PNIPAAm-co-Jeffamine® (PNJ) scaffolds as in vitro models for niche enrichment of glioblastoma stem-like cells.

Glioblastoma (GBM) is the most common adult primary brain tumor, and the 5-year survival rate is less than 5%. GBM malignancy is driven in part by a population of GBM stem-like cells (GSCs) that exhibit indefinite self-renewal capacity, multipotent differentiation, expression of neural stem cell markers, and resistance to conventional treatments. GSCs are enriched in specialized niche microenvironments that regulate stem phenotypes and support GSC radioresistance. Therefore, identifying GSC-niche interactions that regulate stem phenotypes may present a unique target for disrupting the maintenance and persistence of this treatment resistant population. In this work, we engineered 3D scaffolds from temperature responsive poly(N-isopropylacrylamide-co-Jeffamine M-1000® acrylamide), or PNJ copolymers, as a platform for enriching stem-specific phenotypes in two molecularly distinct human patient-derived GSC cell lines. Notably, we observed that, compared to conventional neurosphere cultures, PNJ cultured GSCs maintained multipotency and exhibited enhanced self-renewal capacity. Concurrent increases in expression of proteins known to regulate self-renewal, invasion, and stem maintenance in GSCs (NESTIN, EGFR, CD44) suggest that PNJ scaffolds effectively enrich the GSC population. We further observed that PNJ cultured GSCs exhibited increased resistance to radiation treatment compared to GSCs cultured in standard neurosphere conditions. GSC radioresistance is supported in vivo by niche microenvironments, and this remains a significant barrier to effectively treating these highly tumorigenic cells. Taken in sum, these data indicate that the microenvironment created by synthetic PNJ scaffolds models niche enrichment of GSCs in patient-derived GBM cell lines, and presents tissue engineering opportunities for studying clinically important behaviors such as radioresistance in vitro.

Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology.

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope.

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Comparing high-resolution microscopy techniques for potential intraoperative use in guiding low-grade glioma resections.

BACKGROUND AND OBJECTIVES: Fluorescence image-guided surgery (FIGS), with contrast provided by 5-ALA-induced PpIX, has been shown to enable a higher extent of resection of high-grade gliomas. However, conventional FIGS with low-power microscopy lacks the sensitivity to aid in low-grade glioma (LGG) resection because PpIX signal is weak and sparse in such tissues. Intraoperative high-resolution microscopy of PpIX fluorescence has been proposed as a method to guide LGG resection, where sub-cellular resolution allows for the visualization of sparse and punctate mitochondrial PpIX production in tumor cells. Here, we assess the performance of three potentially portable high-resolution microscopy techniques that may be used for the intraoperative imaging of human LGG tissue samples with PpIX contrast: high-resolution fiber-optic microscopy (HRFM), high-resolution wide-field microscopy (WFM), and dual-axis confocal (DAC) microscopy. MATERIALS AND METHODS: Thick unsectioned human LGG tissue samples (n = 7) with 5-ALA-induced PpIX contrast were imaged using three imaging techniques (HRFM, WFM, DAC). The average signal-to-background ratio (SBR) was then calculated for each imaging modality (5 images per tissue, per modality). RESULTS: HRFM provides the ease of use and portability of a flexible fiber bundle, and is simple and inexpensive to build. However, in most cases (6/7), HRFM is not capable of detecting PpIX signal from LGGs due to high autofluorescence, generated by the fiber bundle under laser illumination at 405 nm, which overwhelms the PpIX signal and impedes its visualization. WFM is a camera-based method possessing high lateral resolution but poor axial resolution, resulting in sub-optimal image contrast. CONCLUSIONS: Consistent successful detection of PpIX signal throughout our human LGG tissue samples (n = 7), with an acceptable image contrast (SBR >2), was only achieved using DAC microscopy, which offers superior image resolution and contrast that is comparable to histology, but requires a laser-scanning mechanism to achieve optical sectioning.

Nutrient modification of the innate immune response: a novel mechanism by which saturated fatty acids greatly amplify monocyte inflammation.

OBJECTIVE: Monocyte/macrophage inflammation is an important contributor to diabetes and cardiovascular disease. Studies have suggested saturated fatty acids (SFA) induce monocyte inflammation in a Toll-like receptor-4-dependent manner, but recent data suggest SFA do not directly interact with Toll-like receptor-4. The present study tests the novel hypothesis that metabolism of SFA cooperatively amplifies Toll-like receptor-4-mediated inflammation. METHODS AND RESULTS: THP-1 monocytes exposed to 100 micromol/L SFA in vitro for 16 hours followed by 1 ng/mL lipopolysaccharide demonstrated enhanced IL-6 and IL-8 mRNA and protein expression (approximately 3-fold higher than the sum of individual responses to SFA and lipopolysaccharide). SFA had similar effects on THP-1 macrophages and primary human monocytes. This amplified lipopolysaccharide response could be blocked by inhibition of SFA metabolism to ceramide and restored by cell-permeable ceramide. Both SFA and ceramide activated PKC-zeta and the mitogen-activated protein kinases Erk, JNK, and p38. Inhibition of these pathways prevented the SFA-induced increase in cytokine expression. CONCLUSIONS: These results provide evidence for potent amplification of monocyte/macrophage innate immune responses by a novel pathway requiring metabolism of SFA to ceramide and activation of PKC-zeta/mitogen-activated protein kinases. These findings demonstrate how nutrient excess may modulate innate immune system activation and possibly contribute to development of diabetes and cardiovascular disease.

Low dose pramipexole is neuroprotective in the MPTP mouse model of Parkinson's disease, and downregulates the dopamine transporter via the D3 receptor.

BACKGROUND: Our aim was to determine if pramipexole, a D3 preferring agonist, effectively reduced dopamine neuron and fiber loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model when given at intraperitoneal doses corresponding to clinical doses. We also determined whether subchronic treatment with pramipexole regulates dopamine transporter function, thereby reducing intracellular transport of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+). METHODS: Ten 12-month old C57BL/6 mice were treated with MPTP (or saline) twice per day at 20 mg/kg s.c. (4 injections over 48 h). Mice were pretreated for 3 days and during the 2-day MPTP regimen with pramipexole (0.1 mg/kg/day) or saline. Stereological quantification of dopamine neuron number and optical density measurement of dopamine fiber loss were carried out at 1 week after treatment, using immunostaining for dopamine transporter (DAT) and tyrosine hydroxylase (TH). Additional wild-type (WT) and D3 receptor knockout (KO) mice were treated for 5 days with pramipexole (0.1 mg/kg/day) or vehicle. The kinetics of [3H]MPP+ and [3H]DA uptake (Vmax and Km) were determined 24 h later; and at 24 h and 14 days dopamine transporter density was measured by quantitative autoradiography. RESULTS: Pramipexole treatment completely antagonized the neurotoxic effects of MPTP, as measured by substantia nigra and ventral tegmental area TH-immunoreactive cell counts. MPTP- induced loss of striatal innervation, as measured by DAT-immunoreactivity, was partially prevented by pramipexole, but not with regard to TH-IR. Pramipexole also reduced DAT- immunoreactivity in non-MPTP treated mice. Subchronic treatment with pramipexole lowered the Vmax for [3H]DA and [3H]MPP+ uptake into striatal synaptosomes of WT mice. Pramipexole treatment lowered Vmax in WT but not D3 KO mice; however, D3 KO mice had lower Vmax for [3H]DA uptake. There was no change in DAT number in WT with pramipexole treatment or D3 KO mice at 24 h post-treatment, but there was a reduction in WT-pramipexole treated and not in D3 KO mice at 14 days post-treatment. CONCLUSION: These results suggest that protection occurs at clinically suitable doses of pramipexole. Protection could be due to a reduced amount of MPP+ taken up into DA terminals via DAT. D3 receptor plays an important role in this regulation of transporter uptake and availability.

Involvement of dopamine D(2)/D(3) receptors and BDNF in the neuroprotective effects of S32504 and pramipexole against 1-methyl-4-phenylpyridinium in terminally differentiated SH-SY5Y cells.

Anti-parkinsonian agents possessing both D(2) and D(3) receptor agonist properties are neuroprotective against 1-methyl-4-phenylpyridinium (MPP(+)) toxicity in a variety of in vitro models. The mechanisms underlying protection by these D(2)/D(3) receptor agonists remain poorly defined. To test if the D(3) receptor preferring agonists S32504 and pramipexole act through D(2) or D(3) receptors and via brain-derived neurotrophic factor (BDNF)-dependent pathways, we utilized a terminally differentiated neuroblastoma SH-SY5Y cell line exhibiting a dopaminergic phenotype. The cytotoxic effects of MPP(+) (LD(50) of 100 microM) were stereospecifically antagonized by S32504 (EC(50) = 2.0 microM) and, less potently, by pramipexole (EC(50) = 64.3 microM), but not by their inactive stereoisomers, R(+) pramipexole and S32601, respectively. Neuroprotective effects afforded by EC(50) doses of S32504 and pramipexole were antagonized by the selective D(3) antagonists S33084, U99194A, and SB269652, and by the D(2)/D(3) antagonist raclopride. However, the preferential D(2) receptor antagonist LY741626 was ineffective as was the D1 antagonist SCH23390. BDNF (1 nM) potently protected against MPP(+)-induced neurotoxicity. Antibody directed against BDNF concentration-dependently blocked both the neuroprotective effects of BDNF and those of pramipexole and S32504 against MPP(+). The protection afforded by BDNF was blocked by the P3K-AKT pathway inhibitor LY249002 and less so by the MEK/MAPKK pathway inhibitor PD98059. LY249002, but not PD98059, blocked the neuroprotective effects of pramipexole and S32504 against MPP(+) toxicity. In conclusion, S32504 and, less potently, pramipexole show robust, stereospecific, and long-lasting neuroprotective effects against MPP(+) toxicity that involve D(3) receptors. Their actions also reflect downstream recruitment of BDNF and via a PK3-AKT pathway.

Terminally differentiated SH-SY5Y cells provide a model system for studying neuroprotective effects of dopamine agonists.

We characterized undifferentiated (UN) and three differentiation conditions of the SH-SY5Y neuroblastoma cell line for phenotypic markers of dopaminergic cells, sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP+), the requirement to utilize the dopamine (DA) transporter (DAT) for MPP+ toxicity, and the neuroprotective effects of pramipexole. Cells were differentiated with retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and RA followed by TPA (RA/TPA). RA/TPA treated cells exhibited the highest levels of tyrosine hydroxylase and DAT but lower levels of vesicular monoamine transporter. The kinetics of [3H]DA uptake and [3H]MPP+ uptake to DAT in RA/TPA differentiated cells were similar to that of rat and mouse caudate-putamen synaptosomes. RA/TPA differentiated cells evidenced high sensitivity to the neurotoxic effects of MPP+ (0.03 to 3.0 mM), and the neurotoxic effects of MPP+ were blocked with the DAT inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909). DA-induced cell death was not more sensitive in RA vs RA/TPA differentiated cells and was not inhibited by transporter inhibitors. RA/TPA differentiated cells exhibited 3-fold and 6-fold higher levels, respectively, of DA D2 and D3 receptors than UN or RA differentiated cells. Pretreatment with pramipexole was protective against MPP+ in the RA/TPA differentiated cells but not in undifferentiated or RA differentiated cells. The neuroprotective effect of pramipexole was concentration-dependent and dopamine D2/D3 receptor dependent. In contrast, protection by pramipexole against DA was not DA receptor dependent. Further characterization of the neuroprotective effects of DA agonists in this model system can provide unique information about DA receptor dependent and independent mechanisms of neuroprotection.