Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue.

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

The Influences of Different Ratios of Biphasic Calcium Phosphate and Collagen Augmentation on Posterior Lumbar Spinal Fusion in Rat Model.

PURPOSE: To determine the influence of different ratios of hydroxyapatite (HA)/beta tricalcium phosphate (ß-TCP) and collagen augmentation for posterior lumbar fusion in a rat model. MATERIALS AND METHODS: We generated a posterior lumbar fusion model in 50 rats and divided it into five groups of equal number as follows; 1) autologous bone graft as group A, 2) 70% HA+30% ß-TCP as group B, 3) 70% HA+30% ß-TCP+collagen as group C, 4) 30% HA+70% ß-TCP as group D, and 5) 30% HA+70% ß-TCP+collagen as group E. Rats were euthanized at 12 weeks after surgery and fusion was assessed by manual palpation, quantitative analysis using microCT and histology. RESULTS: The score of manual palpation was significantly higher in group C than group E (3.1±1.1 vs. 1.8±0.8, p=0.033). However, in terms of microCT analysis, group D showed significantly higher scores than group B (5.5±0.8 vs. 3.1±1.1, p=0.021). According to quantitative volumetric analysis, 30% HA+70% ß-TCP groups (group D and E) showed significantly reduced fusion mass at 12 weeks after surgery (123±14.2, 117±46.3 vs. 151±27.3, p=0.008, 0.003, respectively). Collagen augmentation groups revealed superior results in terms of both microCT score and histologic grade. CONCLUSION: A 7:3 HA/ß-TCP ratio with collagen augmentation rather than a 3:7 HA/ß-TCP ratio could be a more favorable graft substitute for lumbar spinal fusion. There was positive role of collagen as an adjunct for spinal bone fusion process.

Optimization of the optical transparency of rodent tissues by modified PACT-based passive clearing.

Recently, a bio-electrochemical technique known as CLARITY was reported for three-dimensional phenotype mapping within transparent tissues, allowing clearer whole-body and organ visualization with CB-perfusion (CUBIC) and leading to the development of whole-body clearing and transparency of intact tissues with the PACT (passive clarity technique) and PARS (perfusion-assisted agent release in situ) methodologies. We evaluated the structure-function relationships in circuits of the whole central nervous system (CNS) and various internal organs using improved methods with optimized passive clarity. Thus, in the present study, we aimed to improve the original PACT procedure and passive clearing protocols for different intact rodent tissues. We determined the optimal conditions for the passive clarity method that allowed the production of a transparent whole CNS by clearing the brain and spinal cord, as well as various organs. We also improved the tissue transparency using mPACT (modified PACT), a method for direct passive clearing, and whole perfusion-based PARS-mPACT, a method for fusion clearing, and we identified the appropriate experimental conditions. These optimized methods can be used for easy and economical high-resolution mapping and phenotyping of normal and pathological elements within intact tissues.

Improvement Characteristics of Bio-active Materials Coated Fabric on Rat Muscular Mitochondria.

This study surveys the improvement characteristics in old-aged muscular mitochondria by bio-active materials coated fabric (BMCF). To observe the effects, the fabric (10 and 30%) was worn to old-aged rat then the oxygen consumption efficiency and copy numbers of mitochondria, and mRNA expression of apoptosis- and mitophagy-related genes were verified. By wearing the BMCF, the oxidative respiration significantly increased when using the 30% materials coated fabric. The mitochondrial DNA copy number significantly decreased and subsequently recovered in a dose-dependent manner. The respiratory control ratio to mitochondrial DNA copy number showed a dose-dependent increment. As times passed, Bax, caspase 9, PGC-1α and ß-actin increased, and Bcl-2 decreased in a dose-dependent manner. However, the BMCF can be seen to have had no effect on Fas receptor. PINK1 expression did not change considerably and was inclined to decrease in control group, but the expression was down-regulated then subsequently increased with the use of the BMCF in a dose-dependent manner. Caspase 3 increased and subsequently decreased in a dose-dependent manner. These results suggest that the BMCF invigorates mitophagy and improves mitochondrial oxidative respiration in skeletal muscle, and in early stage of apoptosis induced by the BMCF is not related to extrinsic death-receptor mediated but mitochondria-mediated signaling pathway.

Which iodinated contrast media is the least cytotoxic to human disc cells?

BACKGROUND: Iodinated contrast media (CM) is commonly used for various intradiscal injections such as in discography and endoscopic spinal surgery. However, CM has been shown to be toxic to renal tissue due to its ionic strength and osmolarity and as a result of iodine-induced cytotoxicity, which has raised concern over whether there are similar negative effects on disc cells. PURPOSE: This in vitro study was designed to identify the least cytotoxic iodinated CM to the human disc cell among four different physiochemical iodinated contrast dyes. STUDY DESIGN: In vitro laboratory study. METHODS: Intervertebral disc tissue was obtained by discectomy from a total of 10 lumbar disc patients undergoing surgery and disc cells were isolated. The human disc cells were grown in 3D alginate bead culture with 0, 0.1, 10, and 100 mg/mL CM solutions (ionic dimer, ionic monomer, non-ionic dimer, and non-ionic monomer) and mannitol as a control for 2 days. The living cells were analyzed with trypan blue staining. Fluorescence-activated cell sorting analysis was performed using Annexin V and propidium iodide (PI) and 3D alginate bead immunostaining to identify live, apoptotic, and necrotic cells. RESULTS: Human disc cell death was time- and dose-dependent in response to CM and more necrosis was observed than apoptosis. In addition, non-ionic dimeric CM (iodixanol) showed the least toxic effect on human disc cells, followed by non-ionic monomeric (iopromide), ionic dimeric (ioxaglate), and ionic monomeric CM (ioxithalamate). CONCLUSIONS: Contrast media is cytotoxic to human disc cells in a dose- and time-dependent manner. This in vitro study revealed that, among four different CM preparations, non-ionic dimeric CM is the least detrimental to human disc cell viability. Careful attention should be paid to the type of CM chosen for discography and endoscopic spinal surgery. It is also necessary to investigate the detrimental effects of CM on disc cells and disc degeneration in further in vivo studies.

Time- and dose-dependent cytotoxicities of ioxitalamate and indigocarmine in human nucleus pulposus cells.

BACKGROUND CONTEXT: Ioxitalamate (Telebrix 300) is an ionic iodinated contrast medium commonly used for discography or percutaneous endoscopic lumbar discectomy (PELD), though it has side effects such as anaphylactic shock and renal toxicity. Indigocarmine is an organic compound dye with a distinctive blue color that is commonly used during PELD to stain the acidic, degenerated nucleus pulposus (NP). Although ioxitalamate and indigocarmine are widely used in spinal surgery, there have been no reports on their effects on NP cells. We studied the toxicities of both ioxitalamate and indigocarmine to NP cells. PURPOSE: To determine the toxicities of both ioxitalamate and indigocarmine to NP cells in vitro. STUDY DESIGN: In vitro, controlled study of the toxicities of both ioxitalamate and indigocarmine to human NP cells. METHODS: Nucleus pulposus cells were obtained via discectomy from lumbar disc patients and isolated. Nucleus pulposus cells were cultured in three-dimensional (3D) alginate beads with 0.001, 0.1, 10, and 100 mg/mL ioxitalamate, 0.00001, 0.001, 0.1, and 10 mg/mL indigocarmine, or a mixture of both for 1, 2, or 3 days. The living cells were analyzed with trypan blue staining. Fluorescence Activated Cell Sorting analysis using Annexin V and propidium iodide and 3D alginate bead immunostaining was performed to identify live, apoptotic, and necrotic cells. RESULTS: Ioxitalamate, indigocarmine, and their combination induced statistically significant NP cell injury that was both time- and dose dependent (p<.05). Also, at the same concentration, ioxitalamate was more cytotoxic than was indigocarmine or the combination (p<.05). All three treatments also showed dose-dependent cytotoxicity according to flow cytometry and immunostaining. CONCLUSIONS: Ioxitalamate and indigocarmine are toxic to human NP cells in vitro in a time- and dose-dependent manner. We assume that ioxitalamate and indigocarmine may have similar effects in patients undergoing discography and PELD. Thus, we suggest that ioxitalamate and indigocarmine should be used carefully at low concentrations.

Triamcinolone decreases bupivacaine toxicity to intervertebral disc cell in vitro.

BACKGROUND CONTEXT: Local anesthetics combined with corticosteroids are commonly used for management of back pain in interventional spinal procedures. Several recent studies suggest cytotoxicity of bupivacaine, whereas others report protective and cytotoxic effects of corticosteroids on chondrocytes and intervertebral disc cells. Considering the frequent use of these agents in spinal interventions, it is meaningful to know how they affect intervertebral disc cells. PURPOSE: This study was conducted to assess the effects of bupivacaine and triamcinolone, both alone and in combination, on viability of intervertebral disc cells in vitro. STUDY DESIGN: Controlled laboratory study. METHODS: Nucleus pulposus cells were isolated from human disc specimens from patients undergoing surgery because of disc herniation or degenerative disc disease. They were grown in three-dimensional alginate beads for 1 week to maintain their differentiated phenotypes and to allow for matrix formation before analysis. After 1 week of culture, the cells were exposed to bupivacaine (0.1%, 0.25%, 0.5%, and 1%) or bupivacaine (0.1%, 0.25%, 0.5%, and 1%) with 1 mg of triamcinolone for 1, 3, or 6 hours. Cell viability was measured using trypan blue exclusion assay and flow cytometry. Live cell/dead cell fluorescent imaging was assessed using confocal microscopy. RESULTS: Trypan blue exclusion assays demonstrated dose- and time-dependent cytotoxic effects of bupivacaine on human nucleus pulposus cells. Similar but reduced cytotoxicity was observed after exposure to the combination of bupivacaine and 1 mg of triamcinolone. Flow cytometry showed a dose-dependent cytotoxic effect of bupivacaine on nucleus pulposus cells after 3 hours of exposure. The reduced cytotoxicity of bupivacaine combined with 1 mg of triamcinolone was also confirmed in flow cytometry. Confocal images showed that the increase in dead cells correlated with the concentration of bupivacaine. Nevertheless, fewer cells died after exposure to several different concentrations of bupivacaine combined with 1 mg of triamcinolone than did after exposure to bupivacaine alone. CONCLUSIONS: The combination of bupivacaine and triamcinolone induced dose- and time-dependent cytotoxicity on human intervertebral disc cells in vitro, but the cytotoxicity was much weaker than that of bupivacaine alone. This study shows a potential protective influence of triamcinolone on intervertebral disc cells.

Nestin expressing progenitor cells during establishment of the neural retina and its vasculature.

In order to test if nestin is a useful marker for various types of progenitor cells, we explored nestin expression in the retina during development. Nestin expression was co-evaluated with bromodeoxyuridine (BrdU) labeling and Griffonia simplicifolia isolectin B4 (GSIB4) histochemistry. Nestin immunoreactivity appears in cell soma of dividing neural progenitor cells and their leading processes in retinas from embryonic day (E) 13 to E20, in accordance with a BrdU-labeled pattern. At postnatal day (P) 5, it is restricted to the end feet of Müller cells. BrdU-labeled nuclei were mainly in the inner part of the inner nuclear layer in postnatal neonates. The retinal vessels demarcated with GSIB4-positive endothelial cells were first distributed in the nerve fiber layer from P3. Afterward the vascular branches sprouted and penetrated deeply into the retina. The endothelial cells positive for GSIB4 and the pericytes in the microvessels were additionally immunoreactive for nestin. Interestingly, the presumed migrating microglial cells showing only GSIB4 reactivity preceded the microvessels throughout the neuroblast layer during vascular sprouting and extension. These findings may suggest that nestin expression represents the proliferation and movement potential of the neural progenitor cells as well as the progenitor cells of the endothelial cell and the pericyte during retinal development. Thus, Müller glial cells might be potential neural progenitor cells of the retina, and the retinal microvasculature established by both the endothelial and the pericyte progenitor cells via vasculogenesis along microglia migrating routes sustains its angiogenic potential.

Comparative expression of matrix-associated genes and inflammatory cytokines-associated genes according to disc degeneration: analysis of living human nucleus pulposus.

STUDY DESIGN: The 2 groups of living human nucleus pulposus were prospectively compared according to disc degeneration. OBJECTIVES: This study was conducted to investigate the expressions of various genes associated with matrix synthesis and expressions of inflammatory cytokines-associated genes according to degrees of disc degeneration in human discs. SUMMARY OF BACKGROUND DATA: Degenerated discs were obtained from 18 patients who underwent discectomy for lumbar disc herniation. Disc degeneration was graded by T2-weighted magnetic resonance imaging using Pfirrmann's grading system. Discs were allocated to 2 groups: group I (9 patients)-mildly degenerated discs (grades II and III) and group II (9 patients)-severely degenerated discs (grades IV and V). METHODS: Cells from the nucleus pulposus were isolated and then cultured as monolayers. The mRNA expressions of aggrecan, type II collagen, Sox9, type I collagen, alkaline phosphatase, osteocalcin, tumor necrosis factor-α, and interleukin-1ß in the 2 groups were compared by real-time polymerase chain reaction, and production of matrix-associate proteins (aggrecan, type II collagen, Sox9, type I collagen, alkaline phosphatase, and osteocalcin) were compared by Western blotting. RESULTS: mRNA expressions in group I were upregulated versus group II to the following extents: 1.83 times for aggrecan, 1.82 times for type II collagen, 1.80 times for Sox9, 1.41 times for type I collagen, 1.38 times for alkaline phosphatase, and 1.80 times for osteocalcin. Furthermore, Western blotting showed that aggrecan, type II collagen, Sox9, type I collagen, alkaline phosphatase, and osteocalcin were higher in group I. However, the mRNA levels of tumor necrosis factor-α and interleukin-1ß were 1.26 and 1.11-fold, respectively, upregulated in group II. CONCLUSIONS: Mildly degenerated discs showed greater matrix, chondrogenic, and osteoblastic gene expressions than severely degenerated discs, indicating that the ability to produce matrix-associated proteins is greater for cells in mildly degenerated than in severely degenerated discs. However, inflammatory cytokine genes associated with disc degeneration were expressed at higher levels in the severely degenerated group. This study shows that a reduction in matrix synthesis and an increase of inflammatory cytokine levels occurs during disc degeneration at the same time.

The role of KRAS mutations in predicting the efficacy of cetuximab-plus-irinotecan therapy in irinotecan-refractory Korean metastatic colorectal cancer patients.

OBJECTIVE: This study evaluated the clinical relevance of KRAS and BRAF mutational status in 66 irinotecan-refrac- tory Korean metastatic colorectal cancer (mCRC) patients treated with cetuximab-plus-irinotecan-based chemotherapy. METHODS: A total of 66 irinotecan-refractory mCRC patients treated with cetuximab-plus-irinotecan-based chemotherapy were included. Tumors were screened for KRAS mutations (codons 12 and 13) and a BRAF mutation (V600E) using direct sequencing and the Snapshot assay. RESULTS: The objective response rate (RR) for treatment was 21.2% (14/66) and skin rashes were observed in 43 (65.2%) of the 66 patients. A KRAS mutation was detected in 27 (40.9%) tumors, and was associated with lower RR (wild-type vs. mutated KRAS: 33.3 vs. 3.7%, p = 0.005) and shorter progression-free survival (PFS) and overall survival (OS; PFS: 6.4 vs. 2.0 months, p = 0.005; OS: 17.8 vs. 7.1 months, p = 0.001). Severe skin toxicity was associated with better RR and longer PFS and OS. BRAF mutations were not detected. Multivariate analysis revealed that KRAS status and skin toxicity were independent predictive factors of PFS and OS. CONCLUSIONS: This study indicates the clinical relevance of KRAS mutations in predicting the efficacy of cetuximab-plus-irinotecan-based chemotherapy in irinotecan-refractory Korean mCRC patients.

Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer.

Cryopreservation could be a useful technique for providing a steady source of oocytes for nuclear transfer and in vitro embryo production. The purpose of this study was to develop a method for cryopreservation of bovine oocytes while maintaining the developmental potential following subsequent in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Following vitrification-thawing, the surviving oocytes were (a) used for parthenogenetic activation, (b) examined for pronuclear formation after IVF, (c) examined for embryo development after IVF, and (d) used for SCNT employing fetal fibroblasts transfected with green fluorescent protein (GFP) gene. While most of the oocytes survived vitrification when the microdrop method was used (92.50%), the cleavage and blastocyst formation rates after parthenogenetic activation were lower (46.5% and 11.1%) than that in the non-vitrified control (86.6% and 13.5%). After IVF, the pronuclear formation (2PN) of fertilized embryos was lower in the vitrified group than in the control (21.7% and 59.9%). After SCNT, fusion rates were similar in control (58.33%) and vitrified-thawed oocytes (53.19%). However, the cleavage (73.1% and 46.3%) and blastocyst formation rates (22.2%, 7.4%; p<0.05) differed between control and vitrified-thawed oocytes. In vitrified-thawed or control oocytes, all embryos reconstructed using fetal fibroblasts transfected with GFP gene showed GFP expression. To evaluate the complete developmental potential, embryos derived from vitrified-thawed and fresh control oocytes were non-surgically transferred to 27 recipients (16 for control and 11 for vitrified-thawed). In the vitrified-thawed group, two pregnancies were detected at day 60, and one of them lasted until day 222. While in the fresh group, one pregnancy maintained to term. In conclusion, vitrified-thawed bovine oocytes could support development into the subsequent stages after IVF and SCNT. In addition, this study showed the possibility of the vitrified-thawed bovine oocytes in the production of transgenic cloned animals. In addition, further studies are required to increase the efficiency of oocyte vitrification for the practical uses and production of live offspring.

Shifting of parvalbumin expression in the rat retina in experimentally induced diabetes.

The AII amacrine cell, a unique rod signal integrator passing through the cone bipolar cell to ganglion cells, uses parvalbumin as a transducer of cytosolic calcium ion signals in the mammalian retina. For clarification of whether AII amacrine cell network contributes to the early neuropathogenesis of diabetic retinopathy, this study first analyzed alteration of parvalbumin expression in experimental diabetic retinas using immunohistochemical methods. Parvalbumin immunoreactivity was found in AII amacrine cells, some amacrine cells of a wide-field type, and displaced amacrine cells of the normal rat retina. During diabetes, cell density of each parvalbumin immunoreactive amacrine cell type showed no large changes despite decrease in immunoreactivity especially in AII amacrine cells. In addition to these parvalbumin immunoreactive amacrine cell types, a type of cone bipolar cells co-expressing glutamate transporter 1b and connecting electrically with AII amacrine cells appeared clearly by 4 weeks of diabetes, and thereafter sharply increased in number to that of AII amacrine cells. Protein levels of parvalbumin throughout the diabetic retinas also showed no large changes, except a transitional slight increase at 4 weeks of diabetes. These results suggest that the parvalbumin expression propagates from AII amacrine cells to a type of cone bipolar cell through electrical synapses due to dysfunction of biased mechanism in calcium ion buffering, caused by diabetic injury, and thus AII amacrine cells are closely involved in neuropathogenesis of ongoing diabetic retinopathy.

Vitrification of immature bovine oocytes by the microdrop method.

This study was conducted to determine the optimal vitrification conditions for immature bovine oocytes using the microdrop method. In experiment 1, the optimal pre-equilibration period for microdrop vitrification was examined. The maturation rate of vitrified oocytes with a 3 min first pre-equilibration period (41.1%) was higher than that of vitrified oocytes with a 0 min first pre-equilibration period (21.4%), and the values of those with a 1 (33.9%) or 5 min (27.4%) first pre-equilibration period were intermediate. The value for a 1 min second pre-equilibration period (44.4%) was significantly higher (P<0.05) than those for a 0.5 (28.6%) and 2 min (21.4%) second pre-equilibration period. In experiment 2, the distribution of microtubules in matured oocytes was investigated. There was no difference among the first pre-equilibration times in terms of the rates of normal spindles in vitrified oocytes. However, this value was significantly higher (P<0.05) in the 1 min group (52.8%) compared with the 0.5 (16.7%) and 2 min groups (12.3%). In experiment 3, we investigated the developmental capacity of immature bovine oocytes vitrified under optimal pre-equilibration conditions (3 min and 1 min for the first and second pre-equilibrations, respectively). Although the total fertilization rates were significantly lower (P<0.05) in the vitrified oocytes (65.6%) compared with the control oocytes (92.4%), there was no difference in the rate of normal fertilization (2PN) between the vitrified (78.6%) and control (82.0%) oocytes. Cleavage and blastocyst rates were significantly lower (P<0.05) in vitrified oocytes (55.7 and 2.3%) than in control oocytes (84.4 and 34.7%). Thus, these results indicated that immature bovine oocytes can survive after microdrop vitrification and subsequently can be cultured to mature oocytes capable of undergoing fertilization in vitro and developing into blastocysts.