo while monitoring degradation and reconstitution of PNNs. We demonstrate the compatibility of our method to simultaneously monitor neuronal calcium dynamics in vivo and compare the activity of neurons with and without PNNs. Conclusion: Our approach is tailored for studying the intricate role of PNNs in vivo, while paving the road for elucidating their role in different neuropathological conditions.

Intraarterial anti-leptin therapy via ICA protects ipsilateral CA1 neurons subjected to ischemia and reperfusion.

BACKGROUND: Brain reperfusion following an ischemic event is essential for tissue viability, however, it also involves processes that promote neuronal cell death. We have recently shown that local expression of the hormone leptin in cardiovascular organs drives deleterious remodeling. As cerebral ischemia-reperfusion (IR) lesions derive expression of both the leptin hormone and its receptor, we hypothesized that blocking leptin activity in the injured brain area will reduce the deleterious effects of IR injury. METHODS: C57BL6 male mice underwent bilateral common carotid artery and external carotid artery ligation. The right hemisphere was reperfused after 12 minutes, followed by intraarterial injection of either a low-dose leptin antagonist or saline solution via the ipsilateral ICA. The left common carotid artery remained ligated. Fifteen IR/leptin antagonist-injected and fourteen IR/saline-injected mice completed the experiment. Five days after surgery brains were collected and samples of the hippocampal CA1 region were analyzed for cell viability (H&E) and apoptosis (TUNEL and caspase3), for neuroinflammation (Iba1), and for signaling pathways of pSTAT3 and pSmad2. RESULTS: The right hemisphere hippocampal CA1 region subjected to IR and saline injection exhibited increased apoptosis and necrosis of pyramidal cells. Also, increased density of activated microglia/macrophages was evident around the CA1 region. Comparatively, leptin antagonist treatment at reperfusion reduced apoptosis and necrosis of pyramidal cells, as indicated by increased number of viable cells (p < 0.01), and reduced TUNEL (p < 0.001) and caspase3-positive cells (p<0.05). Furthermore, this treatment reduced the density of activated microglia/macrophages (p < 0.001) in the CA1 region. Signaling pathway analysis revealed that while pSTAT3 and pSmad2-positive cells were found surrounding the stratum pyramidal in saline-treated animals, pSTAT3 signal was undetected and pSmad2 was greatly reduced in this territory following leptin antagonist treatment (p < 0.01). CONCLUSIONS: Inhibition of leptin activity in hemispheric IR injury preserved the viability of ipsilateral hippocampal CA1 neurons, likely by preventing apoptosis and local inflammation. These results indicate that intraarterial anti-leptin therapy may have clinical potential in reducing hemispheric brain IR injury.

Blood glutamate scavengers increase pro-apoptotic signaling and reduce metastatic melanoma growth in-vivo.

Inhibition of extracellular glutamate (Glu) release decreases proliferation and invasion, induces apoptosis, and inhibits melanoma metastatic abilities. Previous studies have shown that Blood-glutamate scavenging (BGS), a novel treatment approach, has been found to be beneficial in attenuating glioblastoma progression by reducing brain Glu levels. Therefore, in this study we evaluated the ability of BGS treatment to inhibit brain metastatic melanoma progression in-vivo. RET melanoma cells were implanted in C56BL/6J mice to induce brain melanoma tumors followed by treatment with BGS or vehicle administered for fourteen days. Bioluminescent imaging was conducted to evaluate tumor growth, and plasma/CSF Glu levels were monitored throughout. Immunofluorescence staining of Ki67 and 53BP1 was used to analyze tumor cell proliferation and DNA double-strand breaks. In addition, we analyzed CD8, CD68, CD206, p-STAT1 and iNOS expression to evaluate alterations in tumor micro-environment and anti-tumor immune response due to treatment. Our results show that BGS treatment reduces CSF Glu concentration and consequently melanoma growth in-vivo by decreasing tumor cell proliferation and increasing pro-apoptotic signaling in C56BL/6J mice. Furthermore, BGS treatment supported CD8+ cell recruitment and CD68+ macrophage invasion. These findings suggest that BGS can be of potential therapeutic relevance in the treatment of metastatic melanoma.

Single Cortical Microinfarcts Lead to Widespread Microglia/Macrophage Migration Along the White Matter.

Loss of cognitive function with aging is a complex and poorly understood process. Recently, clinical research has linked the occurrence of cortical microinfarcts to cognitive decline. Cortical microinfarcts form following the occlusion of penetrating vessels and are considered to be restricted to the proximity of the occluded vessel. Whether and how such local events propagate and affect remote brain regions remain unknown. To this end, we combined histological analysis and longitudinal diffusion tensor imaging (DTI), following the targeted-photothrombotic occlusion of single cortical penetrating vessels. Occlusions resulted in distant tissue reorganization across the mouse brain. This remodeling co-occurred with the formation of a microglia/macrophage migratory path along subcortical white matter tracts, reaching the contralateral hemisphere through the corpus callosum and leaving a microstructural signature detected by DTI-tractography. CX3CR1-deficient mice exhibited shorter trail lengths, differential remodeling, and only ipsilateral white matter tract changes. We concluded that microinfarcts lead to brain-wide remodeling in a microglial CX3CR1-dependent manner.

Prevention of liver metastases through perioperative acute CpG-C immune stimulation.

Following excision of colorectal tumors, metastatic disease is prevalent, primarily occurs in the liver, and is highly predictive of poor prognosis. The perioperative period is now recognized as critical in determining the incidence of postoperative metastases and long-term cancer outcomes. Thus, various perioperative prophylactic interventions are currently studied during this time frame. However, immune stimulation during the perioperative period has rarely been attempted due to specific contraindications to surgery and various adverse effects. Here, to prevent liver metastases, we perioperatively employed a TLR-9 agonist, CpG-C, which exhibits minimal pyrogenic and other adverse effects in patients. We found that marginating-hepatic (MH) cells in BALB/c mice contained high percentage of NK cells, but exhibited negligible NK cytotoxicity, as previously reported in humans. However, a single CpG-C administration (25-100 µg/mouse) doubled MH-NK cell numbers, increased NK cell activation and maturation markers (NKp46, CD11b), decreased the inhibitory NKG2A ligand, and dramatically increased MH-NK-cell cytotoxicity against the syngeneic CT26 colon cancer line. Moreover, in operated mice, this innocuous intervention also markedly improved resistance to CT26 and MC38 hepatic metastases in BALB/c and C57BL/6 mice, respectively. Beneficial effects of CpG-C were mediated through activation of MH-NK cells, as indicated by an in vivo NK depletion study. Last, CpG-C protected against surgery-induced suppression of MH-NK cytotoxicity and improved their activation indices. Thus, we suggest that systemic perioperative CpG-C treatment should be considered and studied as a novel therapeutic approach to improve long-term cancer outcomes in colorectal cancer patients.

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Deleterious synergistic effects of distress and surgery on cancer metastasis: Abolishment through an integrated perioperative immune-stimulating stress-inflammatory-reducing intervention.

The perioperative period holds disproportionate impact on long-term cancer outcomes. Nevertheless, perioperative interventions to improve long-term cancer outcomes are not clinical routines, including perioperative stress-reducing or immune-stimulating approaches. Here, mimicking the clinical setting of pre-operative distress, followed by surgery, we examined the separate and combined effects of these events on the efficacy of pre-operative immune stimulation in rats and mice, and on post-operative resistance to tumor metastasis of the syngeneic mammary adenocarcinoma MADB106 in F344 rats and the CT26 colon carcinoma in Balb/C mice. The novel immune stimulating agents, GLA-SE or CpG-C (TLR-4 and TLR-9 agonists, respectively), were employed pre-operatively. Sixteen hours of pre-operative behavioral stressors (i) lowered CpG-C induced plasma IL-12 levels, and reduced resistance to MADB106 and CT-26 experimental metastases, and (ii) worsened the deleterious effects of laparotomy on metastasis in both tumor models. In rats, these effects of pre-operative stress were further studied and successfully abolished by the glucocorticoid receptor antagonist RU-486. Additionally, in vitro studies indicated the dampening effect of corticosterone on immune stimulation. Last, we tested a perioperative integrated intervention in the context of pre-operative stress and laparotomy, based on (i) antagonizing the impact of glucocorticoids before surgery, (ii) activating anti-metastatic immunity perioperatively, and (iii) blocking excessive operative and post-operative adrenergic and prostanoid responses. This integrated intervention successfully and completely abolished the deleterious effects of stress and of surgery on post-operative resistance to experimental metastasis. Such and similar integrated approaches can be studied clinically in cancer patients.

Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation.

Brain metastases are prevalent in various types of cancer and are often terminal, given the low efficacy of available therapies. Therefore, preventing them is of utmost clinical relevance, and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a toll-like receptor (TLR) 9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, natural killer (NK) cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier (BBB) integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG- C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than nonactivated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the antitumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.

Regeneration of Functional Adrenal Tissue Following Bilateral Adrenalectomy.

It is assumed that after complete bilateral adrenalectomy (ADX), no adrenal tissue will redevelop and adrenal hormone levels will remain low and unaffected by stress. However, anecdotal observations in animals and in patients suggest that under some unknown circumstances the opposite can occur. Herein, we studied whether adrenalectomized rats can develop an alternative source of systemic corticosterone after complete bilateral ADX with minimal replacement therapy. Male and female rats underwent either a standard ADX, in which the glands were removed with minimal surrounding adipose tissue, or an extensive ADX, in which glands were removed with most surrounding adipose tissue. Excised glands were histologically tested for completeness, and corticosterone replacement was nullified within 1 to 3 weeks postoperatively. In four experiments and in both excision approaches, some rats gradually reestablished baseline corticosterone levels and stress response in a time-dependent manner, but differences were observed in the reestablishing rates: 80% in standard ADX vs 20% in extensive ADX. Upon searching for the source of corticosterone secretion, we were surprised to find functional macroscopic foci of adrenocortical tissue without medullary tissue, mostly proximal to the original location. Chronic stress accelerated corticosterone level reestablishment. We hypothesized that underlying this phenomenon were preexisting ectopic microscopic foci of adrenocortical-like tissue or a few adrenal cells that were pre-embedded in surrounding tissue or detached from the excised gland upon removal. We concluded that adrenalectomized animals may develop compensatory mechanisms and suggest that studies employing ADX consider additional corticosterone supplementation, minimize stress, and verify the absence of circulating corticosterone.

Sensory Deprivation Triggers Synaptic and Intrinsic Plasticity in the Hippocampus.

Hippocampus, a temporal lobe structure involved in learning and memory, receives information from all sensory modalities. Despite extensive research on the role of sensory experience in cortical map plasticity, little is known about whether and how sensory experience regulates functioning of the hippocampal circuits. Here, we show that 9 ± 2 days of whisker deprivation during early mouse development depresses activity of CA3 pyramidal neurons by several principal mechanisms: decrease in release probability, increase in the fraction of silent synapses, and reduction in intrinsic excitability. As a result of deprivation-induced presynaptic inhibition, CA3-CA1 synaptic facilitation was augmented at high frequencies, shifting filtering properties of synapses. The changes in the AMPA-mediated synaptic transmission were accompanied by an increase in NR2B-containing NMDA receptors and a reduction in the AMPA/NMDA ratio. The observed reconfiguration of the CA3-CA1 connections may represent a homeostatic adaptation to augmentation in synaptic activity during the initial deprivation phase. In adult mice, tactile disuse diminished intrinsic excitability without altering synaptic facilitation. We suggest that sensory experience regulates computations performed by the hippocampus by tuning its synaptic and intrinsic characteristics.

Maintaining unperturbed cerebral blood flow is key in the study of brain metastasis and its interactions with stress and inflammatory responses.

Blood-borne brain metastases are associated with poor prognosis, but little is known about the interplay between cerebral blood flow, surgical stress responses, and the metastatic process. The intra-carotid inoculation approach, traditionally used in animal studies, involves permanent occlusion of the common carotid artery (CCA). Herein we introduced a novel intra-carotid inoculation approach that avoids CCA ligation, namely - assisted external carotid artery inoculation (aECAi) - and compared it to the traditional approach in C57/BL6 mice, assessing cerebral blood flow; particle distribution; blood-brain barrier (BBB) integrity; stress, inflammatory and immune responses; and brain tumor retention and growth. Doppler flowmetry and two-photon imaging confirmed that only in the traditional approach regional and capillary cerebral blood flux were significantly reduced. Corticosterone and plasma IL-6 levels were higher in the traditional approach, splenic numbers of NK, CD3+, granulocytes, and dendritic cells were lower, and many of these indices were more profoundly affected by surgical stress in the traditional approach. BBB integrity was unaffected. Administration of spherical beads indicated that CCA ligation significantly limited brain distribution of injected particles, and inoculation of D122-LLC syngeneic tumor cells resulted in 10-fold lower brain tumor-cell retention in the traditional approach. Last, while most of the injected tumor cells were arrested in extra-cranial head areas, our method improved targeting of brain-tissue by 7-fold. This head versus brain distribution difference, commonly overlooked, cannot be detected using in vivo bioluminescent imaging. Overall, it is crucial to maintain unperturbed cerebral blood flow while studying brain metastasis and interactions with stress and inflammatory responses.

Perioperative treatment with the new synthetic TLR-4 agonist GLA-SE reduces cancer metastasis without adverse effects.

The use of TLR agonists as an anti-cancer treatment is gaining momentum given their capacity to activate various host cellular responses through the secretion of inflammatory cytokines and type-I interferons. It is now also recognized that the perioperative period is a window of opportunity for various interventions aiming at reducing the risk of cancer metastases-the major cause of cancer related death. However, immune-stimulatory approach has not been used perioperatively given several contraindications to surgery. To overcome these obstacles, in this study, we used the newly introduced, fully synthetic TLR-4 agonist, Glucopyranosyl Lipid-A (GLA-SE), in various models of cancer metastases, and in the context of acute stress or surgery. Without exerting evident adverse effects, a single systemic administration of GLA-SE rapidly and dose dependently elevated both innate and adaptive immunity in the circulation, lungs and the lymphatic system. Importantly, GLA-SE treatment led to reduced metastatic development of a mammary adenocarcinoma and a colon carcinoma by approximately 40-75% in F344 rats and BALB/c mice, respectively, at least partly through elevating marginating-pulmonary NK cell cytotoxicity. GLA-SE is safe and well tolerated in humans, and currently is used as an adjuvant in phase-II clinical trials. Given that the TLR-4 receptor and its signaling cascade is highly conserved throughout evolution, our current results suggest that GLA-SE may be a promising immune stimulatory agent in the context of oncological surgeries, aiming to reduce long-term cancer recurrence.