Longitudinal Neuropathological Consequences of Extracranial Radiation Therapy in Mice.

Cancer-related cognitive impairment (CRCI) is a consequence of chemotherapy and extracranial radiation therapy (ECRT). Our prior work demonstrated gliosis in the brain following ECRT in SKH1 mice. The signals that induce gliosis were unclear. Right hindlimb skin from SKH1 mice was treated with 20 Gy or 30 Gy to induce subclinical or clinical dermatitis, respectively. Mice were euthanized at 6 h, 24 h, 5 days, 12 days, and 25 days post irradiation, and the brain, thoracic spinal cord, and skin were collected. The brains were harvested for spatial proteomics, immunohistochemistry, Nanostring nCounter® glial profiling, and neuroinflammation gene panels. The thoracic spinal cords were evaluated by immunohistochemistry. Radiation injury to the skin was evaluated by histology. The genes associated with neurotransmission, glial cell activation, innate immune signaling, cell signal transduction, and cancer were differentially expressed in the brains from mice treated with ECRT compared to the controls. Dose-dependent increases in neuroinflammatory-associated and neurodegenerative-disease-associated proteins were measured in the brains from ECRT-treated mice. Histologic changes in the ECRT-treated mice included acute dermatitis within the irradiated skin of the hindlimb and astrocyte activation within the thoracic spinal cord. Collectively, these findings highlight indirect neuronal transmission and glial cell activation in the pathogenesis of ECRT-related CRCI, providing possible signaling pathways for mitigation strategies.

Glial Cell Activation and Oxidative Stress in Retinal Degeneration Induced by ß-Alanine Caused Taurine Depletion and Light Exposure.

We investigate glial cell activation and oxidative stress induced by taurine deficiency secondary to ß-alanine administration and light exposure. Two months old Sprague-Dawley rats were divided into a control group and three experimental groups that were treated with 3% ß-alanine in drinking water (taurine depleted) for two months, light exposed or both. Retinal and external thickness were measured in vivo at baseline and pre-processing with Spectral-Domain Optical Coherence Tomography (SD-OCT). Retinal cryostat cross sections were immunodetected with antibodies against various antigens to investigate microglial and macroglial cell reaction, photoreceptor outer segments, synaptic connections and oxidative stress. Taurine depletion caused a decrease in retinal thickness, shortening of photoreceptor outer segments, microglial cell activation, oxidative stress in the outer and inner nuclear layers and the ganglion cell layer and synaptic loss. These events were also observed in light exposed animals, which in addition showed photoreceptor death and macroglial cell reactivity. Light exposure under taurine depletion further increased glial cell reaction and oxidative stress. Finally, the retinal pigment epithelial cells were Fluorogold labeled and whole mounted, and we document that taurine depletion impairs their phagocytic capacity. We conclude that taurine depletion causes cell damage to various retinal layers including retinal pigment epithelial cells, photoreceptors and retinal ganglion cells, and increases the susceptibility of the photoreceptor outer segments to light damage. Thus, beta-alanine supplements should be used with caution.

Dose-dependent long-term effects of a single radiation event on behaviour and glial cells.

PURPOSE: The increasing use of low-dose ionizing radiation in medicine requires a systematic study of its long-term effects on the brain, behaviour and its possible association with neurodegenerative disease vulnerability. Therefore, we analysed the long-term effects of a single low-dose irradiation exposure at 10 weeks of age compared to medium and higher doses on locomotor, emotion-related and sensorimotor behaviour in mice as well as on hippocampal glial cell populations. MATERIALS AND METHODS: We determined the influence of radiation dose (0, 0.063, 0.125 or 0.5 Gy), time post-irradiation (4, 12 and 18 months p.i.), sex and genotype (wild type versus mice with Ercc2 DNA repair gene point mutation) on behaviour. RESULTS: The high dose (0.5 Gy) had early-onset adverse effects at 4 months p.i. on sensorimotor recruitment and late-onset negative locomotor effects at 12 and 18 months p.i. Notably, the low dose (0.063 Gy) produced no early effects but subtle late-onset (18 months) protective effects on sensorimotor recruitment and exploratory behaviour. Quantification and morphological characterization of the microglial and the astrocytic cells of the dentate gyrus 24 months p.i. indicated heightened immune activity after high dose irradiation (0.125 and 0.5 Gy) while conversely, low dose (0.063 Gy) induced more neuroprotective features. CONCLUSION: This is one of the first studies demonstrating such long-term and late-onset effects on brain and behaviour after a single radiation event in adulthood.

Red-Light (670 nm) Therapy Reduces Mechanical Sensitivity and Neuronal Cell Death, and Alters Glial Responses after Spinal Cord Injury in Rats.

Individuals with spinal cord injury (SCI) often develop debilitating neuropathic pain, which may be driven by neuronal damage and neuroinflammation. We have previously demonstrated that treatment using 670 nm (red) light irradiation alters microglia/macrophage responses and alleviates mechanical hypersensitivity at 7 days post-injury (dpi). Here, we investigated the effect of red light on the development of mechanical hypersensitivity, neuronal markers, and glial response in the subacute stage (days 1-7) following SCI. Wistar rats were subjected to a mild hemi-contusion SCI at vertebra T10 or to sham surgery followed by daily red-light treatment (30 min/day; 670 nm LED; 35 mW/cm2) or sham treatment. Mechanical sensitivity of the rat dorsum was assessed from 1 dpi and repeated every second day. Spinal cords were collected at 1, 3, 5, and 7 dpi for analysis of myelination, neurofilament protein NF200 expression, neuronal cell death, reactive astrocytes (glial fibrillary acidic protein [GFAP]+ cells), interleukin 1 ß (IL-1ß) expression, and inducible nitric oxide synthase (iNOS) production in IBA1+ microglia/macrophages. Red-light treatment significantly reduced the cumulative mechanical sensitivity and the hypersensitivity incidence following SCI. This effect was accompanied by significantly reduced neuronal cell death, reduced astrocyte activation, and reduced iNOS expression in IBA1+ cells at the level of the injury. However, myelin and NF200 immunoreactivity and IL-1ß expression in GFAP+ and IBA1+ cells were not altered by red-light treatment. Thus, red-light therapy may represent a useful non-pharmacological approach for treating pain during the subacute period after SCI by decreasing neuronal loss and modulating the inflammatory glial response.

Nongenetic optical modulation of neural stem cell proliferation and neuronal/glial differentiation.

Photostimulation has been widely used in neuromodulation. However, existing optogenetics techniques require genetic alternation of the targeted cell or tissue. Here, we report that neural stem cells (NSCs) constitutionally express blue/red light-sensitive photoreceptors. The proliferation and regulation of NSCs to neuronal or glial cells are wavelength-specific. Our results showed a 4.3-fold increase in proliferation and 2.7-fold increase in astrocyte differentiation for cells under low-power blue monochromatic light exposure (455 nm, 300 µW/cm2). The melanopsin (Opn4)/transient receptor potential channel 6 (TRPC6) non-visual opsin serves as a key photoreceptor response to blue light irradiation. Two-dimensional gel electrophoresis coupled with mass spectrometry further highlighted the Jun activation domain-binding protein 1 (Jab1) as a novel and specific modulator in phototransduction pathways induced by blue light exposure. Quiescent adult NSCs reside in specific regions of the mammalian brain. Therefore, we showed that melanopsin/TRPC6 expressed in these regions and blue light stimulation through optical fibers could directly stimulate the NSCs in vivo. Upconversion nanoparticles (UCNPs) converted deep-penetrating near-infrared (NIR) light into specific wavelengths of visible light. Accordingly, we demonstrated that UCNP-mediated NIR light could be used to modulate in vivo NSC differentiation in a less invasive manner. In the future, this light-triggered system of NSCs will enable nongenetic and noninvasive neuromodulation with therapeutic potential for central nervous system diseases.

Са2+- and NF-κB-dependent generation of NO in the photosensitized neurons and satellite glial cells.

Photodynamic therapy (PDT) is used for killing of malignant cells in tumors including brain cancer. It can also damage normal neurons and glial cells. Nitric oxide (NO) is known to control PDT-induced cell death. To study the mechanisms that regulate NO generation in photosensitized neurons and glial cells, we used a simple model object - isolated crayfish mechanoreceptor that consists of a single sensory neuron surrounded by glial cells. PDT induced NO generation in glial cells, neuronal dendrites, and, less, in soma and axon. Using modulators of the cytosolic Ca2+ level and nuclear factor-kappa B (NF-κB) activity, we showed that Ca2+ and NF-κB regulate NO generation in the photosensitized neurons and glia. Actually, NO production was stimulated by 4-fold cadmium chloride (CdCl2) concentration in the saline, Ca2+ ionophore ionomycine, or inhibition of Ca2+-ATPase in the endoplasmic reticulum by 2,5-ditert-butylbenzene-1,4-diol (tBuBHQ). Oppositely, CdCl2 or nifedipine, blockers of Ca2+ channels in the plasma membrane, decreased NO generation. NO production was also inhibited by S-methylthiouronium sulfate (SMT), inhibitor of Ca2+-independent inducible NO synthase. SMT also prevented the stimulation of PDT-induced NO generation by NF-κB activator prostratin. This suggests the involvement of both Ca2+-dependent neuronal NO synthase and Ca2+-independent inducible NO synthase, which is regulated by NF-κB, in NO production in the crayfish neurons and glia.

RCC2 promotes proliferation and radio-resistance in glioblastoma via activating transcription of DNMT1.

Regulator of chromosome condensation 2 (RCC2) is a regulator of cell-cycle progression linked in multiple cancers to pro-tumorigenic phenomena including promotion of tumor growth, tumor metastases and poorer patient prognoses. However, the role of RCC2 in GBM remains under-investigated. Here, we sought to determine the relevance of RCC2 in GBM, as well as its roles in GBM development, progression and prognosis. Initial clinical evaluation determined significant RCC2 enrichment in GBM when compared to normal brain tissue, and elevated expression was closely associated with a poorer prognosis in glioma patients. Via shRNA inhibition, we determined that RCC2 is essential to tumor proliferation and tumorigenicity in vitro and in vivo. Additionally, RCC2 was determined to promote radioresistance of GBM tumor cells. Investigation of the underlying mechanisms implicated DNA mismatch repair, JAK-STAT pathway and activated transcription of DNA methyltransferase 1 (DNMT1). For validation, pharmacologic inhibition via administration of a DNMT1 inhibitor demonstrated attenuated GBM tumor growth both in vitro and in vivo. Collectively, this study determined a novel therapeutic target for GBM in the form of RCC2, which plays a pivotal role in GBM proliferation and radio-resistance via regulation of DNMT1 expression in a p-STAT3 dependent manner.

Sodium sulfide selectively induces oxidative stress, DNA damage, and mitochondrial dysfunction and radiosensitizes glioblastoma (GBM) cells.

Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na2S), a hydrogen sulfide (H2S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na2S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na2S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na2S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na2S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ0) was not altered by Na2S whereas the survival of LN-18 ρ+ cells was compromised supports this conclusion. When cells were treated with Na2S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H2S being a radiosensitizer. Therefore, this study provides the first evidence that H2S donors could be used in GBM therapy to potentiate radiation-induced killing.

Neurotrophin receptor tyrosine kinases regulated with near-infrared light.

Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.

Glial ensheathment of the somatodendritic compartment regulates sensory neuron structure and activity.

Sensory neurons perceive environmental cues and are important of organismal survival. Peripheral sensory neurons interact intimately with glial cells. While the function of axonal ensheathment by glia is well studied, less is known about the functional significance of glial interaction with the somatodendritic compartment of neurons. Herein, we show that three distinct glia cell types differentially wrap around the axonal and somatodendritic surface of the polymodal dendritic arborization (da) neuron of the Drosophila peripheral nervous system for detection of thermal, mechanical, and light stimuli. We find that glial cell-specific loss of the chromatin modifier gene dATRX in the subperineurial glial layer leads to selective elimination of somatodendritic glial ensheathment, thus allowing us to investigate the function of such ensheathment. We find that somatodendritic glial ensheathment regulates the morphology of the dendritic arbor, as well as the activity of the sensory neuron, in response to sensory stimuli. Additionally, glial ensheathment of the neuronal soma influences dendritic regeneration after injury.

The functional synergism of microRNA clustering provides therapeutically relevant epigenetic interference in glioblastoma.

MicroRNA deregulation is a consistent feature of glioblastoma, yet the biological effect of each single gene is generally modest, and therapeutically negligible. Here we describe a module of microRNAs, constituted by miR-124, miR-128 and miR-137, which are co-expressed during neuronal differentiation and simultaneously lost in gliomagenesis. Each one of these miRs targets several transcriptional regulators, including the oncogenic chromatin repressors EZH2, BMI1 and LSD1, which are functionally interdependent and involved in glioblastoma recurrence after therapeutic chemoradiation. Synchronizing the expression of these three microRNAs in a gene therapy approach displays significant anticancer synergism, abrogates this epigenetic-mediated, multi-protein tumor survival mechanism and results in a 5-fold increase in survival when combined with chemotherapy in murine glioblastoma models. These transgenic microRNA clusters display intercellular propagation in vivo, via extracellular vesicles, extending their biological effect throughout the whole tumor. Our results support the rationale and feasibility of combinatorial microRNA strategies for anticancer therapies.

Retinal Neuron Is More Sensitive to Blue Light-Induced Damage than Glia Cell Due to DNA Double-Strand Breaks.

Blue light is a major component of visible light and digital displays. Over-exposure to blue light could cause retinal damage. However, the mechanism of its damage is not well defined. Here, we demonstrate that blue light (900 lux) impairs cell viability and induces cell apoptosis in retinal neurocytes in vitro. A DNA electrophoresis assay shows severe DNA damage in retinal neurocytes at 2 h after blue light treatment. γ-H2AX foci, a specific marker of DNA double-strand breaks (DSBs), is mainly located in the Map2-posotive neuron other than the glia cell. After assaying the expression level of proteins related to DNA repair, Mre11, Ligase IV and Ku80, we find that Ku80 is up-regulated in retinal neurocytes after blue light treatment. Interestingly, Ku80 is mainly expressed in glia fibrillary acidic protein (GFAP)-positive glia cells. Moreover, following blue light exposure in vivo, DNA DSBs are shown in the ganglion cell layer and only observed in Map2-positive cells. Furthermore, long-term blue light exposure significantly thinned the retina in vivo. Our findings demonstrate that blue light induces DNA DSBs in retinal neurons, and the damage is more pronounced compared to glia cells. Thus, this study provides new insights into the mechanisms of the effect of blue light on the retina.

Blue light exposure in vitro causes toxicity to trigeminal neurons and glia through increased superoxide and hydrogen peroxide generation.

Today the noxiousness of blue light from natural and particularly artificial (fluorescent tubes, LED panels, visual displays) sources is actively discussed in the context of various ocular diseases. Many of them have an important neurologic component and are associated with ocular pain. This neuropathic signal is provided by nociceptive neurons from trigeminal ganglia. However, the phototoxicity of blue light on trigeminal neurons has not been explored so far. The aim of the present in vitro study was to investigate the cytotoxic impact of various wavebands of visible light (410-630 nm) on primary cell culture of mouse trigeminal neural and glial cells. Three-hour exposure to narrow wavebands of blue light centered at 410, 440 and 480 nm of average 1.1 mW/cm2 irradiance provoked cell death, altered cell morphology and induced oxidative stress and inflammation. These effects were not observed for other tested visible wavebands. We observed that neurons and glial cells processed the light signal in different manner, in terms of resulting superoxide and hydrogen peroxide generation, inflammatory biomarkers expression and phototoxic mitochondrial damage. We analyzed the pathways of photic signal reception, and we proposed that, in trigeminal cells, in addition to widely known mitochondria-mediated light absorption, light could be received by means of non-visual opsins, melanopsin (opn4) and neuropsin (opn5). We also investigated the mechanisms underlying the observed phototoxicity, further suggesting an important role of the endoplasmic reticulum in neuronal transmission of blue-light-toxic message. Taken together, our results give some insight into circuit of tangled pain and photosensitivity frequently observed in patients consulting for these ocular symptoms.

TGF-ß/Smads Signaling Affects Radiation Response and Prolongs Survival by Regulating DNA Repair Genes in Malignant Glioma.

To understand the molecular mechanism underlying the causal relationship between aberrant upregulation of transforming growth factor beta (TGF-ß) and radio-resistance in glioma. The mouse glioma cell GL261 was irradiated, and relative expression of TGF-ß/Smad signaling genes was determined by real-time PCR and western blotting. The DNA repair response on exogenous TGF-ß or LY2109761 was evaluated by quantification of diverse genes by real-time PCR and western blotting. Xenograft mice were employed for in vivo investigation to assess the response to irradiation and LY2109761 either alone or in combination. The expression of DNA repair genes was further determined in the xenograft tumor. The TGF-ß/Smad signaling pathway was activated by radiation in the GL261 cell line. The exogenous complement of TGF-ß significantly stimulated DNA repair response. Administration of LY2109761 suppressed DNA repair genes. Simultaneous treatment with LY2109761 abrogated the upregulation of DNA repair genes in GL261. In the xenograft tumor model, LY2109761 synergistically improved the therapeutic effect of radiation via improvement of sensitivity. Our data suggested that LY2109761 treatment re-sensitized glioma to radiation via antagonizing TGF-ß/Smad-induced DNA repair.

Evaluation of early acute radiation-induced brain injury: Hybrid multifunctional MRI-based study.

PURPOSE: Radiation injury is a serious threat to humans that requires prompt and accurate diagnosis and assessment. Currently, there is no effective imaging method to evaluate acute radiation injury in the early stage. We used hybrid multifunctional MRI to evaluate acute radiation-induced brain injury. MATERIALS AND METHODS: Different extents of brain injury were created by exposing SD rats to different radiation doses, namely, 0, 10, 20, 30 and 40 Gy. DCE, IVIM-MRI and MRS were performed on the 5th day after irradiation. Immunohistochemistry, western blotting and electron microscopy were used to determine histopathological changes in neurons and glial cells. RESULTS: The Ktrans, Ve, and iAUC values in DCE and the S0, f and D* values in IVIM showed significant positive correlations with injury grade. In particular, Ktrans, iAUC and S0 showed very good correlations with injury grade (r > 0.5, P < 0.05), and the values in the 30 Gy group were significantly higher than those in the other groups (P < 0.05). The NAA/Cr ratio in the 30 Gy group was significantly lower than those in the other groups, whereas the NAA/Cho ratio increased from the 10 Gy to the 20 Gy group and decreased significantly in the 30 Gy group (P < 0.05). VEGF, Caspase-3 and GFAP increased with irradiation dose increasing from 10 Gy to 30 Gy (P < 0.05). ROC analysis demonstrated that multifunctional MRI was more effective for diagnosing the 30 Gy group than it was for the 10 Gy and 20 Gy groups. CONCLUSION: Hybrid multifunctional MRI can noninvasively evaluate acute radiation-induced brain injury in the early stage, particularly high-dose radiation exposure.

Upregulation of Connexin-43 is Critical for Irradiation-induced Neuroinflammation.

BACKGROUND: Radiation therapy is widely used for the treatment of pituitary adenomas. Unfortunately, it might raise the risk of ischemic stroke, with neuroinflammation being a major pathological process. Astrocytes are the most abundant cell type in the central nervous system and have been reported for playing important roles in ischemic stroke. OBJECTIVE: Here we studied how γ-radiation would introduce astrocytes into a detrimental state for neuroinflammation and provide new theory evidence and target for the clinical management of inflammation- related neural damage after radiation-induced ischemic stroke. METHOD: HA-1800 cells were treated with γ-radiation and then the protein and mRNA levels of Connexin (Cx)-43 were evaluated by western and q-PCR. The culture supernatant was collected and the concentrations of the inflammatory factors were determined by ELISA. MiRNA complementary to Cx-43 was designed through the online tools. RESULTS: Cx-43 is upregulated in the treatment of γ-radiation in astrocytes and γ-radiation introduced the detrimental function of astrocytes: cell viability was reduced while the apoptotic cells were increased. Inflammatory factors like tumor necrosis factor alpha, interferon gamma, interleukin-6, interleukin 1-beta were dramatically up-regulated by the irradiation. MiR-374a rescued irradiation induced Cx-43 up-regulation of astrocytes and eliminated detrimental function triggered by γ-radiation. CONCLUSION: Cx-43 expression level may play an important role in the inflammation-related neural damage after irradiation-induced ischemic stroke.

Focal brain lesions induced with ultraviolet irradiation.

Lesion and inactivation methods have played important roles in neuroscience studies. However, traditional techniques for creating a brain lesion are highly invasive, and control of lesion size and shape using these techniques is not easy. Here, we developed a novel method for creating a lesion on the cortical surface via 365 nm ultraviolet (UV) irradiation without breaking the dura mater. We demonstrated that 2.0 mWh UV irradiation, but not the same amount of non-UV light irradiation, induced an inverted bell-shaped lesion with neuronal loss and accumulation of glial cells. Moreover, the volume of the UV irradiation-induced lesion depended on the UV light exposure amount. We further succeeded in visualizing the lesioned site in a living animal using magnetic resonance imaging (MRI). Importantly, we also observed using an optical imaging technique that the spread of neural activation evoked by adjacent cortical stimulation disappeared only at the UV-irradiated site. In summary, UV irradiation can induce a focal brain lesion with a stable shape and size in a less invasive manner than traditional lesioning methods. This method is applicable to not only neuroscientific lesion experiments but also studies of the focal brain injury recovery process.

TET1 deficiency attenuates the DNA damage response and promotes resistance to DNA damaging agents.

Recent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G2/M check point and expression of cyclin B1 were greatly diminished in TET1-deficient cells, and TET1-deficient cells displayed lower levels of γH2A.x following exposure to IR. Levels of DNA-PKcs, which are DNA-PK complex members, were lower in TET1-deficient cells compared with control cell lines. However, levels of ATM were similar in both cell lines. Cyclin B1, DNA-PKcs, and γH2A.x levels were each rescued by reintroduction of the TET1 catalytic domain. Finally, cytosine methylation within intron 1 of PRKDC, the gene encoding DNA-PKcs, was significantly higher upon depletion of TET1. Taken together, this study illustrates the involvement of TET1 in the different arms of the DDR and suggests its loss results in the continued survival of cells with genomic instability.

Surpassing light-induced cell damage in vitro with novel cell culture media.

Light is extensively used to study cells in real time (live cell imaging), separate cells using fluorescence activated cell sorting (FACS) and control cellular functions with light sensitive proteins (Optogenetics). However, photo-sensitive molecules inside cells and in standard cell culture media generate toxic by-products that interfere with cellular functions and cell viability when exposed to light. Here we show that primary cells from the rat central nervous system respond differently to photo-toxicity, in that astrocytes and microglia undergo morphological changes, while in developing neurons and oligodendrocyte progenitor cells (OPCs) it induces cellular death. To prevent photo-toxicity and to allow for long-term photo-stimulation without causing cellular damage, we formulated new photo-inert media called MEMO and NEUMO, and an antioxidant rich and serum free supplement called SOS. These new media reduced the detrimental effects caused by light and allowed cells to endure up to twenty times more light exposure without adverse effects, thus bypassing the optical constraints previously limiting experiments.

Low level laser therapy alters satellite glial cell expression and reverses nociceptive behavior in rats with neuropathic pain.

BACKGROUND: Nerve injury often results in persistent or chronic neuropathic pain characterized by spontaneous burning pain accompanied by allodynia and hyperalgesia. Low level laser therapy (LLLT) is a noninvasive method that has proved to be clinically effective in reducing pain sensitivity and consequently in improving the quality of life. Here we examined the effects of LLLT on pain sensitivity induced by chronic constriction injury (CCI) in rats. CCI was performed on adult male rats, subjected thereafter to 10 sessions of LLLT, every other day, and starting 14 days after CCI. Over the treatment period, the animals were evaluated for nociception using behavioral tests, such as allodynia, thermal and mechanical hyperalgesia. Following the sessions, we observed the involvement of satellite glial cells in the dorsal root ganglion (DRG) using immunoblotting and immunofluorescence approaches. In addition we analyzed the expression levels of interleukin 1 (IL-1ß) and fractalkine (FKN) after the same stimulus. RESULTS: LLLT induced an early reduction (starting at the second session; p ≤ 0.001) of the mechanical and thermal hyperalgesia and allodynia in CCI rats, which persisted until the last session. Regarding cellular changes, we observed a decrease of GFAP (50%; p ≤ 0.001) expression after LLLT in the ipsilateral DRG when compared with the naive group. We also observed a significant increase of pro-inflammatory cytokines after CCI, whereas LLLT dramatically inhibited the overexpression of these proteins. CONCLUSIONS: These data provide evidence that LLLT reverses CCI-induced behavioral hypersensitivity, reduces glial cell activation in the DRG and decreases pro-inflammatory cytokines; we suggest that this involvement of glial cells can be one potential mechanism in such an effect.